A comparative analysis of micafungin and caspofungin for empirical antifungal therapy in antibiotic-unresponsive febrile patients with hematologic malignancies.

This study was retrospectively carried out to compare the efficacy of echinocandins such as micafungin (MCFG) and caspofungin (CPFG) in the treatment of antibiotic-unresponsive febrile patients with hematologic malignancies. A total of 163 patients received either MCFG or CPFG. We evaluated the efficacy of echinocandin against fever decline in all patients. Fever decline, defined as a body temperature of less than 37.5 °C sustained for more than 48 h without scheduled antipyretic medication. Efficacy assessments showed that the incidence of fever decline was not significantly different between the MCFG and CPFG groups (P=0.599). The median number of days from the start of echinocandin administration to fever decline was 5 in both the MCFG and CPFG groups. Multivariate analysis showed that the use of anti-MRSA drugs (HR, 0.64; 95%CI, 0.45-0.90; P=0.011) and a change from echinocandins to voriconazole or liposomal-amphotericin B (HR, 0.50; 95%CI, 0.30-0.74; P<0.001) are significant risk factors for sustained fever. A significant difference (P=0.002) in incidence of fever decline was however associated with differences in the timing of anti-MRSA drug administration. The median number of days from the start of echinocandin administration to fever decline was 5 when administration of the anti-MRSA drug occurred "simultaneously or prior to echinocandin start" and 11 in the "next day or later of echinocandin start" group. In other words, starting anti-MRSA drug treatment after echinocandin treatment is a risk factor. In conclusion, MCFG and CPFG have similar efficacy as empirical antifungal agents in the treatment of antibioticunresponsive febrile patients with hematopoietic malignancies.

Overexpression of the activated form of the AtAREB1 gene (AtAREB1ΔQT) improves soybean responses to water deficit.

Abscisic acid-responsive element binding protein (AREB1) is a basic domain/leucine zipper transcription factor that binds to the abscisic acid (ABA)-responsive element motif in the promoter region of ABA-inducible genes. Because AREB1 is not sufficient to direct the expression of downstream genes under non-stress conditions, an activated form of AREB1 (AREB1ΔQT) was created. Several reports claim that plants overexpressing AREB1 or AREB1ΔQT show improved drought tolerance. In our studies, soybean plants overexpressing AREB1ΔQT were characterized molecularly, and the phenotype and drought response of three lines were accessed under greenhouse conditions. Under conditions of water deficit, the transformed plants presented a higher survival rate (100%) than those of their isoline, cultivar BR 16 (40%). Moreover, the transformed plants displayed better water use efficiency and had a higher number of leaves than their isoline. Because the transgenic plants had higher stomatal conductance than its isoline under well-watered conditions, it was suggested that the enhanced drought response of AREB1ΔQT soybean plants might not be associated with altered transpiration rates mediated by ABA-dependent stomatal closure. However, it is possible that the smaller leaf area of the transgenic plants reduced their transpiration and water use, causing delayed stress onset. The difference in the degree of wilting and percentage of survival between the 35S-AREB1ΔQT and wildtype plants may also be related to the regulation of genes that protect against dehydration because metabolic impairment of photosynthesis, deduced by an increasing internal CO2 concentration, was not observed in the transgenic plants.

Pharmacokinetics of oxaliplatin in gastrointestinal cancer patients with malignant ascites.

The pharmacokinetics of oxaliplatin in plasma and ascitic fluid was investigated in 5 gastrointestinal cancer patients with malignant ascites. Oxaliplatin was administered at 85 mg/m² by 2-hour infusion in the FOLFOX4 regimen, and the concentrations of total and free platinum were measured. There was a trend of lower plasma C(max) values of total platinum in patients with a larger volume of ascitic fluid. The AUC(0-t) values of mean concentration curves of total plasma platinum, total ascites platinum, free plasma platinum, and free ascites platinum were 31.15, 7.96, 4.93 and 2.93 mg•h/ml, respectively. The concentrations of free ascites platinum were similar to those of free plasma platinum at the last sampling time of 26 h in each patient. The decrease or disappearance of ascitic fluid was observed in 4 patients. These results suggest that oxaliplatin exerted a beneficial effect in gastrointestinal cancer patients with malignant ascites, even when administered intravenously.

