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The dependence of the low molecular weight chemoattractants (LMCs) formylmethionyl-leucylphenylalanine (f-MLP), C5f, and leukotriene B4 (LTB4) on albumin to express their chemotactic activity towards granulocytes (PMNs) was investigated in order to study the required qualities of albumin and if albumin could be replaced by any other proteins. The results demonstrated that the supporting effect of isolated albumin was dependent on the method of purification. Only isolated albumin exposed to ethanol precipitation during the purification procedure supported the chemotactic effect of LMCs. The albumin preparation that supported the effect of LMCs also mediated a chemokinetic effect on PMN migration. Albumin isolated by methods other than ethanol precipitation neither exerted a chemokinetic effect nor supported the chemotactic effect of LMCs. Heated, normal serum and isolated alpha 1-antitrypsin supported the chemotactic activity of LMCs and also mediated a chemokinetic effect on PMN migration. The present investigation suggests an important role for the chemokinetic factors, since it is indicated that their presence is necessary for the chemotactic response of PMNs to the low molecular weight chemoattractants C5f; LTB4, and f-MLP.

Cigarette smoking results in variable degrees of inflammation in the lower respiratory tract. Furthermore, smoking produces oxidant-mediated changes in the lung, important to the pathogenesis of emphysema. Since glutathione can neutralize reactive oxygen species and prevent peroxidation of unsaturated lipids, it may constitute an important component of the lung's defense against oxidant and inflammatory injury. In the present study, broncholaveolar lavage (BAL) was performed in 27 smokers, and the concentrations of total glutathione as well as the cellular and humoral markers of inflammatory activity were studied. There were significant correlations between total glutathione and neutrophils; two neutrophil granule components, myeloperoxidase and elastase; and chemotactic activity for neutrophils. Moreover, the total glutathione correlated with the eosinophil cationic protein (ECP), a granule constituent of the eosinophil, with two locally produced antiproteases, secretory leukocyte protease inhibitor (SLPI) and antichymotrypsin (ACHY), but not with an alpha 1-protease inhibitor and albumin. These data suggest that the total glutathione levels in BAL fluid may reflect a degree of oxidative and inflammatory stress caused by cigarette smoke, and they are therefore likely to contribute to the protection against this stress.

Bronchoalveolar lavage (BAL) was performed on eleven healthy smokers before and after eight weeks of oral treatment with N-acetylcysteine (NAC) 200 mg t.i.d. The concentrations of selected eosinophil and neutrophil granule constituents and of selected proteases and protease inhibitors, albumin and endotoxin were determined in the recovered BAL fluid and in plasma or serum samples. In addition, in vitro chemotactic activities for neutrophils and eosinophils were assessed in the BAL fluid. Significant reductions in BAL fluid content of lactoferrin (LF), eosinophil cationic protein (ECP), antichymotrypsin (ACT) and chemotactic activity for neutrophils were recorded after NAC treatment. The levels of other examined markers tended to be reduced or were not affected. In serum/plasma, the concentrations of myeloperoxidase (MPO) and elastase were reduced after NAC treatment whereas concentrations of other constituents examined were unaltered. These data, together with previously reported findings, suggest that oral NAC may influence the activity of "inflammatory" cells in the bronchoalveolar space of smokers.

Twenty patients with palmoplantar pustulosis (PPP) and 21 patients with psoriasis were compared with respect to the chemotactic response and random migration of their neutrophils (PMNs) and to the chemotactic and chemokinetic activities of their serum. The Boyden chamber technique was used. Compared with the reference group, the PPP neutrophils showed a significant decrease in random migration (p = 0.036) and chemotactic response to casein (p = 0.031), but not to zymosan-activated serum. The psoriasis PMNs had normal chemotactic response, but decreased random migration (p = 0.001). The chemokinetic effects of serum from PPP and psoriatic patients on normal neutrophils were increased (for PPP, p = 0.019 and for psoriasis p = 0.024). The chemokinetic activity of the heated sera was decreased and inversely correlated to the serum level of IgA in both disorders (PPP, p = 0.021; psoriasis, p = 0.013). The chemotactic activity of serum on normal PMNs was decreased in both conditions (PPP, p = 0.001; psoriasis, p = 0.047); this was due to the presence of chemotactic factor inhibitors in 34% of the sera. It seems likely that the high chemokinetic effect of serum both from the PPP and from the psoriasis patients compensates for the slightly low or normal migratory activity of neutrophils from these patients.

