Increased basal phosphorylation of mitogen-activated protein kinases and reduced responsiveness to inflammatory cytokines in neutrophils from patients with rheumatoid arthritis.

OBJECTIVE: We studied the functions of peripheral blood (PB) and synovial fluid (SF) neutrophils from patients with rheumatoid arthritis (RA), focusing the molecular basis for the activated state and the functional responsiveness of RA neutrophils to inflammatory cytokines. METHODS: Paired samples of PB neutrophils and SF neutrophils from the inflamed knee joint were obtained from 18 RA patients (5 males and 13 females). RESULTS: RA neutrophils exhibited increased spontaneous superoxide (O2-) release and adherence, increased basal phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase, accelerated spontaneous apoptosis, and enhanced O2- release in response to N-formyl-methionyl-leucyl-phenylalanine as compared with healthy normal PB neutrophils. When challenged with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF) or tumor necrosis factor alpha (TNF-alpha), RA neutrophils exhibited reduced responses to these cytokines, which included O2- release, adherence, priming for enhanced O2- release, and phosphorylation of ERK and p38. The functional alterations were greater in SF neutrophils than in PB neutrophils from RA. Reduced responsiveness to cytokines in RA neutrophils was closely associated with increased serum and SF levels of GM-CSF and TNF-alpha. RF and RAHA titers were closely correlated with increased TNF-alpha level in SF. CONCLUSION: These findings indicate that RA neutrophils are in the activated state with increased basal phosphorylation of ERK and p38, and exhibit reduced responsiveness to inflammatory cytokines (G-CSF, GM-CSF and TNF-alpha) and accelerated spontaneous apoptosis.

Activation of human neutrophil by cytokine-activated endothelial cells.

Cytokine activation of vascular endothelial cells renders the hyperadhesiveness for neutrophils. During the processes of inflammation and atherosclerosis, the production of reactive oxygen species by neutrophils contributes to endothelial cell (EC) damage and injury. However, the precise mechanisms for neutrophil activation by ECs remain unknown. Thus, we investigated what kinds of pathophysiological factors synthesized by inflammatory cytokine-activated ECs potentiated the activity of neutrophil functions. The magnitude of O(2)(-) release from neutrophils, which is one of pivotal neutrophil functions, was measured as an indicator potentiated by activated ECs. Neutrophils release massive amounts of O(2)(-) on coculture with activated ECs. Anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody (Ab) or specific platelet-activating factor (PAF)-receptor antagonist suppressed the O(2)(-) release from neutrophils on coculture with the activated ECs by 50% to 70%. The supernatants from activated ECs also induced O(2)(-) release by neutrophils. This stimulatory effect of activated EC supernatants on O(2)(-) release by neutrophils was abolished by anti-GM-CSF Ab or by PAF-receptor antagonist. As we previously reported, we demonstrated the expression of GM-CSF mRNA by Northern blotting and protein synthesis of GM-CSF by ELISA on tumor necrosis factor as well as interleukin-1-activated ECs. Although phosphorylation of mitogen-activated protein kinases was observed in ECs stimulated by tumor necrosis factor and interleukin-1, treatment of ECs with PD98059 (MEK1 inhibitor) and SB203580 (p38 mitogen-activated protein kinase inhibitor) in the presence of the cytokine failed to attenuate the stimulatory effect of activated ECs on neutrophil activation. We found that activated ECs regulated neutrophil function on coculture. We show here for the first time, to our knowledge, that the collaboration between GM-CSF and PAF synthesized by activated ECs markedly potentiated neutrophil activation.

Possible inhibitory effects of promotion stage treatment with thymosin fraction 5 on N-butyl-N-(4-hydroxybutyl)nitrosamine-initiated urinary tract carcinogenesis in NON/Shi mice.

The modifying effects of thymosin fraction 5 (TF5), a dialyzable glycoprotein extract obtained from calf thymus, on second stage urinary tract carcinogenesis after N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) initiation were investigated in NON/Shi male mice. BBN was administered for 8 weeks and then TF5 was administered subcutaneously twice a week for 12 weeks. The incidence of urinary bladder carcinomas was decreased in the TF5 treatment group, but the malignancy of the renal pelvic carcinomas appeared to be enhanced. We repeated the experiment to obtain confirmatory evidence, but found no significant effect, although TF5 did stimulate the immune surveillance system. The reason why this discrepancy occurred might be related to the degree of hydronephrosis or the confounding influence of renal pelvic carcinomas.