Pattern of lymph node metastases of esophageal squamous cell carcinoma based on the anatomical lymphatic drainage system.

The recent anatomical studies of the esophagus showed that submucosal longitudinal lymphatic vessels connect to the superior mediastinal and the paracardial lymphatics and lymphatic routes to periesophageal nodes originate from the muscle layer. Using clinical data for lymph node metastasis, we verify these anatomical bases to clarify the rational areas of lymph node dissection in esophageal cancer surgery. Analysis was performed on 356 consecutive patients who underwent esophagectomy with three-field dissection. Patients were divided into those with tumor limited within the submucosal layer and those with tumor invading or penetrating the muscle layer. Frequency of node metastasis was compared according to supraclavicular, upper mediastinum, mid-mediastinum, lower mediastinum, perigastric and celiac areas. In patients with tumor limited to the submucosal layer, node metastasis was more frequent in the upper mediastinum and perigastric area than the mid- or lower mediastinum. Even in patients with tumor located in the lower esophagus, node metastasis was more frequent in the upper mediastinum than the mid-mediastinum or lower mediastinum. In patients with tumor located in the mid-esophagus, node metastasis was more frequent in the supraclavicular area than the mid-mediastinum or lower mediastinum. In patients with tumor invading or penetrating the muscle layer, node metastasis in the mid- and lower mediastinum increased dramatically, but was still less frequent than those in the upper mediastinum or the perigastric area. Postoperative survival curves did not differ among the involved areas. The most predictive factor associated with lymph node metastasis for postoperative survival was not the area of involved nodes, but the number of involved nodes by multivariate analyses. These clinical results verify recent anatomical observations. The lack of difference in survival rates among the involved areas suggests that these areas should be staged equivalently. For adequate nodal staging, the upper mediastinum should be dissected for the lower esophageal tumor and supraclavicular areas should be dissected for the mid-esophageal tumor even in patients with tumor limited to within the submucosal layer.

[FLEP therapy for advanced and recurrent gastric cancer].

The FLEP regimen (5-FU, LV, ETP and CDDP) is a combination chemotherapy administered regionally and systemically for the control of both local and disseminated disease in intra- and extra-abdominal regions in patients with advanced and recurrent gastric cancer. Sixty-one patients with advanced and recurrent gastric cancer were entered into this study. The treatment regimen consisted of 5-FU at 370 mg/m2 (days 1 to 5, i.v. 24 h); LV at a dose of 30 mg (days 1 to 5, i.v. bolus); and ETP and CDDP each at 70 mg/m2 (days 7 and 21, ia 2 h). This regimen was repeated every four weeks. The overall response rate was 36.1% (22/61) and the 50% and median survival times were 10.23 and 11.80 months, respectively. The adverse events were Grade 3/4 leukocytopenia (18.0%), Grade 3/4 thrombocytopenia (4.9%), Grade 3 nausea and/or vomiting (3.3%) and Grade 3 stomatitis (1.6%). Of the 17 NAC patients, the six curability B patients showed a statistically higher survival rate than the curability C and unresected patients. Based on the encouraging response rate and the improvement in prognosis, we recommend the FLEP regimen for patients with primary gastric cancer. Neoadjuvant chemotherapy using the FLEP regimen should be performed with curative resection as an objective.

Induction of C-reactive protein, serum amyloid P component, and kininogens in the submandibular and lacrimal glands of rats with experimentally induced inflammation.

The mRNAs for acute-phase proteins and kininogens were found to be increased in the submandibular gland (SMG) and extraorbital and intraorbital lacrimal gland (ELG and ILG) in response to experimentally induced inflammation in rats; i.e., 24 hours after subcutaneous injection of turpentine oil, mRNAs for C-reactive protein (CRP), serum amyloid P component (SAP), and H- and T-kininogens were induced in the SMG, ELG, and ILG of rats, whereas these mRNAs were not detected in the same tissues of normal control rats. The induction of mRNAs for these inflammatory proteins by turpentine oil was preceded by a transient increase in the level of mRNA for tumor necrosis factor-alpha (TNF-alpha) at 6 hours after subcutaneous injection of the oil. This was confirmed by injection of another inflammation inducer, lipopolysaccharide (LPS), which induced the TNF-alpha mRNA in the same way at 6 hours as turpentine oil did. The up-regulation of acute-phase proteins including kininogens in the SMG, ELG, and ILG suggest the existence of a strict defense system in the exocrine glands.

