Biomarkers for intracellular pathogens: establishing tools as vaccine and therapeutic endpoints for visceral leishmaniasis.

Visceral leishmaniasis in South Asia is a serious disease affecting children and adults. Acute visceral leishmaniasis develops in only a fraction of those infected individuals, the majority being asymptomatic with the potential to transmit infection and develop disease. We followed 56 individuals characterized as being asymptomatic by seropositivity with rk39 rapid diagnostic test in a hyperendemic district of Bangladesh to define the utility of Leishmania-specific antibodies and DNA in identifying infection. At baseline, 54 of the individuals were seropositive with one or more quantitative antibody assays and antibody levels persisted at follow up. Most seropositive individuals (47/54) tested positive by quantitative PCR at baseline, but only 16 tested positive at follow up. The discrepancies among the different tests may shed light on the dynamics of asymptomatic infections of Leishmania donovani, as well as underscore the need for standard diagnostic tools for active surveillance as well as assessing the effectiveness of prophylactic and therapeutic interventions.

The safety and efficacy of oral docosahexaenoic acid supplementation for the treatment of primary sclerosing cholangitis - a pilot study.

BACKGROUND: Primary sclerosing cholangitis (PSC) is characterised by progressive inflammatory and fibrotic destruction of the biliary ducts. There are no effective medical therapies and presently high dose ursodeoxycholic acid is no longer recommended due to significant adverse events in a recent clinical trial. Cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction is associated with PSC in both children and adults. Since CFTR dysfunction leads to altered fatty acid metabolism, specifically reduced docosahexaenoic acid (DHA), we hypothesised that DHA supplementation might be an effective therapy for patients with PSC. AIM: To determine the safety and efficacy of oral DHA supplementation for the treatment of PSC. METHODS: We conducted a 12 month open-label pilot study to evaluate safety of oral DHA and its effects on serum alkaline phosphatase as a primary outcome measure in 23 patients with PSC. DHA was administered orally at 800 mg twice per day. Secondary outcomes included changes in other liver function tests and fibrosis biomarkers. RESULTS: A 1.7-fold increase in serum DHA levels was observed with supplementation. The mean alkaline phosphatase level (±S.E.) at baseline was 357.8 ± 37.1 IU compared to 297.1 ± 23.7 IU (P < 0.05) after 12 months of treatment. There were no changes in other liver function tests and fibrosis biomarkers. No adverse events were reported. CONCLUSIONS: Oral DHA supplementation is associated with an increase in serum DHA levels and a significant decline in alkaline phosphatase levels in patients with PSC. These data support the need for a rigorous trial of DHA therapy in PSC.

Cysteine-rich regions of pig gastric mucin contain von willebrand factor and cystine knot domains at the carboxyl terminal(1).

In order to sequence the cysteine-rich regions of pig gastric mucin (PGM), we used our previously identified pig gastric mucin clone PGM-2A to screen a pig stomach cDNA library and perform rapid amplification of cDNA ends to obtain two cysteine-rich clones, PGM-2X and PGM-Z13. PGM-2X has 1071 base pairs (bp) encoding 357 amino acids containing five serine-threonine-rich 16 amino acid tandem repeats, downstream from a cysteine-rich region similar to human and mouse MUC5AC. PGM-Z13 encodes the complete 3'-terminus of PGM and is composed of 3336 bp with a 2964 bp open reading frame encoding 988 amino acids with four serine-threonine-rich tandem repeats upstream from a cysteine-rich region similar to the carboxyl terminal regions of human and rat MUC5AC and human MUC5B. This region is homologous to von Willebrand factor C and D domains involved in acid induced polymerization, and to the carboxyl terminal cystine-knot domain of various mucins, TGF-beta, vWF and norrin, which is involved in dimerization. These newly sequenced cysteine-rich regions of pig gastric mucin may be critical for its gelation and for its observed increased viscosity induced by low pH.

pH-dependent conformational change of gastric mucin leads to sol-gel transition.

