Differential immunomodulatory effects by Tripterygium wilfordii Hook f-derived refined extract PG27 and its purified component PG490 (triptolide) in human peripheral blood T cells: potential therapeutics for arthritis and possible mechanisms explaining in part Chinese herbal theory “Junn-Chenn-Zuou-SS”
© Ho et al.; licensee BioMed Central Ltd. 2013
Received: 28 July 2013
Accepted: 19 November 2013
Published: 21 November 2013
For thousands of years, it remains unclear why Chinese prefer complex herbal remedy and seldom try to purify it. One of the reasons is that they believe Chinese herbs compared to Western drugs are relatively less toxic and better tolerated. The so called “Junn-Chenn-Zuou-SS” theory illustrates a concept of coordinated effects from a combination of different Chinese herbs. PG27, a refined extract from a well-known Chinese antirheumatic herb Tripterygium wilfordii Hook f (TwHf), is effective in attenuating transplantation rejection and extending survival of cardiac xenografts.
Experiments were conducted in human primary T lymphocytes isolated from buffy coat. The activities of the inhibitor of kappaB alpha kinase-inhibitor of kappaB alpha-nuclear factor kappaB (IKK-IκBα-NF-κB) and mitogen activated protein kinase-activator protein-1 (MAPK-AP-1) signaling pathways were determined via electrophoretic mobility shift assays, immunoprecipitation kinase assays, Western blots, and transfection assays.
We showed that PG27 inhibited IKKα-IκBα-NF-κB and MAPK-AP-1 signaling pathways; however, IKKβ activity was less susceptible to inhibition by PG27. In contrast, the purified component of TwHf, PG490 (triptolide), reduced both MAPK-AP-1 and IKK-IκBα-NF-κB signaling pathways, including both IKKα and IKKβ, with similar potency. By means of high performance liquid chromatography analysis, it was estimated that PG490 constituted 1.27 ± 0.06% of the total PG27 content. Further analysis demonstrated that compared to PG490 alone, PG27 that contained an equal amount of PG490 was less toxic and less immunosuppressive, suggesting the presence of cytoprotective ingredient(s) in the non-PG490 components of PG27.
In addition to demonstrating the immunomodulatory capacity of PG27 as the potential therapeutics for arthritis and prevention of transplantation rejection, the differential regulatory effects and mechanisms by PG27 and PG490 further support in part a possibly-existing Chinese herbal theory “Junn-Chenn-Zuou-SS”.
KeywordsTripterygium wilfordii Hook f Nuclear factor kappaB I-kappaBalpha kinase-beta PG27 PG490 (Triptolide) T Cells
According to the concept of Chinese herbal therapy, the greatest therapeutic effects come from a combination of several ingredients; some of them are effective in treating diseases and some of them modulate the function of these active components through enhancing their efficacy, reducing their side effects, or manipulating their delivery into the target organs. The record about this concept called “Junn-Chenn-Zuou-SS” is first published in Shen Nong Ben Cao Jing, an earliest medical material dictionary composed at the era of Qin and Han dynasties. The theory “Junn-Chenn-Zuou-SS” has been generally adopted in Chinese medicine practice where “Junn” represents the active effective ingredient(s); “Chenn” is the adjunctive ingredient(s) enhancing the effectiveness of active ingredient(s); “Zuou” is the complementary ingredient(s) reducing the side effects of active effective ingredient(s) and “SS” is the ingredient(s) helpful in delivering the active effective ingredient(s) into the target organs. The “Junn-Chenn-Zuou-SS” theory is presumably achieved by administering different herbs (called “Danfang” for each herb) with known functions together called a “Fufang” at a time. The concept of “Fufang” in fact has already been applied in medicine for a long time including the medications from Western societies. Typical examples include the treatments for rheumatoid arthritis (RA)  and HIV infection . In these examples, a combination of several drugs preserving different inhibitory effects and mechanisms on specific molecules or pathways may work together and achieve synergistic effects. By doing this, the dosages of individual drugs may be greatly reduced to attenuate the potential toxic effects from each drug and yet the effects are synergistically enhanced. Nevertheless, there has been no scientific evidence showing that “Junn-Chenn-Zuou-SS” therapeutic theory may exist and work by different components in a single herb.