Expression of kininogens in the connective tissue-type mast cells of the rat.

The connective tissue-type mast cells present in the submandibular gland (SMG) and peritoneal cavity of rats were found to express kininogens (KGs), the expression of which was demonstrated by Western blotting, reverse transcription-polymerase chain reaction (RT-PCR), RT-PCR Southern blotting, and light- and electron-microscopic immunocytochemistry. In the SMG, the analysis of cDNA amplified by RT-PCR revealed that the molecular species of mRNAs expressed were high-molecular-weight (HMW)-K KG and T-I KG. Light microscopic immunocytochemistry exclusively localized the KG protein(s) in the mast cells present in the SMG. The signals in the mast cells were very strong, but no positive reaction was observed in the granular convoluted tubular cells, acinar cells or striated duct cells. As determined by using electron microscopy, extremely strong labelling with immunogold was observed in the secretory granules of the mast cells, but no labelling in their nucleus or cytoplasm. Analysis by Western blotting and RT-PCR Southern blotting indicated that both protein and mRNA of KGs were present in the mast cells separated from the peritoneal cavity, indicating de novo synthesis of KG in these cells. Preliminary experiments implied that the connective tissue-type mast cells in other rat tissues also expressed KG.

[Neoadjuvant chemotherapy for advanced gastric cancer using FLEP therapy].

Combination chemotherapy with 5-FU, LV, ETP and CDDP (FLEP) for advanced gastric cancer uses a combination of regional and systemic delivery for the control of both local and disseminated disease in the intra- and extra-abdominal regions. We performed this regimen as neoadjuvant chemotherapy (NAC). Fifteen patients with unresectable primary advanced gastric cancer underwent FLEP. The treatment regimen was 5-FU at 370 mg/m2, LV at 30 mg/body (days 1 to 5, i.v. 24 h) and ETP and CDDP each at 70 mg/m2 (days 7 and 21, ia 2 h). This regimen was repeated every four weeks. The overall response rate was 46.7% (7/15), and the 50% and median survival times were 11.43 and 12.35 months, respectively. The adverse events were Grade 3 leukocytopenia, Grade 3 thrombocytopenia, and Grade 3 stomatitis in 20.0%, 13.3%, and 6.7% of the patients, respectively. The 50% and median survival time overall were 11.43 and 12.35 months, respectively. Of the 15 NAC patients, curability B patients showed a statistically higher survival rate than curability C and unresected patients. In conclusion, FLEP was effective for unresectable advanced gastric cancer.

Highly regulated expression of subtilisin-like proprotein convertase PACE4 (SPC4) during dentinogenesis.

Expressions of mRNAs for four subtilisin-like proprotein convertases (SPCs: furin, PACE4, PC6, and PC8) and bone morphogenetic protein 4 (BMP4) in the rat molar tooth during development were analyzed by Northern blotting, reverse transcriptase-polymerase chain reaction (RT-PCR), and in situ hybridization to explore the possible involvement of SPCs in the processing of proBMPs. We found a temporospacial expression of PACE4, but not one of the other SPCs, in this tissue; i.e., RT-PCR analysis revealed that the level of PACE4 mRNA, but not that of the other SPC mRNAs became high around the second postnatal day. This increase was in good accordance with the increase in BMP4 mRNA, indicating an apparent association of these molecules with the differentiation and establishment of functional ameloblasts and odontoblasts. During dentinogenesis, PACE4 mRNA was localized in the ameloblasts and odontoblasts. These observations suggest that PACE4 plays a crucial role in dentinogenesis, especially via the activation of BMPs.

Involvement of vesicle-cytoskeleton interaction in AQP5 trafficking in AQP5-gene-transfected HSG cells.

A cDNA of rat aquaporin 5 (AQP5) was used to transfect to HSG (human salivary gland cells), and the trafficking mechanism was studied in vitro by confocal laser microscopy. The trafficking of AQP5 to the plasma membrane was induced by stimulation of AQP5-gene-transfected human salivary gland cells (HSGAQP5 cells) with thapsigargin, an inhibitor of endoplasmic Ca(2+)-ATPase, and or with A-23187, a calcium ionophore. Pretreatment of these cells with colchicine or vinblastine, microtubule inhibitors, prevented the trafficking induced by thapsigargin or A-23187. The trafficking event was not completely inhibited by cytochalasin B, a microfilament inhibitor. These results demonstrate that the trafficking of AQP5 vesicles to the plasma membrane is triggered by an increase in intracellular Ca(2+) and that the interaction of AQP5-containing vesicles with the cytoskeleton is involved in this trafficking.

