Properdin deficiency protects from 5-fluorouracil-induced small intestinal mucositis in a complement activation-independent, interleukin-10-dependent mechanism.

Intestinal mucositis is a serious complication of chemotherapy that leads to significant morbidity that may require dose or drug adjustments. Specific mitigating strategies for mucositis are unavailable, due partly to an incomplete understanding of the pathogenic mechanisms. We have previously shown an effect of properdin, a positive regulator of complement activation, in models of colitis. Here we use properdin-deficient (PKO ) mice to interrogate the role of properdin and complement in small intestinal mucositis. Mucositis was induced by five daily injections of 5-fluorouracil (5-FU) in wild-type (WT), PKO , interleukin (IL)-10-/- and properdin/IL-10-/- double knock-out (DKO) mice. At the time of euthanasia their jejunum was collected for histology, immunohistochemistry and cytokine and complement activation measurements. Complement became activated in mice receiving 5-FU, indicated by increased intestinal levels of C3a and C5a. Compared to WT, PKO mice experienced significantly less mucositis, despite C3a levels as high as inflamed WT mice and slightly less C5a. Conversely, PKO mice had higher intestinal levels of IL-10. IL-10 expression was mainly by epithelial cells in both uninflamed and inflamed PKO mice. IL-10-/- mice proved to be highly susceptible to mucositis and DKO mice were equally susceptible, demonstrating that a lack of properdin does not protect mice lacking IL-10. We interpret our findings to indicate that, to a significant extent, the inflammation of mucositis is properdin-dependent but complement activation-independent. Additionally, the benefit achieved in the absence of properdin is associated with increased IL-10 levels, and IL-10 is important in limiting mucositis.

Detachment-induced upregulation of XIAP and cIAP2 delays anoikis of intestinal epithelial cells.

Detachment of normal epithelial cells from the extracellular matrix triggers apoptosis, a phenomenon called anoikis. Conversely, carcinoma cells tend to be relatively more anoikis-resistant than their normal counterparts, and this increased resistance represents a critical feature of the malignant phenotype. Mechanisms that control susceptibility and resistance to anoikis are not fully understood. It is now known that detachment of non-malignant epithelial cells triggers both pro- and antiapoptotic signals, and it is the balance between these signals and the duration of detachment that determine further fate of the cells. Detachment-induced antiapoptotic events delay anoikis and if cells reattach relatively soon after detachment they survive. Direct regulators of apoptosis responsible for this delay of anoikis are unknown. We found that detachment of non-malignant intestinal epithelial cells triggers upregulation of inhibitors of apoptosis protein (IAP) family, such as X-chromosome-linked inhibitor of apoptosis protein and cellular inhibitor of apoptosis-2 (cIAP2). We demonstrated that this upregulation requires detachment-dependent activation of the transcription factor nuclear factor-kappaB. We further observed that various IAP antagonists accelerate anoikis, indicating that upregulation of the IAPs delays detachment-triggered apoptosis. We conclude that the IAPs are important regulators of the balance between detachment-triggered life and death signals. Perhaps, not by coincidence, these proteins are often upregulated in carcinomas, tumors composed of cells that tend to be anoikis-resistant.

The trypanosome leucine repeat gene in the variant surface glycoprotein expression site encodes a putative metal-binding domain and a region resembling protein-binding domains of yeast, Drosophila, and mammalian proteins.

We have identified a new variant surface glycoprotein expression site-associated gene (ESAG) in Trypanosoma brucei, the trypanosome leucine repeat (T-LR) gene. Like most other ESAGs, it is expressed in a life cycle stage-specific manner. The N-terminal 20% of the predicted T-LR protein resembles the metal-binding domains of nucleic acid-binding proteins. The remainder is composed of leucine-rich repeats that are characteristic of protein-binding domains found in a variety of other eucaryote proteins. This is the first report of leucine-rich repeats and potential nucleic acid-binding domains on the same protein. The T-LR gene is adjacent to ESAG 4, which has homology to the catalytic domain of adenylate cyclase. This is intriguing, since yeast adenylate cyclase has a leucine-rich repeat regulatory domain. The leucine-rich repeat and putative metal-binding domains suggest a possible regulatory role that may involve adenylate cyclase activity or nucleic acid binding.

