Nuclear receptor activation and interaction with morphine

Journal of Neuroimmunology 157 (2004) 61 – 65 www.elsevier.com/locate/jneuroim

Nuclear receptor activation and interaction with morphine Walter Royal IIIa,*, Michelle Leander a, Yuqing E. Chenb, Eugene O. Major c, Reid P. Bissonnetted a

Neuroscience Institute, Morehouse School of Medicine, MRC 214, 720 Westview Drive, S.W., Atlanta, GA 30310, USA b Cardiovascular Research Institute, Morehouse School of Medicine, USA c National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD, USA d Ligand Pharmaceuticals, San Diego, CA, USA Accepted 30 August 2004

Abstract Nervous system disease in HIV infection is associated with toxic damage induced by effects from proinflammatory responses and oxidative stress, and such effects may be more prominent among opioid abusers. In these studies, the effects of activating retinoid receptor (retinoic acid receptor (RAR) and retinoid X receptor (RXR)) and peroxisome proliferator activated receptor (PPAR) g, which belong to the steroid-lipid nuclear receptor family, on tumor necrosis factor (TNF)-a production and inducible nitric oxide synthase (iNOS) gene expression by stimulated U937 and SVG cells, respectively, were examined. Also studied were the effects of morphine on these responses. These studies showed that, in stimulated cells, the observed responses were suppressed by activation of the nuclear receptors as compared to non-stimulated control cells. Moreover, in phytohemagglutinin (PHA)-stimulated U937 cells, morphine reversed the TNF-a suppression that was induced by LG101305 and ciglitazone. Preliminary data in SVG cells suggest a tendency for morphine to have a similar effect on LG101305-exposed SVG cells stimulated with a combination of lipopolysaccharide (LPS) and interferon-g, whereas this effect was not induced when these cells were incubated with ciglitazone. Therefore, specific nuclear receptor activation may be potentially beneficial in the treatment of neurological disease associated with HIV infection and may show specific interactions with opioids. The mechanisms that underlie these effects require further study. D 2004 Elsevier B.V. All rights reserved. Keywords: Nuclear receptor; Morphine; HIV infection

1. Introduction In HIV dementia, the cause of damage to nervous system tissue appears to be related to the effects of neurotoxic compounds released by activated macrophage/microglial cells, astrocytes, and endothelial cells and to toxicity associated with HIV proteins, such as gp120, tat, and nef. Among the inflammatory mediators and other soluble factors that have been associated with HIV-related neurotoxicity are the pro-inflammatory cytokines tumor necrosis factor (TNF)a, interleukin (IL)-1h, and IL-6, quinolinic acid, nitric oxide, platelet-derived growth factor, and prostaglandins (Lipton and Gendelman, 1995). In addition, there is also increased production and release of pro-inflammatory chemokines, * Corresponding author. Tel.: +1 404 756 5792; fax: +1 404 752 1041. E-mail address: [email protected] (W. Royal). 0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2004.08.023

including macrophage inflammatory protein (MIP)-1a, MIP1h, RANTES, and monocyte chemoattractant protein (MCP)1, which can further promote the infiltration of mononuclear phagocytes into nervous system tissue (McManus et al., 1998; Conant et al., 1998). Among HIV-infected opioid users, both in vitro and in vivo studies suggest that such responses may be more prominent and result in an increased risk of HIV dementia (Bell et al., 1998; Nath et al., 2000). Several members of the steroid and lipid nuclear receptor families, including the retinoid receptors and peroxisomal proliferators activated receptors, have been demonstrated to induce modulatory effects on proinflammatory responses by immune and other cells (Cantorna et al., 1994; Brinckerhoff et al., 1980). The retinoid receptors, which bind vitamin A and related molecules, have been the most extensively studied members of this family of receptors. These receptors, identified as retinoic acid receptor (RAR) and

62

W. Royal III et al. / Journal of Neuroimmunology 157 (2004) 61–65

retinoic X receptor (RXR) (Burri and Kutnick, 1987; Mangelsdorf et al., 1990; Heyman et al., 1992; Levin et al., 1992), can exist as RAR–RXR heterodimers or RXR homodimers. RXR can also function as a partner receptor for other nuclear receptors, including the peroxisomal proliferator activated receptor (PPAR) and the thyroid hormone and vitamin D receptors (Mangelsdorf et al., 1994a). The interaction between these receptors and RXR is required for normal function of the receptor partners. In these studies, the effects of retinoid receptor and PPARg activation in mononuclear and astroglial cell lines were examined. These studies suggest that these agents may be useful in suppressing cellular responses that may enhance toxicity to nervous system cells in the context of HIV infection.

