Spermine inhibits proinflammatory cytokine synthesis in human mononuclear cells: a counterregulatory mechanism that restrains the immune response.

The local production of proinflammatory cytokines mediates the host response to inflammation, infection, and injury, whereas an overexpression of these mediators can injure or kill the host. Recently, we identified a class of multivalent guanylhydrazone compounds that are effective inhibitors of proinflammatory cytokine synthesis in monocytes/macrophages. The structure of one such cationic molecule suggested a molecular mimicry with spermine, a ubiquitous endogenous biogenic amine that increases significantly at sites of inflammation and infection. Here, we addressed the hypothesis that spermine might counterregulate the innate immune response by downregulating the synthesis of potentially injurious cytokines. When spermine was added to cultures of human peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), it effectively inhibited the synthesis of the proinflammatory cytokines tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6, MIP-1alpha, and MIP-1beta. The inhibition of cytokine synthesis was specific and reversible, with significant inhibition of TNF synthesis occurring even when spermine was added after LPS. The mechanism of spermine-mediated cytokine suppression was posttranscriptional and independent of polyamine oxidase activity. Local administration of spermine in vivo protected mice against the development of acute footpad inflammation induced by carrageenan. These results identify a distinct molecular counterregulatory role for spermine in downregulating the monocyte proinflammatory cytokine response.

Suppression of proinflammatory cytokines in monocytes by a tetravalent guanylhydrazone.

An overproduction of proinflammatory cytokines by activated macrophages/monocytes mediates the injurious sequelae of inflammation, septic shock, tissue injury, and cachexia. We recently synthesized a tetravalent guanylhydrazone compound (CNI-1493) that inhibits cytokine-inducible arginine transport and nitric oxide (NO) production in macrophages, and protects mice against lethal endotoxemia and carrageenan-induced inflammation. During these investigations we noticed that CNI-1493 effectively prevented lipopolysaccharide (LPS)-induced NO production, even when added in concentrations 10-fold less than required to competitively inhibit L-arginine uptake, suggesting that the suppressive effects of this guanylhydrazone compound might extend to other LPS-induced responses. Here, we report that CNI-1493 suppressed the LPS-stimulated production of proinflammatory cytokines (tumor necrosis factor [TNF], interleukins 1beta and 6, macrophage inflammatory proteins 1alpha and 1beta) from human peripheral blood mononuclear cells. Cytokine suppression was specific, in that CNI-1493 did not inhibit either the constitutive synthesis of transforming growth factor beta or the upregulation of major histocompatibility complex class II by interferon gamma (IFN-gamma). In contrast to the macrophage suppressive actions of dexamethasone, which are overridden in the presence of IFN-gamma, CNI-1493 retained its suppressive effects even in the presence of IFN-gamma. The mechanism of cytokine-suppressive action by CNI-1493 was independent of extracellular L-arginine content and NO production and is not restricted to induction by LPS. As a selective inhibitor of macrophage activation that prevents TNF production, this tetravalent guanylhydrazone could be useful in the development of cytokine-suppressive agents for the treatment of diseases mediated by overproduction of cytokines.

KIT oncoprotein interactions in gastrointestinal stromal tumors: therapeutic relevance.