Thymosin fraction 5 does not influence urinary tract carcinogenesis by phenacetin and N-butyl-N-(4-hydroxybutyl)nitrosamine in NON/Shi mice.

The effect of thymosin fraction 5 (TF5) on the promotion and progression phases of urinary tract carcinogenesis induced by consecutive administration of phenacetin and N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in NON/Shi mice was investigated. The study was carried out twice with a minor modification to the protocol in the second experiment. Fifty-seven male NON/Shi mice in experiment 1 and 100 mice in experiment 2 were each divided into four groups. Phenacetin was administered for 8 weeks in experiment 1 and 12 weeks in experiment 2, and subsequently BBN was given for 6 weeks in both cases, for total observation periods of 30 and 34 weeks, respectively. Sixty micrograms of TF5 per mouse was inoculated subcutaneously twice a week during (group 2) or after (group 3) BBN exposure, or both periods (group 4). Group 1 served as a control group without TF5 treatment. Histopathological examination revealed no effects on either induction of urinary tract carcinomas or distant metastasis from renal pelvic carcinomas in either experiment.

A study of antitumor effects of thymosin on rat and mouse urinary bladder carcinoma induced by N-butyl-N-(4-hydroxybutyl)-nitrosamine.

N-Butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), which induces bladder carcinoma in female Wistar rats and female C3H/HeJ mice, was given as 0.025% (w/v) solution to rats and 0.05% to mice in the drinking water. Thymosin fraction 5 (TF5) or thymosin alpha 1 (T alpha 1) dissolved in normal saline, and normal saline (control) respectively were injected into the right thigh IM twice a week in the experimental rats. At the 30th week of BBN oral administration, weights of urinary bladder were different among the three groups. Bladder weights and incidence of bladder carcinoma were significantly lower in the TF5-treated group as compared with control group. Natural killer cell activity of splenic lymphoid cells was significantly elevated in the TF5-treated group as compared with the control group. However, these parameters were not significantly different between the T alpha 1-treated group and control saline-treated group. On the other hand, bladder weights and incidence of bladder carcinoma were not significantly different among TF5-, T alpha 1- and saline-treated groups at 10, 15, 20, and 25 weeks of BBN administration in mice according to the same protocol as rats.

CD71 antibody enhances iron uptake by mouse bone marrow cells and the survival potential of erythroid progenitor cells.

Transferrin has been shown to have cytoprotective functions. The aim of this study was to evaluate the effect of antibody against CD71 (transferrin receptor) on survival potential of erythroid progenitor cells. After anti-CD71 IgG or control IgG was reacted with cells for 20 min on ice, the uptake of transferrin-bound 59Fe by the MEL cells or the mouse bone marrow cells during 2-day incubation without erythropoietin was measured. Erythropoiesis was evaluated by addition of erythropoietin after 4-5 weeks' culture without erythropoietin or beta-mercaptoethanol, and production of cells containing hemoglobin was compared by immunocytological methods. MEL cells reacted with anti-CD71 IgG incorporated more transferrin-bound 59Fe than MEL cells reacted with control IgG. Uptake of transferrin-bound 59Fe by mouse bone marrow cells reacted with anti-CD71 IgG was also greater than by cells reacted with control IgG. Erythrocytes developed from cells reacted with anti-CD71 IgG and cultured for 4-5 weeks, but few erythrocytes developed from cells reacted with control IgG. Bone marrow cells depleted of TER119+ cells, CD34+ cells, or CD45+ cells and reacted with CD71 antibody did not differentiate into erythrocytes. Survival potential of erythroid progenitor cells were enhanced by reaction with anti-CD71 antibody.

Cellular and molecular interactions of thymus with endocrine organs and nervous system.