Expression of a retroposon-like sequence upstream of the putative Trypanosoma brucei variant surface glycoprotein gene expression site promoter.

We have cloned the region spanning the putative promoter from two variant surface glycoprotein gene expression sites that are at each end of chromosome M4 of Trypanosoma brucei IsTat 7. Both expression sites contain a retroposon-like sequence (ESR) pseudogene whose 3' end is approximately 30 bp upstream of the putative expression site promoter. The ESRs from both expression sites share considerable sequence homology and are related to LINE-like elements, especially the T. brucei ingi retroposon. Other ESRs are located on large, but not intermediate or mini-, chromosomes in the IsTaR 1 serodeme, and the total copy number is 10 to 20, similar to that estimated for variant surface glycoprotein expression sites. No DNA rearrangements in the vicinity of the ESR and putative expression site promoter were detected following antigenic switches in the IsTaR 1 serodeme. ESR transcripts are present in bloodstream, but not procyclic, forms. Variation in transcript size and sequence between bloodstream variant antigenic types implies that only the ESR from the active expression site is transcribed. This pattern of expression reflects that of sequences downstream of the putative expression site promoter, suggesting that the region of coordinately controlled expression extends upstream of this promoter.

Neutrophil migration stimulates rat intestinal epithelial cell cytokine expression during helminth infection.

We are interested in understanding the role of epithelial cells during inflammation, and we previously reported that rat small intestinal epithelial cells express interleukin-1beta (IL-1beta) during infection by Trichinella spiralis. We now report that the epithelium also produces the potent neutrophil chemotactic factor, macrophage inflammatory protein-2 (MIP-2), and an IL-1 antagonist: the type II IL-1 receptor. Consequently we investigated the pattern of neutrophil infiltration into the infected intestine, which closely paralleled the epithelial cytokine expression. Speculating that neutrophil infiltration may provoke epithelial cytokine expression, neutrophil migration into the infected gut was reduced by depleting circulating cells through the use of a specific antibody, or by preventing migration through the use of a function-blocking anti-CD18 monoclonal antibody. Either treatment reduced the number of neutrophils recoverable from the small intestinal epithelium and was paralleled by reduced mRNA levels for epithelial cytokines. These results demonstrate that neutrophil infiltration of the small intestinal epithelium contributes to the stimulation of epithelial cell cytokines.

CD43-, but not CD43+, IL-10-producing CD1dhiCD5+ B cells suppress type 1 immune responses during Chlamydia muridarum genital tract infection.

Regulatory B (Breg) cells are known to modulate immune responses through predominantly interleukin-10 (IL-10)-dependent mechanisms and can be hypothetically divided into innate and adaptive subsets based on the nature of their activating signals. However, the specific role of different Breg subsets in modulating immune responses remains ambiguous. Here we have shown that Chlamydia induces IL-10-producing splenic B-cell populations consisting of CD43(+) and CD43(-) subsets of IgM(hi)IgD(lo) innate-like B (ILB) cells in vitro. While CD43(+)IL-10-producing B cells displayed innate type features and were readily induced by Chlamydia via Toll-like-receptor (TLR) signaling, CD43(-)IL-10-producing B cells required additional B-cell activating factor (BAFF)-mediated signals from dendritic cells (DCs) for their differentiation and activation, thereby classifying them as adaptive type Bregs. Importantly, CD43(-), but not CD43(+), IL-10-producing ILB cells displayed bona fide Breg activity by potently suppressing interferon-γ (IFN-γ) production in vitro in an IL-10-dependent manner. Furthermore, a novel CD43(-)CD1d(hi)CD5(+) IL-10-producing Breg population was predominantly induced by Chlamydia genital infection in vivo. Correspondingly, mixed bone marrow chimeric mice with B-cell-specific IL-10 deficiency exhibited significantly increased type 1 immune responses, decreased bacterial burden, and reduced oviduct pathology upon infection. Our data demonstrate for the first time a distinct role for CD43(-)CD1d(hi)CD5(+)-adaptive Bregs over CD43(+) innate counterparts in controlling mucosal responses against intracellular bacterial infection.

Interleukin-4 and IFN-gamma differentially stimulate macrophage chemoattractant protein-1 (MCP-1) and eotaxin production by intestinal epithelial cells.

