Stimulation of the respiratory burst in peripheral blood monocytes by lipoteichoic acid. The involvement of calcium ions and phospholipase A2

Lipoteichoic acid (LTA) from Streptococcus faecalis stimulates the respiratory burst in peripheral blood monocytes (mon), as measured by cytochrome C reduction. The effect of LTA was time and dose dependent. LTA stimulated the respiratory burst in a biphasic manner within a range of 1 to 1000 ng/ml.10(6) mon, with maximal activity at 50 ng/ml. At this concentration LTA increased the activity from 0.97 +/- 0.2 to 4.88 +/- 0.2 nmol.10(6) mon/20 min. The role of calcium ions in the effect of LTA in stimulating respiratory burst was studied by changing the availability of calcium ions in the medium, and by measuring the effect of LTA on 45Ca2+ uptake and on intracellular Ca2+ levels. Removal of extracellular calcium ions in the presence of the calcium chelator EGTA, abolished the LTA-stimulated respiratory burst. LTA (50 ng/ml) was found to increase 45Ca2+ uptake into monocytes within seconds (from 2200 +/- 242 in the untreated cells to 4642 +/- 365 cpm/min in the LTA-treated mon). At this concentration, LTA stimulated an immediate rise in the intracellular free Ca2+ concentration to 155 +/- 15 nM as compared with 120 +/- 14 nM in the unstimulated monocytes. LTA caused a specific release of arachidonic acid indicating the involvement of phospholipase A2 in the transduction signal stimulating the respiratory burst by LTA.     

Induction of proliferation of human follicular (B type) lymphoma cells by cognate interaction with CD4+ T cell clones

We examined stimuli which are required for the induction of in vitro proliferation of follicular lymphoma cells, a low grade non-Hodgkin's B cell lymphoma characterized by a specific chromosomal translocation, t(14;18)(q32;q21), and by in vivo growth of the lymphoma cells in germinal center-like follicles infiltrated with CD4+ T cells. The purified follicular lymphoma cells, which are morphologically uniform, small, and dense, did not respond to stimulation with soluble lymphokines in the absence of T cells. Vigorous in vitro proliferation of follicular lymphoma cells was induced, however, when the follicular lymphoma cells were cultured with a CD4+ T cell clone which recognized alloantigens expressed by the lymphoma cells. This response required B-T cell contact, and was inhibited by anti-class II but not by anti-class I MHC mAb, indicating that these neoplastic B cells behaved as normal B cells and responded to normal activation and differentiation signals from T cells. After the cognate B lymphoma-T cell interaction occurred in culture, addition of IL-2 or IL-4 enhanced the proliferation of the tumor cells. These results, with a monoclonal and homogeneous population of B cells, affirm the idea that cognate interaction between B cells and Th cells is required for the effective activation of resting B cells. Moreover, these results suggest that a critical host-tumor interaction occurs in vivo, and that the polyclonal CD4+ T cells that infiltrate follicular lymphomas play a role in sustaining rather than inhibiting tumor growth in vivo. If so, therapies directed not only against the neoplastic cell but also against specific T cells and their cognate interactions with tumor cells may have a rationale.     

The viral envelope gene is involved in macrophage tropism of a human immunodeficiency virus type 1 strain isolated from brain tissue.

Human immunodeficiency virus type 1 (HIV-1) strains isolated from the central nervous system (CNS) may represent a subgroup that displays a host cell tropism different from those isolated from peripheral blood and lymph nodes. One CNS-derived isolate, HIV-1SF128A, which can be propagated efficiently in primary macrophage culture but not in any T-cell lines, was molecularly cloned and characterized. Recombinant viruses between HIV-1SF128A and the peripheral blood isolate HIV-1SF2 were generated in order to map the viral gene(s) responsible for the macrophage tropism. The env gene sequences of the two isolates are about 91.1% homologous, with variations scattered mainly in the hypervariable regions of gp120. Recombinant viruses that have acquired the HIV-1SF128A env gene display HIV-1SF128A tropism for macrophages. Furthermore, the gp120 variable domains, V1, V2, V4, and V5, the CD4-binding domain, and the gp41 fusion domain are not directly involved in determining macrophage tropism.     

Two epitopes and one agretope map to a single HLA-A2 peptide recognized by H-2-restricted T cells

Mice immunized with syngeneic cells transfected with cloned genes coding for HLA class I molecules could recognize the human MHC Ag in the context of their own H-2 molecules. We obtained CTL clones from DBA/2 mice (H-2d) which had been immunized with P815 cells (a mastocytoma of DBA/2 origin) expressing either HLA-A2 or HLA-A3 or two different molecules containing recombined sequences of HLA-A2 and HLA-A3. Fourteen of these clones recognized a synthetic peptide corresponding to the region 170-185 of HLA-A2 in the context of H-2Kd. Moreover, from their activity on P815 cells expressing HLA-Cw3, two subpatterns could be distinguished: subpattern Cw3+, defined by those clones which lysed P815-Cw3, and subpattern Cw3- defined by those clones which did not lyse P815-Cw3. By testing the activity of clones of each subpattern on a series of modified synthetic peptides, we were able to define two epitopes on the same 170-185 peptide of HLA-A2. One of them was dependent on amino acids at positions 173 and 177, whereas the other was dependent on amino acid 177 alone. By using competition experiments, we were also able to define an agretopic region strongly dependent on the amino acid at position 178. Furthermore, experiments with L cells expressing molecules containing recombined sequences between H-2Kd and H-2Dd demonstrated the determinant role of residues 152, 155, and 156 from H-2Kd in the presentation to murine T cells of the 170-185 peptide of HLA-A2.     