HA affects neutrophil function both in vitro and in vivo at picomolar concentrations. The present data suggest that the activity of HA is directed against some very basic metabolic mechanisms in the cell involving the increased synthesis of ATP and increased glucose metabolization. A preliminary clinical trial suggests that subcutaneous injections of HA may be used in the therapy of patients with undue susceptibility to bacterial infections but firm conclusions as to this point has to await regular controlled clinical trials.

Chemotactic and chemokinetic activity was studied in sera from 57 patients with various stages of sarcoidosis. Reduced chemotactic activity was observed in 39% of the sera, mainly in sera from patients with parenchymal densities on chest radiographs. Increased chemokinetic activity was noted in more than one third of the fresh sarcoid sera. Asymptomatic patients showed higher chemokinetic activity in their sera than patients with symptoms. The presence of heat-stable chemokinetic inhibitors was suggested in one third of the cases. No relationship was found between these inhibitors and the inhibitors of neutrophil phagocytosis previously demonstrated in sarcoid sera. Although many of the findings show minor differences to those of normal sera, the liability in sarcoidosis to produce a variety of inhibitors directed against various functions of neutrophils would theoretically hamper the elimination of putative antigens. The presence of serum mediated stimulation and inhibition of neutrophil locomotion could be significant for the pathogenesis of the disease.

1,2,3,4-tetrahydro-9-aminoacridine (THA) is a cholinesterase inhibitor presently under investigation in clinical trials for treatment of Alzheimer's disease, senile dementia of Alzheimer type (AD/SDAT). To further analyse the underlying mechanisms for its effect in human brain, an in vitro model which allows measurement of acetylcholine (ACh) release from human postmortem brain slices has been used. In control cortical tissue THA induces a decreased release of ACh probably due to negative feedback mechanisms mediated via presynaptic muscarinic autoreceptors. In AD/SDAT cortex THA enhances the release of ACh to control level. This effect is prevented by nicotinic or muscarinic receptor antagonists, which suggest receptor mechanisms involving both nicotinic and muscarinic receptors. Subchronic treatment of rats with THA (10 mg/kg sc twice daily) or physostigmine (0.9 mg/kg sc five times daily) causes a significant increase in the number of high affinity nicotinic receptors in the cortex of THA treated rats whereas no change is found in the physostigmine treated rats. The number of muscarinic receptors are decreased following both THA and physostigmine treatment.

Neutrophil and eosinophil chemotactic activities (NCA and ECA) were measured in serum from twenty-two patients with urticaria pigmentosa or systemic mastocytosis. NCA was also measured after heating serum to 56 degrees C (heat-stable NCA). Although these factors were increased in about half of the patients there was no correlation with histamine release as estimated by the excretion of the main histamine metabolite methylimidazoleacetic acid (MelmAA) in urine. A significant increase in heat-stable NCA, however, was found in patients with pruritus and abnormal high values of MelmAA. It is concluded that only heat-stable NCA is a specific mast cell mediator, but that the heat-labile NCA and ECA are dependent on mast cells for their production by a different cell, tentatively identified as the macrophage.

The influence of a copper and a plastic IUD on the localization and intensity of alkaline and acid phosphatase and beta-glucuronidase activity in the rabbit endometrium during early pseudopregnancy was investigated. The activities of the two lysosomal enzymes, acid phosphatase and beta-glucuronidase were increased in surface epithelial cells in the presence of both types of IUDs. The alkaline phosphatase activity was almost completely abolished in surface and glandular epithelial cells in uterine horns.

The therapeutic effect of absorbing plasma from tumour patients with immobilized staphylococcal protein A was tested. Plasma prepared by centrifugation was passed over protein A-Sepharose and then reinfused into the patient. Five patients were thus treated. One with malignant melanoma and one with renal adenocarcinoma showed measurable regression of metastatic lesions. In another with malignant melanoma a subcutaneous metastasis showed histopathological changes compatible with a therapeutic effect. In two patients, one with malignant melanoma and one with renal adenocarcinoma, no signs of regression were found. No severe adverse effects of the treatment were observed.