The most commonly used Chinese medicine for autoimmune disorders, such as RA, is Tripterygium wilfordii Hook f (TwHf; known as Thunder God Vine), which has potent immunosuppressive effects [3, 4]. Currently, different TwHf extracts are prescribed to treat autoimmune disorders in mainland China. Aside from extensive clinical trials conducted in oriental populations, the double blinded studies in RA patients of Western populations also confirm its effectiveness [5, 6]. In our previous work, we demonstrated that TwHf is an effective immunomodulatory drug, which acts by inhibiting T-cell activation and inducing T-cell apoptosis [7, 8]. Although the usefulness of each ingredient of TwHf extracts has not been studied in detail, the major therapeutic effects of TwHf have been suggested to be from some of the ingredients such as PG490 (triptolide), tripdiolide, triptonide and triptophenolide [9–11].
Because the commonly prescribed TwHf preparations are considered to have toxicities, this greatly reduces the usefulness of this drug for clinical purposes. In order to minimize drug toxicity yet reserve drug efficacy, further purification of TwHf leads to the refined extract called PG27 that shows promising effects in prevention of bone marrow transplantation rejections . Importantly, a combination of both PG27 and cyclosporine results in strong synergistic effects in extending the survival of hamster-to-rat cardiac xenograft model . In this context, the TwHf purified product PG490 also preserves strong immunosuppressive effects [14, 15]. In the light of the current therapeutic strategy for autoimmune disorders with a combination of several disease-modifying antirheumatic drugs to increase efficacy and to reduce adverse events , the exploration of effects and mechanisms of Chinese antirheumatic drugs should bring more alternatives for the therapy of autoimmune disorders.
The nuclear factor kappaB (NF-κB) family consists of Rel-domain-containing proteins that are crucial for the regulation of inflammation and immune responses [16, 17]. In resting cells, these proteins are retained in the cytosol by a group of inhibitory proteins such as inhibitor of kappaB alpha (IκBα). After activation, IκBα is phosphorylated by IκBα kinases (IKKs) such as IKKα and IKKβ, and undergoes ubiquitination and subsequent proteosome-mediated degradation . This leads to the nuclear translocation of NF-κB from the cytosol and induces the activation of a variety of genes, leading to diseases such as RA . Similar to NF-κB, the activating protein-1 (AP-1) transcription factors, which consist of Jun and Fos family proteins, extensively participate in regulating cell proliferation, transformation and death, and serve as good therapeutic targets for the control of inflammation .
In this report, we investigated the effects and mechanisms of PG27-mediated immunomodulation in primary T cells. We observed that PG27 had differential immunosuppressive potency for IKKα and IKKβ, a phenomenon not observed for the purified compound PG490. In addition, PG27 that contained an equivalent amount of PG490 was less toxic than PG490 alone. These observations explain in part a possible mechanism of “Junn-Chenn-Zuou-SS” theory and provide evidence suggesting that PG27 may be assessed for potential use as a disease-modifying antirheumatic drug for autoimmune disorders like RA.
Materials and methods
Preparation of PG27 and PG490
PG27 powder was prepared by Pharmagenesis (La Jolla, California) and was kindly provided by PhytoHealth, Inc, Taipei, Taiwan. PG490 was purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). Both drugs were dissolved in DMSO to generate 100 mg/mL (PG27) or 100 ng/mL (PG490) stock solutions. For experiments, the required concentrations of each drug were made by dilution of the concentrated stock solution with culture medium, which contained RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, and 1,000 U/mL penicillin-streptomycin (Gibco-BRL, Gaithersberg, MD).