T-cell ontogenesis has been disclosed to depend on the interactions of thymus with endocrine glands and nervous system as follows: i/ Thymic deprivation not only impaired the immunological development but also brought about the dysgenesis of pituitary anterior lobe. Conversely, hypophysectomy resulted in thymus atrophy with the disturbed immune responses. ii/ Binding of pituitary acidophilic cell hormones to their receptors on thymus epithelial cells (TECs) augmented the release of thymic hormonal peptides (THPs) in vitro. iii/ Elevation of blood glucocorticoid level after stress caused atrophy of thymus cortex through double positive thymocyte apoptosis. Morpho-molecular alterations of cytoplasm preceded nuclear damage in the apoptotic thymocytes. iv/ Administration of thymosin to the streptozotocin-induced diabetic mice repressed mononuclear cell infiltration to the pancreatic islets. v/ Autonomic nerve fibers innervate thymic parenchyma. Binding of acetylcholines (Achs) to Ach receptors on TECs enhanced protein synthetic activity which seemed to connect with THP production. vi/ Thymectomy not only depressed the immune responses but also accelerated the reduction of leaming and memory ability with aging. The operation appears to disturb the brain adrenoceptor functions and to suppress the regulatory roles of hypothalamus to other nervous tissues. vii/ Several kinds of THPs, separated from the culture supernatant of TEC line by high performance liquid chromatography, showed a favorable effect on the thymocytes at different stage of differentiation and maturation. viii/ Thymosin, thymulin and THPs were capable of proliferating and differentiating thymocytes in vitro. However, the administration of each thymic product to the thymus-deprived animals could not restore from their "wasting disease". Since TECs are composed of a heterogeneous population, it would be one of essential ways for isolating "true thymus hormone" (TTH) to use the material which consists of functionally homogeneous subset of TECs. ix/ An additional grafting of pituitary gland to the thymus-grafted nude mice improved the disturbed T-cell ontogeny. Accordingly, the administration of "TTH" and pituitary acidophilic cell hormones might be more hopeful procedure for rescuing the thymus-deprived animals from "wasting disease".

Functional diversity of polypeptides in primary culture supernatant of thymus epithelial cells.

Polypeptide fractions A-C (M.W., 7 kd, 4.7 kd, and 3 kd) were obtained from the primary culture supernatant of thymus epithelial cells from Wistar rats by high-pressure liquid chromatography with a gel-filtration column. Changes in the mitogen responses of rat thymocytes and their subpopulations with addition of a fraction were studied. One subpopulation was rich in non-rosette-forming cells (non-RFCs), and the other was cortisone resistant thymocytes (CRTs). These subpopulations were incubated with a fraction for 24 hrs. before mitogen stimulation. Fractions A and C increased the response of the non-RFCs to concanavalin A (Con A) and that of total thymocytes and CRTs to phytohemagglutinin (PHA). Fraction B inhibited Con A and PHA response of total thymocytes and CRTs. Fraction B was cytotoxic toward total thymocytes and CRTs when viability was evaluated by [3H]uridine prelabelling. This cytotoxicity was suppressed by treatment with trypsin. Subfractions B3 and B4 obtained by reversed-phase column chromatography were cytotoxic toward CRTs. The effects of the fractions on thymocyte maturation were different, showing their functional diversity.

Surface antigenic analysis of rosette-forming cells in rat thymus--using monoclonal antibodies against T cell surface markers.

We have assumed that rat thymocytes capable of rosette-formation with guinea pig erythrocytes were double positive (CD4+8+) cells. Therefore surface-binding erythrocytes on rosette-forming lymphocytes (RFLs), separated in highly pure state, were eliminated by a hypotonic shock. The surface markers of RFLs, without surface erythrocytes, were analyzed by flow cytometry using two kinds of monoclonal antibodies (anti-CD4, anti-CD8) labelled with different fluorescence materials. About 96.2% of cells in the RFL population were sorted in two areas, into which double positive cells were separated. This result substantiates the assumption mentioned above. It was, furthermore, discussed that the RFLs without surface erythrocytes would be one of the valuable materials for disclosing the mechanisms of differentiation and maturation from double positive thymocytes to single positive ones.

Surface characteristics of thymocytes in glucocorticoid treated rats using rosette-formation technique and surface marker analysis.