2. Materials and methods 2.1. Cell cultures U937 cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in medium containing RPMI 1640 (BioWhittaker, Frederick, MD) supplemented with 10% fetal bovine serum (BioWhittaker), 50 units/ml of penicillin, 50 Ag/ml of streptomycin, and 2 mM l-Glutamine (ICN Biomedical, Costa Mesa, CA). The cell cultures were incubated with either 10 nM TTNPB (4-[E-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid; Biomol, Plymouth Meeting, PA), which is a RAR agonist, 100 nM LG101305 (Ligand Pharmaceuticals; San Diego, CA), a RXR agonist, 5 AM ciglitazone (Biomol), a PPARg agonist, or medium alone for 1 h. Selected cultures were then stimulated with phytohemagglutinin (PHA; MP Biomedicals, Irving, CA) alone or in the presence of 100 nM morphine sulfate (Sigma-Aldrich, St. Louis, MO) for 4 h. The culture supernatants were then collected and frozen at 808 Celsius for further analysis. SVG cells were grown in Eagles Minimum Essential Medium with Earle’s salts supplemented with 50 Al/ml gentamicin, 2 mM l-glutamine, and 10% inactivated fetal calf serum. The astroglial cells were then seeded onto 12 well plates then, when about 70% confluent, incubated with retinoid and PPARg ligands as described for the U937 cells except that cell stimulation was performed using 500 ng/ml lipopolysaccharide (LPS) and 150 IU interferon-g (BD Pharmingen, San Jose, CA) for 16 h in serum-free medium. Selected cultures were also exposed to morphine alone as described above or to morphine in the presence of 250 ng/ ml naloxone. 2.2. Flow cytometry Intracytoplasmic staining of U937 cells for TNF-a was performed following permeabilization with Cytoperm/

Cytofix solution with GolgiStop (BD Pharmingen) and incubation with FITC-conjugated anti-human TNF-a monoclonal antibody (BD Pharmingen) as previously described.(Mou et al., 2004). 2.3. Polymerase chain reaction assays Total cellular iNOS and 18s mRNA produced by the SVG cells were detected using the polymerase chain reaction assay with primers obtained from Ambion (Austin, TX). The cells were washed in PBS then scraped from the surface of the wells using a rubber policeman. Total RNA was then extracted using RNeasy Mini Columns with Qiashredder column inserts (Qiagen, Valencia, CA) according to the product instructions. RT-PCR and subsequent PCR amplification were performed in an iCycler (Bio-Rad, Hercules, CA) using the Qiagen OneStep RT-PCR Kit (Qiagen) according to the manufacturer’s directions. Reverse transcription was using performed using random hexamers for 30 min at 50 8C. Amplification was initiated by enzyme activation for 15 min at 95 8C followed by 35 cycles of denaturation at 94 8C for 15 s, annealing at 58 8C for 30 s, and extension at 72 8C for 30 s with SYBR green detection at 37 8C. The PCR products were then resolved on ethidium bromide-stained 1.5% agarose gels. 2.4. ELISA assays U937 cell culture supernatants were analyzed for TNF-a concentration in the human TNF-a BD OptEIA ELISA Set as previously described (Mou et al., 2004). 2.5. Statistics Comparisons between U937 treatment groups were examined using the Mann–Whitney U-test.

3. Results 3.1. TNF-a expression by U937 Cells Studies were performed to examine the effects of morphine on intracytoplasmic TNF-a production by PHA-activated U937 cells exposed to retinoid and PPARg receptor agonists and antagonists and the effects of morphine on these responses. In these studies, TNF-a was examined by flow cytometry of cells stained for intracytoplasmic cytokine. These studies revealed that exposure to PHA alone increased intracellular TNF-a, an effect that was suppressed in the presence of all of the receptor agonists (Fig. 1). In the presence of morphine, continued suppression was noted from treatment with TTNBP. In contrast, LG101305- and ciglitazone-induced TNF-a suppression was reversed by morphine.