Preparation of peripheral blood T cells
The use of buffy coat purchased from the blood bank (Taipei, Taiwan) was approved by the Institutional Review Board, Tri-Service General Hospital, through a fast review tract. Human peripheral blood T cells were purified from whole blood via negative selection, according to our previous report . Briefly, buffy coat was mixed with Ficoll-Hypaque, and the layer of mononuclear cells was collected after centrifugation. After lysis of red blood cells, the peripheral blood mononuclear cells were incubated with antibodies (Abs), including L243 (anti-DR; American Type Culture Collection [ATCC], Rockville, MD), OKM1 (anti-CD11b; ATCC), and LM2 (anti-Mac1; ATCC) for 30 min at 4°C. The cells were washed with medium containing 10% fetal bovine serum and incubated with magnetic beads conjugated to goat anti-mouse IgG (R & D, Minneapolis, MN). The antibody-stained cells were then removed with a magnet. Following a repeat of the above procedures, T cells were obtained with a purity of more than 98%, as determined by the percentage of CD3+ cells in flow cytometry (Beckton Dickinson, Mountain View, CA).
Cell stimulation, cytokine determination, and cell survival measurement
To activate T cells, the following stimuli were used: phorbol 12-myristate 13-acetate (PMA; Sigma) at 10 ng/mL, ionomycin (Sigma) at 1 μM, immobilized monoclonal antibody (mAb) anti-CD3 (OKT3; ATCC) at 10 μg/mL, soluble anti-CD28 mAb (Beckton Dickinson) at 1 μg/mL, and TNF-α at 10 ng/mL. The cells were incubated with the various stimuli, and the cell pellets or supernatants were collected for analysis. The determination of cytokine concentrations was performed via ELISA as described previously . IC50 was the concentration of drug (PG27 or PG490) that inhibited half the cytokine production from different stimuli-activated T cells and was calculated by linear regression using Microsoft Excel. Cell viability was measured by either the trypan blue exclusion assay or the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) colorimetric assay, as described in our previous report . Similarly, the 50% lethal concentrations (LC50) of PG27 and PG490 were calculated.
Preparation of cytoplasmic and nuclear extracts
Cytoplasmic and nuclear extracts were prepared according to our published work . Briefly, treated cells were incubated at 4°C in 50 μL of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 1 mM PMSF, 3.3 μg/mL aprotinin) for 15 min, with occasional gentle vortexing. The swollen cells were centrifuged at 15,000 rpm for 3 min. After removal of the supernatants (cytoplasmic extracts), the pelleted nuclei were washed with 50 μL of buffer A, and subsequently, the cell pellets were resuspended in 30 μL of buffer C (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 1 mM DTT, 0.5 mM PMSF, 3.3 μg/mL aprotinin) and incubated at 4°C for 30 min, with occasional vigorous vortexing. The mixtures were then centrifuged at 15,000 rpm for 20 min, and the supernatants were used as nuclear extracts.
Electrophoretic mobility shift assays (EMSAs)
EMSAs were performed as detailed in our previous report . Oligonucleotides containing the NF-κB-binding site (5′-AGT TGA GGG GAC TTT CCC AGG C-3′), the AP-1-binding site (5′-CGC TTG ATG AGT CAG CCG GAA-3′), and the Oct-1-binding site (5′-TGT CGA ATG CAA ATC ACT AGA A-3′) were purchased and used as DNA probes (Promega, Madison, WI). The DNA probes were radio-labeled with [γ-32P]ATP using T4 kinase, according to the manufacturer’s instructions (Promega). For the binding reactions, the radio-labeled probes were incubated with 5 μg of nuclear extracts. The binding buffer contained 10 mM Tris–HCl, pH 7.5, 50 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 1 mM MgCl2, 4% glycerol, and 2 μg poly(dI-dC). The reaction mixtures were incubated at room temperature for 20 min prior to the binding reaction. For supershift assays, different mAbs were preincubated with nuclear extracts for 30 min before the addition of the radiolabeled probes. The final reaction mixtures were analyzed in 6% non-denaturing polyacrylamide gels with 0.25× Tris-borate/EDTA as an electrophoresis buffer.
ECL Western blotting (Amersham, Arlington Heights, IL) was performed as described previously . Briefly, equal amounts of whole cell lysates and cytoplasmic or nuclear extracts were analyzed by 10% SDS-PAGE and transferred to a nitrocellulose filter. For immunoblotting, the nitrocellulose filter was incubated with Tris-buffered saline containing 5% nonfat milk (milk buffer) for 2 h, and then blotted with antisera against IκBα, IKKα, IKKβ (Santa Cruz Biotechnology), or β-actin overnight at 4°C. After washing twice with milk buffer, the filter was incubated with donkey anti-mouse IgG conjugated to horseradish peroxidase at a concentration of 1:5,000 for 30 min. The filter was then incubated with the substrate and exposed to X-ray film.
