Thymocyte activation induces the association of phosphatidylinositol 3-kinase and pp120 with CD5.

CD5 is a glycoprotein expressed on thymocytes, T cells, and a subset of B cells. Antibody-mediated cross-linking studies or studies on CD5 knockout mice implicate CD5 as a co-stimulatory or negative regulatory molecule. CD5 is rapidly phosphorylated on tyrosine (Y) residues following Tcell activation. Y429 and Y441 occur in an imperfect immunoreceptor tyrosine-based activation motif (ITAM)-like sequence. We investigated whether phosphatidylinositol (PI) 3-kinase, which binds to tyrosine-phosphorylated ITAM, interacts with CD5 following T cell activation. PI 3-kinase activity and the regulatory p85 subunit of PI 3-kinase associated with CD5 in pervanadate-stimulated, but not in unstimulated thymocytes. Cellular p85 as well as the recombinant Src homology 2 (SH2) domains of p85 bound a tyrosine-phosphorylated peptide encompassing Y463 with approximately threefold greater affinity than a doubly tyrosine-phosphorylated Y429-Y441 peptide. Binding of the C-SH2 domain to the Y463 phosphopeptide, together with preferential binding of the N-SH2 domain to the Y429-Y441 phosphopeptide, suggests a bivalent interaction. A 120-kDa phosphoprotein (pp120) associated with CD5 and specifically with the Y429-Y441 phosphopeptide in stimulated thymocytes. We conclude that stimulation of thymocytes with pervanadate induces the recruitment of PI 3-kinase and pp120 to CD5.

Characterisation of phosphoprotein complexes associated with rat thymocyte Thy-1.

We have previously demonstrated the non-covalent association of the protein tyrosine kinases p56lck and p60fyn together with a number of substrates for phosphorylation with rat thymocyte Thy-1. Here we present evidence that one of these associated phosphoproteins, p85, is associated by disulphide bridging with another polypeptide, demonstrating that it is an integral membrane protein with an extracellular domain. We also show that phosphatidylinositol 3 kinase activity may be coprecipitated with Thy-1 in Brij 96 thymocyte lysates.

Characterisation of phosphoprotein complexes associated with rat thymocyte Thy-1

We have previously demonstrated the non-covalent association of the protein tyrosine kinases p56lck and p60fyn together with a number of substrates for phosphorylation with rat thymocyte Thy-1. Here we present evidence that one of these associated phosphoproteins, p85, is associated by disulphide bridging with another polypeptide, demonstrating that it is an integral membrane protein with an extracellular domain. We also show that phosphatidylinositol 3 kinase activity may be coprecipitated with Thy-1 in Brij 96 thymocyte lysates

Multimolecular associations of the T-cell antigen receptor.

T cells are activated when the T-cell receptor for antigen (TCR) interacts with an antigenic peptide bound to a major histocompatibility complex (MHC) molecule on the surface of another cell. It is often assumed that T-cell activation is induced by the crosslinking of TCRs. In this article, Albertus Beyers, Louise Spruyt and Alan Williams argue that this mechanism is not generally applicable. They hypothesize that the key event in T-cell activation is the formation of multimolecular complexes consisting of the TCR and several other polypeptides, including CD4 or CD8, CD2, CD5 and the associated tyrosine kinases p59(fyn) and p56(lck).

Molecular associations between the T-lymphocyte antigen receptor complex and the surface antigens CD2, CD4, or CD8 and CD5.

The T-cell antigen receptor (TCR) complex is the key structure involved in signal transduction in T cells. To analyze associations between the TCR complex and other molecules, immunoprecipitations were carried out, followed by phosphorylation of molecules in vitro by tyrosine kinases associated with the precipitated molecules. This provided a sensitive assay for molecular complexes, and associations were demonstrated between the TCR complex and the surface antigens CD2, CD4, or CD8 and CD5 in normal rat T cells. The complexes were readily seen in immunoprecipitates from Brij 96 but not Nonidet P-40 detergent extracts. The multimolecular complexes are associated with the internal tyrosine kinases p56lck and p59fyn. The presence of p56lck associated with CD4 or CD8 was also examined in early thymocytes, natural killer cells, and macrophages. The kinase was present in all cases except that of normal macrophages.