Circulating immune complexes (CIC) were determined in tumour patient sera using three methods. One is based on PEG-precipitation, one on C1q-reactivity, and one on protein A-reactivity. About 25-30% of the sera were positive in at least one of the tests. Incubation of serum with protein A-Sepharose in vitro removed PEG-precipitable CIC from most sera, whereas C1q-reactive CICs had a much lower affinity to protein A. The protein A-reactive complexes showed considerable variation in their binding to protein A-Sepharose, and in some sera the amount of these CICs was actually increased. Similar changes in protein A-reactive CIC were also found during ex vivo treatment of tumour patients with immune adsorption. It is proposed that the binding of immune complexes to protein A can result in remodelling of protein A itself. Results from ultracentrifugation and fractionated PEG-precipitation support this hypothesis.

A new application of the Boyden chamber method for the measurement of eosinophil migration, without the need of eosinophil isolation, has been developed. A cell suspension containing a mixture of granulocytes, neutrophils to the greater part, was used. The eosinophils were identified by staining their granules with Chromotrope 2R. The method made it possible to study the migration of eosinophils from normal individuals without eosinophilia. Control experiments demonstrated that the chemotactic and chemokinetic response of eosinophils in the granulocyte mixture was in accordance with the response of isolated eosinophils from the same donor. Normal eosinophils demonstrated a significant chemotactic response to C5f, platelet-activating factor (PAF), leukotriene B4 (LTB4), and f-meth-leu-phe (f-MLP). Furthermore, PAF was demonstrated to be significantly more eosinophil chemotactic than neutrophil chemotactic.

The function of alveolar macrophages (AM phi s) was studied in terms of the secretion of various chemotactic factors. Human AM phi s were obtained by BAL from healthy non-smokers and smokers. One of the chemotactic factors was LTB4, an arachidonic acid metabolite of the lipoxygenase pathway. The amount of LTB4 was determined in culture medium, in cell homogenate and in BAL-fluid. The total chemotactic activity for neutrophils was measured in culture medium and in BAL-fluid. AM phi s from smokers showed an impaired secretion of LTB4. The spontaneous secretion in vitro was inhibited by 90% (p less than 0.05) and the stimulated one was blocked by 84% (p less than 0.05). This impairment was not followed by a decrease in total chemotactic activity, indicating the existence of other chemotactic factors than LTB4. Preliminary characterization of the chemotactic activity by gel filtration demonstrated at least four different chemotactic factors. Budesonide inhibited both the release of LTB4 and the total chemotactic activity in medium from stimulated AM phi s.

Immune complexes in the serum of tumor-bearing patients were adsorbed from whole blood or plasma on a protein A-Sepharose column. The adsorbed material was eluted, precipitated and analyzed for gangliosides. All precipitates obtained from eight patients at different treatment occasions contained gangliosides at concentrations varying from 0.1 to 12.2 nmol sialic acid/mg protein. The compositions of gangliosides were similar among the patients, regardless of the type of cancer, and quite different from that of normal serum. Most (75-85% of total sialic acid) belonged to the gangliotetraose series, of which 26-33% was GM1, 26-34% GD1a, 8-17% GD1b, and 5-13% GT1b. However, the dominant ganglioside in normal serum, GM3, was present in only trace amounts, which ruled out a nonspecific adsorption of serum ganglioside by protein A-Sepharose. Similar results were obtained for whole blood and plasma treatments, and these results suggest a specific interaction between gangliosides of the gangliotetraose series and serum immunoglobulins, either by the gangliosides acting as antigens and forming immune complexes or by their binding to already formed complexes.

The sensitivity of adenocarcinomas of the stomach to various cytostatic drugs has been tested in short-term incubations. A suspension of single cells and small tissue fragments was prepared. It was used for incubation with the following drugs: melphalan, vinblastine sulphate, amethopterin, and 5-fluoro-uracil. The effect of the cytostatic drugs was measured as the difference in incorporation of labelled precursors in treated tubes and untreated control tubes. The different tumours were found to vary significantly in the their sensitivity. Moreover, a correlation of the effects between melphalan and vinblastine sulphate, and between amethopterin and vinblastine sulphate was observed.

