Identification of a tumor-specific allo-HLA-restricted γδTCR.

γδT cells are key players in cancer immune surveillance because of their ability to recognize malignant transformed cells, which makes them promising therapeutic tools in the treatment of cancer. However, the biological mechanisms of how γδT-cell receptors (TCRs) interact with their ligands are poorly understood. Within this context, we describe the novel allo-HLA-restricted and CD8α-dependent Vγ5Vδ1TCR. In contrast to the previous assumption of the general allo-HLA reactivity of a minor fraction of γδTCRs, we show that classic anti-HLA-directed, γδTCR-mediated reactivity can selectively act on hematological and solid tumor cells, while not harming healthy tissues in vitro and in vivo. We identified the molecular interface with proximity to the peptide-binding groove of HLA-A*24:02 as the essential determinant for recognition and describe the critical role of CD8 as a coreceptor. We conclude that alloreactive γδT-cell repertoires provide therapeutic opportunities, either within the context of haplotransplantation or as individual γδTCRs for genetic engineering of tumor-reactive T cells.

Biochemical characterization and worldwide distribution of serologically distinct lipopolysaccharides of Haemophilus influenzae type b.

The sugar composition and the electrophoretic mobility in SDS-polyacrylamide gel electrophoresis of the various lipopolysaccharides (LPS) from clinical isolates of Haemophilus influenzae type b (Hib) were determined to correlate epidemiologic data with compositional data. Rabbit sera specific in Ouchterlony immunodiffusion for 10 different LPS (LPS 1-10) reacted with 647 or 690 Hib strains isolated from patients with invasive disease in various continents. Serotype 1 was predominant and was found in 550 isolates (80%). None of the Hib isolates reacted with antisera specific for LPS of two nonencapsulated isolates (LPS 5 and 6). Sugar analysis by gas-liquid chromatography of trimethylsilylated methyl glycosides revealed that the LPS of the 10 serotypes contained glucose, galactose, L-glycero-D-mannoheptose, and glucosamine in various proportions. LPS 1, 2, 8, and 9 contained the highest amounts of glucose and galactose relative to L-glycero-D-mannoheptose, which is considered present in constant amounts in H. influenzae LPS. LPS 1, 2, and 9 were most frequently found in invasive disease isolates.

Comparison of antibiotic resistant and sensitive strains of Haemophilus influenzae type b in The Netherlands by outer-membrane protein subtyping.

The percentage of beta-lactamase producing Haemophilus influenzae strains from patients with meningitis in The Netherlands increased from 0% in 1975/1976 to 4.6% in 1985/1986 (n = 1559). Twenty-three isolates resistant to ampicillin, penicillin, chloramphenicol, rifampicin and/or tetracycline were subtyped to determine if one resistant strain was spreading. (Sub)typing was performed by capsular typing, analysis of the major outer membrane protein patterns on sodium dodecylsulfate gels (SDS-PAGE subtypes), lipopolysaccharide serotyping and biotyping. The (sub)types of the resistant strains were similar to those of sensitive strains, thus indicating that antibiotic resistant strains develop at random.

Clinical experience with transfusion of leukocyte-poor platelet concentrates prepared by filtration with prostacyclin.

Repeated transfusions with platelets from randomly selected donors lead to HLA alloimmunization in about 50% of patients due to lymphocyte contamination of platelet concentrates. Attempts to remove the leukocytes from the platelet concentrates by additional centrifugation steps led to substantial loss of platelets. We report a new procedure for removal of almost all leukocytes with excellent platelet recoveries. Single donor concentrates are treated with 50 ng/mL prostacyclin to inactivate the platelets transiently. The concentrates are then passed through a cellulose-acetate filter to remove the leukocytes. In 30 concentrates this treatment reduced the contamination by leukocytes to less than 0.1 million per concentrate with a platelet recovery of 89% +/- 1% (mean +/- SEM). Thirty filtered platelet concentrates transfused to ten thrombocytopenic patients within one hour after filtration were well tolerated and led to corrected count increments of (22.0 +/- 1.1) X 10(6)/mL blood after one hour and normal survival thereafter. In four of five patients these concentrates reduced the bleeding time. We conclude that transient inactivation of platelets by prostacyclin enables optimal removal of leukocytes and may help to reduce alloimmunization during frequent transfusions with platelet concentrates.