Neuronal Activation Regulates the Expression of Opioid Receptors: Possible Role of Glial-Derived Factors and Voltage-Dependent Ion Channels

The previous observation that a continuous chemical depolarization of aggregating rat brain cells with KCl alters the expression of opioid receptors was examined in more detail. In contrast to its significant and converse effect on forebrain and hindbrain cells cultured in serum-containing medium, KCl had only a small and transient effect in serum-free cultures of both types. The basal receptor density in serum-free cultures was similar to the receptor density in KCl-treated serum-containing cultures, but medium conditioned by glial cells restored partially the effect of KCl in serum-free cultures. The effect of KCl in serum-containing forebrain cultures was enhanced by the voltage-dependent calcium channel blocker verapamil, and magnesium and cadmium had a similar, though smaller, effect. The sodium channel activator veratridine had a profound and dose-dependent inhibitory effect on the expression of the receptors in forebrain and hindbrain cultures, and tetrodotoxin blocked the veratridine effect. Information about the selectivity of the effect of neuronal activation on the various opioid receptor subtypes was obtained with the neuroblastoma X glioma hybrid M8 cells that possess only delta type opioid receptors. A Scatchard analysis of [3H]etorphine binding to these cells has shown that depolarization increased the Bmax, but had little, if any, effect on the affinity (KD) of the ligand to the receptors. The significance of depolarization and voltage-dependent sodium and calcium channels on the expression of different opioid receptor subtypes is discussed.     

The envelope gp120 gene of human immunodeficiency virus type 1 determines the rate of CD4-positive T-cell depletion in SCID mice engrafted with human peripheral blood leukocytes.

We have used envelope recombinant viruses generated between two molecular clones of human immunodeficiency virus type 1 (HIV-1), T-cell-tropic HIV-1SF2 and macrophage-tropic HIV-1SF162, to assess pathogenic potential in the human peripheral blood leukocyte-reconstituted severe combined immune deficiency mouse model. Recombinant HIV-1SF2 viruses expressing the envelope gp120 gene of HIV-ISF162 caused as rapid a CD4+ T-cell depletion as did HIV-1SF162. The reciprocal HIV-1SF162 recombinant virus with the HIV-1SF2 envelope caused slower CD4+ T-cell loss. Although changing the V3 loop sequence of HIV-1SF162 to that of HIV-1SF2 did not change the rate of CD4+ T-cell depletion, replacing the V3 of HIV-1SF2 with the sequence of HIV-1SF162 resulted in virus that was poorly infectious in vivo but not in vitro. These studies suggest that the envelope gene determines properties important for pathogenesis in vivo as well as for cell tropism in vitro. HIV-1 infection in vivo may have more stringent requirements for envelope conformation.     

The dynamics of sparse random networks

Recurrent neural networks with full symmetric connectivity have been extensively studied as associative memories and pattern recognition devices. However, there is considerable evidence that sparse, asymmetrically connected, mainly excitatory networks with broadly directed inhibition are more consistent with biological reality. In this paper, we use the technique of return maps to study the dynamics of random networks with sparse, asymmetric connectivity and nonspecific inhibition. These networks show three qualitatively different kinds of behavior: fixed points, cycles of low period, and extremely long cycles verging on aperiodicity. Using statistical arguments, we relate these behaviors to network parameters and present empirical evidence for the accuracy of this statistical model. The model, in turn, leads to methods for controlling the level of activity in networks. Studying random, untrained networks provides an understanding of the intrinsic dynamics of these systems. Such dynamics could provide a substrate for the much more complex behavior shown when synaptic modification is allowed.     

Cloning and comparative mapping of a gene from the commonly deleted region of DiGeorge and Velocardiofacial syndromes conserved in C. elegans

We have identified and cloned a gene, ES2, encoding a putative 476 amino acid protein with a predicted M _r of 52,568. The gene is localized within the DiGeorge/Velocardiofacial syndrome locus on 22q11.2 and is deleted in all the patients in which a deletion within 22q11 could be demonstrated, with the exception of one patient. ES2 is expressed in all the tissues studied. Sequence comparison showed identity with five ESTs and at the amino acid level the sequence was highly similar to, and collinear with, a hypothetical C. elegans protein of unknown function. Mutation analysis was performed in 16 patients without deletion, but no mutation has been found. The cDNA sequence is conserved in mouse and is localized on MMU16B1-B3, known to contain a syntenic group in common with HSA 22q11.2. Received: 25 March 1996 / Accepted: 15 May 1996