An intrauterine device (IUD) consisting of either a plain or a copper-coated polyethylene catheter was inserted in one of the uteri of rabbits. Sham-operated uteri served as controls. The norepinephrine (NE) content was compared only between uteri within the same animal. Both copper and plastic IUDs markedly reduced the NE content in the uterus, but not in the oviduct, within 1 week after insertion. NE in both organs was present exclusively in the adrenergic nerve system. There was no difference in the action of the two types of IUDs. The effect of an IUD was probably eliminated by interruption of the preganglionic nerve connections. The results showed that the uterine adrenergic neurons are influenced by the presence of an IUD.

Cell suspensions of either mouse thymocytes or chick embryo cells were incubated with labelled precursors after preincubation with different cytostatic drugs. The total time of in vitro incubation was four hours. The ratio between the amount of incorporated labelled precursors and the total amount of DNA was determined and used to measure the cytostatic effect. All drugs used caused a marked depression of this ratio. The cytostatic drugs had no effect on the phosphorylation of thymidine.

This study compared the relative efficacy of a therapist-assisted relaxation treatment programme with a self-help approach and a self-monitoring condition in the treatment of chronic headaches in adolescents within a school setting. The subjects were 46 high school students, 16-18 years of age, who were randomly assigned to the three treatment conditions. The results indicated that the self-help approach was as effective as the therapist-assisted relaxation condition in reducing the students' headache, and that these findings were maintained at a 5-month follow-up evaluation. The results suggest that the self-help relaxation procedure is a potential low-cost alternative treatment to a more traditional therapist-based intervention condition.

The present investigation studied the influence of different types of intra-uterine devices (IUDs) especially that of a copper IUD, on the incorporation of 3-H-thymidine and 5-3-H-uridine into the endometrium of the rabbit. "Inert" IUD. In non-stimulated rabbits, the incorporation of 3-H-thymidine was increased in the copper influenced horn. The incorporation of 3H-thymidine in control and Cu-IUD-containing horns reached a maximum at 48 hours after HCG stimulation, but was significantly lower in the copper-containing horn than in the control horn. On the fifth day of pseudopregnancy, the incorporation of 3-H-thymidine was significantly higher in the copper IUD-containing horn. The total amount of DNA in the endometrium increased during early pseudopregnancy, but this increase was markedly reduced in the presence of copper. The copper IUD had no influence on the rate of incorporation of 5-3-H-uridine in non-stimulated rabbits, whereas it caused a higher incorporation on the fifth day of pseudopregnancy.

The expression of five antigens, associated with transitional cell carcinoma (TCC) of the urinary bladder on biopsies of tumors or normal urothelium, was studied by immunostaining with the corresponding monoclonal antibodies. Both tissue sections and single cell preparations were investigated with either indirect immunoperoxidase staining or immunofluorescence. All 5 antigens were expressed on the majority (70-90%) of sectioned tumor specimens from 44 TCC patients, and 4 of them were similarly expressed on single cell tumor preparations from 26 additional patients. However, in both types of preparation, the degree of expression of these antigens varied from scattered staining of less than 25% of the tumor cells to homogenous staining of all or almost all cells. This degree of expression varied individually for each of the antigens and was not related to the malignancy grade of the tumors. However, as most of the tumors were of grades II or III, no conclusions regarding the relationship of antigen expression to the aggressiveness of the tumors can be drawn. In any event, all tumors expressed at least one and mostly several of these antigens. Antigen expression on biopsies of normal bladder mucosa from TCC patients or on urothelial biopsies from patients with prostate hyperplasia was also observed on single cell specimens (34 patients) but not on sectioned material (9 patients). However, the frequency of positive specimens was much lower (4-20%). Moreover, the number of cells expressing one or, occasionally, several of the antigens in normal urothelium was small (usually less than 5%). Because of these marked differences in antigen expression between tumors and normal tissue, the results indicate that a combination of 3-5 of the antibodies used in this study may be suitable for diagnostic purposes.

