A new immunomodulating compound (AS-101) with potential therapeutic application

There has been interest in the potential of synthetic compounds to modify immune responses by imitation of cytokine action. Direct administration of interleukin 2 (IL-2) in conjunction with adoptive transfer of lymphokine activated killer cells has been used in the treatment of cancer, but there are toxic effects resulting from the high doses of IL-2 required. We have developed a new synthetic compound, ammonium tri-chloro(dioxoethylene-O,O'-)tellurate (AS-101), which has immunomodulating properties and minimal toxicity. The effects of AS-101 on the activation and function of immunocompetent cells have been assessed. We have found that AS-101 induces proliferation and IL-2 production by human lymphocytes in vitro, and enhances the production of IL-2 and colony-stimulating factor by mouse spleen cells. Splenocytes of BALB/c mice injected with AS-101 increased production of IL-2 and CSF in vitro in the presence of mitogen. Mononuclear cells of normal donors acquired responsiveness to recombinant IL-2 and bound monoclonal antibody to IL-2 receptor after incubation with AS-101. Splenocytes of mice treated in vivo with AS-101 expressed high levels of IL-2 receptor. The stimulation of lymphocytes by AS-101 apparently involves an increase in intracellular free calcium. AS-101 administered systemically to mice mediated antitumour effects which could be attributable to its immunomodulatory properties. In addition, AS-101 could directly enhance the ratio of OKT4 to OKT8-positive cells in cultured mononuclear cells from AIDS (acquired immune deficiency syndrome) patients. These results indicate that AS-101 is potentially useful in the treatment of clinical conditions involving immunosuppression.     

HIV F/3' orf encodes a phosphorylated GTP-binding protein resembling an oncogene product

Apart from the retroviral gag, pol and env the HIV genome contains the F (3' orf) gene which encodes a polypeptide of 206 amino acids which is myristylated at the N-terminal and whose function is unknown. We have expressed the F gene in Escherichia coli and from a recombinant vaccinia virus, VVTGfHIV. The F-protein produced in VVTGfHIV-infected mammalian cells is myristilated, and is phosphorylated by protein kinase C at a residue close to the N-terminus like pp60-src (ref. 5). Purified bacterial F-protein also shows the GTPase, autophosphorylation and GTP-binding activities reported for the ras gene product. Furthermore, we show that expression of F in a CD4+ cell line down-regulates the CD4(T4) antigen. These results suggest that F is important in the pathophysiology of AIDS (acquired immune deficiency syndrome).     

Micromolar concentrations of Zn2+ antagonize NMDA and GABA responses of hippocampal neurons

NMDA (N-methyl-D-aspartate) receptors serve as modulators of synaptic transmission in the mammalian central nervous system (CNS) with both short-term and long-lasting effects. Divalent cations are pivotal in determining this behaviour in that Mg2+ blocks the ion channel in a voltage-dependent manner, and Ca2+ permeates NMDA channels. Zn2+ could also modulate neuronal excitability because it is present at high concentrations in brain, especially the synaptic vesicles of mossy fibers in the hippocampus and is released with neuronal activity. Both proconvulsant and depressant actions of Zn2+ have been reported. We have found that zinc is a potent non-competitive antagonist of NMDA responses on cultured hippocampal neurons. Unlike Mg2+, the effect of Zn2+ is not voltage-sensitive between -40 and +60 mV, suggesting that Zn2+ and Mg2+ act at distinct sites. In addition, we have found that Zn2+ antagonizes responses to the inhibitory transmitter GABA (gamma-aminobutyric acid). Our results provide evidence for an additional metal-binding site on the NMDA receptor channel, and suggest that Zn2+ may regulate both excitatory and inhibitory synaptic transmission in the hippocampus.     

Millisecond X-ray diffraction and the first electron density map from Laue photographs of a protein crystal

Attempts to use X-ray crystallography to extract three-dimensional information on transient phenomena in crystals have been hampered primarily by long data collection times. Here we report on the first difference Fourier map obtained from Laue diffraction photographs of a protein crystal, glycogen phosphorylase b. Data collection time was 3 s using the high-intensity white X-radiation generated on the wiggler magnet of the Daresbury Synchrotron Radiation Source (SRS), but data acquisition in the millisecond-submillisecond range is possible. The method presented here uses a simple difference technique and was designed to analyse structural changes relative to a known starting structure. The combination of this approach with cine techniques allows the recording of three-dimensional motion pictures at atomic resolution and opens up new areas in structural biology and chemistry.     

Cross-talk between cellular signalling pathways suggested by phorbol-ester-induced adenylate cyclase phosphorylation

Receptor-mediated activation of both adenylate cyclase and phosphatidylinositide hydrolysis systems occurs through guanine nucleotide regulatory proteins and ultimately leads to specific activation of either cyclic AMP-dependent protein kinase A or Ca2+/phospholipid-dependent protein kinase C. Given the remarkable diversity of agents that influence cellular metabolism through these pathways and the similarities of their components, interactions between the two signalling systems could occur. In fact, stimulation of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a phorbol ester that activates protein kinase C, influences hormone-sensitive adenylate cyclase. In some cells TPA induces desensitization of receptor-mediated stimulation of adenylate cyclase, whereas in others, such as frog erythrocytes, phorbol ester treatment results in increased agonist-stimulated as well as basal, guanine nucleotide- and fluoride ion-stimulated adenylate cyclase activities. We show here that TPA produces phosphorylation of the catalytic unit of adenylate cyclase in frog erythrocytes. Moreover, purified protein kinase C can directly phosphorylate in vitro the catalytic unit of adenylate cyclase purified from bovine brain. These results suggest that phosphorylation of the catalytic unit of adenylate cyclase by protein kinase C may be involved in the phorbol ester-induced enhancement of adenylate cyclase activity. In addition to providing the first direct demonstration of a covalent modification of the catalytic unit of adenylate cyclase, these results provide a potential biochemical mechanism for a regulatory link between the two major transmembrane signalling systems.     