When the intestine becomes infected by pathogenic organisms, intestinal epithelial cells (IEC) respond with the production of chemokines, which then attract and activate specific subsets of leukocytes. During chronic inflammation, the panel of IEC chemokines produced likely represents the net effect of a plethora of mediators present in the milieu, including cytokines from activated T lymphocytes. To explore the influence of T lymphocyte cytokines, we treated IEC-18 cells with interferon-y (IFN-gamma) and interleukin-4 (IL-4) and measured the effect on production of the CC chemokines, monocyte chemoattractant protein-1 (MCP-1) and eotaxin, and the CXC chemokine, macrophage inflammatory protein-2 (MIP-2). Both IFN-gamma and IL-4 enhanced MCP-1 mRNA levels but with different kinetics. IFN-gamma stimulated a transient increase in MCP-1 mRNA levels, which peaked at 2 h, whereas IL-4-stimulated MCP-1 mRNA levels were markedly increased at 1 h and remained elevated at all time points studied. With each stimulus, the increase in MCP-1 mRNA levels was accompanied by a steady time-dependent increase in MCP-1 secretion. In addition, treatment with IFN-gamma or IL-4 enhanced IL-1beta-stimulated MCP-1 mRNA production and protein secretion. Eotaxin mRNA was detectable in unstimulated IEC-18 cells, and IL-4 but not IFN-gamma caused a rapid enhancement in levels, which remained elevated for 24 h after treatment. Finally, IL-1beta but not IFN-gamma or IL-4 enhanced MIP-2 mRNA levels. Knowledge gained from studying the outcome of T lymphocyte-derived stimuli will help understand the complex sequence of events during chronic intestinal inflammation.

Monocyte chemoattractant protein-1 production by intestinal epithelial cells in vitro: a role for p38 in epithelial chemokine expression.

The intestinal epithelial cell (IEC) represents the first cellular barrier to infection. Consistent with this sentinel role, IEC are known to produce a variety of chemokines in response to bacterial infection or proinflammatory cytokines. These chemokines act as potent leukocyte activators and chemoattractants in vivo. In this report, we begin to characterize the regulation of expression of the chemokine monocyte chemoattractant protein-1 (MCP-1) in the rat small intestinal IEC-18 line. Following stimulation with either interleukin-1beta (IL-1beta) or lipopolysaccharide (LPS), IEC-18 cells produced MCP-1, with IL-1 proving a more effective stimulus than LPS at both the mRNA and protein levels. Expression of MCP-1 due to either stimulus was inhibited by tyrosine kinase inhibitors, prompting us to investigate potential phosphotyrosine-dependent targets responsible for MCP-1 expression. We detected activation of p38, a member of the mitogen-activated protein kinase family, following either IL-1 or LPS treatment. Specific inhibition of this kinase using the compound SB203580 caused a destabilization of MCP-1 mRNA. These data point to a role for p38 in the regulation of MCP-1 mRNA expression by the IEC.

Isolation and characterization of highly purified rat intestinal intraepithelial lymphocytes.

The study of intestinal intraepithelial lymphocytes (IEL) has been hindered by the difficulty of isolating a population of lymphocytes which is free of epithelial cell or lamina propria cell contaminants and representative of the in vivo population of IEL in both phenotype and function. We describe an improved technique for the extraction and purification of IEL from the proximal small intestine of the rat. This technique rapidly and reproducibly isolates 5-10 x 10(6) IEL/rat with 90-95% purity and viability without the use of enzymes which affect lymphocyte function. The resulting cell population, which is 75% alpha beta T cell receptor (TCR)+, 70% CD8+, and 33% CD4+ T cells, and only 5% B cells and 2% macrophages, is of suitable purity to allow for flow cytometric analysis of the entire population of cells without requiring gating on lymphocytes. IEL are comprised of a unique T cell repertoire in that 27% of cells co-express the CD4 and CD8 molecules, but only 11% of CD4+ cells co-express CD45RC. All CD4+ cells express the alpha beta TCR, but 9% of IEL are CD8+ CD4- alpha beta TCR-. The adhesion molecules alpha 4 integrin and L-selectin are expressed on 57% and less than 1% of IEL, respectively. The isolated IEL population contains mRNA for IL-1 alpha, IL-1 beta, IL-1R, IL-1RA, IL-2, IL-6R, IFN-gamma, TGF-alpha, TGF-beta 1, and TNF-alpha. Mesenteric lymph node cells (MLNC) were examined in parallel. This technique allows for the isolation of rat IEL appropriate for phenotypic analysis by flow cytometry and for cytokine analysis by reverse transcription/polymerase chain reaction.