The relationship between psoriasis and palmoplantar pustulosis (PPP) is uncertain, as is the role of the neutrophil granulocyte in these conditions. In a previous comparative study of the rate of polymorphonuclear leucocyte (PMN) phagocytosis of IgG- and IgG-C3b-coated particles, an increased uptake rate was found in both diseases. Further information on the in vivo activity of PMNs in these conditions may be obtainable by determining the level of lactoferrin (LF) in serum from such patients, since LF serves as a specific marker of the turnover and activity of the circulating pool of neutrophils. In this study on 19 patients with psoriasis and 20 patients with PPP, elevated levels of LF were found in both conditions. In contrast, the levels of lysozyme and beta 2-microglobulin, which are markers of monocyte-macrophage and lymphocyte activity, respectively, were normal. This suggests the selective activation of neutrophils in these disorders. LF was significantly correlated (P less than 0.05 and 0.001, respectively) to the rates of phagocytosis of IgG- and IgG-C3b-coated particles, but not to the chemotaxis of isolated PMNs. There was no correlation between the severity of the disease and the levels of serum LF. The data suggest the increased in vivo activity of neutrophils in psoriasis and PPP.

The eosinophil granulocyte is a pro-inflammatory cell which in its granules contains an abundance of highly cytotoxic proteins such as eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, eosinophil protein X and major basic protein. Upon stimulation of the cell, these proteins as well as a number of lipid mediators such as leukotriene C4, prostaglandin and platelet-activating factor are released to the exterior. The molecules which are produced during inflammatory reactions of the allergic type attract eosinophils to the target organ and stimulate them to liberate their products. The physiological meaning of this reaction is probably to combat invading parasites; however, in their absence accumulation and activation of eosinophils may cause disease, and one such disease may be chronic asthma.

Degradable starch microspheres (DSM) mixed and injected with a cytostatic drug might improve intra-arterial chemotherapy by increasing the local drug concentration. Several factors are of importance for an optimal effect of the microspheres, e.g. size and vascularity of the tumour, arterial blood flow and arterio-venous shunts. Therefore, the dose of DSM has to be individualized. A method for continuous monitoring of the effect of DSM was developed. A radiolabelled marker was injected intra-arterially mixed with DSM and mitomycin C. Two kinetic parameters--Passing Fraction and Marker Flow Rate--were found to be influenced by the microspheres and thus seemed to be useful for monitoring the DSM-treatment. Arterio-venous shunting was measured as passage of 99Tcm-labelled macroaggregated albumin through the liver to the lungs. Significant increase of shunting after injection of DSM was demonstrated in 15 out of 19 patients. Almost no effect of the microspheres was seen in patients with marked arterio-venous shunting or minimal reduction of the marker flow rate, but in others the passage of the labelled marker could generally be significantly reduced.

Neutrophil chemotactic activity (NCA) in serum is raised in subjects with asthma after inhalation of an allergen. Two kinds of NCA have been demonstrated, heat-stable and heat-labile. In addition, inhibitory activity is generated after inhalation challenge. In the present study we have investigated the relationship of heat-labile NCA to the development of the late asthmatic reaction (LAR) in 13 subjects with asthma after allergen challenge and the effects of asthma medication on the formation of this activity. Heat-labile NCA peaked 120 to 240 minutes after challenge and demonstrated at this time significant (p less than 0.001) quantitative relationships to the ensuing LAR. The inhalant corticosteroid, budesonide, significantly inhibited (p less than 0.001) the generation of heat-labile NCA and the development of LAR both after a single dose and after 4 weeks of pretreatment. Single-dose disodium cromoglycate pretreatment, initially, slightly enhanced (p less than 0.05) heat-labile NCA but, after 120 minutes, slightly inhibited (p less than 0.05) the activity. Disodium cromoglycate also slightly abrogated the development of LAR. Single-dose pretreatment with the beta 2-agonist, terbutaline, inhibited generation of heat-labile NCA (p less than 0.001) but was without effect on LAR. It is concluded that the generation of heat-labile NCA is related to the development of the LAR and may be of importance for the attraction of inflammatory cells to the lung in the development of the inflammatory reaction probably responsible for LAR. However, the pharmacologic control of heat-labile NCA indicates that the process is multifactorial and not solely dependent on the generation of NCAs detected in serum.