Immunoprecipitation kinase assay
The immunoprecipitation kinase assay was described in detail in our previous report . The GST-IκBα fusion protein was used as a substrate for IKKα and IKKβ. The JNK substrate, a GST-c-Jun fusion protein, was a kind gift from Dr. S.-F. Yang (Academia Sinica, Taiwan). Myelin basic protein (MBP), which was used as a substrate for both ERK and p38, was purchased from Sigma. The Abs used for the kinase assays were purchased from Cell Signaling (Beverly, MA; for anti-JNK and anti-p38 polyclonal Abs) and Santa Cruz Biotechnology (for anti-ERK, anti-IKKα and anti-IKKβ polyclonal Abs). To perform the immunoprecipitation kinase assay, 50–100 μg of whole cell extract was incubated with 5 μL of specific Abs in an incubation buffer (25 mM HEPES, pH 7.7, 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton-X-100, 20 mM β-glycerophosphate, 0.1 mM Na3VO4, 2 μM leupeptin, 400 μM PMSF) overnight. The mixture was then immunoprecipitated by the addition of protein A beads and rotated at 4°C for 2 h. After extensive washing, (twice with a HEPES washing buffer [20 mM HEPES, pH 7.7, 50 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100]; twice with an LiCl washing buffer [500 mM LiCl, 100 mM Tris, pH 7.6, 0.1% Triton X-100, 1 mM DTT]; and twice with a kinase buffer [20 mM MOPS, pH 7.2, 2 mM EDTA, 10 mM MgCl2, 0.1% Triton X-100 and 1 mM DTT], the beads were resuspended in 40 μL of kinase buffer, along with cold ATP (30 μM) and 10 μCi of [γ-32P]ATP. The mixture was incubated at 30°C with occasional gentle mixing for 30 min. The reaction was then terminated by resuspending the samples in a 1% SDS solubilizing buffer and boiling for 5 min. The samples were then analyzed by SDS-PAGE.
Transfection assay in purified human peripheral blood T cells
The transfection assay was performed by electroporation with an Amaxa Nucleofector apparatus, according to the manufacturer’s instructions (Amaxa, Cologne, Germany). In brief, primary T cells were mixed with 5 μg of the reporter plasmid pNF-κB-luciferase (Luc) or pAP-1-Luc (Stratagene, La Jolla, CA) in 100 μL of the provided electroporation buffer. After electroporation, the cells were transferred to 2 mL of pre-warmed RPMI medium. After transfection for 48 h, the cells were aliquoted equally for testing the individual conditions described in the figure legends. For treatment, the drugs were added 2 h before the addition of stimuli. After stimulation with TNF-α for 18 h, the cell pellets were collected, total cell lysates were prepared, and the luciferase activity, after normalization to the total protein amounts, was determined according to the manufacturer’s instructions (Promega).
Analysis of the PG490 content in PG27 by HPLC
Both PG490 (1.6 mg) and PG27 (2.4 mg) were dissolved in DMSO (1.0 mL) to generate stock solutions. PG490 was spiked into the mobile phase to create a series of standards consisting of 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 mg/mL concentrations. High Performance Liquid Chromatography (HPLC) was performed on an Agilent Model 1100 Quat pump system equipped with an Agilent 1100 VWD Detector and an Agilent 1100 ALS auto-injector. The detector was set to 218 nm. A reversed phase column (Cosmosil 5C18-AR-II, 25 cm × 4.6 mm I.D.) was applied. The mobile phase was 45% MeOH/55% H2O eluted at a flow rate of 0.8 mL/min. The injection volume was 20 μL for each sample.
The results were expressed as means ± standard deviations. One-way ANOVA analysis was used to evaluate the differences, which were considered to be statistically significant at a P value of <0.05.