We present dynamic light scattering (DLS) and hydrophobic dye-binding data in an effort to elucidate a molecular mechanism for the ability of gastric mucin to form a gel at low pH, which is crucial to the barrier function of gastric mucus. DLS measurements of dilute mucin solutions were not indicative of intermolecular association, yet there was a steady fall in the measured diffusion coefficient with decreasing pH, suggesting an apparent increase in size. Taken together with the observed rise in depolarized scattering ratio with decreasing pH, these results suggest that gastric mucin undergoes a conformational change from a random coil at pH >/= 4 to an anisotropic, extended conformation at pH < 4. The increased binding of mucin to hydrophobic fluorescent with decreasing pH indicates that the change to an extended conformation is accompanied by exposure of hydrophobic binding sites. In concentrated mucin solutions, the structure factor S(q, t) derived from DLS measurements changed from a stretched exponential decay at pH 7 to a power-law decay at pH 2, which is characteristic of a sol-gel transition. We propose that the conformational change facilitates cross-links among mucin macromolecules through hydrophobic interactions at low pH, which in turn leads to a sol-gel transition when the mucin solution is sufficiently concentrated.

Dysregulation of proteoglycan production by intrahepatic biliary epithelial cells bearing defective (delta-f508) cystic fibrosis transmembrane conductance regulator.

Hepatic dysfunction in cystic fibrosis (CF) has been attributed to accumulation of viscous mucoid secretions in intrahepatic bile ducts. The purpose of our study was to compare glycoconjugate secretion by intrahepatic biliary epithelial (IBE) cells derived from normal livers and livers of CF patients with the delta F508 mutation of the cystic fibrosis transmembrane conductance regulator (CFTR). Confluent cells were incubated with 3H-glucosamine (GlcN) for 16 hours, and radiolabeled macromolecules were analyzed for the amount and type of glycoconjugates. Incorporation of 3H-GlcN into macromolecular glycoconjugates was two- to threefold higher in CF cells versus normals, as was uptake of 3H-Glcn into the cytoplasm of CF cells. Gel exclusion chromatography on Sepharose Cl 4B revealed that the secreted glycoconjugates from CF cells eluted entirely in the excluded fraction (molecular weight > 2 x 10(6)), while, in the normal cells, 60% of the glycoconjugates eluted as lower-molecular-weight species. The high-molecular-weight glycoconjugates in both CF and normal cells were identified as chondroitin sulfates, as evidenced by susceptibility to beta elimination, chondroitinase digestion, and amino acid composition. Western blotting of IBE cell secretions with a polyclonal antibody to chondroitin sulfate revealed proteoglycan bands at 100 and 210 kd. Our results indicate that secretion of chondroitin sulfate is markedly increased in CF biliary epithelium in vitro compared with non-CF cells. Increased uptake of precursor 3H-GlcN may contribute to enhanced glycosylation of chondroitin sulfate in CF cells.

Interaction of an aluminum-magnesium containing antacid and gastric mucus: possible contribution to the cytoprotective function of antacids.

BACKGROUND: Antacids are generally thought to protect the gastric mucosa from damage primarily by their ability to neutralize hydrochloric acid, but recently other mechanisms of antacid cytoprotection have been suggested. The aim of our study was to determine if the antacid hydrotalcit (Mg6Al2(OH)16CO3 x 4H2O) and its clinical formulations Talcid (suspension and tablet) can influence the acid barrier properties of pig gastric mucus (PGM). METHODS: Viscosities, flow patterns of injected HCl, and permeability to HCl were assayed in solutions of PGM with and without added antacid. RESULTS: Talcid-suspension markedly increased mucin viscosity between pH 2 and 7. In contrast, powdered Talcid-tablet and hydrotalcit noticeably reduced mucin viscosity at pH 5 and below. HCl barely diffused through PGM-Talcid-suspension, whereas the acid was able to quickly penetrate a PGM-Talcid-tablet powder or PGM-hydrotalcit mixture. When injected into a mixture of PGM-Talcid-suspension, HCl travelled in a single distinct channel whereas in both PGM-Talcid-tablet powder or PGM-hydrotalcit mixtures, the acid mixed irregularly throughout. Experiments with antacids alone revealed that Talcid-suspension, but not Talcid-tablet nor hydrotalcit, had barrier properties similar to PGM. CONCLUSION: Talcid-suspension has viscoelastic features similar to gastric mucin and may afford mucosal protection by its ability to maintain or mimic the barrier properties of gastric mucus gel. In contrast, powdered Talcid-tablets and hydrotalcit reduce the barrier function of gastric mucus.