Most gastrointestinal stromal tumors (GISTs) express oncogenic and constitutively active forms of the KIT or platelet-derived growth factor receptor alpha (PDGFRA) receptor tyrosine kinase proteins, and these kinase oncoproteins serve as targets for effective therapies. Given that mutant KIT oncoproteins serve crucial transforming roles in GISTs, we evaluated interactions with the KIT oncoproteins and determined signaling pathways that are dependent on KIT oncogenic activation in GISTs. Tyrosine-phosphorylated KIT oncoproteins interacted with PDGFRA, PDGFRB, phosphatidylinositol 3-kinase (PI3-K) and PKCtheta in GIST cells, and these interactions were abolished by KIT inhibition with imatinib or PKC412 or KIT RNAi. Notably, tyrosine-phosphorylated PDGFRA was prominent in frozen GIST tumors expressing KIT oncoproteins, suggesting that KIT-mediated PDGFRA phosphorylation is an efficient and biologically consequential mechanism in GISTs. Activated signaling intermediates were identified by immunoaffinity purification of tyrosine-phosphorylated proteins in GIST cells before and after treatment with KIT inhibitors, and these analyses show that GRB2, SHC, CBL and MAPK activation are largely KIT dependent in GISTs, whereas PI3-K, STAT1 and STAT3 activation are partially KIT dependent. In addition, we found that phosphorylation of several tyrosine kinase proteins - including JAK1 and EPHA4 - did not depend on KIT activation. Likewise, paxillin activation was independent of the KIT oncogenic signal. These studies identify signaling pathways that can provide both KIT-dependent and KIT-independent therapeutic synergies in GIST, and thereby highlight clinical strategies that might consolidate GIST therapeutic response to KIT/PDGFRA inhibition.

Chromogranin A expression in normal and malignant human tissues.

We used a recombinant cDNA probe for human chromogranin A to measure the expression of mRNA encoded by this gene in a variety of normal human tissues and tumor specimens using Northern blot and in situ hybridization analysis. With few exceptions, the expression of chromogranin A mRNA appears to be restricted to normal tissues and tumors of neuroendocrine lineage. However, we have detected mRNA expression of this gene in 1 of 14 cell lines and 2 of 13 tumor specimens of colon adenocarcinoma. The finding of chromogranin A expression in some colon carcinomas suggests that a previously unrecognized subgroup of these tumors has neuroendocrine features. The detection of this subgroup demonstrates the potential for improving tumor classification through the use of techniques and reagents developed by recombinant DNA technology.

Regulation of c-myb expression in human neuroblastoma cells during retinoic acid-induced differentiation.

We detected expression of the c-myb proto-oncogene, which was initially thought to be expressed in a tissue-specific manner in cells of hematopoietic lineage, in human tissues of neuronal origin. Since the level of c-myb expression declined during fetal development, we studied the regulation of its expression in human neuroblastoma cell lines induced to differentiate by retinoic acid. The expression of c-myb declined during the maturation of neuroblastoma cells, and this change was mediated by a decrease in c-myb transcription.

Monoclonal antibody and enzymatic profiles of human malignant T-lymphoid cells and derived cell lines.

Recently, four distinct cell lines were established from patients whose malignancies had been defined by immunological and biochemical markers. Each patient had a distinct subtype of a T-cell cancer, and each possessed elevated adenosine deaminase and reduced nucleoside phosphorylase activity. Cell lines cultured in vitro possessed the same basic immunophenotype and biochemical enzyme activity as the patients' original malignant cells. In a direct comparison of the immunophenotype of the cell lines and the patients' malignant cells, full concordance existed for 48 of 52 paired antibody tests performed. However, when compared to the corresponding patient's sample, each cell line showed some minor changes in antigen expression or enzyme level. Antigen loss, de novo antigen expression, or elevated adenosine deaminase levels occurred in the cell lines, and these changes were stable on repeated analysis. While there was good general concordance between the patient's cancer and the established cell line, minor biological differences in the cell lines may reflect cellular maturation or subpopulation selection in vitro.

Neuropeptide Y expression in the developing adrenal gland and in childhood neuroblastoma tumors.