Inhibition of Na+/H+ exchange reduces Ca2+ mobilization without affecting the initial cleavage of phosphatidylinositol 4,5-bisphosphate in thrombin-stimulated platelets.

Stimulation of human platelets increases cytoplasmic pH (pHi) via activation of Na+/H+ exchange. We have determined the effect of inhibiting Na+/H+ exchange on (i) thrombin-induced Ca2+ mobilization and (ii) turnover of 32P-labelled phospholipids. Blocking Na+/H+ exchange by removal of extracellular Na+ or by ethylisopropylamiloride (EIPA) inhibited Ca2+ mobilization induced by 0.2 U/ml thrombin, whereas increasing pHi by NH4Cl enhanced the thrombin-induced increase in cytosolic free Ca2+. The effect of EIPA was bypassed after increasing pHi by moneasin. The thrombin-induced cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) was unaffected by treatments that blocked Na+/H+ exchange or increased pHi. It is concluded that activation of Na+/H+ exchange is a prerequisite for Ca2+ mobilization in human platelets but not for the stimulus-induced hydrolysis of PIP2.

Characteristics of major outer membrane proteins of Haemophilus influenzae.

Several properties of Haemophilus influenzae outer membrane proteins were analyzed to define related proteins in various isolates. H. influenzae type b 760705 had six major outer membrane proteins with the following characteristics. Protein a (Mr, 47,000) demonstrated heat modifiability in sodium dodecyl sulfate; its apparent molecular weight was 34,000 at temperatures below 60 degrees C. This protein was extracted from cell envelopes by using Triton X-100-10 mM MgCl2; in cell envelope preparations, the protein was degraded by trypsin. Proteins b (Mr, 41,000) and c (Mr, 40,000) were insensitive to trypsin degradation, were not heat modifiable in sodium dodecyl sulfate, and were peptidoglycan associated in 0.5% Triton X-100-0.2% sodium dodecyl sulfate. The amount of protein b was reduced in ultrasonically obtained cell envelopes. Protein d (Mr, 37,000) was heat modifiable in sodium dodecyl sulfate with an Mr of 28,000 at temperatures below 100 degrees C and was degraded by trypsin, leaving a membrane-bound fragment of Mr, 27,000. Both the intact and degraded proteins were immunologically cross-reactive with the heat-modifiable OmpA protein of Escherichia coli K-12. Protein d was absent in LiCl-EDTA extracts of cells. Protein e (Mr, 30,000), invariably present in all H. influenzae strains tested, was insensitive to trypsin and absent in LiCl-EDTA extracts of cells. Protein k (Mr, 58,000) was extracted from cell envelopes with 2% Triton X-100-10 mM MgCl2 and, in cell envelopes, appeared to be sensitive to trypsin degradation. Proteins with similar properties to those of proteins a to k were found in 10 other H. influenzae b strains, reference strains with serotype a, c, d, e, and f capsules, and 18 of 20 nonencapsulated strains. Their relative molecular weights, however, varied.

Homogeneity of cell envelope protein subtypes, lipopolysaccharide serotypes, and biotypes among Haemophilus influenzae type b from patients with meningitis in The Netherlands.

Eighty strains of Haemophilus influenzae type b were randomly selected from 531 strains collected between 1975 and 1982 from patients with meningitis in The Netherlands. Subtyping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that 67 of 80 isolates had identical major outer membrane protein patterns (subtype 1). Among the 13 other isolates four different polyacrylamide gel electrophoresis patterns were observed, two of which closely resembled subtype 1. Lipopolysaccharides were characterized immunologically by immunoprecipitation (Ouchterlony technique) and the gel-immuno-radio-assay. Four serotypes were found among the 80 selected strains, leaving one strain not typable. Seventy-four strains (93%) belonged to the same lipopolysaccharide serotype; 77 (97%) of 80 of the strains belonged to biotype I. Sixty strains (75%) had identical major outer membrane protein patterns (subtype 1), lipopolysaccharide serotypes (serotype 1), and biotypes (I).