PG27 inhibited IL-2 production from activated T cells
PG27 inhibited NF-κB and AP-1 activation induced by various stimuli
Effects of PG27 on IκBα degradation and IKK activity
PG490 inhibited NF-κB and AP-1 activation
PG490 inhibited both IKKα and IKKβ activities
PG27 and PG490 downregulated MAPK activity
Side-by-side comparisons of the effects of PG27 and PG490 on IL-2 production and cytotoxicity in activated T cells
PG27 attracts the attention of scientists since one decade ago with its promising immunomodulatory effects in inducing antigen-specific tolerance in bone marrow transplanted mice  and the extending survival of cardiac xenograft models . The following work was postponed because of consideration of the priority of clinical trials by the pharmaceutical company. In considering the potential applications of PG27 for autoimmune disorders, in the present study, we conducted molecular experiments to examine the mechanisms of PG27-mediated immunomodulation in human peripheral blood T cells. PG27, at therapeutic concentrations, not only suppressed various stimuli-induced transcription factor DNA-binding activities but also suppressed the transcriptional activities of both NF-κB and AP-1 (Figure 2). To our surprise, PG27 differentially regulated IKKα and IKKβ kinase activities induced by various stimuli. In contrast, the purified TwHf component PG490 inhibited both IKKα and IKKβ activities with similar potency. HPLC analysis determined that PG490 constituted 1.27 ± 0.06% of PG27 content (Figure 7). Compared to PG490 alone, PG27 that contained an equal amount of PG490 was not only less potent in immunosuppressive activity but also less cytotoxic in activated T cells (Figure 8).
Based upon the “Junn-Chenn-Zuou-SS” theory, the evidence for PG490 working as “Junn”, the main active ingredient, can be supported by its apoptosis-inducing effects and potent immunosuppressive effects demonstrated in a variety of tissue cells stimulated by different stimuli as well as in animal models of autoimmune disorders [11, 14, 22, 23]. In addition to PG490, there are many uncharacterized components in PG27 and compared to PG490 alone, PG27 was less toxic. It seems probable that the IKKβ-suppressive effect of PG490 was masked or neutralized by other non-PG490 components in PG27, resulting in the reduction of both immunosuppressive potency and cytotoxic effects. Accordingly, some of these non-PG490 components may function as “Zuou”. Because there have been no reports simultaneously examining the combinatorial effects of two or more than two different components of TwHf, the components in PG27 that work as “Zuou” are currently unclear. Further purification and examination of PG27 components can help solve the question.
Because NF-κB transcription factors can up-regulate many genes involved in inflammatory responses, targeting NF-κB signaling events has been one of the major therapeutic goals in preventing graft rejections and in controlling autoimmune diseases [24, 25]. The commonly prescribed disease modifying antirheumatic drugs also preserve inhibitory effects against NF-κB activation [26, 27]. Regarding the significance of NF-κB in transplantation immunology, the inhibition of NF-κB by IκBα gene transfer is shown to improve oxygenation of the transplanted lung . Transfection with NF-κB decoy into the donor lung effectively reduces lung injury during acute allograft rejections . Like NF-κB, MAPK-AP-1 signaling pathway is also a critical and excellent target to block in developing therapy for inflammation-related disorders [30, 31]. The inhibition of IKKα-IκBα-NF-κB and MAPK-AP-1 signaling pathways by PG27 and PG490 should lead them to potential candidates of promising immunomodulatory drugs for the therapy of autoimmune disorders and for the prevention of graft rejections.
Three major classes of stimuli, including PMA + ionomycin, the CD28 costimulatory molecule, and TNF-α, were used to activate T cells for the investigation of the immunomodulatory effects of PG27 and PG490 in human peripheral blood T cells. Different types of stimuli did not seem to affect the experimental outcomes, as the results were consistent and reproducible regardless of the stimuli being evaluated. PMA + ionomycin mimics a T cell receptor-mediated stimulus that bypasses the requirement for an antigen- or lectin-induced signal . The pro-inflammatory cytokine, TNF-α, is considered to be an important molecule for the regulation of upstream cytokine cascades in inflammatory responses. The blockade of TNF-α-mediated events has been found to have significant therapeutic effects on active RA and seronegative spondyloarthropathies . Given the significance of CD28 signaling in T cell activation, blockade of the CD28 signaling pathway is another promising therapeutic strategy for RA, even for those who are refractory to anti-TNF therapy . Considering the critical roles of T cells in autoimmune disorders, we examined these crucial stimuli-activated T cells and demonstrated the broad-spectrum immunosuppressive capacities of both PG27 and PG490.