Signal transduction by the CD2 antigen in T cells and natural killer cells: requirement for expression of a functional T cell receptor or binding of antibody Fc to the Fc receptor, Fc gamma RIIIA (CD16).

Crosslinking of CD2 antigen on T lymphocytes and natural killer (NK) cells leads to a rise in cytoplasmic-free Ca2+ concentration ([Ca2+]i). However, CD2 seems unlikely to interact directly with the second messenger pathways since signaling via CD2 is poor in T cells that lack the T cell receptor (TCR) and is absent in L cells or insect cells that express CD2. In contrast, NK cells that are also TCR- can be triggered via CD2, but it is unclear as to whether the CD16 Fc receptor (FcR) may facilitate this effect. The CD16 transmembrane molecule is expressed in a complex with the zeta homodimer or the zeta/gamma heterodimer and these dimers are also associated with the TCR complex. Thus, it seemed that zeta chains may provide the link between signaling on NK cells and T cells. This could be tested on TCR- cells since when CD16 is transfected into T cells it is expressed in a complex with TCR zeta homodimer or the zeta/gamma heterodimer. At first, potentiation of CD2 signaling was seen on TCR- Jurkat cells expressing CD16, but this was found to be dependent on trace levels (1%) of IgG in F(ab')2 antibody preparations. With pure F(ab')2, the effect was lost. Signaling on a rat NK cell line was also re-examined with F(ab')2 antibodies that had no IgG contamination, and again no signal transduction via CD2 was seen. We thus conclude that there is no clear evidence for potent signaling via CD2 on cells that lack a TCR complex and that TCR zeta chain expressed at the cell surface is not sufficient to potentiate signaling via CD2 as measured by an increase in [Ca2+]i.

A prospective study of long-term use of amikacin in a paediatrics department. Indications, administration, side-effects, bacterial isolates and resistance.

Amikacin (Amikin; B-M) was used as the only aminoglycoside for 18 months in a paediatric department within a general hospital because of high levels of resistance of Klebsiella pneumoniae, Pseudomonas aeruginosa and Enterobacter cloacae isolates to tobramycin, gentamicin and netilmicin. Between 1 February 1987 and 31 July 1988, 816 children were treated with a slow intravenous injection at a standardised dose adjusted for weight and age. Respiratory disease was present in 35.8% of 537 neonates, 56.4% of 190 infants and 70.9% of 89 older children. Escherichia coli (65 isolates), Klebsiella species (59 isolates), Enterobacter species (26 isolates) and P. aeruginosa (22 isolates) constituted the most common Gram-negative pathogens. The positive blood culture yield was 7.8%. Satisfactory median peak and trough serum amikacin levels were achieved. No significant renal side-effects were noted. Severe bilateral hearing loss in 1 low-birthweight infant resulted from inadvertent overdosage. At the end of this 18-month surveillance period 97.7% of E. coli, 98.6% of K. pneumoniae, 96.3% of E. cloacae, and 98.0% of P. aeruginosa isolates remained sensitive to amikacin, while resistance of K. pneumoniae to tobramycin, netilmicin and gentamicin decreased significantly (P less than 0.003, P less than 0.001 and P less than 0.007 respectively; chi-square test).

Subsets of thymopoietic rat thymocytes defined by expression of the CD2 antigen and the MRC OX-22 determinant of the leukocyte-common antigen CD45.

The MRC OX-22 monoclonal antibody recognizes a restricted determinant of the rat leukocyte-common antigen (L-CA, CD45), which is expressed on most peripheral T cells and all B cells. In contrast only 2%-3% of thymocytes are OX-22+ and these are now shown to be mostly of the immature CD4-CD8- (double-negative, DN), phenotype with very few of the mature phenotype cells being OX-22+. Analysis of immunoperoxidase sections suggests that the DN OX-22+ cells are located in the cortex. Among the DN cells about 60% are OX-22+ and a similar percentage are positive for CD2 antigen. Double staining showed that OX-22+CD2-, OX-22+CD2+, and OX-22-CD2+ populations can be defined and that these three sets account for approximately 95% of the DN cells. Measurement of the thymopoietic activity of DN subsets showed that OX-22+CD2- and OX-22+CD2+ cells have regenerative capacity whilst OX-22-CD2+ cells do not.

[The existence of an immunoreactive digitalis-like substance in normal people and hypertensive people]. / Die voorkoms van 'n immuunreaktiewe digitalis-soortige stof in 'n normale bevolking en persone met hipertensie.