W. Royal III et al. / Journal of Neuroimmunology 157 (2004) 61–65

Fig. 1. Effect of retinoid receptor and PPARg activation and morphine on TNF-a production by U937 cells. After a 1-h incubation of the cell cultures with TTNPB, LG101305, ciglitazone, or medium alone for one hour, indicated cultures were stimulated with PHAFmorphine. RAR, RXR, and PPARg activation suppressed PHA-induced increases in TNF-a production. Morphine reversed this effect of LG101305 and ciglitazone. Asterisks denote comparisons between morphine- and morphine+groups (**pb0.01; ***pb0.001).

63

been demonstrated to be an important factor that underlies the cellular damage in the nervous system that is associated with the development of HIV dementia (Dewhurst et al., 1996; Anderson et al., 2002). One of these products is nitric oxide (NO), which is synthesized by the enzyme inducible nitric oxide synthase (iNOS) in monocytes/macrophages and astrocytes (Anderson et al., 2002; Liu et al., 2002; Torre et al., 2002; Zhao et al., 2001; Adamson et al., 1996, 1999; Lipton, 1996; Rostasy et al., 1999). To model the effects of nuclear receptor activation on glial cell iNOS production, we examined the effects of the RAR, RXR, and PPARg agonists on iNOS mRNA produced by the SVG cells. These cells were stimulated with LPS and interferon-g, which has been shown to increase iNOS production by astrocytes (Nakagawa et al., 2000). The studies showed that, for non-stimulated cells, a tendency for a slight relative decrease in iNOS gene expression with activation of the nuclear receptors in the presence of morphine (Fig. 2). These effects were inhibited in the presence of naloxone. In contrast, for stimulated morphine-exposed cells, LG101305 induced a slight relative increase in iNOS expression whereas TTNPB and ciglitazone both suppressed iNOS mRNA production. Naloxone slightly suppressed iNOS expression by LG101305- and ciglitazonetreated cells.

4. Discussion 3.2. Effect of nuclear receptor activation on iNOS expression by SVG cells Oxidative stress, with the development of reactive oxygenated species, free radicals, and other reactive products, has

The steroid and lipid nuclear receptor family is comprised of an ever expanding list of molecules that play critical roles in the regulation of cell metabolism and other functions. In addition to having immunomodulatory effects,

Fig. 2. PCR analysis of iNOS and 18s mRNA expression by SVG cells exposed to retinoid receptor and PPARg agonists, morphine, and naloxone. Cells were incubated as described in Fig. 1 except stimulation was for performed for 16 h using LPS and interferon-g. (A) Agarose electrophoresis of the PCR products. (B) Bar graph showing absorbance measures of the iNOS bands relative to those for the 18s internal control. Non-stimulated cells showed slightly decreased relative expression of iNOS with LG101305, TTNPB, and ciglitazone exposure as compared to controls. This effect was reversed by naloxone. For stimulated cells, LG101305 increased relative iNOS expression relative to control cells and, in the presence of naloxone, there was relative suppression of iNOS expression by LG101305 and ciglitazone.

