Importance of immunoglobulin isotype in therapy of experimental autoimmune encephalomyelitis with monoclonal anti-CD4 antibody

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease mediated by CD4+ T cells. Prior studies have established that monoclonal anti-CD4 antibodies can reverse EAE. To determine whether immunoglobulin isotype plays a role in the therapy of EAE with anti-CD4 antibody, an isotype switch variant family of the mouse IgG1 anti-rat CD4 antibody W3/25 was isolated with the fluorescence-activated cell sorter. The IgG1, IgG2b, and IgG2a W3/25 isotype variants all had identical binding capacities for rat CD4+ T cells. Although all three W3/25 isotypes showed some beneficial effects in the amelioration of EAE, the IgG1 and IgG2a W3/25 antibodies were superior to the IgG2b W3/25 in the treatment of EAE. Multiparameter fluorescence-activated cell sorter analysis of T cell subpopulations from treated rats showed that none of the antibodies of the W3/25 isotype switch variant family substantially depleted CD4+ target cells in vivo. These experiments demonstrate that immunoglobulin isotype is important in the monoclonal antibody therapy of autoimmune disease. They indicate that therapy of EAE may be successful without a major depletion of CD4+ lymphocytes. Immunotherapy may be optimized by selecting an appropriate isotype of a monoclonal antibody.     

Conformation of Leu-Arg-Arg-Ala-Ser-Leu-Gly bound in the active site of adenosine cyclic 3',5'-phosphate dependent protein kinase

Studies utilizing NMR spectroscopy have shown that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) probably binds Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) in one of two extended coil conformations (A or B). The relative reactivities of a series of N-methylated peptides based on the structure of peptide 1 might, therefore, be related to how well each can assume the A or B conformation. From estimates of the magnitude of steric interactions that would be induced by N-methylation of an amide in peptide 1 that is locked in either conformation, the ability of each peptide to form that conformation was predicted. The ability of A-kinase to catalyze phosphorylation of the N-methylated peptides correlated well with the ability of each peptide to form conformation A, but not conformation B. In accord with these findings, the reactivity of an unreactive N-methylated peptide was partially restored by a second change, which allowed the peptide to assume conformation A. These results suggest that, when bound in the enzymatic active site, peptide 1 has a conformation that resembles structure A much more closely than structure B.     

Genetic analysis of murine strains C57BL/6J and C3H/HeJ to confirm the map position ofAth-1, a gene determining atherosclerosis susceptibility

Previous results suggested that strains C57BL/6J and C3H/HeJ differed in a single gene for atherosclerosis susceptibility, calledAth-1. Based on data from recombinant inbred strainsAth-1 was tentatively assigned to chromosome 1 linked toAlp-2. In this report, a cross between C57BL/6 and C3H/HeJ was carried out in order to test whether the tentative map position was correct. Parental strains and F₁ and F₂ progeny were examined. Susceptible alleles ofAth-1, found in C57BL/6, are associated with relatively low levels of high-density lipoprotein (HDL)-cholesterol in animals fed an atherogenic diet; resistant alleles ofAth-1 are associated with relatively high levels of HDL-cholesterol. F₁ progeny have HDL levels that are intermediate between these of the two parental strains. Among the F₂ progeny,Alp-2 andAth-1 cosegregated, providing confirmatory evidence thatAth-1 is linked toAlp-2 on chromosome 1. Three mice recombinant forAlp-2 andAth-1 were found among the 60 chromosomes tested, giving an estimated map distance between these two genes of 5.0±2.8 (SE) cM. The phenotypic characteristics ofAth-1 resemble a genetic trait in humans, hyperalphalipoproteinemia, which is characterized by elevated levels of HDL-cholesterol, reduced risk of heart disease, and increased longevity.This work was supported by Grant HL-32087 from the Heart, Lung, and Blood Institute, National Institutes of Health, Grant 1858 from the Council for Tobacco Research, Grant 86-1387 from the American Heart Association with funds contributed in part by the Alameda, Orange, and Santa Barbara County Chapters, and Grants 85-N132A and 85-N136A from the California Affiliate of the American Heart Association.     

p82H identifies sequences at every human centromere

Summary A cloned alphoid sequence, p82H, hybridizes in situ to the centromere of every human chromosome. After washing under stringent conditions, no more than 8% of the grains are located on any specific chromosome. p82H thus differs from other centromeric sequences which are reported to be chromosome specific, because it detects sequences that are conserved among the chromosomes. Two experimental approaches show that the p82H sequences are closely associated with the centromere. First, p82H remains with the relocated centromeres in an inv(19) and an inv(6) chromosome. Second, p82H hybridizes at the centromere but not to the centromeric heterochromatin of chromosomes 1, 9 and 16 that have elongated 1qh, 9qh and 16qh regions produced by short growth in 5-azacytidine. The only noncentromeric site of hybridization is at the distal end of the 9qh region.     

