Activation of acceptor-suppressor hybridoma with antigen-specific suppressor T cell factor of two-chain type: requirement of the antigen- and the I-J-restricting specificity

The molecular mechanisms of activation of immunoregulatory T cells were characterized by using two complementary suppressor T cell hybridoma systems: the KLH-specific monoclonal suppressor factor (KLH-TsF), and the inducible acceptor-suppressor hybridoma line with anti-idiotypic receptor for KLH-TsF. It was demonstrated that the identity of the KLH specificity and genetic specificity was required for the TsF-acceptor interaction. These specificities were found to be mediated by the two polypeptide chains of TsF: KLH-binding, Ct-bearing heavy chain and I-J+ light chain. These two chains were essential for stimulation of the acceptor hybridoma. The results were also confirmed by the findings that the mixture of the 11S and 13S mRNA translation products reconstituted the active TsF to stimulate the acceptor hybridoma. Furthermore, the genetic restriction observed was found to be mediated by the I-J+ light chain and to be governed by the gene linked to the H-2 complex but not to the Igh genes. The gene controlling the restriction specificity was strongly suggested to be in the intra-H-2 complex, but not outside of the H-2 complex.     

Serological discrimination of DR4 haplotypes by radioimmunoassay

Three new alloantigenic specificities of human major histocompatibility complex class 11 molecules have been defined by testing the reactivity of alloantisera at the molecular level. Two of these specificities identify different DR4 haplotypes. The Fe75 specificity is associated with the DR4/DW10 haplotype and the CBC/MRG6 specificity with the DR4/DKT2 haplotype. Both are supertypic specificities and are associated with other DR specificities as well. Both specificities are carried by class 11 molecules belonging to the first DR subset. Together with previously described determinants, these specificities contribute to serological discrimination of the different DR4 haplotypes.     

Effects of W-7 on catecholamine release and 45Ca2+ uptake in cultured adrenal chromaffin cells.

Effects of N-(6-aminohexyl)-5-chloro-1-naph-thalenesulfonamide (W-7), a calmodulin antagonist, on catecholamine (CA) release and ⁴⁵Ca²⁺ uptake were studied using cultured bovine adrenal chromaffin cells. W-7 inhibited the carbamylcholine (CCh)-evoked CA release and ⁴⁵Ca²⁺ uptake in a concentration-dependent manner. The inhibitory effect of W-7 on CCh-evoked CA release was not overcome either by an increase in extracellular calcium or CCh concentration. Although W-7 inhibited the high K⁺-evoked CA release and ⁴⁵Ca²⁺ uptake, potency of the drug was approximately 50–100 fold less than when inhibiting the CCh-evoked CA release and ⁴⁵Ca²⁺ uptake. The inhibitory effects of W-7 were observed both in norepinephrine release and epinephrine release. Moreover, W-7 inhibited the CCh-evoked ⁴⁵Ca²⁺ efflux. These results suggest that the inhibition of CA release by W-7 in adrenal chromaffin cells is mainly due to its inhibition of calcium uptake. W-7 may influence the linkage between acetylcholine-receptor and calcium uptake with higher potency than depolarization-dependent calcium entry.     

Cerebral Blood Flow and Oxidative Metabolism in Conscious Fischer-344 Rats of Different Ages

Abstract: The cerebral metabolic rates for O₂ and for glucose were measured in conscious, fasted male Fischer-344 rats at the ages of 3, 12, and 24 months, and cerebral blood flow was determined with ¹⁴C-iodoantipyrine. The metabolic rates for oxygen and glucose were obtained by multiplying blood flow by the O₂ and glucose concentration differences, respectively, between blood in the femoral artery and in the superior sagittal sinus. Mean cerebral blood flow and the metabolic rates for oxygen and glucose did not differ significantly (p > 0.05) between 3 and 12 or between 12 and 24 months. Nor did the arteriovenous differences for O₂ and for glucose change significantly with age. Because the superior sagittal sinus drains blood mainly from the cerebral cortex, the results indicate that average cerebral cortical oxidative metabolism, and the coupling ratios between the cerebral metabolic rate for oxygen and cerebral blood flow and between the cerebral metabolic rate for glucose and cerebral blood flow, do not change significantly with age in the Fischer-344 rat.     