Are there non-monoaminergic paradoxical sleep-off neurons in the brainstem?

Using extracellular single unit recording in the medulla of freely moving cats, we have found a population of PS-off ("Type II") neurons that are distinct from the classically described monoaminergic PS-off ("Type I") neurons. The presumed non-monoaminergic Type II PS-off neurons (n=22) showed a relatively high rate of tonic discharge during both quiet waking and slow-wave sleep (10.4+/-4.1 and 9.3+/-3.1 spikes/sec, mean +/- S.D., respectively) and a marked overall decrease in discharge rate during PS (0.3+/-0.4 spikes/sec). In contrast to the presumed monoaminergic PS-off neurons (n=62), Type II PS-off neurons showed short-lasting phasic discharges during PS, often in association with rapid eye movement and PGO wave bursts. These Type II neurons were all characterized by a short action potential which was significantly different from that of the monoaminergic PS-off neurons described so far. Five out of 22 neurons were identified antidromically by stimulation of the ventrolateral reticulospinal tract (vlRST) at the caudal medulla, while 2 of the 22 were identified antidromically by stimulation of the peri-locus coeruleus alpha of the mediodorsal pontine tegmentum. Their mean conduction velocity (7.2+/-1.9 m/sec) was significantly higher than that (0.9+/-0.3 m/sec) of the presumed monoaminergic PS-off neurons which were identified exclusively by stimulation of the vlRST. In addition, when examined during the sleep-waking cycle, the antidromic responses of Type II PS-off neurons were either completely blocked or reduced, with a prolongation of antidromic latency during PS. Most of these neurons were located in medullary structures containing no, or virtually no, monoaminergic neurons, and none responded by inhibition to systemic administration of serotonergic or adrenergic autoreceptor agonists. These findings indicate the existence, in the medulla, of non-monoaminergic PS-off neurons that would play an important role in PS generation.

Subtilisin-like proprotein convertase PACE4 (SPC4) is a candidate processing enzyme of bone morphogenetic proteins during tooth formation.

The temporospatial expression of PACE4, a member of the mammalian subtilisin-like proprotein convertase family, in the developing rat molar tooth was determined by in situ hybridization. At the initiation stage of tooth development, PACE4 mRNA was weakly expressed in the dental lamina, whereas the mesenchymal cells intensely expressed the PACE4 transcript. At the bud stage, high-level expression of PACE4 mRNA was found in the dental epithelium and condensed dental mesenchyme. Its expression became more localized in the differentiating ameloblasts during cap and early bell stages. In the newborn rats, PACE4 mRNA was localized in the ameloblasts and odontoblasts, but its expression became weaker with advancing development, showing apparent association with the differentiation and establishment of functional ameloblasts and odontoblasts. These expression patterns of PACE4 were very similar to those of several bone morphogenetic proteins (BMPs) reported previously. Because BMPs, which are primarily involved in the morphogenesis in tooth formation, are synthesized as inactive precursors and activated by limited proteolysis at the consensus Arg-X-X-Arg maturation site, the present observations suggest that PACE4 is possibly a candidate proBMP convertase that acts during tooth formation.

Salivary gland tissue kallikrein family and processing of growth factor precursors and proenzymes.

Four major enzymes of the tissue kallikrein family were purified from the mouse submandibular gland and characterized. The sequences indicated that they were mK1, mK9, mK13, and mK22. All four enzymes showed kinin-releasing activity, with mK1 exhibiting the highest activity. Like mK13, mK9 and mK22 also processed prorenin to give renin and/or arginyl renin, although their activities were less than that of mK13. The results suggest that tissue kallikrein family enzymes bearing higher kinin-releasing activity have lower prorenin-converting activity and vice versa. These enzymes may possibly have a physiological role in the tissue renin-angiotensin system.

Expression of an allozyme of prorenin-converting enzyme in the submandibular gland of DBA/2N mice.