Acute stress causes mucin release from rat colon: role of corticotropin releasing factor and mast cells.

We determined the effects of immobilization stress on rat colonic mucus release and mast cell degranulation and examined whether corticotropin releasing factor (CRF) was involved in these responses. After 30-min immobilization, rats were killed, colonic mucosal explants were cultured, and levels of rat mast cell protease II (RMCP II) and prostaglandin E2 (PGE2) were measured. Mucin release from explants was assayed by incorporation of [3H]glucosamine into colonic mucin and by histological evaluation of goblet cell depletion. Stress caused significant increases of colonic RMCP II, PGE2, and mucin release and fecal pellet output and caused an approximately 10-fold increase in colonic mucosal levels of cyclooxygenase-2 (COX-2) mRNA. These stress-associated changes were reproduced by intravenous or intracerebral injection of CRF in conscious, nonstressed rats. Pretreatment of rats with the CRF antagonist alpha-helical-CRF9-41, hexamethonium, atropine, or bretylium, or the mast cell stabilizer lodoxamide inhibited stress-induced release of RMCP II, PGE2, and mucin, whereas indomethacin prevented mucin release but not mast cell degranulation. Hexamethonium and CP-96,345, a substance P antagonist, inhibited fecal pellet output caused by stress. We conclude that CRF released during immobilization stress increases colonic transit via a neuronal pathway and stimulates colonic mucin secretion via activation of neurons and mast cells.

Isolation and characterization of cDNA clones encoding pig gastric mucin.

Polyclonal antibodies raised to deglycosylated pig gastric mucin were used to screen a cDNA library constructed with pig stomach mucosal mRNA. Immunocytochemistry indicated that the antibody recognizes intracellular and secreted mucin in surface mucous cells of pig gastric epithelium. A total of 70 clones producing proteins immunoreactive to this antibody were identified, two of which (PGM-2A,9B) were fully sequenced from both ends. Clone PGM-9B hybridized to a polydisperse mRNA (3-9 kb) from pig stomach, but not liver, intestine or spleen, nor to mRNA from human, mouse, rabbit or rat stomach. Sequence analysis indicated that PGM-9B encodes 33 tandem repeats of a 16-amino-acid consensus sequence rich in serine (46%) and threonine (17%). Using the restriction enzyme MwoI, which has a single target site in the repeat, it was demonstrated that PGM-9B consists entirely of this tandem repeat. Southern-blot analysis indicated that the repeat region is contained in a 20 kb HindIII-EcoRI fragment, and BamHI digestion suggested that most of the repeats are contained in a 10 kb fragment. In situ hybridization with an antisense probe to PGM-9B showed an intense signal in the entire gastric gland. Clone PGM-2A also contains the same repeat sequence as 9B, but, in addition, has a 64-amino-acid-long non-repeat region at its 5' end. Interestingly the non-repeat region of PGM-2A has five cysteine residues, the arrangement of which is identical with that reported for human intestinal mucin gene MUC2.

Viscous fingering of HCl through gastric mucin.

The HCl in the mammalian stomach is concentrated enough to digest the stomach itself, yet the gastric epithelium remains undamaged. One protective factor is gastric mucus, which forms a protective layer over the surface epithelium and acts as a diffusion barrier Bicarbonate ions secreted by the gastric epithelium are trapped in the mucus gel, establishing a gradient from pH 1-2 at the lumen to pH 6-7 at the cell surface. How does HCl, secreted at the base of gastric glands by parietal cells, traverse the mucus layer without acidifying it? Here we demonstrate that injection of HCl through solutions of pig gastric mucin produces viscous fingering patterns dependent on pH, mucin concentration and acid flow rate. Above pH 4, discrete fingers are observed, whereas below pH 4, HCl neither penetrates the mucin solution nor forms fingers. Our in vitro results suggest that HCl secreted by the gastric gland can penetrate the mucus gel layer (pH 5-7) through narrow fingers, whereas HCl in the lumen (pH 2) is prevented from diffusing back to the epithelium by the high viscosity of gastric mucus gel on the luminal side.

Profound increase in viscosity and aggregation of pig gastric mucin at low pH.

