Differential interaction of estrogen receptor and thyroid hormone receptor isoforms on the rat oxytocin receptor promoter leads to differences in transcriptional regulation.

Both the estrogen receptor (ER) and thyroid hormone receptor (TR) are members of the nuclear receptor superfamily. Two isoforms of the ER, alpha and beta, exist. The TRalpha and beta isoforms are products of two distinct genes that are further differentially spliced to give TRalpha1 and alpha2, TRbeta1 and beta2. The TRs have been shown to interfere with ER-mediated transcription from both the consensus estrogen response element (ERE) and the rat preproenkephalin (PPE) promoter, possibly by competing with ER binding to the ERE or by squelching coactivators essential for ER-mediated transcription. The rat oxytocin receptor (OTR) gene is thought to be involved in several facets of reproductive and affiliative behaviors. 17beta-Estradiol-bound ERs upregulate the OTR gene in the ventromedial hypothalamus, a region critical for the induction of lordosis behavior in several species. We investigated the effects of the ligand-binding TR isoforms on the ER-mediated transcription from a physiological promoter of a behaviorally relevant gene such as the OTR. Only ERalpha could induce the OTR gene in two cell lines tested, the CV-1 and the SK-N-BE2C neuroblastoma cell lines. ERbeta was incapable of inducing the gene in either cell line. ERalpha is therefore not equivalent to ERbeta on this physiological promoter. Indeed, in the neural cell line, ERbeta can inhibit ERalpha-mediated induction from the OTR promoter. While the TRalpha1 isoform inhibited ERalpha-mediated induction in the neural cell line, the TRbeta1 isoform stimulated induction, thus demonstrating isoform specificity in the interaction. The use of a DNA-binding mutant, the TR P box mutant, showed that inhibition of ERalpha-mediated induction of the rat OTR gene promoter by the TRalpha1 isoform does not require DNA-binding ability. SRC-1 overexpression relieved TRalpha1-mediated inhibition in both cell lines, suggesting that squelching for coactivators is an important molecular mechanism in TRalpha-mediated inhibition. Such interactions between TR and ER isoforms on the rat OTR promoter provide a mechanism to achieve neuroendocrine integration.

Antisense RNA gene therapy for studying and modulating biological processes.

Agents that produce their effects through an antisense mechanism offer the possibility of developing highly specific alternatives to traditional pharmacological antagonists, thereby providing a novel class of therapeutic agents, ones which act at the level of gene expression. Among the antisense compounds, antisense RNA produced intracellularly by an expression vector has been used extensively in the past several years. This review considers the advantages of the antisense RNA approach over the use of antisense oligodeoxynucleotides, the different means by which one may deliver and produce antisense RNA inside cells, and the experimental criteria one should use to ascertain whether the antisense RNA is acting through a true antisense mechanism. Its major emphasis is on exploring the potential therapeutic use of antisense RNA in several areas of medicine. For example, in the field of oncology antisense RNA has been used to inhibit several different target proteins, such as growth factors, growth factor receptors, proteins responsible for the invasive potential of tumor cells and proteins directly involved in cell cycle progression. In particular, a detailed discussion is presented on the possibility of selectively inhibiting the growth of tumor cells by using antisense RNA expression vectors directed to the individual calmodulin transcripts. Detailed consideration is also provided on the development and potential therapeutic applications of antisense RNA vectors targeted to the D2 dopamine receptor subtype. Studies are also summarized in which antisense RNA has been used to develop more effective therapies for infections with certain viruses such as the human immunodeficiency virus and the virus of hepatitis B, and data are reviewed suggesting new approaches to reduce elevated blood pressure using antisense RNA directed to proteins and receptors from the renin-angiotensin system. Finally, we outline some of the problems which the studies so far have yielded and some outstanding questions which remain to be answered in order to develop further antisense RNA vectors as therapeutic agents.

Effect of antisense oligodeoxynucleotides directed to individual calmodulin gene transcripts on the proliferation and differentiation of PC12 cells.