Neuropeptide Y (NPY) expression is limited to tissues of the central and peripheral nervous system. In the adrenal gland, NPY is found in a subset of cells of the adrenal medulla. Using in situ hybridization analysis, NPY mRNA expression was characterized during human fetal adrenal medullary development. We found a biphasic pattern of NPY mRNA expression during the development of the human adrenal medulla. NPY mRNA is detectable at the earliest evaluable time point (7.5 weeks of gestational age) through 18 weeks of gestational age, and is then not detectable until 8 months after birth. We also analyzed NPY mRNA expression in neuroblastoma tumors, which often arise in the adrenal medulla. Thirty-eight neuroblastoma tumors were analyzed for NPY mRNA expression using in situ hybridization. We found NPY mRNA expression in 30 of 38 tumors; 15 of 15 Stage IVS tumors from children under 1 year of age at diagnosis expressed NPY mRNA, whereas 0 of 4 Stage IV tumors from children less than 1 year of age at diagnosis expressed NPY mRNA. These data suggest that in children under 1 year of age at diagnosis, Stage IVS and Stage IV neuroblastoma may be marked by the presence or absence, respectively, of NPY mRNA expression. Moreover, since NPY is expressed for only a short period of time during embryogenesis, these tumors may arise from different neuroblast populations occurring during the course of adrenal medullary development.

Analysis of nerve growth factor receptor expression in human neuroblastoma and neuroepithelioma cell lines.

A series of 22 neuroepithelioma and neuroblastoma cell lines were screened for expression of nerve growth factor receptor (NGFR) by flow cytometry, Western blotting, and Northern blotting. All 5 neuroepithelioma cell lines expressed cell surface NGFR, with 30-69% of cells NGFR positive, but the 17 neuroblastoma cell lines tested had a smaller percentage of cell surface NGFR-positive cells (0-21%) and 10 lines were completely lacking cell surface NGFR. SY5Y, a variant line with a neuronal phenotype derived from neuroblastoma line SKNSH, expressed much more NGFR than SHEP, a variant line with an epithelial-like phenotype also derived from SKNSH. By Western blotting, the Mr approximately 69,000 NGFR band was detected for all four neuroepithelioma cell lines tested but was visible for only 8 of 15 neuroblastoma cell lines tested. The band was most intense for neuroepithelioma cell lines SKNMC and TC32. For these two lines, a Mr approximately 56,000 and a Mr approximately 60,000 band were also detected. By Northern blotting, all three neuroepithelioma cell lines tested were positive for the 3.8 kilobase NGFR mRNA, but only 8 of 15 neuroblastoma cell lines were positive. Neuroepithelioma cell line TC32 and neuroblastoma cell line GICAN had the strongest expression of NGFR mRNA. These results demonstrate that NGFR is a biological marker for neuroepithelioma and that NGFR expression is heterogeneous for neuroblastoma cell lines. This series of neural cell lines differing in NGFR expression will be useful for future studies of regulation of NGFR expression and neuronal differentiation.

Induction of transforming growth factor beta 1 and its receptors during all-trans-retinoic acid (RA) treatment of RA-responsive human neuroblastoma cell lines.

Recent work on a variety of normal and malignant cell lines has shown that induction and secretion of biologically active TGF-beta may occur after exposure to all-trans-retinoic acid (RA), coincident with decreased growth rate and/or differentiation. This study evaluates the expression and regulation of transforming growth factor beta (TGF-beta) and its receptors during RA-induced cell growth arrest and induction of differentiation in the RA-sensitive human neuroblastoma cell line SMS-KCNR and the RA-resistant neuroblastoma cell line SK-N-AS. RA treatment of SMS-KCNR cells results in a 40-fold increase in TGF-beta 1 mRNA after 4 days of RA, a dose-dependent increase in TGF-beta 1 secretion, an increase in types I (TBRI) and III (TBRIII) TGF-beta receptor proteins, and an increase in type II TGF-beta receptor (TBRII) mRNA coincident with RA-responsiveness of the cells. However, in the RA-resistant line SK-N-AS, TGF-beta 1 is constitutively secreted at levels that are unchanged after RA treatment, and although TBRI and TBRIII mRNA is expressed in untreated SK-N-AS cells, levels of TBRI and TBRIII protein and TBRII mRNA decrease after RA treatment. Thus, in RA-sensitive neuroblastoma cells, RA treatment may result in the induction of a negative autocrine TGF-beta 1 growth regulatory loop. These results suggest the hypothesis that: (a) induction of a TGF-beta 1 negative autocrine growth loop may be a necessary component for RA-responsiveness of neuroblastoma cells in vivo; and (b) the inability to induce or maintain this TGF-beta 1 negative autocrine growth loop may be a mechanism of RA resistance in neuroblastoma.