Epithelial mucins are glycoproteins of very large molecular weight that provide viscoelastic and gel-forming properties to mucus, the jellylike protective layer covering epithelial organs. In the mammalian stomach the mucus gel layer protects the underlying epithelial cells from HCl in the lumen. We report here that pig gastric mucin undergoes a 100-fold increase in viscosity in vitro when pH is lowered from 7 to 2. Sedimentation velocity and dynamic light-scattering measurements revealed the formation of extremely large aggregates at low pH consistent with the observed increase in viscosity. Aggregation of mucin at low pH was prevented by increasing the ionic strength, suggesting the involvement of electrostatic interactions. Trypsin digestion and thiol reduction, but not enzymatic removal of neuraminic acid, prevented aggregation at low pH. This implies that the peptide core rather than the oligosaccharide side chains of the molecule is involved in the aggregation of mucin at low pH. Increased aggregation and viscosity at low pH were also observed in a solvent made to mimic the ionic composition of gastric juice, indicating the physiological relevance of our findings. Our observations suggest that one mechanism of gastric protection may be the ability of gastric mucin to undergo aggregation with a marked increase in viscosity at low pH.

Bronchial mucus hypersecretion in acute quadriplegia. Macromolecular yields and glycoconjugate composition.

In acute quadriplegia we have noted that about one in five patients develops unexplained production of markedly excessive and tenacious bronchial mucus. Spontaneous recovery from mucus hypersecretion usually occurs within weeks to months. Mucus samples collected from 12 patients have been found to be abnormal. Macromolecular contents of single aspirates yielded as much as 500 mg. Analytical ultracentrifuge analysis showed the mucus to contain considerable epithelial glycoprotein (GP) of typical buoyant density; its amino acid and carbohydrate compositions were characteristic of the GP from hypersecretory bronchial mucus such as in chronic bronchitis and cystic fibrosis. In five patients studied after recovery from hypersecretion, there tended to be relatively less GP. The mucus samples contained a high density glycoconjugate (GC): this had sugars of GP but also reacted positively with a monoclonal antibody to keratan sulfate. Its amino acid composition was different from that of GP: threonine was lower and glycine was higher than in GP. In mucus from one patient who died, chondroitin sulfate ABC and hyaluronic acid were identified as well. This suggests proteoglycans are involved in the pathophysiology of mucus hypersecretion. The sudden onset and spontaneous recovery of hypersecretion suggests that it is not due to gland hypertrophy. We speculate that in acute quadriplegia it is due to disturbed neuronal control of bronchial mucus gland secretion, perhaps related to initial disappearance and later reappearance of peripheral sympathetic nervous system tone.

Lipid binding to gastric mucin: protective effect against oxygen radicals.

Gastric mucus forms a viscous gel overlying the gastric mucosa and is thought to protect the underlying mucosa from noxious agents such as acid, proteases, and bile salts. A common property of mucin, the principal glycoprotein in mucous secretions, is its ability to bind lipids. The purpose of this study was to determine if lipids bound to gastric mucin protect the mucin from oxygen radical attack. Pig gastric mucin, partially purified by Sepharose 4B gel chromatography, was found to contain large amounts of free fatty acids and cholesterol as well as lesser amounts of sphingomyelin and phospholipids. Purified mucin obtained by density-gradient ultracentrifugation in a CsCl gradient contained only trace amounts of fatty acids but no other lipids. Exposure to the oxygen radical-generating system iron/ascorbate caused a marked reduction in viscosity of purified mucin but did not affect partially purified mucin, suggesting that bound lipids shielded the mucin from attack by oxygen radicals. Using discontinuous sucrose-gradient ultracentrifugation in the presence of liposomes containing [3H]palmitic acid, we demonstrated that mucin is capable of binding fatty acids. We also observed a striking increase in solution viscosity of gastric mucin at low pH, a feature that might contribute to the ability of mucin to form a protective diffusion barrier for the underlying epithelium.

Lipids in airway mucus of acute quadriplegic patients.