64

W. Royal III et al. / Journal of Neuroimmunology 157 (2004) 61–65

the retinoid and PPAR groups of receptors have been demonstrated to exert control over transcription of HIV-1 (Skolnik et al.; Hanley et al., 2004;Yamaguchi et al., 1994; Lee et al., 1994). The net effects of these influences include the reduction of chronic inflammation and levels of virus produced, which could potentially be of benefit to the host. The data presented in this report obtained by exposure of stimulated U937 cells to LG101305 and morphine are similar to what we previously described (Mou et al., 2004). We now report a similar effect with exposing U937 cells to ciglitazone and in studies of RXR activation in LPS/IFN-gstimulated SVG cells. The mechanisms that underlie this response would appear to be mediated by RXR activation, since morphine did not inhibit TNF-a and iNOS suppression that was induced by TTNPB and heterodimerization with RXR is required for PPAR activity. However, morphine had no effect on the suppression of iNOS gene expression that was induced by ciglitazone in stimulated SVG cells. Therefore, other factors are likely also important in determining the effects of these agonist agents in various cell types. The natural ligands for RAR are all-trans retinoic acid and 9-cis retinoid acid. 9-cis retinoic acid can also bind RXR. Formation of the RAR-RXR heterodimer is preferred by high ATRA and low 9-cis retinoic acid levels (Mangelsdorf et al., 1994b). Therefore, processes that are activated by RXRspecific ligands will likely require sustained biologically active levels of the RXR agonist and its receptor. The DNAbinding regions of the receptors recognize response elements that are located within the promoter region of target genes and can interact with components of the gene transcriptional complex to either enhance or suppress activation of the promoter (Goldman et al., 1997). Both retinoid receptor and PPARg agonists have been considered for use in the treatment of immune-mediated nervous system disease. For example, experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis (MS) that can be induced in mice, rats, guinea pigs and some primates, and the associated secretion of TNF-a and IFN-g can be suppressed in mice by the administration of various retinoids (Racke et al., 1995; Massacesi et al., 1987; Vladutiu and Cringulescu, 1968). In addition, studies of retinol levels in patients with MS showed levels in blood and in cerebrospinal fluid (CSF) to, respectively, be lower than what was observed among control subjects and to correlate with CSF CD4+CD45RO+ cell percentages (Royal et al., 2002). The PPARg agonist ciglitazone as well as pioglitazone, rosiglitazone, troglitazone, and GW34785 have also been observed to suppress murine EAE (Diab et al., 2004; Feinstein et al., 2002). Similar effects were induced by the natural PPARg ligand PGJ2 (Diab et al., 2002; Natarajan and Bright, 2002; Niino et al., 2001); however, this molecule can also act by blocking degradation of the NK-nB inhibitor molecule, InB (Feinstein et al., 2002). Notably, simultaneous activation of RXR agonist and PPARg can have additive effects in the EAE model. There

are no RXRE or PPARE described in the TNF-a or the MOR promoters, however, which suggests that these receptors might indirectly affect TNF-a and MOR production by interacting with transcriptional co-factors required for the production of these gene products or by directly influencing the production of these proteins. Therefore, future studies examining the specific influences of these agents on cytokine production and opioid responses will be of great interest.

References Adamson, D.C., Wildemann, B., Sasaki, M., Glass, J.D., McArthur, J.C., Christov, V.I., Dawson, T.M., Dawson, V.L., 1996. Immunologic NO synthase: elevation in severe AIDS dementia and induction by HIV-1 gp41. Science 274, 1917 – 1921. Adamson, D.C., McArthur, J.C., Dawson, T.M., Dawson, V.L., 1999. Rate and severity of HIV-associated dementia (HAD): correlations with Gp41 and iNOS. Mol. Med. 5, 98 – 109. Anderson, E., Zink, W., Xiong, H., Gendelman, H.E., 2002. HIV-1associated dementia: a metabolic encephalopathy perpetrated by virusinfected and immune-competent mononuclear phagocytes. J. Acquir. Immune Defic. Syndr. 31 (Suppl. 2), S43 – S54. Bell, J.E., Brettle, R.P., Chiswick, A., Simmonds, P., 1998. HIV encephalitis, proviral load and dementia in drug users and homosexuals with AIDS. Effect of neocortical involvement. Brain 121, 2043 – 2052. Brinckerhoff, C.E., McMillan, R.M., Dayer, J.-M., Harris Jr., E.D., 1980. Inhibition by retinoic acid of collagenase production in rheumatoid synovial cells. N. Engl. J. Med. 303, 432 – 436. Burri, B.J., Kutnick, M.A., 1987. High performance liquid chromatographic test for retinol binding protein. Clin. Chem. 33, 935. Cantorna, M.T., Nashold, F.E., Hayes, C.E., 1994. In vitamin A deficiency multiple mechanisms establish a regulatory T helper cell imbalance with excess Th1 and insufficient Th2 function. J. Immunol. 152, 1515 – 1522. Conant, K., Garzino-Demo, A., Nath, A., McArthur, J.C., Halliday, W., Power, C., Gallo, R.C., Major, E.O., 1998. Induction of monocyte chemoattractant protein-1 in HIV-1 Tat-stimulated astrocytes and elevation in AIDS dementia. Proc. Natl. Acad. Sci. U. S. A. 95, 3117 – 3121. Dewhurst, S., Gelbard, H.A., Fine, S.M., 1996. Neuropathogenesis of AIDS. Mol. Med. Today 2, 16 – 23. Diab, A., Deng, C., Smith, J.D., Hussain, R.Z., Phanavanh, B., LovettRacke, A.E., Drew, P.D., Racke, M.K., 2002. Peroxisome proliferatoractivated receptor-gamma agonist 15-deoxy-Delta(12,14)-prostaglandin J(2) ameliorates experimental autoimmune encephalomyelitis. J. Immunol. 168, 2508 – 2515. Diab, A., Hussain, R.Z., Lovett-Racke, A.E., Chavis, J.A., Drew, P.D., Racke, M.K., 2004. Ligands for the peroxisome proliferator-activated receptor-gamma and the retinoid X receptor exert additive antiinflammatory effects on experimental autoimmune encephalomyelitis. J. Neuroimmunol. 148, 116 – 126. Feinstein, D.L., Galea, E., Gavrilyuk, V., Brosnan, C.F., Whitacre, C.C., Dumitrescu-Ozimek, L., Landreth, G.E., Pershadsingh, H.A., Weinberg, G., Heneka, M.T., 2002. Peroxisome proliferator-activated receptorgamma agonists prevent experimental autoimmune encephalomyelitis. Ann. Neurol. 51, 694 – 702. Goldman, P.S., Tran, V.K., Goodman, R.H., 1997. The multifunctional role of the co-activator CBP in transcriptional regulation. Recent Prog. Horm. Res. 52, 103 – 119. Hanley, T.M., Kiefer, H.L., Schnitzler, A.C., Marcello, J.E., Viglianti, G.A., 2004. Retinoid-dependent restriction of human immunodeficiency virus type 1 replication in monocytes/macrophages. J. Virol. 78, 2819 – 2830.