Neuropeptide Y expression distinguishes malignant from benign pheochromocytoma.

No reliable gross or microscopic features distinguish benign from malignant pheochromocytomas. The diagnosis of malignant pheochromocytoma is based solely on the presence of regional or distant metastases. This study evaluated the expression of neuropeptide Y messenger RNA (mRNA) in nine benign and 11 malignant pheochromocytomas and has found that neuropeptide Y mRNA was expressed in all nine benign tumors but in only four of 11 malignant tumors (P = .0084). These data suggest that the determination of neuropeptide Y expression in the evaluation of patients with pheochromocytoma may have prognostic significance.

Aggression induced by intermittent positive reinforcement.

Mammalian and non-mammalian species engage in aggressive behavior toward animate and inanimate targets when exposed to intermittent access to a positive reinforcer. This behavior, called extinction- or schedule-induced aggression, typically includes a biting or striking topography that inflicts damage on a target. This paper critically reviews research and theoretical issues concerning such aggression and suggests directions for future investigation.

The leuX-encoded tRNA5(Leu) but not the pathogenicity islands I and II influence the survival of the uropathogenic Escherichia coli strain 536 in CD-1 mouse bladder mucus in the stationary phase.

The uropathogenic Escherichia coli strain 536 carries two pathogenicity islands, each of which is associated with either of the tRNA genes selC or leuX, respectively. Growth competition in CD-1 mouse mucus between the wild-type strain E. coli 536, its leuX mutant 536 delta 102 and its mutant 536R3, lacking both pathogenicity islands but expressing a functional tRNA5(Leu), revealed a major impact of leuX on E. coli survival in bladder mucus. The impaired survival in CD-1 mouse mucus observed upon deletion of the leuX gene was abolished after complementation with the leuX gene. The survival of bacteria in bladder mucus was not influenced by the presence of pathogenicity islands I and II.

A Salmonella typhimurium mutant unable to utilize fatty acids and citrate is avirulent and immunogenic in mice.

Salmonella typhimurium SR-11 is extremely virulent at a dose as low as 10(5) colony forming units (cfu) when administered perorally to BALB/c mice. Utilizing mini-transposon mutagenesis, a mutant of S. typhimurium SR-11 was isolated that was unable to utilize oleate and citrate as carbon sources. This mutant, designated S. typhimurium SR-11 Fad- (Fatty acid), was found to utilize sugars under cya/crp control as sole carbon sources, suggesting that the mutation is not in either of these genes. In addition, SR-11 Fad- utilized pyruvate and succinate, but was unable to utilize either acetate or isocitrate as sole carbon source. In contrast to SR-11, SR-11 Fad- was found to be avirulent, i.e. BALB/c mice were completely healthy after oral infection with 10(9) S. typhimurium SR-11 Fad- cells. Moreover, 21 days after SR-11 Fad- infection, BALB/c mice were found to be protected against an oral challenge with 10(9) cells of S. typhimurium SR-11.

Stimulation of Escherichia coli F-18Col- type-1 fimbriae synthesis by leuX.

Escherichia coli F-18, a normal human fecal isolate, is an excellent colonizer of the streptomycin-treated mouse large intestine. E. coli F-18Col-, a derivative of E. coli F-18 which no longer makes the E. coli F-18 colicin, colonizes the large intestine as well as E. coli F-18 when fed to mice alone but is eliminated when fed together with E. coli F-18. Recently we randomly cloned E. coli F-18 DNA into E. coli F-18Col- and let the mouse intestine select the best colonizer. In this way, we isolated a 6.5-kb E. coli F-18 DNA sequence that simultaneously stimulated synthesis of type 1 fimbriae and enhanced E. coli F-18Col- colonizing ability. In the present investigation we show that the gene responsible for stimulation of type 1 fimbriae synthesis appears to be leuX, which encodes a tRNA specific for the rare leucine codon UUG. Moreover, it appears that expression of leuX may be regulated by two proteins (22 kDa and 26 kDa) encoded by genes immediately adjacent to leuX.