Glucocorticoid (GC) treatment is known to induce destruction of cortical thymocytes and then their reconstitution. By using the rats treated with GC, we examined the relationship between rosette-formation and surface markers (CD4 and CD8) for clarifying the processes of differentiation and maturation in rat thymocytes. Thymus weight and thymocyte count began to decrease immediately after GC administration and became minimal on 5-7 days, followed gradual recovery. The percentage of rosette-forming thymocytes began to decrease immediately after GC treatment and became minimal on 5 days, followed by recovery to the normal level by the 10th to 14th day after treatment. During the analysis of the changes in the percentage of 4 subsets (CD4-8-, CD4+8+, CD4+8+, CD4-8+) of rat thymocytes after GC treatment, the percentage of CD4+8+ cells was found to change in close relation to the change in the percentage of rosette-forming lymphocytes, suggesting that rosette-forming thymocytes are CD4+8+ cells. These results suggest that the treatment induces destruction of GC-sensitive thymocytes, possibly rosette-forming cells, followed by migration of precursor T cells (CD4-8- cells) in the thymus, and that the precursors change into rosette-forming cells (CD4+8+ cells) in the thymus, followed by differentiation and maturation into non-rosette-forming cells (CD4+8- or CD4-8+ cells).

Enhancement of DNA synthetic activity of thymic lymphocytes by the culture supernatant of thymus epithelial cells stimulated by growth hormone.

The authors examined the effect of the culture supernatant of growth hormone (GH)-stimulated thymus epithelial cells (TECs) on DNA synthetic activity of thymic lymphocytes (TLs) and then examined TL proliferation-inducing factors released from the TECs. TEC line, IT-45R1 derived from Wistar strain rat, was used. It was revealed that the supernatant from TECs treated with GH enhanced significantly DNA synthetic activity of TLs and that the activity of the least dense subset of TLs, containing undifferentiated lymphoid cells and the most immature TLs, was significantly increased by the supernatant as compared with other subsets. Anti-insulin like growth factor-I (IGF-I) monoclonal antibody (MAb) binding specifically to C region of IGF-I molecule was added to the culture supernatant from the GH-treated TECs, and then the supernatant was treated with ultrafiltration (MW cutting off; more than 50 kDa). When TLs were incubated with the ultrafiltered supernatant, the enhancement of TL proliferation induced by the supernatant of GH-treated TECs was significantly suppressed. However, the suppression did not descend to the level of TL-proliferative response observed in the supernatant of GH non-stimulated TECs. These results suggested that IGF-I released into the supernatant from GH-stimulated TECs enhances markedly the DNA synthetic activity of TLs and that the TL-proliferation-inducing factors (PIFs) other than IGF-I possibly exist in the supernatant of GH-stimulated TECs.

Enhancement of thymic lymphocyte proliferation by the culture supernatant of thymus epithelial cells stimulated by prolactin.

Culture supernatant of thymus epithelial cells (TECs) stimulated by prolactin (PRL) enhanced markedly DNA synthetic activity of thymic lymphocytes (TLs) as compared with the hormone-non-stimulated TECs. The supernatant, which was treated with anti-insulin-like growth factor-I (IGF-I) monoclonal antibody (MAb) (binding specifically to C region of IGF-I) has still the capacity of enhancing remarkably TL-proliferation. However, further treatment by ultrafiltration of the MAb-treated supernatant, removing the immune complex (IGF-I and anti-IGF-I MAb) from the supernatant, suppressed significantly the enhanced proliferation of TLs. It is assumed that PRL, like growth hormone (GH), promotes the release of IGF-I from TECs which induces a marked TL-proliferation. Moreover, it seems that the active site for inducing the proliferation is a region different from C, possibly the A or the B regions. TLs, present at different maturation steps, were separated into three subsets by discontinuous density gradient centrifugation and then treated with the supernatant of PRL-stimulated TECs. The least dense subset containing precursor T-cells and the most immature TLs showed the highest proliferative response to the supernatant in the comparison with other subsets and whole TLs. It is possible that the target cells to one of TL-proliferation-inducing factors (PIFs), namely IGF-I, in the supernatant exist in a greater concentration in the most immature step of TL population.