Cellulases, hemicelluloses and auxin-stimulated growth: a possible relationship

The plant growth promoter, auxin, may loosen the primary cell wall by increasing the activity of extracellular cellulases – a group of enzymes that cleave hemicellulose chains in the walls of both monocotyledons and dicotyledons. Evidence is reviewed that suggests that these hemicellulose chains tether adjacent microfibrils, and that by cleaving such chains the cellulases facilitate cell expansion. On the basis of this structural arrangement a mechanism for elastic and plastic wall extension is proposed.     

Developmental Control of Apiogalacturonan Biosynthesis and UDP-Apiose Production in a Duckweed

Vegetative fronds of Spirodela polyrrhiza were induced to form dormant turions by the addition of 1 micromolar abscisic acid or by shading. The cell wall polymers of fronds contained a high proportion of the branched-chain pentose, d-apiose (about 20% of total noncellulosic wall sugar residues), whereas turion cell walls contained only trace amounts (about 0.2%). When the fronds were fed d-[³H]glucuronic acid for 30 minutes, the accumulated UDP-[³H]apiose pool accounted for about 27% of the total phosphorylated [³H]pentose derivatives; in turions, the UDP-[³H]apiose pool accounted for only about 4% of the total phosphorylated [³H]pentose derivatives. We conclude that the developmentally regulated decrease in the biosynthesis of a wall polysaccharide during turion formation involves a reduction in the supply of the relevant sugar nucleotide. One controlling enzyme activity is suggested to be UDP-apiose/UDP-xylose synthase. However, since there was a 100-fold decrease in the rate of polysaccharide synthesis and only a 9-fold decrease in UDP-apiose accumulation, there is probably also control of the activity of the relevant polysaccharide synthase.     

Antigen recognition in autoimmune encephalomyelitis and the potential for peptide-mediated immunotherapy

Peptide binding and lymph node T cell activation studies have been used to characterize T cell recognition of an encephalitogenic T cell autoantigen from myelin basic protein in (PL/J x SJL)F1 mice. Amino acids that determine interactions with either the restriction element of the major histocompatibility complex (MHC) or the encephalitogenic T cell receptor are defined. This information enables the design of peptides that bind MHC yet do not cross-react with the autoantigen. A peptide analog of the encephalitogenic epitope is shown to be "heteroclitic" for MHC binding and activation of encephalitogenic T cells in vitro. This analog is not immunogenic for encephalitogenic T cells in vivo and is shown to inhibit disease that is induced by the autoantigen itself.     

Interleukin-1 stimulates prostaglandin biosynthesis by human amnion

The purpose of these studies was to determine if Interleukin-1 (IL-1) alters the rate of prostaglandin biosynthesis by human amnion. Primary monolayer cultures of amnion cells were established from women undergoing elective cesarean section before the onset of labor. Natural purified and recombinant human IL-1 alpha and IL-1 beta were incubated with amnion cells in culture, and prostaglandin E2 (PGE2) biosynthesis was measured by radioimmunoassay in cell-free media. A concentration-dependent increase in PGE2 production by amnion cells occurred in response to natural purified and recombinant IL-1 preparations. No differences in the parameters of the dose-response curves between the two IL-1 gene products could be determined (p greater than 0.05). Indomethacin blocked the effect of IL-1 in prostaglandin biosynthesis by human amnion. Interleukin-1, a fever mediator, could serve as a signal for the initiation of labor in cases of intrauterine or systemic infection.     

Novel ligands for the affinity labelling of luteinizing hormone releasing hormone receptors.

A number of novel luteinizing hormone releasing hormone (LHRH) analogues incorporating biotin together with potential covalent attachment sites have been synthesized. Those based on the des-Gly10-[D-Lys6]-LHRH ethylamide peptide backbone resulted in the most useful characteristics of binding to the LHRH receptor in rat anterior pituitary gland membranes. Of these, des-Gly10-[biotinyl-aminoethylglycyl-D-Lys6]-LHRH ethylamide (XBAL) gave the best specific: non-specific binding ratio, with 44 +/- 6% (+/- S.E.M.) of total binding being specific with a Kd of 131 +/- 16 pM (+/- S.E.M., n = 4) as determined by Scatchard analysis. Two methods have been used to covalently crosslink these analogues with the LHRH receptor; photoaffinity labelling and the use of homobifunctional N-hydroxysuccinimide ester crosslinkers. The photoaffinity analogues gave poor specific: non-specific binding ratios. Of the chemical crosslinkers tested, ethylene glycolbis(succinimidylsuccinate) (EGS) was found to be the most efficient at covalently linking the 125I-XBAL bound to the LHRH receptor site. At an EGS concentration of 5 mM, 23 +/- 3% (+/- S.E.M.) of the specific binding of 125I-XBAL was covalently crosslinked.