Two surface antigens expressed on proliferating mouse T lymphocytes defined by rat monoclonal antibodies

A hybrid cell line resulting from the fusion of a Con A-activated normal mouse spleen cell and a transformed mouse T cell (EL-4BU) has been used to prepare and select rat monoclonal antibodies reactive with molecules expressed on the surface of proliferating, as opposed to resting, mouse T cells. In this report, the characterization of two such antigens identified in this way is described. One antigen is a membrane component common to mitogen-activated T and B cells, some bone marrow cells, and various transformed cell lines but is not detectable on either normal thymocytes or the majority of spleen cells by radioimmunoassay or FACS analysis. It has a m.w. of approximately 200,000 daltons under nonreducing conditions and 100,000 daltons under reducing conditions. Antibodies to this antigen precipitate cell-bound transferrin but do not react directly with transferrin itself. It would thus appear that the antigen is the transferrin receptor molecule. The second antigen is not detectable on normal thymocytes, spleen cells, bone marrow cells, or mitogen-stimulated spleen cells but is expressed at high levels on some transformed T cell lines. It, too, appears to be a dimer, with a m.w. of 95,000 daltons under nonreducing conditions, decreasing to 50,000 daltons under reducing conditions. Although the function of the 95,000-dalton antigen is not yet known, its lack of expression on adult T cell populations both before and after activation suggests either a short-lived role at a very early stage of T cell development and/or an association with T cell transformation.     

Expression cloning of a cDNA encoding M1/69-J11d heat-stable antigens

The differentiation Ag identified by the mAb M1/69 and J11d (commonly referred to as heat-stable Ag) are found in structurally heterogeneous forms on the surfaces of many types of murine hemopoietic cells. The extinction of expression of these antigens is associated with thymocyte maturation and Ig class switching in B cells, as well as terminal differentiation of macrophages. A cDNA encoding the M1/69-J11d peptide was cloned from a hemopoietic progenitor cell line by immunoselection of COS cells transfected with expression libraries. The cloned cDNA is a copy of a gene that is transcribed in M1/69-J11d+ lymphoid, myeloid, and erythroid cells. This gene could be responsible for the expression of all forms of the M1/69-J11d Ag, although there are homologous genes that may encode some forms of the Ag that are specifically expressed in bone marrow. The cloned cDNA encodes a surprisingly small peptide, predicted to contain only 30 amino acids after removal of a signal sequence and displacement of the C-terminal region by the glycosyl-phosphatidylinositol group that anchors the peptide to the cell surface. Almost all of the mass of the M1/69-J11d Ag accumulates through extensive N- and O-linked glycosylation at multiple sites in the short peptide. These carbohydrates are likely to execute the functions of M1/69-J11d Ag, which could be specialized to each cell type as a consequence of differential glycosylation.     

Regulation of Histamine Turnover via Muscarinic and Nicotinic Receptors in the Brain

To clarify the regulation of central histaminergic (HAergic) activity by cholinergic receptors, the effects of drugs that stimulate the cholinergic system on brain histamine (HA) turnover were examined, in vivo, in mice and rats. The HA turnover was estimated from the accumulation of tele-methylhistamine (t-MH) during the 90-min period after administration of pargyline (65 mg/kg, i.p.). In the whole brain of mice, oxotremorine, at doses higher than 0.05 mg/kg, s.c., significantly inhibited the HA turnover, this effect being completely antagonized by atropine but not by methylatropine. A large dose of nicotine (10 mg/kg, s.c.) also significantly inhibited the HA turnover. This inhibitory effect was antagonized by mecamylamine but not by atropine or hexamethonium. A cholinesterase inhibitor, physostigmine, at doses higher than 0.1 mg/kg, s.c., significantly inhibited the HA turnover. This effect was antagonized by atropine but not at all by mecamylamine. None of these cholinergic antagonists used affected the steady-state t-MH level or HA turnover by themselves. In the rat brain, physostigmine (0.1 and 0.3 mg/kg, s.c.) also decreased the HA turnover. This inhibitory effect of physostigmine was especially marked in the striatum and cerebral cortex where muscarinic receptors are present in high density. Oxotremorine (0.2 mg/kg, s.c.) and nicotine (1 mg/kg, s.c.) also decreased the HA turnover in the rat brain. However, these effects showed no marked regional differences. These results suggest that the stimulation of central muscarinic receptors potently inhibits the HAergic activity in the brain and that strong stimulation of central nicotinic receptors can also induce a similar effect.     