Marked differences in proliferative response to stimulation of growth hormone and insulin-like growth factor-I between thymoma- and normal thymus-derived epithelial cell lines.

Two kinds of thymus epithelial cell lines have been utilized: the TAD3 from lymphocyte-dominant rat thymoma and the IT-45R1 derived from normal rat thymus. In the preconfluent state, the percent increase of DNA synthetic activity of TAD3 cells stimulated by GH or IGF-I was significantly higher than that of IT-45R1 cells. In the confluent state, at the contrary, no any noticeable modification of the activity by GH could be detected and that in both cell lines. However, a treatment of the confluent TAD3 cells with IGF-I caused a significant increase of their DNA synthetic activity, whereas the confluent IT-45R1 cells treated with these molecules did not. It was observed that TAD3 cells in the preconfluent state proliferate markedly after stimulation by GH or IGF-I as compared with IT-45R1 cells and that the former is able to proliferate even in the confluent state, after an exposure of IGF-I, whereas the latter did not. Thymus epithelial cells treated with GH were reported to release more IGF-I molecules what had as a consequence a marked proliferative response of thymic lymphocytes. It is therefore assumed that the very particular nature of TAD3 cells treated with IGF-I, able of proliferating by crossing over the contact inhibition, might have a close relationship in the formation of the lymphocyte-dominant thymoma.

Rat thymus reconstitution after thymocyte destruction by glucocorticoid treatment--from the view of endogenous DNA synthesis and soybean lectin responsiveness in thymocytes.

The dynamic process in rat thymocyte restoration after their destruction by glucocorticoid (GC) administration was examined. Thymus weight and thymocyte count became minimal 4-5 days after the administration. Then the thymus took a course of recovery. Endogenous DNA synthesis in thymocytes, reflecting their proliferation within thymus, decreased for 4 days but began to increase 6-8 days after GC treatment. Thymocyte responsiveness to soybean lectin (SBL), a possible stimulator for T-cell-precursors, showed elevation 4-5 days after the treatment. A marked decrease of lymphocytes in the cortex and unclearness of cortico-medullary junction were observed 2-3 days after GC treatment. Clusters of small lymphoid cells, which possibly contained SBL-responding cells, were found in the subcapsular area 4 days after the treatment and successively, large lymphocytes became visible in the same area. Thereafter, small lymphocytes in the cortical mid and deep zones increased, and cortico-medullary junction was restored. These histological features are discussed from the view of correspondence with the dynamic changes of endogenous DNA synthesis and SBL responsiveness in the thymocytes after GC administration.

The in vitro proliferation of thymus epithelial cells stimulated with growth hormone and insulin-like growth factor-I.

The experimental system to scrutinize the in vitro proliferation of thymus epithelial cells (TECs) was established on the basis of enhancing their mitotic index and DNA synthetic activity. The TEC line, TAD3 derived from lymphocyte-dominant thymoma, was used as the cell material. Growth hormone (GH) induced a significant proliferation of the cultured cell line in the preconfluent state. The optimal concentration of and the duration of the incubation with GH, in this system, were 50 ng/ml and 18 hrs., respectively. Furthermore, insulin-like growth factor-I (IGF-I), the mediator of somatotropic action of GH, also enhanced the DNA synthetic activity of the cultured cells in the preconfluent state. The authors recently found that the TECs, stimulated with GH, released significantly more IGF-I than the cells without GH. It is possible that there are two systems in the TEC proliferation, namely, direct induction by GH itself and indirect induction by IGF-I released from the GH-stimulated TECs. The data showing that GH could directly or indirectly induce proliferation of TECs might possibly be related to the formation of epithelial thymic rudiment in the fetal stage.

The indirect participation of growth hormone in the thymocyte proliferation system.