Accurate three-dimensional reconstruction of neuronal distributions in brain: reconstruction of the rat nucleus locus coeruleus.

A technique is described which permits construction of accurate, quantified 3-dimensional maps of the distribution of neuronal cell groups in brain. The cartesian coordinates of landmarks and individual neurons are obtained from serial histological sections utilizing a computer-linked digitizing microscope. The digitized images of these sections are displayed on a computer graphics picture system where they are aligned so that spatial relationships within the nucleus are essentially identical to those of the intact brain. This is accomplished using information about landmarks obtained from photomicrotomy. As a consequence of the alignment procedure, each neuron is assigned a 3-dimensional coordinate representing its position in the reconstituted nucleus, and the reconstruction is oriented in a stereotaxic coordinate system. Nuclei from different brains can then be registered to one another, assigned coordinates relative to this standard coordinate space, and be compared statistically. Differences between nuclei in the spatial distribution of neurons in toto, or in the distribution of anatomically or physiologically defined subpopulations of neurons, can then be visualized with greater accuracy and in more detail than that permitted by traditional techniques. In addition, such comparisons can easily be quantified and statistically evaluated using, for example, analysis-of-variance techniques. For illustrative purposes, the technique is applied to the rat nucleus locus coeruleus as reconstructed from serial Nissl-stained sections.

In vitro and in vivo chemotherapy screening of the divalent cation chelator 1,10-orthophenanthroline.

1,10-Orthophenanthroline (OP) is a divalent cation chelating agent with known cytotoxicity to human normal and malignant T-lymphocytes. To determine whether OP might be a useful anticancer agent with specific T cell toxicity, OP's effect on cell growth was determined on colony-forming cells. The assay used supported growth of both malignant lymphoid and normal myeloid colony-forming cells (CFU-C) and thus a direct comparison of OP's antilymphoid and antimyeloid toxicity was obtained. The malignant lymphoid cells tested were established from patients at relapse and were resistant to conventional chemotherapeutic agents in vitro. While OP was found to be toxic to all cells tested, some selective kill of malignant cells over CFU-C occurred. OP's cytotoxicity was time-dependent and a three-log enhanced kill occurred when the drug exposure time was increased from 1 to 24 h. When test cells were continously exposed to OP, the ID50 was less than 1 micrograms/ml for malignant lymphoid cells and the sensitivity index (SI = x ID50 CFU-C divided by x ID50 cell line) ranged from 1.5 to 3.0. The National Cancer Institute currently screens new compounds for antitumor activity by determining whether the test drug is toxic to a mouse lymphocytic leukemia cell line (P388). While the mouse P388 cells were sensitive to OP in vitro, no effect was seen when OP was administered in vivo, even when schedules designed to take advantage of OP's time-dependent toxicity were used. Since malignant cells were sensitive to OP (ID50 less than 1 micrograms/ml), and some selectivity over CFU-C occurred (SI greater than 1), OP may be a useful agent for control of leukemic cell growth in vitro. However, since OP did not control the growth of P388 cells in vivo, additional studies designed to enhance the therapeutic index of OP in vivo are needed.

Temporal patterns of reinforcer-induced general activity and attack in pigeons.

Three White King pigeons were exposed to fixed-time schedules ranging from 60 to 240 sec. General activity and attack against a conspecific target were measured separately at each interreinforcer interval value. Comparison of the temporal distributions of activity and attack revealed significant differences in their temporal locus and overall distribution between reinforcer presentations. At all interval values, attack was localized to the postreinforcer period, and peaked at the same absolute time (5-10 sec) following each reinforcer presentation. General activity was more uniformly distributed between reinforcers, and the peak level occurred later in the interval. As the interval length increased, peak levels of activity shifted to later times following reinforcement. These differences question traditional accounts which posit a unitary process underlying induced behaviors, while supporting Cohen, Looney, Campagnoni and Lawler's recent two-state model of induced behaviors.