Heat-stable (HS) and heat-labile (HL) neutrophil chemotactic activities (NCAs) have been demonstrated in serum after allergen challenge of subjects with asthma. In this investigation, we have studied the possible occurrence of similar activities in 20 atopic individuals on natural exposure to allergen, that is, during the birch-pollen season. Since eosinophil accumulation is a hallmark of an ongoing allergic inflammation in the respiratory tract also, the possible production of eosinophil chemotactic activity (ECA) was examined in serum after allergen challenge and at natural exposure to pollen. Both HL-NCA and HL-ECA were produced to a significant extent (p less than 0.001) during the season, with the peak of activities occurring simultaneously with the peak pollen count. HL-ECA was produced after allergen challenge of subjects with asthma in the laboratory, as has been demonstrated for NCA previously. The activity of the HS-NCA was unaltered during season. Gel-filtration studies of the major HL-NCA and HL-ECA indicated a molecular weight for both activities of 100 to 150,000, and the activities produced during season cochromatographed with the HL-NCA and HL-ECA produced after allergen challenge in the laboratory, suggesting that all these activities are due to one and the same molecule. The results suggest that the heat-labile chemotactic activity found in serum of atopic subjects and subjects with asthma after allergen exposure may be involved in the attraction of eosinophils and neutrophils to the site of allergic inflammation.

The influence of a copper IUD and two "inert" IUDs on the histological picture of the rabbit endometrium during early pseudopregnancy and early pregnancy was studied. The copper IUD markedly inhibited the endometrial maturation during the first five days of both pseudopregnancy and pregnancy. The "inert" IUDs had no such effect. The copper IUD prevented the normal response of the rabbit uterus to 17-beta-estradiol in castrated animals. The observed effects of the copper IUD might seriously interfere with the normal process of implantation.

Hyaluronic acid (HA) stimulates normal neutrophil function both in vitro and in vivo Stimulation was also achieved by subcutaneous administration of HA to patients with extreme susceptibility to bacterial infections. Clinical improvement of some patients was obtained in connection to the administration. It is premature at this time to conclude any therapeutic effect of HA in patients with extreme infection propensity. The data presented here, however, for certain merit further investigation on this matter.

Degradable starch microspheres (DSM) injected intra-arterially together with cytostatic drugs increase the regional uptake of the drug and as a result reduce the systemic drug concentration. Previous studies have indicated that fixed doses of DSM result in different degrees of vascular occlusion and therefore variable systemic concentration of the co-injected drug. Continuous registration of the systemic concentration of 99Tcm-hydroxymethylene diphosphonate (99Tcm-HDP) co-injected intra-arterially with DSM was earlier used to monitor treatment sessions and to optimize the dose of microspheres. Further to investigate the mechanism of DSM-induced retention, the effect of DSM on the passage of this low molecular weight marker and of labelled erythrocytes (a marker confined to the blood vessels), was compared in 10 patients with liver metastases. DSM reduced the amount of 99Tcm-HDP passing through the liver by 6 to 47 per cent while the amount of erythrocytes eventually passing the liver was much less reduced (0-14%). The rate of passage, however, was significantly reduced for both labelled markers. These results indicate that substances retained by co-injection of DSM are not to a significant extent lodged within the blood vessels but diffuse into extra-vasal tissue compartments.

A truncated form of IGF-1 which lacks the aminoterminal tripeptide Gly-Pro-Glu (GPE) is found in human brain. It was proposed that GPE may result from neural specific processing and also have a function within the CNS. GPE was synthesized and shown to inhibit glutamate binding to the N-methyl-D-aspartate (NMDA) receptor. Whilst the carboxyterminal glutamate was necessary for NMDA receptor binding, the aminoterminal glycine potentiated receptor crossreaction. Furthermore, GPE had a potent stimulatory effect on the potassium induced release of acetylcholine from rat cortical slices. A less potent stimulation of dopamine release from striatum was also observed. The specific competitive NMDA receptor antagonist, (+/-)2-amino-7-phosphonoheptanoate (AP7), inhibited the action of GPE on dopamine but not on acetylcholine release. These studies have identified GPE as a novel neuroactive peptide with a potent action on acetylcholine release and support the general concept that the proteolytic products of the IGF-1 precursor play a role in the regulation of brain function.

In the present paper a test model was used to examine if human adenocarcinomas of the colon and the stomach are heterogenous as regards the sensitivity to cytosine arabinoside, melphalan, vinblastine sulphate, amethopterin and 5-fluorouracil in vitro. The effects of the five drugs were measured as the differences in incorporation of tritiated thymidine and deoxyuridine in drug-containing tubes and control tubes. It was found, that different parts of eleven colon and five stomach cancers differ significantly in their sensitivity to the same cytostatic treatment in vitro.