W. Royal III et al. / Journal of Neuroimmunology 157 (2004) 61–65 Heyman, R.A., Mangelsdorf, D.J., Dyck, J.A., Stein, R.B., Eichele, G., Evans, R.M., Thaller, C., 1992. 9-cis-Retinoic acid is a high affinity ligand for the retinoid X receptor. Cell 68, 397 – 406. Lee, M.O., Hobbs, P.D., Zhang, X.K., Dawson, M.I., Pfahl, M., 1994. A synthetic retinoid antagonist inhibits the human immunodeficiency virus type 1 promoter. Proc. Natl. Acad. Sci. U. S. A. 91, 5632 – 5636. Levin, A.A., Sturzenbecker, L.J., Kazmer, S., Bosakowski, T., Huselton, C., Allenby, G., Speck, J., Kratzeisen, C., Rosenberger, M., Lovey, A., Grippo, J.F., 1992. 9-cis-Retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha. Nature 355, 359 – 361. Lipton, S.A., 1996. Similarity of neuronal cell injury and death in AIDS dementia and focal cerebral ischemia: potential treatment with NMDA open-channel blockers and nitric oxide-related species. Brain Pathol. 6, 507 – 517. Lipton, S.A., Gendelman, H.E., 1995. Seminars in medicine of the Beth Israel Hospital, Boston. Dementia associated with the acquired immunodeficiency syndrome. N. Engl. J. Med. 332, 934 – 940. Liu, X., Jana, M., Dasgupta, S., Koka, S., He, J., Wood, C., Pahan, K., 2002. Human immunodeficiency virus type 1 (HIV-1) tat induces nitricoxide synthase in human astroglia. J. Biol. Chem. 277, 39312 – 39319. Mangelsdorf, D.J., Ong, E.S., Dyck, J.A., Evans, R.M., 1990. Nuclear receptor that identifies a novel retinoic acid-response pathway. Nature 345, 224 – 229. Mangelsdorf, D.J., Umesono, K., Evans, R.M., 1994a. Retinoid receptors. In: Sporn, M.B., Roberts, A.B., Goodman, D.S. (Eds.), The Retinoids. Raven Press, New York, pp. 319 – 350. Mangelsdorf, D.J., Umesono, K., Evans, R.M., 1994b. The retinoid receptors. In: Sporn, M.B., Roberts, A.B., Goodman, D.S. (Eds.), The Retinoids. Raven Press, New York, pp. 319 – 350. Massacesi, L., Abbamondi, A.L., Giorgi, C., Sarlo, F., Lolli, F., Amaducci, L., 1987. Suppression of experimental allergic encephalomyelitis by retinoic acid. J. Neurol. Sci. 80, 55 – 64. McManus, C.M., Brosnan, C.F., Berman, J.W., 1998. Cytokine induction of MIP-1 alpha and MIP-1 beta in human fetal microglia. J. Immunol. 160, 1449 – 1455. Mou, L., Lankford-Turner, P., Leander, M.V., Bissonnette, R.P., Donahoe, R.M., Royal, W., 2004a. RXR-induced TNF-alpha suppression is reversed by morphine in activated U937 cells. J. Neuroimmunol. 147, 99 – 105. Nakagawa, H., Moritake, T., Tsuboi, K., Ikota, N., Ozawa, T., 2000. Induction of superoxide in glioma cell line U87 stimulated with