Prolongation of survival of primary renal allografts by feeding of donor spleen cells.

We have found that feeding Brown Norway (BN) rat spleen cells to Lewis rats prior to transplanting BN kidneys prolongs allograft survival (mean: 8.8 days in unfed rats, 21 days in the BN cell-fed rats; longest survival: 11 days without allo-feeding vs. 37 days with feeding). We have also found that feeding BN cells both before and after transplantation further extends survival (mean: 38 days; longest survival: 105 days). We also examined the cells infiltrating the grafts during the early stages of the allograft response (day 5). Using flow cytometry, we found a significant decrease in the number of leukocytes infiltrating the transplanted kidneys of fed animals. This decrease was mainly due to a drop in the number of infiltrating T cells. We also found that cytokine mRNA production by the graft-infiltrating lymphocytes, assessed by reverse transcription polymerase chain reaction, showed a significant increase in interleukin-4 and transforming-growth factor-beta mRNA in the graft-infiltrating lymphocytes of fed animals compared with the controls.

Characterization of nonspecific esterase activity in macrophages and intestinal epithelium of the rat.

We determined the histochemical characteristics of nonspecific esterase in different populations of rat macrophages. The cells included alveolar and peritoneal macrophages recovered by lavage and a mixed cell population obtained by collagenase digestion of the small intestine. The histochemically localized enzyme activity of alveolar and peritoneal macrophages was cytoplasmic, diffuse, and inhibited by sodium fluoride. Both populations were effectively stained using alpha-naphthyl acetate and alpha-naphthyl butyrate as the esterase substrate. When the intestinal cells were examined for activity, a greater percentage of cells showed positive nonspecific esterase than would be predicted by differential counts for macrophages on the basis of morphological criteria. We confirmed, using cell smears and tissue sections, that rat intestinal epithelial cells, a prominent component of the isolated cell population, possessed esterases that react similarly to macrophage esterases with histochemical procedures.

Class II MHC expression on rat intestinal intraepithelial lymphocytes.

The expression of class II major histocompatibility complex (MHC) on rat intestinal intraepithelial lymphocytes (IELs) and mesenteric lymph node cells (MLNCs) were examined by immunofluorescence and flow cytometry. As expected, MLNCs contain eight small populations of CD4+ class II MHC+ T-cells in addition to classical antigen presenting cells. In contrast, rat IELs include a significant population of class II MHC+ T-cells, predominantly in the CD8+ CD4-alpha beta TCR+ subset. IEL samples with a relatively high percentage of class II MHC+ cells also include some CD4+ class II MHC+ cells; IEL samples with a low percentage of class II MHC+ cells also include some CD4- CD8- class II MHC+ cells. The role of lymphocyte subpopulations in the intestinal epithelium may need to be revisited in consideration of these findings.

Cytokine mRNA in the joints and draining lymph nodes of rats with adjuvant arthritis and effects of cyclosporin A.

TNF-alpha and IL-1beta promote leukocyte recruitment to arthritic joints and may contribute to cartilage degradation while regulatory cytokines such as IL-4 and IL-1RA may in part determine the course of arthritis. Here we report the pattern of TNF-alpha, IL-1beta, IL-6, IFN-gamma, IL-1RA, and IL-4 mRNA expression, detected by RT/PCR, in the talar joint and draining popliteal lymph node (PLN) of rats with adjuvant arthritis (AA). Levels of TNF-alpha and IFN-gamma mRNA were increased in the PLN before clinical signs of arthritis. This was followed by increases in IL-1beta and IL-1RA mRNA at d9 and IL-6 mRNA at d12. PLN IL-1RA mRNA levels were positively correlated with those of IL-1beta and TNF-alpha throughout d5-d20. IL-4 mRNA levels were highest on days 7 and 20. In the synovium, a small increase in TNF-alpha, IL-1beta, and IL-6 mRNA was detected on d5 then again on d12. Maximal synovial TNF-alpha levels were reached on d20, while IL-1beta peak expression was on d16 and IL-6 on d14. IL-4, IL-1RA, and IFN-gamma mRNA was undetectable in the synovium. Cyclosporin treatment for 4 days, initiated at the height of arthritis, rapidly decreased clinical disease, and decreased migration of neutrophils and T lymphocytes into the joints. Yet no significant effect of CyA was observed on inflammatory cytokine expression, although the correlation between PLN IL-1RA and IL-1beta or TNF-alpha was lost in treated animals. Thus there is a variable pattern of cytokine gene expression in rat AA, the undetectable IL-4 and IFN-gamma mRNA in synovium being analogous to human rheumatoid arthritis.