A protein product of the tissue kallikrein gene family was isolated from the submandibular gland of DBA/2N mice. Amino acid sequencing showed this protein to be highly homologous to two tissue kallikreins, mK13 and mK26, also known as prorenin-converting enzymes PRECE and PRECE-2, respectively. The cDNA corresponding to the present enzyme was cloned, and its complete nucleotide sequence was determined. The cloned cDNA was different in 6 and 12 bases out of 783 nucleotides from those of mK1k-13 and mK1k-26 cDNAs, respectively, the homologies being 99.2 and 98.5% (nucleotide), or 98.3 and 96.2% (amino acid). Upon incubation with either bovine kininogens or mouse Ren 2 prorenin, this tissue kallikrein generated bradykinin and renin, respectively, as judged by Western blotting and protein sequence analysis. Isoelectric focusing analysis of the submandibular gland tissue kallikreins suggested that the present enzyme was not expressed in CD-1 or ICR mice and that no mK13 protein was present in DBA/2N mice. These data suggest that the enzyme is an allozyme of mK13, a prorenin-converting enzyme highly expressed in the submandibular gland of DBA/2N mice. The mK1k-13 gene in mice is therefore suggested to be polymorphic, having at least two allelic forms with a high sequence homology. The designation mK13(b) and mK1k-13(b) for the protein and gene of this tissue kallikrein is proposed.

[Two patients with obstructive jaundice due to intra-abdominal lymph-node metastases of gastric cancer responding to combination chemotherapy with 5-FU and CDDP].

Combination chemotherapy with 5-FU and CDDP was given to two patients with obstructive jaundice due to intra-abdominal lymph-node metastases of advanced and recurrent gastric cancer. One patient was a primary case associated with lymph-node metastases of portal fissure and periaorta, and the other was a recurrent case associated with lymph-node metastases of hepatoduodenal ligament and periaorta. The regimen consisted of 5-FU 1,000 mg/ m2 (day 1-5, continuous infusion) and CDDP 100 mg/m2 (day 3, 1 hr drip infusion). The interval was from the 6th to 21st day. The response to chemotherapy showed shrinking of intra-abdominal lymph-nodes and reopening of the biliary tract. The patients could be discharged from the hospital without PTBD tube and enjoyed a better quality of life (QOL). This therapy is thought to be effective against obstructive jaundice due to intra-abdominal lymph-node metastases of advanced and recurrent gastric cancer.

Prorenin processing and restricted endoproteolysis by mouse tissue kallikrein family enzymes (mK1, mK9, mK13, and mK22).

Four members of the tissue kallikrein family, mK1, mK9, mK13, and mK22, all of which exhibit extensive homology in amino acid sequence among themselves, were obtained from the submandibular gland of ICR mice and examined for their ability to cleave prorenin. Tissue kallikrein mK13 was confirmed to be a prorenin-converting enzyme; and mK9, which was earlier shown to be an EGF-binding protein, was found to cleave mouse Ren 2 prorenin specifically and convert it to mature renin with an activity of approximately 1/10 of that of mK13. With the same substrate, mK22 (beta-NGF endopeptidase) gave two products, renin and arginyl-renin; whereas mK1 (true tissue kallikrein) did not process it at all. The endoproteolytic activity of tissue kallikreins was examined with various peptide-MCA substrates. The substrates contained three key structures; X(Y)-Arg-Arg, X(Y)-Lys-Arg and X-Lys-Lys motifs (where X and Y are hydrophilic and hydrophobic amino acids, respectively). We found that mK1, mK9 and mK13 preferentially cleaved the former two types of substrate, except Y-Arg-Arg-MCA. The substrate X-Lys-Lys-MCA was hardly cleaved by these three tissue kallikreins but was preferentially cleaved by mK22. The four tissue kallikreins seem to have the ability to process precursor proteins containing a pair of basic amino acid residues; the specificities of three of the enzymes (mK1, mK9 and mK13) were similar to each other but were different from that of mK22.

rHuEPO enhances the production of plasminogen activator inhibitor-1 in cultured endothelial cells.