Calmodulin (CaM) is encoded by three different genes that collectively give rise to five transcripts. In the present study, we used antisense oligodeoxynucleotides targeted to unique sequences in the transcripts from the individual CaM genes to selectively block the expression of the different genes and to investigate the roles these individual genes play in the proliferation and nerve growth factor (NGF)-induced differentiation of PC12 cells. Culturing PC12 cells in the presence of oligodeoxynucleotide antisense to the transcripts from CaM genes I and II caused a significant decrease in the proliferation and a significant delay in the NGF-induced differentiation of PC12 cells when compared with untreated cells and with cells treated with the corresponding randomized oligodeoxynucleotides. However, an oligodeoxynucleotide antisense to CaM gene III did not significantly alter the proliferation or the NGF-induced differentiation of PC12 cells. The inhibition of cell proliferation could be reversed by washing out the antisense oligodeoxynucleotides. The levels of CaM in cells treated with oligodeoxynucleotides antisense to CaM genes I or II were reduced 52% or 63%, respectively, of the levels found in the control cells. However, the levels of CaM were not significantly reduced in PC12 cells treated with CaM gene III antisense oligodeoxynucleotide. None of the randomized oligodeoxynucleotides had any effect on the levels of CaM in PC12 cells. The reduced levels of CaM in PC12 cells treated with an oligodeoxynucleotide antisense to CaM gene I were accompanied by a reduction in the levels of the CaM gene I mRNAs, supporting a true antisense mechanism of action for these oligodeoxynucleotides. These results suggest that altering the level of CaM by using antisense oligodeoxynucleotides targeted to the dominant CaM transcripts in a particular cell type will specifically inhibit their proliferation and, in the case of neuronal cells, alter the course of their differentiation.

D2 dopamine antisense RNA expression vector, unlike haloperidol, produces long-term inhibition of D2 dopamine-mediated behaviors without causing Up-regulation of D2 dopamine receptors.

Long-term inhibition of D2 dopamine receptors using classic D2 dopamine receptor antagonists such as haloperidol often causes a compensatory up-regulation of the D2 dopamine receptors. We investigated whether the long-term inhibition of D2 dopamine receptors using an eukaryotic expression vector housing a cDNA sequence encoding an antisense RNA directed to the D2 dopamine receptor transcript (D2 antisense vector) would also produce up-regulation of the D2 receptors. Single, bilateral injections of the D2 antisense vector into the corpora striata of mice inhibited the stereotypy induced by acute challenge injections with the D2/D3 dopamine receptor agonist quinpirole but did not inhibit the grooming induced by acute challenge injections with the D1 agonist SKF 38393. Similar treatment with the D2 antisense vector produced a long-term (>1 month) cataleptic response without producing tolerance to challenge injections with haloperidol. By contrast, catalepsy induced by a single injection of haloperidol lasted only approximately 2 days, and tolerance developed to its effects after long-term treatment. Repeated treatment of mice with haloperidol resulted in an inhibition of apomorphine-induced climbing behavior throughout the time of treatment with haloperidol, but the climbing behavior markedly increased to levels significantly higher than that of the control mice immediately after withdrawal from haloperidol treatment. This increased climbing was accompanied by increased levels of D2 dopamine receptors in the striatum. By contrast, single, bilateral intrastriatal injections of the D2 antisense vector significantly inhibited apomorphine-induced climbing for approximately 30 days but failed to increase the climbing behavior or the levels of D2 dopamine receptors in striatum over those of the control values. These results suggest that a single injection of a D2 antisense RNA expression vector into mouse striatum produces specific, long-term inhibition of D2 dopamine receptor behaviors without causing a compensatory increase in the levels or function of D2 dopamine receptors.

Expression of a D2 dopamine receptor antisense RNA in brain inhibits D2-mediated behaviors.

Drugs currently used to treat disorders of dopamine-mediated behaviors in the central nervous system are non-selective in that they interact not only with more than one isoform of dopamine receptor but also with receptors for other neurotransmitters. A new strategy to inhibit the actions of individual dopamine receptor subtypes is to inhibit the synthesis of the receptors through the use of oligonucleotides antisense to the transcripts encoding the different receptors. Earlier studies showed that oligodeoxynucleotides antisense to the D1 or D2 dopamine receptor messenger RNAs specifically inhibited the biological actions mediated by these individual isoforms of the dopamine receptor. However, these actions were relatively short-lasting. To determine whether one can achieve long-lasting inhibition of dopamine responses, while still taking advantage of the highly selective nature of an antisense strategy, an expression vector was employed that generates antisense RNA to the transcript encoding the D2 dopamine receptor. A single intrastriatal injection of this vector generated an antisense RNA to the D2 dopamine receptor, selectively reduced the levels of D2 dopamine receptors, and caused selective, long-term inhibition of behaviors mediated by D2 dopamine agonists. Such an antisense RNA strategy may find use in studying the function of dopaminergic receptors and in disorders associated with dopaminergic hyperactivity.