Quadriplegic patients have difficulty in clearing lung mucus due to paralysis of muscles of respiration. In about 25% of these patients, excessive mucus in the airway necessitates tracheostomy, and in some patients it is fatal. In others there is spontaneous recovery. To determine if the excessive mucus results from secretion of abnormal mucus or from accumulation of normal mucus, we analyzed the lipids in mucus from eight quadriplegic patients. Lipids were separated from other constituents of the mucus by density gradient ultracentrifugation, extracted with chloroform-methanol (2:1), and examined by high-performance thin-layer chromatography (HPTLC). Cholesterol was the major neutral lipid; phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were the main phospholipids. Glycolipids were predominant, lactosylceramide (CDH) being the highest in amount. Two-dimensional HPTLC as well as high-performance lipid chromatography also revealed the presence of gangliosides: comparison with standards indicated the presence of GM1, GM2, GM3, and some unidentified gangliosides. In normal mucus, cholesterol is the predominant lipid; phospholipid is present in smaller amounts but glycolipids are not identified. Thus, results of our lipid analysis show that mucus from the quadriplegic patients is abnormal and similar to that in hypersecretory diseases such as chronic bronchitis and cystic fibrosis. Unlike these latter cases, hypersecretion in the quadriplegic has a rapid onset and, often, spontaneous recovery, suggesting that this is due to abnormal stimulation rather than an increase in the population of secretory cells.

Recovery of an epitope recognized by a novel monoclonal antibody from airway lavage during experimental induction of chronic bronchitis.

Prolonged exposure of dogs to high concentrations of SO2 gas results in a syndrome with many of the characteristics of human chronic bronchitis, including cough and chronic mucous hypersecretion as well as airway obstruction. We developed and used a novel monoclonal antibody, GB-4B, raised against epithelial glycoprotein isolated from human hypersecretory mucus to probe airway lavage samples from dogs before and during prolonged exposure to SO2 gas. There were relatively low mean titers of the epitope recognized by GB-4B in airway lavage fluid as evidenced by enzyme-linked immunosorbent assay before exposure to SO2 gas. After 25 to 50 wk of SO2 exposure, the dogs showed a significant increase in pulmonary resistance and there was a significant increase in the titer of the epitope in the airway lavage fluid. Using the same antibody immunohistochemical analysis of airway tissues from SO2-exposed dogs revealed patchy staining of the mucous glands and airway secretory cells and dense staining along the airway surface; airway tissue from control dogs and one SO2-exposed dog whose lavage fluid did not contain the epitope showed little or no staining. These data demonstrate that similar mucin epitopes appear in airway lavage fluid under hypersecretory conditions in both animals and humans. The epitope may have utility as a marker of chronic mucous hypersecretion.

Secretions from primary hamster tracheal surface epithelial cells in culture: mucin-like glycoproteins, proteoglycans, and lipids.

Surface epithelial cells dissociated from hamster tracheas and grown on a thick collagen gel in the presence of 5% fetal bovine serum become highly enriched with secretory cells at confluence. In the present communication, we have analyzed secretory products from this primary hamster tracheal surface epithelial (HTSE) cell culture. The secreted glycoconjugates included high-molecular-weight mucin-like glycoproteins (HMW MLGP) and proteoglycans that comprised 22% and 5% of the total [3H]glycoconjugates secreted when [3H]glucosamine was added as a metabolic precursor. Among the proteoglycans were hyaluronic acids (53%), heparan sulfate proteoglycans (29%), and chrondroitin sulfate proteoglycans (18%). Chondroitin sulfates were mostly 4-sulfated. On the other hand, the secreted lipids included cholesterol, phospholipids, and glycolipids, and most of them were associated with HMW MLGP.

Macromolecular and lipid constituents of bronchial epithelial mucus.

