B cell antigen receptor desensitization: disruption of receptor coupling to tyrosine kinase activation.

Antigen binding to the B cell receptor (BCR) induces receptor desensitization, a condition characterized by cellular unresponsiveness to subsequent Ag stimulation despite the continued ability to bind Ag. To better understand the molecular mechanism of this unresponsiveness, we have used complementary lymphoma (K46 mu) and Ig transgenic (3-83 mu delta) mouse models to study regulation of BCR signaling. Our findings in the lymphoma model show that an initial Ag encounter renders receptors unresponsive to subsequent Ag challenge, as measured by their inability to mobilize Ca2+ and to mediate phosphorylation of receptor-proximal kinases, including Lyn, Blk, and Syk. Most importantly, the Ig alpha and Ig beta components of desensitized receptors are not phosphorylated, and receptor-associated kinases are not activated upon Ag challenge. The molecular defect does not appear to result from Lyn inactivation, sequestration, or repression, since Lyn from desensitized cell lysates is activated in vitro by synthetic doubly phosphorylated immunoreceptor tyrosine-based activation motif peptides. A similar deficit in Ag-induced receptor phosphorylation was observed in desensitized B cells from 3-83 mu delta transgenic mice. These studies indicate that Ag receptor desensitization reflects an inability to initiate activation of receptor-associated kinases that normally phosphorylate receptor Ig alphabeta subunits, leading to signal propagation.

[Fluidity of hepatic microsomal membrane and its relation with aging]. / Fluidificación de la membrana microsomal hepática y su relación con el envejecimiento.

The effect of aging on hepatic microsomal membrane phospholipid composition was studied composition was studied in both young (2 months) and mature (6 months) Wistar rats. When total microsomal phospholipid content was analysed the aged group showed a significant increment (73%). Microsomal phospholipid pattern also showed a different behavior between both groups, with a significative increase in phosphatidylcholine (62%), phosphatidylserine (124%), phosphatidylinositol (31%) and sphingomyelin (10%) and appearance of phosphatidylethanolamine and phosphatidylglycerol in the six-month group. A higher microsomal membrane fluidity in the aged animals was revealed by the increase in PC/EM index (47%). This increment jin fluidity during aging process may reflect an adaptative response resulting in changes on the enzyme activities responsible for drug and carcinogen metabolism.

[Phospholipid composition of the hepatic microsomal membrane and its relationship to bilirubin UDP glucuronyltransferase in human cholestasis]. / Composición fosfolipídica de la membrana microsomal hepática y su relación con la actividad de la bilirrubina UDP-glucuroniltransferasa en colestasis humana.

A number of morphological and functional changes on liver cells were reported during experimental cholestasis. Some specific metabolic pathways catalyzed by "membrane bound" enzymes were described to be altered by lipid microenvironment changes. The purpose of he present study is to establish Bilirubin UDP-Glucuronyltransferase activity--a microsomal integral enzyme responsible for bilirubin conjugation--and microsomal phospholipid profile in cholestatic and normal patients. Surgical liver biopsies were taken fron five patients suffering prolonged extrahepatic cholestasis, and five patients submitted to abdominal surgery excluding hepato-biliary diseases that were considered as controls. The following biochemical parameters were determined in both groups: bilirubin concentration, alkaline phosphatase, gamma-glutamyltranspeptidase, oxalacetic and pyruvic transaminases, and pseudo-cholinesterase activities. Serum cholestatic markers showed significative increments in cholestatic patients (Table 1). Total Bilirubin UDP-Glucuronyltransferase activity was similar comparing normal and cholestatic individuals (1.11 +/- 0.66 and 1.93 +/- 0.82 nmol conjugated bilirubin/mg protein in 10 min. respectively). When final reaction product was analysed, the normal group showed 80% of bilirubin diglucuronide; but resulted undetectable in cholestatic patients yielding 100% of bilirubin monoglucuronide. Microsomal phospholipid analysis showed a decrease in phosphatidylcholine and phosphatidylethanolamine contents in the cholestatic group; probably due to the action of bile acids accumulated into the hepatic cells. Simultaneously we found an increment in phosphatidylserine and sphingomyelin levels in cholestatic patients compared to normals (Figure 1). This fact could be explained by the existence of special sites in the membrane for the latter phospholipids, protected against bile acids detersive action.(ABSTRACT TRUNCATED AT 250 WORDS)

Demethylated CD8 gene in CD4+ T cells suggests that CD4+ cells develop from CD8+ precursors.