Antisense strategies in neurobiology.

The use of antisense oligodeoxynucleotides, targeted to the transcripts encoding biologically active proteins in the nervous system, provides a novel and highly selective means to further our understanding of the function of these proteins. Recent studies of these agents also suggest the possibility of their being used therapeutically for a variety of diseases involving neuronal tissue. In this paper we review studies showing the in vitro and in vivo effects of antisense oligodeoxynucleotides as they relate to neurobiological functions. Particular attention is paid to the behavioral and biochemical effects of antisense oligodeoxynucleotides directed to the various subtypes of receptors for the neurotransmitter dopamine. An example is also provided showing the effects of a plasmid vector expressing an antisense RNA targeted to the calmodulin mRNAs in the PC12 pheochromocytoma cell line. The advantages of antisense oligodeoxynucleotides over traditional pharmacological treatments are assessed, and the advantages of using vectors encoding antisense RNA over the use of antisense oligodeoxynucleotides are also considered. We also describe the criteria that should be used in designing antisense oligodeoxynucleotides and several controls that should be employed to assure their specificity of action.

Selective usage of VH genes in adult human B lymphocyte repertoires.

The authors have compared the VH gene utilization patterns among small resting immunocompetent B cells and large naturally activated B lymphocytes of healthy human adults. They employed a non-radioactive RNA in situ hybridization technique that allows detection of VH gene family expression at the single cell level. Pokeweed mitogen stimulated and unmanipulated mononuclear cells from peripheral blood and spleen of unrelated individuals were hybridized to digoxigenin-labelled antisense RNA probes specific for human VH families 1-6 and for the constant region genes C mu and C gamma. The observed VH gene family utilization patterns did not correlate with the genomic complexity of human VH genes. The VH3 gene family was most frequently used among resting B cells in both peripheral blood and spleen. Among naturally activated lymphocytes the VH6 gene was markedly over-represented, while expression of the VH1 and VH3 gene families was decreased. The data show that V-region mediated selection participates in shaping the peripheral antibody repertoire in healthy adults.

Reduced level of calmodulin in PC12 cells induced by stable expression of calmodulin antisense RNA inhibits cell proliferation and induces neurite outgrowth.

The role calmodulin plays in the growth and differentiation of nerve cells was assessed by altering the levels of calmodulin in the PC12 rat pheochromocytoma cell line and determining the effects of altering these levels on cellular proliferation and differentiation. Calmodulin levels in the PC12 cells were increased or decreased by transfecting the cells with a mammalian expression vector into which the rat calmodulin gene I had been cloned in the sense or antisense orientation, respectively. The cells transfected with the calmodulin sense gene showed increased levels of calmodulin immunoreactivity and increased levels of calmodulin messenger RNA as ascertained by immunocytochemistry and slot-blot analysis, respectively. Cells transfected with the calmodulin antisense construct showed reduced levels of calmodulin immunoreactivity. Reducing the levels of calmodulin by expression of antisense calmodulin messenger RNA resulted in a marked inhibition of cell growth, whereas increasing the levels of calmodulin by overexpressing calmodulin messenger RNA resulted in an acceleration of cell growth. Transfected PC12 cells having reduced levels of calmodulin immunoreactivity exhibited spontaneous outgrowth of long, stable and highly branched neuritic processes. PC12 cells in which calmodulin was overexpressed showed no apparent changes in cell morphology, but did show an altered response to the addition of nerve growth factor. While nerve growth factor slowed cellular proliferation and induced extensive neurite outgrowth, in parental PC12 cells nerve growth factor induced little or no neurite outgrowth and little inhibition of cell proliferation in transfected cells overexpressing calmodulin. These results indicate that calmodulin is essential for the proliferation of nerve cells and for the morphological changes that nerve cells undergo during differentiation. The study also suggests the possibility that a calmodulin antisense approach may be used to inhibit the proliferation of neuronal tumors.

Stimulating effect of DEODAN (an oral preparation from Lactobacillus bulgaricus "LB51") on monocytes/macrophages and host resistance to experimental infections.