The physical and chemical properties of bronchial epithelial mucus depend on its special mix of macromolecules and lipid constituents: these are different in the normal airway under baseline conditions from one stimulated acutely, and show major modification in disease. Since the last conference, density gradient ultracentrifugation has been extended to the study of normal bronchial mucus in addition to that of sputum from patients with chronic bronchitis, cystic fibrosis and asthma, and has revealed striking differences between the chemical profiles of normal and hypersecretory mucus. Normal mucus represented individual bronchial aspirates, obtained at fiberoptic bronchoscopy from healthy human volunteers (non-smokers), aspirates from normal dogs (before SO2 exposure in a canine model of SO2 induced bronchitis) and secretions released in vitro by human bronchial and canine tracheal explants. Mucus 'in transition' included aspirates from otherwise healthy smokers and from dogs early in irritation. Hypersecretory mucus included, besides those mentioned above, aspirates from dogs that had developed bronchitis and the excessive mucus produced by some patients with acute quadriplegia. Lipids. In normal mucus, human (unpooled) and canine, neutral lipids are the predominant species, with lesser amount of phospholipids: no glycolipids are detected. The first qualitative change on irritation, even before macromolecular yield increases, is appearance of glycolipids. In hypersecretory mucus, human (including quadriplegics) or canine, glycolipids are detected in appreciable amounts and often are the predominant species: they include complex forms such as sialic acid containing gangliosides. Organ and cell culture studies establish that these lipids are produced by airway epithelial cells. These lipids are important to gel formation. Glycoconjugates. A major recent advance is the recognition that normal mucus does not contain typical epithelial glycoprotein. Its glycoconjugate is of higher density with sugars typical of both glycoprotein (GP) and proteoglycan (PG) and with an amino acid profile more akin to PG (glycine greater than serine greater than threonine). In transition to hypersecretion, a 'mixed molecule' changes its sugar mix to produce a density typical of GP. In hypersecretion, the epithelial GP develops a typical buoyant density and amino acid profile (threonine greater than serine greater than glycine). Organ culture of bronchial explants, and more recently cell culture, establish that the PGs are major products of airway secretory cells. The normal airway is capable of producing glycoprotein on cholinergic stimulation. The range of glycoconjugates present in the secretion support the wide range of granule and cell features identified in vivo. Monoclonal antibody raised to a pure preparation of bronchial epithelial glycoprotein reacts with mucous cells in the surface epithelium and also in the submucosal gland in both human and canine airways.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies on the peptide core of human bronchial mucus glycoprotein.

We have employed thiol-Sepharose chromatography following deglycosylation to analyze the protein core of bronchial epithelial mucus glycoprotein (MGP), isolated by a two stage density gradient ultracentrifugation. Deglycosylation using triflouromethanesulfonic acid resulted in loss of greater than 90% of carbohydrate. The deglycosylated core protein was reduced and the sulfhydryl residues activated with 2-2'dipyridyl disulfide. This preparation was then bound covalently to thiol-Sepharose, and eluted specifically with reducing agents. Our results demonstrate that bronchial MGP contains cysteine residues potentially capable of forming disulfide bonds. Pepsin digestion studies suggest that cysteine residues are present near both the heavily glycosylated region and the naked peptide region. Thiol-Sepharose chromatography resolved several mucin-associated proteins (MAPS) that did not bind to the column. Amino acid analysis showed that the largest of these (200 kDa) is enriched in serine/threonine, like MGP that absorbed to the column: the two smallest (20 kDa and 60 kDa) are similar to the proline rich proteins reported in salivary mucin. These associated proteins, although not linked by disulfide bonds to the MGP, are, nevertheless, tightly bound to it, since they were only recovered after deglycosylation and thiol chromatography.

Transition from normal to hypersecretory bronchial mucus in a canine model of bronchitis: changes in yield and composition.

Density-gradient analysis was used to follow the transition from normal to hypersecretory bronchial mucus in a model of bronchitis induced in dogs by chronic exposure to SO2 gas. Aspirates of saline bronchial lavage were obtained by fiberoptic bronchoscopy from dogs before, during a 6- to 9-month exposure period to SO2 gas, and during a recovery period of similar duration. Prior to SO2 exposure, aspirates from all animals had a low yield of nondialyzable macromolecules (15 +/- 6 mg/aspirate) and similar composition. Specifically, epithelial glycoprotein of typical buoyant density was not detected; rather a glycoconjugate of higher buoyant density with features of both proteoglycan and glycoprotein was identified. Neutral lipids were predominant with lesser amounts of phospholipids; no glycolipids were detected. During the SO2 exposure period, aspirates from five of the eight dogs contained components similar in buoyant density to human bronchitic glycoprotein. Glycoprotein isolated from the canine aspirates was similar to glycoprotein isolated from human chronic bronchitic sputum, having the same carbohydrate composition and range of oligosaccharide size. Further, during and after SO2 exposure some aspirates contained appreciable amounts of glycolipids. These data demonstrate substantial similarities in composition between normal human and canine mucus and in mucus isolated from dogs with chronic airway inflammation induced by repeated irritant exposure and from human patients with chronic bronchitis.