The specific binding sites for growth hormone was recognized on both thymic lymphocytes (thymocytes) and thymus epithelial cells. The somatotropic action of GH is considered to be mediated mainly by insulin-like growth factor-1 (IGF-1) and partially by GH itself. In this study, effect of GH and IGF-1 on DNA synthetic activity of thymocytes was examined. By 48-hrs. culture with IGF-1 and 24-hrs. 3H-TdR pulse labeling, significant enhancement of DNA synthetic activity of thymocytes was detected, while the enhancement in the culture with GH was very weak. It is considered that IGF-1 acts on the cells in G0/G1 phase in cell cycle. The effect of IGF-1 on thymocyte proliferation was examined by using the thymocytes incubated 12 hrs. before IGF-1 stimulation and 3-hrs. 3H-TdR pulse labeling. The optimal condition to induce thymocyte proliferation was 15-hrs. culture with 380 ng/ml of IGF-1. Furthermore, 10 ng/ml or higher concentration of GH significantly increases IGF-1 release from TECs in confluent state. These results suggest that GH indirectly participates in thymus growth by increasing IGF-1 release from TECs, which enhances thymocyte proliferation.

Effect of exogenous growth hormone on in vitro proliferation of thymic lymphocytes from the hypophysectomized rats.

Thymus weight and total thymic lymphocyte (TL) number decreased markedly after hypophysectomy (HX). Serum level of insulin-like growth factor-I (IGF-I), one of mediators brought about by growth hormone (GH), reduced considerably after the operation. Exogenous GH stimulation enhanced significantly DNA synthetic activity of TLs from HX group only at lower cell concentrations, less than 5 x 10(5) cell/ml. However, there was not a significant difference in the percentage increase of the TL-DNA synthetic activity with exogenous GH between HX and sham-operated (SO) groups. Since GH-responding TLs appeared to be mature cells and to exist in thymus medulla, the results suggest that rate of the responding cells in the thymus medulla of HX animals is similar to that of SO ones. Furthermore, there was not a significant difference in the percentages of rosette-forming cells and non-rosette-forming cells, reflecting nearly the maturation steps of TLs, between groups. It is difficult to explain such an aspect in spite of severe reduction of TLs after hypophysectomy, since the proliferative-responding rate of TLS to exogenous GH stimulation and the constitutive ratio of each maturation step of TLs in the HX group were almost same as those of SO group. The contradictory data found in thymus after hypophysectomy are discussed from the point of view of the compensatory effects of growth hormone-releasing hormone on TLs from the operated animals.

Cytokine-specific activation of distinct mitogen-activated protein kinase subtype cascades in human neutrophils stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha.

To clarify the differences of the signaling pathways used by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor- (TNF), we investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by these cytokines. G-CSF exclusively tyrosine-phosphorylated extracellular signal-regulated kinase (ERK). GM-CSF tyrosine-phosphorylated ERK strongly and p38 MAPK weakly, whereas TNF tyrosine-phosphorylated p38 MAPK strongly and ERK weakly. Consistent with these findings, MEK, an upstream kinase of ERK, was phosphorylated by G-CSF, GM-CSF, and TNF, whereas MKK3/MKK6, an upstream kinase of p38 MAPK, was phosphorylated by GM-CSF and TNF, but not by G-CSF. The potency of these cytokines to phosphorylate ERK and MEK was GM-CSF > G-CSF > TNF, whereas that to phosphorylate p38 MAPK and MKK3/MKK6 was TNF > GM-CSF. C-Jun amino-terminal kinase (JNK) was not tyrosine-phosphorylated by any cytokine despite the existence of JNK proteins in human neutrophils, whereas it was tyrosine-phosphorylated by TNF in undifferentiated and all-trans retinoic acid-differentiated HL-60 cells. Increased phosphorylation of ERK or p38 MAPK was detected within 1 to 5 minutes after stimulation with each cytokine and was dependent on the concentrations of cytokines used. MEK inhibitor (PD98059) reduced tyrosine phosphorylation of ERK, but not p38 MAPK, induced by G-CSF, GM-CSF, or TNF. GM-CSF- or TNF-induced superoxide (O2-) release was inhibited by p38 MAPK inhibitor (SB203580) in a dose-dependent manner, suggesting the possible involvement of p38 MAPK in GM-CSF- or TNF-induced O2- release. The results indicate that G-CSF, GM-CSF, and TNF activate the overlapping but distinct MAPK subtype cascades in human neutrophils and suggest that the differential activation of ERK and p38 MAPK cascades may explain the differences of the effects of these cytokines on human neutrophil functions.