Prevention of vaccinia virus infection in immunodeficient mice by vector-directed IL-2 expression

Recombinant vaccinia viruses have been proposed as live vaccines against a variety of infectious diseases, including AIDS (acquired immune deficiency syndrome). Objections have been concerned primarily with side effects of the vaccinia virus vector itself. Recently it has been shown that inactivation of the vaccinia virus thymidine kinase gene or deletion of certain other non-essential genes is associated with a marked reduction in pathogenicity. Nevertheless, the ability of vaccinia virus to produce a progressive infection in immunodeficient individuals remains a most serious problem. Indeed, an incident of this type in a vaccinated man seropositive for human immunodeficiency virus was recently reported. We have used immunodeficient athymic nude mice to establish a model of disseminated vaccinia virus infection, and to demonstrate a novel approach to virus attenuation which involves insertion of a gene encoding human interleukin-2 into the genome of vaccinia virus vectors.     

T-cell receptors of human suppressor cells

Cells which can suppress the immune response to an antigen (TS cells) appear to be essential for regulation of the immune system. But the characterization of the TS lineage has not been extensive and many are sceptical of studies using uncloned or hybrid T-cell lines. The nature of the antigen receptor on these cells is unclear. T cells of the helper or cytotoxic lineages appear to recognize their targets using the T-cell receptor (TCR) alpha beta-CD3 complex. TCR beta-gene rearrangements are also found in some murine and human suppressor cell lines but others have been shown not to rearrange or express the beta-chain or alpha-chain genes. We previously established TS clones derived from lepromatous leprosy patients which carry the CD8 antigen and recognize antigen in the context of the major histocompatibility complex (MHC) class II molecules in vitro. We here report the characterization of additional MHC-restricted TS clones which rearrange TCR beta genes, express messenger RNA for the alpha and beta chains of the TCR and express clonally unique CD3-associated TCR alpha beta structures on their cell surface but do not express the gamma chain of the gamma delta TCR on the cell surface. We conclude that antigen recognition by at least some human CD8+ suppressor cells is likely to be mediated by TCR alpha beta heterodimers.     

AIDS virus-specific cytotoxic T lymphocytes in lung disorders

Human immunodeficiency virus (HIV) is implicated in the development of AIDS (acquired immune deficiency syndrome). HIV infection leads to the generation of HIV-specific thymus-derived (T) lymphocytes in humans and apes. We describe an experimental system permitting the quantitative and systematic analysis of HIV-specific cytotoxic T lymphocytes (CTL). Functional, HIV-specific CTL are obtained by broncho-alveolar lavage (BAL) from the lungs of seropositive patients with lymphocytic alveolitis. These alveolar CTL: (1) recognize and kill HIV-infected alveolar macrophages in vitro under autologous, but not heterologous, conditions; (2) correspond to standard CTL as they express the CD3 and CD8 surface markers, but not the CD4 marker; and (3) are restricted by class I HLA transplantation antigens in their cytotoxic activities. We propose the hypothesis that interactions between HIV-specific CTL and infected macrophages induce major inflammatory reactions in seropositive patients.     

Ligand binding to the beta-adrenergic receptor involves its rhodopsin-like core

Recently the genes for several hormone receptors that interact with guanine nucleotide binding proteins (G proteins) have been cloned, including the hamster beta 2-adrenergic receptor (beta 2AR), a human beta AR, the turkey erythrocyte beta AR and the porcine muscarinic acetylcholine receptor (MAR). All these receptors share some amino-acid homology with rhodopsin, particularly in 7 hydrophobic stretches of residues that are believed to represent transmembrane helices. To determine whether differences in ligand specificity result from the divergence in the sequences of the hydrophilic regions of these receptors, we have expressed in mammalian cells genes for the wild-type hamster and human beta AR proteins, and a series of deletion mutant genes of the hamster beta 2AR. The pharmacology of the expressed receptors indicates that most of the hydrophilic residues are not directly involved in the binding of agonists or antagonists to the receptor. In addition, we have identified a mutant receptor that has high agonist affinity but does not couple to adenylate cyclase.     

The role of clonal selection and somatic mutation in autoimmunity

Polyclonal activation has been proposed as the reason that autoantibodies are produced during autoimmune disease. This model denies a role for specific antigen selection of B cells and predicts instead a multiclonal population of unmutated or randomly mutated autoantibodies. We have found that the genetic features and clonal composition of spontaneously derived immunoglobulin G (IgG) antiself-IgG (rheumatoid factor (RF] autoantibodies derived from the autoimmune MRL/lpr mouse strain are inconsistent with both the predictions of this model and the actual outcome of experimental polyclonal activation. Instead we have found that MRL/lpr RFs are oligoclonal or even monoclonal in origin. They harbour numerous somatic mutations which are distributed in a way that suggests immunoglobulin-receptor-dependent selection of these mutations. In this sense, the MRL/lpr RFs resemble antibodies elicited by exogenous antigens after secondary immunization. The parallels suggest that, like secondary immune responses, antigen stimulation is important in the generation of MRL/lpr RFs.