Characterization of Nippostrongylus brasiliensis infection in different strains of mice.

Five mouse strains, CBA/J, BALB/c, C3H/HeJ, A/J, and C57Bl/6J-bg-bg, all showed similar expulsion kinetics for Nippostrongylus brasiliensis (infective dose = 500 L3). Typically, parasite recovery was maximal on day 2 in the lungs and by day 4 in the small intestine. Few worms (less than 5% infective dose) were recovered on day 14 in all strains. These same mouse strains exhibited immune depression on day 5 of infection with mesenteric lymph node cells (MLN) showing reduced (10-30% normal) IgM, IgG, and IgA responses against heterologous antigen. The intestinal mast cell numbers and tissue histamine levels were examined in CBA/J mice. Mast cell numbers increased (normal = less than 1/villous crypt unit; VCU) from day 5 and peaked on day 12 (greater than 15/VCU). Intestinal histamine levels did not completely correlate with mast cell numbers with maximum concentrations (240 +/- 73 ng/g, 2-fold over normal) reached by day 8. Histamine concentrations in the intestine returned to normal levels by day 20.

The C5a receptor antagonist PMX205 ameliorates experimentally induced colitis associated with increased IL-4 and IL-10.

BACKGROUND AND PURPOSE: Anti-complement therapies have not been advanced for treating the inflammatory bowel diseases (IBDs) despite a growing body of evidence that blocking C5a protects against induced colitis in rodents. The purpose of this study was to further build on this evidence by examining the efficacy, mechanism and specificity of a potent, non-competitive and orally active C5a receptor (CD88) antagonist, PMX205, in the dextran sulphate sodium (DSS) model of murine innate colitis. EXPERIMENTAL APPROACH: Mice with DSS added to their drinking water were orally administered 100 or 200 µg day(-1) PMX205 in prophylactic and therapeutic regimens. Clinical illness, colon histology and local generation of inflammatory mediators were measured to evaluate the impact of PMX205 on disease. KEY RESULTS: PMX205 significantly prevented DSS-induced colon inflammation in both regimens, associated with lower pro-inflammatory cytokine production and nitrotyrosine staining in colon sections. Additionally, the levels of anti-inflammatory cytokines IL-4 and IL-10 were increased. PMX205 had no significant effect on C5a levels. The beneficial effect of PMX205 was seen in two strains of mice of differing sensitivities to DSS inflammation, but was inactive in mice lacking CD88. CONCLUSIONS AND IMPLICATIONS: Pharmacological inhibition of C5a activity by PMX205 is efficacious in preventing DSS-induced colitis, providing further evidence that targeting CD88 in IBD patients could be a valuable therapeutic option.

Prostaglandins and activation of AC/cAMP prevents anoikis in IEC-18.

Recent data indicates that chronic inflammation of the intestine such as Crohn's or ulcerative colitis puts those individuals at heightened risk for colorectal adenocarcinoma. In this study, we examine the effect of the inflammatory mediator PGE(2) and associated signalling on detachment-induced cell death (anoikis) in intestinal epithelial cells. Treatment of detached IEC-18 with 0.01-0.05 microM PGE(2) increased cell viability as well as induced aggregation. As EP4 prostaglandin receptors on IEC are coupled to adenylate cyclase, we next treated cells with agents that promote cAMP signalling (Forskolin, dbcAMP, and etazolate), all of which promoted IEC aggregation as well as survival. We next treated detached IECs with specific inhibitors of adenylate cyclase or PKA, which accelerated anoikis. To explore the mechanism of cell-cell adhesion, we next treated detached IECs with an anti-E-cadherin blocking antibody which dispersed aggregates induced by dbcAMP, and an adenovirus expressing a dominant negative E-cadherin (EcadDeltaEC) prevented aggregate formation. Interestingly EcadDeltaEC prevented aggregation of IEC induced by dbcAMP but did not significantly reduce viability. This suggests that cAMP signalling is important in both aggregate formation and promoting viability but these are distinct events. Taken together, these data support a mechanism whereby elevated PGE(2) levels characteristic of colitis prevent anoikis by activating an AC-, cAMP-, and PKA-dependent signalling pathway. The delay of apoptosis by PGE(2) may be one mechanism by which inflammation may contribute to carcinogenesis.