VH sequence of a human anti-Sm autoantibody. Evidence that autoantibodies can be unmutated copies of germline genes

The utilization of germline genes for the synthesis of autoantibodies has been suspected for many years based on the presence of cross-reactive idiotypes among patients as well as in some healthy first-degree relatives of patients with several autoimmune diseases including SLE. One such system of idiotypes involves anti-Sm antibodies, which are highly specific for SLE. To definitively establish the utilization of germline genes in the Sm system, we produced human-human B cell hybridomas from a patient with SLE who had circulating anti-Sm antibodies. One stable hybridoma designated 4B4 secretes an IgM-kappa mAb that binds Sm and shares idiotypic determinants with other anti-Sm antibodies. A second anti-Sm antibody (3C3), isolated from the same patient was also studied. Oligo(dT) priming was used to produce cDNA corresponding to full length IgM. Sequence analysis revealed that the VH gene segment (1-96) of 4B4 is identical to a VH sequence previously detected in a fetal liver cDNA library by Schroeder and his co-workers as well as a germline VH recently described by Berman and his associates. The identity of a lupus mAb and sequences derived from unrelated individuals provides strong evidence that this autoantibody is a direct copy of a germline gene.     

Arg74 in HLA-DRBI and DRB3 controls a DR3-related epitope

TR81 is a specificity closely related to or identical with DR3. In Caucasoids two amino acids, Tyr at position 26 and Arg at position 74 of HLA class II DR chains, have been found to be associated with the presence of TR81. Recently, a variant of DRBI *03 identified in American Blacks has been shown to possess Arg at position 74 but Phe at position 26. This codon combination is found to be present in four other cell lines where it still specifies the TR81 determinant. This suggests that the TR81 specificity is uniquely dependent on the presence of Arg at position 74.     

Carbachol-Induced Cosecretion of Immunoreactive Atrial Natriuretic Peptides with Catecholamines from Cultured Bovine Adrenal Medullary Cells

The distribution and secretion of atrial natriuretic peptides (ANPs) were investigated in bovine adrenal medulla. (1) Cultured bovine adrenal medullary cells (2 x 10(6)/dish) contained 100.4 +/- 6.0 fmol of immunoreactive ANP (IR-ANP) and 207.3 +/- 6.6 nmol of catecholamines as epinephrine plus norepinephrine. (2) Stimulation of nicotinic but not muscarinic acetylcholine receptors caused a cosecretion of IR-ANP and catecholamines corresponding to the ratio of IR-ANP to catecholamines in cultured bovine adrenal medullary cells. (3) Carbachol-stimulated secretion of IR-ANP was dependent on the presence of extracellular Ca2+. (4) Chromaffin granules isolated from bovine adrenal medulla contained large amounts of IR-ANP and catecholamines, in the same ratio as did cultured adrenal medullary cells. (5) Reverse-phase HPLC analysis showed that both stored and secreted IR-ANP consisted of two components, which eluted at the position of ANP(99-126) or ANP(1-126). These results indicate that ANPs are stored as ANP(99-126) and ANP(1-126) in chromaffin granules, and are cosecreted in parallel with catecholamines in a Ca2+-dependent manner by the stimulation of nicotinic acetylcholine receptors.