Reduced serum cholesterol with dietary change using fat-modified and oat bran supplemented diets.

A low-fat, low-cholesterol diet and oat bran supplementation for treatment of hypercholesterolemia were studied for their effectiveness in lowering blood lipids and their impact on dietary intake. Seventy-one free-living men and women with hypercholesterolemia (serum cholesterol greater than 75th percentile) were randomly assigned to one of the following four groups: low-fat, low-cholesterol diet (LFLC); low-fat, low-cholesterol diet plus 50 gm/day oat bran (LFLC + OB); 50 gm/day oat bran supplemented diet (OB); or 42.5 gm/day processed oat bran (ready-to-eat cereal containing beta-glucan concentrated from oat bran) (POB). Subjects assigned to regimens OB and POB were requested to add the oat supplement without making additional changes in their diet. Serum cholesterol and high-density lipoprotein cholesterol analyses were performed at 4-week intervals, and diet records were assigned and analyzed. All groups experienced significant decreases in cholesterol from original levels (p less than .05). The average decrease in total serum cholesterol varied from 10% to 17%, with no significant differences among the four groups. High-density lipoprotein cholesterol concentrations decreased in all groups except group 4, in which there was a slight increase; however, no differences were found between groups. Energy, fat, and cholesterol intakes decreased in all groups, suggesting that displacement of higher fat foods from the diet may be one of the many mechanisms whereby oat supplements lower serum cholesterol. In addition, all groups reduced their intakes of calcium, copper, folic acid, and potassium from marginal levels at the beginning of the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Punishment: the interactive effects of delay and intensity of shock.

A discrete-trial punishment procedure, with rats, was used to examine how delay-of-shock intervals of 0 to 28 sec and shock intensity interact to decrease the frequency and increase the latency of a positively reinforced response. For delay-of-shock intervals of 0, 7, 14, and 28 sec, there was a range of shock intensities, for some subjects, over which the punishing effect of shock was an increasing, monotonic function of shock intensity. For other subjects this transition was abrupt. Functions relating response frequency and latency measures to shock intensity were displaced toward higher values on the shock intensity axis with an increase in delay-of-shock interval. The effects of "gradual" and "abrupt" introduction to "severe" shock, as well as re-exposure to previously used shock intensities, were examined under both the immediate and delay-of-shock conditions. With delay-of-shock intervals of 7, 14, or 28 sec, shock intensities of approximately 0.50 milliamperes or greater were necessary to decrease substantially the number and increase the latency of the lever-pressing response. For the immediate punishment group this intensity was approximately 0.20 ma. These facts were related to Annau and Kamin's (1961) conditioned emotional response experiment in which a shock intensity of 0.49 ma or greater was required to suppress the rate of a positively reinforced response.

DRL escape: effects of minimum duration and intensity of electric shock.

Three dogs were exposed to a DRL-escape procedure that required them to endure a minimum duration of electric shock without responding in order for a response to terminate that shock. When this minimum duration increased from 0 to either 2.25 or 7.00 sec, response latencies increased proportionately. With the minimum duration held constant at 2.25 sec, a gradual increase in shock intensity to 5.0 ma had no systematic effect upon latencies. Even under the highest shock intensity, 5.0 ma, latency and interresponse-time distributions were unimodal with very few latencies and interresponse times less than the minimum duration. Three additional dogs were exposed to an escape procedure in which every response was immediately reinforced. For these subjects, the same increase in shock intensity to 5.0 ma was accompanied by a decrease in latencies. The precise temporal spacing of responses obtained with the DRL-escape procedure may in part be due to the fact that every response latency and interresponse time that did not meet the minimum duration was not only extinguished but was also punished.