The direct effects of recombinant human erythropoietin(rHuEPO) on coagulation and fibrinolysis factors were evaluated in a cultured endothelial cell (EC) system. Confluent quiescent ECs were incubated with or without 5.0 U/ml rHuEPO for 1, 6, and 18 hours, and supernatant concentrations of plasminogen activator inhibitor-1 (PAI-1): antigen (Ag), tissue plasminogen activator and thrombomodulin, and supernatant activities of tissue factor pathway inhibitor and von Willebrand factor were measured. The results showed that only PAI-1 levels were increased by the presence of rHuEPO. In order to assess the effect of rHuEPO on PAI-1 production by EC more precisely, confluent ECs were incubated with various doses of rHuEPO (0, 1.0, 2.5, 5.0, 10.0 U/ml) for 1, 6, 12, and 18 hours, and PAI-1:Ag concentrations in the supernatants of media were measured. PAI-1:Ag in the supernatants were increased by the presence of rHuEPO at all incubation times (P < 0.01) and the increase in PAI-1:Ag was dependent on rHuEPO concentration. The increases in PAI-1:Ag by 5.0 U/ml rHuEPO were comparable to those by 0.1 U/ml tumor necrosis factor-alpha, 1.0 microgram/ml lipopolysaccharide, and 0.5 U/ml thrombin. The increase in PAI-1:Ag by rHuEPO was suppressed by pre-incubation with 10 micrograms/ml cycloheximide (P < 0.01) or 0.2 microgram/ml actinomycin D (P < 0.01). These results indicate that rHuEPO directly stimulates PAI-1 production in cultured EC via de novo protein and RNA syntheses.

Effects of rHuEpo on cellular proliferation and endothelin-1 production in cultured endothelial cells.

BACKGROUND: Although elevation of blood pressure is considered to be the main adverse effect under rHuEpo therapy in haemodialysis patients, the precise mechanism remains obscure. The direct effect of rHuEpo on endothelial cells (EC) has been suggested as one of contributing factors of rHuEpo-induced hypertension. METHODS: EC were incubated with various concentrations of rHuEpo (0, 1000, 5000, 10,000 mU/ml) for up to 7 days, and cell numbers, DNA and protein synthesis by EC and supernatant concentrations of immunoreactive endothelin-1 (ET) were determined by haemocytometer, 3H-thymidine and 3H-leucine incorporation, and RIA, respectively. The effect of rHuEpo on EC proliferation was confirmed by anti-rHuEpo rabbit antiserum. The effect of cycloheximide or acinomycin D was also examined on the increase in ET production by rHuEpo. RESULTS: rHuEpo dose-dependently stimulated the proliferation of cultured EC, and this proliferative effect was inhibited by anti-rHuEpo rabbit antiserum. DNA and protein syntheses by EC were also increased by rHuEpo. The supernatant concentrations of ET cultured with rHuEpo at 5000 mU/ml or more showed significantly greater values than those without rHuEpo and the increase in ET in the supernatants of media containing 5000 mU/ml rHuEpo was inhibited by incubation with 0.2 microgram/ml actinomycin D or 10 micrograms/ml cycloheximide. Further, rHuEpo increased DNA synthesis by EC which had been cultured in E-BM medium containing 0.5 or 2% FBS for 3 h and which were recultured in E-BM medium containing 5% FBS for 15 h. CONCLUSIONS: rHuEpo directly stimulates EC proliferation as a competence factor, and it also accelerates endothelin-1 production in association with stimulation of DNA and protein syntheses by EC.

[Pharmacokinetic study of intraperitoneal cisplatin chemotherapy for gastric cancer as an adjuvant setting].

The pharmacokinetic studies of intraperitoneal cisplatin (CDDP) for gastric cancer were discussed elsewhere, but those studies were investigated in patients with ascites. The purpose of this study is to compare the difference in pharmacokinetics between patients with malignant ascites and those curatively resected without ascites. One hundred mg of CDDP and 300 ml of saline were administered intraperitoneally for 9 curatively resected patients by catheter just after operation, and the same doses of CDDP were administered for 3 advanced or recurrent patients with ascites just after removal of whole fluid. Blood samples were corrected at 6 points after administration. Results were as follows: The 0-t area under the curve (AUC) and the Cmax of both total and free CDDP in the patients without ascites was higher than in the patients with ascites. The 0-infinity AUC and MRT of the ascites patients were higher than in the patients without ascites. These data suggest that intraperitoneal CDDP chemotherapy for gastric cancer as an adjuvant setting is more effective than chemotherapy for advanced malignant ascites patients.