Mature T cells and medullary thymocytes bear either the CD4 or CD8 differentiation antigen. Precursor cells in the thymus express neither CD4 nor CD8 (CD4-8-), but most cortical thymocytes are CD4+8+. Whether CD4+ and CD8+ mature T cells arise directly from CD4-8- precursors or from a CD4+8+ intermediate remains unresolved. In this study, methylation of the CD8 gene in murine T cells and thymocytes was examined. There was progressive demethylation of the CD8 gene in the thymus during the transition from CD4-8- to CD4+8+. A similar pattern of demethylation of the CD8 gene was seen in CD4+ mature T cells, suggesting previous expression of CD8 in the CD4+ lineage.

Remethylation at sites 5' of the murine Lyt-2 gene in association with shutdown of Lyt-2 expression.

We have used hybridomas made by fusing the Lyt-2- AKR thymoma, BW5147, to Lyt-2+ SJL/J lymph node cells to study the regulation of Lyt-2 expression. Fusions of this type yielded hybridomas, the majority of which failed to express Lyt-2. In the minority of hybridomas that did express surface Lyt-2, expression was transient and greatly diminished in terms of molecules of Lyt-2 per cell. Lack of Lyt-2 expression was not due to loss of the gene encoding this cell surface molecule; rather, negative regulation of Lyt-2 appeared to be at the level of transcription (i.e., no Lyt-2 transcripts were detected in these hybridomas). We have shown that the Lyt-2 gene is undermethylated in normal Lyt-2+ T cells, whereas the gene is heavily methylated in Lyt-2- liver cells and in BW5147. Loss of Lyt-2 expression in (BW5147 x Lyt-2+ SJL/J lymph node cell) hybridomas was associated with remethylation of DNA within the Lyt-2 gene and at sites 5' of the Lyt-2 gene.

Role of macrophage-myelin basic protein interaction in the induction of experimental allergic encephalomyelitis in Lewis rats.

Stimulated peritoneal exudate cells (PEC) containing activated macrophages (M phi) of Lewis (Le) rats were exposed for 1 hr in vivo or in vitro to radioiodinated soluble myelin basic protein (MBP) or MBP incorporated into magnetic microspheres (MBP-microspheres). The uptake by M phi of the dose of microsphere-bound MBP averaged 6.2%, whereas the average uptake of soluble MBP was 0.17%. Naive rats were sensitized with M phi-associated MBP or M phi-associated MBP-microspheres via the hind footpads without the aid of conventional immunologic adjuvants. Draining lymph node cells (LNC) or spleen cells from sensitized rats were cultured for 3 days with guinea pig MBP (GPMBP) alone or in combination with concanavalin A (Con A), then injected i.v. into naive recipients. Clinical signs of experimental allergic encephalomyelitis (EAE) appeared 6 days after transfer of LNC or spleen cells, and typical CNS lesions were seen in recipients sacrificed 10 to 14 days after transfer. The challenge of MO-MBP-sensitized rats with MBP-CFA resulted in severe clinical signs of EAE marked by an accelerated onset of neurologic symptoms.

Albumin magnetic microspheres: a novel carrier for myelin basic protein.

Myelin basic protein (MBP) of guinea pig origin was incorporated into magnetically responsive albumin microspheres. Protein-protein bonding and stabilization of the GPMBP microspheres by heating at 120 degrees C did not adversely influence their capacity to bind anti-MBP antibodies or demonstrably alter the encephalitogenic activity of the incorporated GPMBP. The magnetic properties of the particles and the fact that immunodeterminants of some of the incorporated MBP fortuitously were distributed on the exterior surfaces of the microspheres allowed a number of experiments to be carried out in Lewis rats for the first time: (a) selective capture and deletion of that particular subpopulation of lymphoid cells responsible for transfer of experimental allergic encephalomyelitis (EAE) represented within the lymph node cells (LNC) of donor animals sensitized to neutral antigen, (b) enhancement of in vivo uptake of MBP by macrophages (M phi s) contained in oil-induced peritoneal cell exudates and exposed briefly to MBP microspheres, and (c) preparation of cell suspensions specifically enriched with respect to MBP-containing M phi s.