65

lipopolysaccharide and interferon-gamma: ESR using a new flow-type quartz cell. FEBS Lett. 471, 187 – 190. Natarajan, C., Bright, J.J., 2002. Peroxisome proliferator-activated receptorgamma agonists inhibit experimental allergic encephalomyelitis by blocking IL-12 production. IL-12 signaling and Th1 differentiation. Genes Immun. 3, 59 – 70. Nath, A., Haughey, N.J., Jones, M., Anderson, C., Bell, J.E., Geiger, J.D., 2000. Synergistic neurotoxicity by human immunodeficiency virus proteins Tat and gp120: protection by memantine. Ann. Neurol. 47, 186 – 194. Niino, M., Iwabuchi, K., Kikuchi, S., Ato, M., Morohashi, T., Ogata, A., Tashiro, K., Onoe, K., 2001. Amelioration of experimental autoimmune encephalomyelitis in C57BL/6 mice by an agonist of peroxisome proliferator-activated receptor-gamma. J. Neuroimmunol. 116, 40 – 48. Racke, M.K., Burnett, D., Pak, S.H., Albert, P.S., Cannella, B., Raine, C.S., McFarlin, D.E., Scott, D.E., 1995. Retinoid treatment of experimental allergic encephalomyelitis. IL-4 production correlates with improved disease course. J. Immunol. 154, 450 – 458. Rostasy, K., Monti, L., Yiannoutsos, C., Kneissl, M., Bell, J., Kemper, T.L., Hedreen, J.C., Navia, B.A., 1999. Human immunodeficiency virus infection, inducible nitric oxide synthase expression, and microglial activation: pathogenetic relationship to the acquired immunodeficiency syndrome dementia complex. Ann. Neurol. 46, 207 – 216. Royal III, W., Gartner, S., Gajewski, C.D., 2002. Retinol measurements and retinoid receptor gene expression in patients with multiple sclerosis. Mult. Scler. 8, 452 – 458. Skolnik, P.R., Rabbi, M.F., Mathys, J.M., and Greenberg, A.S., Stimulation of peroxisome proliferator-activated receptors alpha and gamma blocks HIV-1 replication and TNFalpha production in acutely infected primary blood cells, chronically infected U1 cells, and alveolar macrophages from HIV-infected subjects. Torre, D., Pugliese, A., Speranza, F., 2002. Role of nitric oxide in HIV-1 infection: friend or foe? Lancet, Infect. Dis. 2, 273 – 280. Vladutiu, A., Cringulescu, N., 1968. Suppression of experimental allergic encephalomyelitis by vitamin A. Experimentia 24, 718 – 719. Yamaguchi, K., Groopman, J.E., Byrn, R.A., 1994. The regulation of HIV by retinoic acid correlates with cellular expression of the retinoic acid receptors. AIDS 8, 1675 – 1682. Zhao, M.L., Kim, M.O., Morgello, S., Lee, S.C., 2001. Expression of inducible nitric oxide synthase, interleukin-1 and caspase-1 in HIV-1 encephalitis. J. Neuroimmunol. 115, 182 – 191.