DEODAN is a lysozyme lysate from Lactobacillus bulgaricus for oral administration which has shown antitumor activity in mice and humans. The effects of this preparation on some functions of monocytes/macrophages and on host resistance to experimental infections were examined. The oral administration to mice of DEODAN-150 mg/kg daily (the recommended dose in humans) caused an increase of the spreading ability and phagocytic activity of peritoneal macrophages, which showed morphological signs of cell activation. The level of Interleukin-1 (IL-1) secreted in the culture supernatant of peritoneal macrophages of DEODAN-treated mice was found to be slightly increased only when the mice were treated with 150 mg/kg DEODAN for 10 days. However, the in vitro incubation of human blood monocytes with DEODAN resulted in induction of membrane-bound and cytoplasmic IL-1 and Tumor Necrosis Factor (TNF)-alpha. The oral treatment of mice with DEODAN also caused a decrease in mortality after experimental infections with Klebsiella pneumoniae and Listeria monocytogenes. These results indicate that DEODAN activates the phagocytic and secretory functions of mononuclear cells and increases host resistance to bacterial infections.

Endogenous production of tumor necrosis factor in normal mice orally treated with Deodan--a preparation from Lactobacillus bulgaricus "LB51".

The ability of orally administered Deodan, a product from the cell wall of Lactobacillus bulgaricus strain "I. Bogdanov patent strain tumoronecroticance B51" ATCC #21815, shortly called "LB51", to induce endogenous tumor necrosis factor-alpha (TNF alpha) production in normal mice was evaluated. The priming and triggering activities of the preparation were investigated in combination with lipopolysaccharide (LPS) and live BCG vaccine. Deodan was applied at a dose of 150 mg/kg and various treatment schedules were employed. The serum levels of TNF alpha in treated mice were quantified by ELISA. Oral administration of Deodan at a dose of 150 mg/kg for 1, 3, 10 or 20 consecutive days only enhanced serum TNF alpha levels in treated mice. Maximal TNF alpha levels were reached 6 h after the last application of Deodan. Deodan was effective in priming TNF alpha in mice triggered intravenously (i.v.) with LPS. Deodan triggered the production of TNF alpha in BCG-primed mice. The preparation, however, was not an effective trigger of mice primed intradermally (i.d.) with 1 microgram/mouse LPS. These findings suggest that Deodan is both a primer and trigger of endogenous TNF alpha. The advantages of treatment of neoplastic disease with agents which induce endogenous TNF alpha is discussed.

Determination of cytokine release after in vivo and in vitro administration of Deodan (a preparation from Lactobacillus bulgaricus "LB51") by the rabbit pyrogen test.

We investigated the in vivo and in vitro cytokine inducing effects of Deodan, an oral preparation from Lactobacillus bulgaricus "LB-51", using the rabbit pyrogen test. In the first experimental approach we administered Deodan, or its chromatographically purified fraction, via the i.m. or i.v. routes. Low doses of Deodan i.m. caused the formation of a single temperature peak, whereas large doses produced a biphasic temperature curve. Intravenous injection of Deodan produced a monophasic fever in all tested doses. Chromatographically purified Deodan injected i.v. to rabbits caused a febrile response with a dose-dependent pattern, strikingly similar to that of lipopolysaccharide. LAL-testing of Deodan, however, showed that the preparation does not contain endotoxin. In in vivo neutralization studies we demonstrated that IL-1, TNF alpha, and IL-6 mediate the rabbit febrile response to Deodan. Interestingly, the effects of Deodan on the production of TNF alpha and IL-6 were more pronounced than its IL-1 inducing activity. In the second approach, we injected supernatants from mononuclear cells incubated with nonpyrogenic doses of Deodan, intravenously to rabbits ("monocyte type" of pyrogen test). Rapid-onset monophasic fevers were observed, typical for the rabbit pyrogen reaction to i.v. administration of exogenous IL-1 and TNF. Finally, we demonstrated the presence of pyrogenic cytokines in the supernatants from macrophages of Deodan-treated mice. Together, these results indicate that Deodan induces the production of cytokines with endogenous pyrogenic activity.

[The cytofluorimetric characteristics of monoclonal antibodies for use in humans in respect of their activity and cross reactivity]. / Tsitofluorimetrichna kharakteristika na monoklonalni antitela za prilozhenie pri khora po otnoshenie na aktivnost i krustosana reaktivnost.

A cytofluorimetric method for determination of the reactivity of antimyeloma MkAt 225,28S with myeloma cells and blood elements is described in the present article. The obtained results show that the antibody possesses high specific activity and does not bind with normal human lymphocytes, monocytes, granulocytes, thrombocytes and erythrocytes in the examined dilutions. The method is suitable for control of cross reactivity with blood cells of MkAt preparations for usage in subjects.