The differential inhibitory potency of PG27 against IKKα and IKKβ is interesting. Studies of many different compounds against IKK activity have indicated that for arthritis therapeutics, the suppression of either IKKα or IKKβ may be sufficient to block NF-κB activation . The concentrations of IC50 for PG490 and PG27 with equivalent content of PG490 on PMA + ionomycin-induced and CD3/CD28-induced IL-2 production indicate that PG490 preserved more potent immunosuppressive activity than PG27. It suggests that the counteraction of PG490-mediated IKKβ suppression by other components resulted in reduction of immunosuppressive potency of PG27. Nevertheless, the selective suppression of IKKα but not IKKβ by PG27 may potentially lead to some benefits therapeutically. For example, in knockout studies, the targeted deletion of IKKβ results in early embryonic lethality due to extensive apoptosis of fetal hepatocytes [36, 37]. In contrast, the deficiency of IKKα which results in abnormal development of skin and skeleton is relatively less fatal . In addition, IKKβ plays a requisite role in B cell activation and maintenance . Furthermore, the preservation of intact IKKβ-NF-κB signaling pathway is important for protecting T cells from TNF-α-induced apoptosis . According to Egan et al. , in an in vivo system, the preservation of IKKβ-dependent NF-κB activation pathway is crucial for protection against radiation-induced apoptosis in intestinal epithelium. It is therefore possible that given an already suppressed IKKα-NF-κB signaling pathway, the preservation of IKKβ-NF-κB signaling pathway may help to reduce the potential side effects of PG27 in T cells and in other tissue cells. This suggestion was supported by a much reduced cytotoxic effect, as compared to PG490 alone, in PG27 containing equivalent amount of PG490 content.
In this study, we observed that PG27 and PG490 had differential suppressive effects on IKKα and IKKβ activities induced by a variety of stimuli in T cells. The results also suggest that compared to PG490 alone, PG27 that contained an equivalent amount of PG490 caused less cell death. In light of the current therapeutic strategy for autoimmune disorders, which involves the combination of several disease-modifying antirheumatic drugs to increase efficacy and reduce adverse events , the exploration of the effects and mechanisms of Chinese antirheumatic drugs such as PG27 should provide additional alternatives for the therapy of autoimmune disorders like RA.
There are several limitations in this report. Firstly, it remains unclear the components responsible for counteracting PG490-mediated IKKβ-suppressive effects. Secondly, this in vitro study can not exactly reflect the in vivo situations, especially the situations in humans. Thirdly, whether the observations in T cells may happen in other tissue cells requires additional experiments to examine. Lastly, only a head-to-head comparison in clinical trials but not in this study can really tell us whether the observed benefit/risk of PG27 compared to PG490 does exist. Evidently, more studies are needed to answer these questions.
Traditional Chinese medicine prescriptions “Fufang” usually contain several herbs (each called “Danfang”). The traditional Chinese medicinal doctors will modify and adjust the ingredients and doses of each “Danfang” according to the need of individual patients. This formulation is based on the principle of “Junn-Chenn-Zuou-SS”. Therefore, the commonly accepted working concept of “Junn-Chenn-Zuou-SS” illustrates the specific coordinated effects from a combination of different Chinese herbs. In this study, we provide novel and interesting observations demonstrating that the “Junn-Chenn-Zuou-SS” theory may also work in a refined extract PG27 from a single herb TwHf. It is anticipated that with the inclusion of more molecular studies on Chinese herbs, the concept of “Junn-Chenn-Zuou-SS” will gain more scientific support.
This work is supported in part by grants from the National Health Research Institutes (NHRI-EX94-9208SI), the Department of Health (CCMP92-RD-112) and the National Science Council (NSC 101-2314-B-182A-103-MY3), Taipei, Taiwan, R.O.C. The kind gift from Dr. S.F. Yang is greatly appreciated.
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