A study confirmed the existence of an immune reactive digitalis-like substance in normotensive and hypertensive people between the ages of 15 years and 64 years. In 13.6% of the population examined, values higher than 0.15 ng/ml of digitalis-like substances were obtained. The possible presence of this substance in a large proportion of patients should be borne in mind when interpreting digitalis measurements.

Therapeutic monitoring as an aid in rationalizing aminoglycoside dosage techniques in the neonate.

General pharmacokinetic parameters applicable to adults are not suitable in neonatal practice owing to wide interpatient variations in respect of fluid balance, renal clearance and metabolic rates. We attempted to determine whether acceptable blood levels of gentamicin or tobramycin are obtained with dosage regimens and dosage techniques which are generally recommended. Forty neonates receiving aminoglycosides were studied. After administration of the drug as a slow, constant intravenous infusion into the 'Y' connection of the infusion set, peak levels were found to be subtherapeutic. Trough levels were also very low. After administration of the same dose of gentamicin or tobramycin as a bolus into the butterfly connection of the infusion set, however, high therapeutic levels were obtained. We therefore recommend that gentamicin and tobramycin be administered as an intravenous bolus injection and that blood levels be monitored constantly in order to individualize therapy.

Immunoreactive digitalis-like substance in pre-eclampsia.

An endogenous digitalis-like substance (DLS) may be involved in the pathogenesis of essential hypertension and pre-eclampsia. The digoxin levels in maternal and cord blood of 504 randomly selected patients were determined. Since none of the patients received digoxin, these levels indicated a cross-reacting substance (immunoreactive DLS). DLS levels were significantly higher in the cord blood of pre-eclamptic patients than in the cord blood of controls. DLS levels in cord blood increased with the severity of pre-eclampsia, and levels were higher in primigravidas than in multigravidas. The structure and biological activity of DLS must be determined before definite conclusions about its role in the pathogenesis of pre-eclampsia can be made.

Endogenous immunoreactive digitalis-like substance in neonatal serum and placental extracts.

Therapeutic levels of digoxin in the serum of untreated neonates delivered to mothers who had not received the drug prenatally were detected by radio-immunoassay. Digoxin levels in neonates should be interpreted with care because of the unknown contribution by the endogenous digitalis-like substance (DLS) to the level of the drug. Three commercially available radio-immunoassay kits were compared with regard to their sensitivity and reproducibility in detecting the endogenous DLS. The kit from Clinical Assays (Cambridge, Mass., USA) was selected for further investigations. In a series of 35 paired samples of maternal and cord blood the average DLS values in terms of digoxin were 0,52 +/- 0,07 and 0,81 +/- 0,27 ng/ml respectively. This difference is statistically highly significant. In the case of infants with DLS values of 1-1,5 ng/ml in terms of digoxin, approximately 1 week was required to reach nontherapeutic digoxin levels, i.e. below 0,5 ng/ml. Gel chromatography showed that the DLS in neonatal serum was more closely associated with protein than is authentic digoxin. In placental extracts it followed the elution profile of the protein completely, but it shifted to fractions with a lower molecular weight than haemoglobin after trypsinization. The level of DLS in neonatal serum was also increased by more than half its original value by trypsinization. Proteolysis therefore seems to have a releasing effect on DLS. The molecular size of this substance is probably in the same range as that of polypeptides, since it was not dialysable from trypsinized and untreated samples through a membrane with a 22 000 dalton molecular weight cut-off point.

The possible role of endogenous digitalis-like substance in the causation of pre-eclampsia.

Digoxin levels have been reported in neonatal blood when neither the mother nor the baby had received digoxin. An endogenous digoxin-like substance (DLS) that may be causally related to hypertension has been described. Using a commercially available radioimmunoassay kit, we investigated the presence of an immunoreactive DLS in 21 pre-eclamptic mothers, 36 mothers with normal blood pressure (the control group) and their infants. We found mean DLS levels to be higher in cord blood from infants born to the pre-eclamptic mothers than in cord blood from those born to mothers in the control group. Levels were also higher in cord blood than in maternal blood in both the pre-eclamptic and the control groups. DLS seems to be associated with pre-eclampsia. Although further work is needed for verification, a hypothesis on the possible role of DLS in the causation of pre-eclampsia is presented.