Cytokine production by epithelial cells.

The epithelium has long been regarded as a passive barrier intended to protect the underlying tissues. Many regulatory signals, including cytokines, that control epithelial cell proliferation and differentiation and cell function during inflammation were thought to be nonepithelial-derived. Now there is a growing appreciation that epithelial cells provide some of the impetus for their own growth and differentiation, and may also regulate the function of other cells through the elaboration of certain cytokines. Moreover, because epithelial cells serve as the interface between the organism and environment, they are in a position to signal changes in the environment. It is now clear that epithelial cells respond to injury or infection with cytokine secretion. Various approaches to detecting cytokines in normal and diseased tissue have been undertaken during the past few years to establish cytokine synthesis by different epithelial. This review will examine these recent investigations in various general contexts of epithelial cell function.

Established murine lupus nephritis does not respond to exogenous interleukin-1 receptor antagonist; a role for the endogenous molecule?

Interleukin 1 beta (IL-1 beta) is a potent inflammatory cytokine and IL-1 beta gene expression is elevated in the kidneys of mice with lupus nephritis. This study was designed to examine whether pharmacological administration of the IL-1 receptor antagonist (IL-1ra) would reduce the inflammation in MRL lpr/lpr mice with lupus nephritis. Human recombinant IL-1ra (RA) or saline (SA) was infused by intraperitoneal osmotic minipumps in 16 week old mice (n = 9, group RA or n = 12, group SA, respectively). Age matched MRL +/+ mice served as normal controls. At the end of 4 weeks of treatment glomerular filtration rates (5.4 +/- 0.4 vs 5.6 +/- 0.4 ml/min/kg BW), proteinuria (6.0 +/- 1.0 vs 5.5 +/- 1.2 micrograms IgG/day) glomerular volumes (571 +/- 30 vs 509 +/- 25 microns3 x 10(3)), mesangial volumes (172 +/- 23 vs 158 +/- 17 microns3 x 10(3)), and cells/glomerulus (519 +/- 51 vs 506 +/- 47) were not significantly different between RA and SA groups respectively. There was also no significant differences in spleen sizes, plasma IgG and anti-dsDNA antibody levels despite achieving levels of IL-1ra of over 0.8 microgram/ml in RA mice. Circulating IL-1 was not detected by bioassay in the plasma of diseased or normal mice. In fact, diseased, saline treated mouse plasma inhibited the cell proliferation assay in the presence of IL-1, and dilution studies showed that the endogenous inhibitors were of high titre. Although IL-1 may play a role in the renal injury of lupus nephritis, pharmacological inhibition with IL-1ra in animals with established injury is without effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Pattern of proinflammatory cytokine mRNA expression during Trichinella spiralis infection of the rat.

Trichinella spiralis occupies an intramulticellular niche in the small intestinal epithelium, and thus we examined the intestine and gut-associated tissues for proinflammatory cytokines during the infection. We document the patterns of interleukin-1 (IL-1), IL-6, gamma interferon, and tumor necrosis factor alpha mRNA expression in the duodenum, jejunum, Peyer's patches, mesenteric lymph node, spleen, and liver in T. spiralis-infected rats. By reverse transcription-PCR detection of mRNAs, IL-1beta was found increased in the jejunum but only on day 2. The jejunal IL-1beta increase was attributed to the epithelium by isolating epithelial cells and then depleting them of intraepithelial lymphocytes prior to analysis. The only cytokine for which mRNA was substantially increased in tissues later in infection was tumor necrosis factor alpha in the spleen and, to a lesser extent, in the mesenteric lymph node. In fact mRNA levels for some cytokines declined below uninfected levels in some organs during the infection. IL-1 may be important in the initiation of the intestinal inflammatory response to this infection.