Course Of Cholestasis Research Articles

Indole-3-Carboxaldehyde Restores Gut Mucosal Integrity and Protects from Liver Fibrosis in Murine Sclerosing Cholangitis.

Primary sclerosing cholangitis (PSC) is a long-term liver disease characterized by a progressive course of cholestasis with liver inflammation and fibrosis. Intestinal barrier dysfunction has been implicated in the pathogenesis of PSC. According to the “leaky gut” hypothesis, gut inflammation alters the permeability of the intestinal mucosa, with the translocation of gut-derived products that enter the enterohepatic circulation and cause hepatic inflammation. Thus, the administration of molecules that preserve epithelial barrier integrity would represent a promising therapeutic strategy. Indole-3-carboxaldehyde (3-IAld) is a microbial-derived product working at the interface between the host and the microbiota and is able to promote mucosal immune homeostasis in a variety of preclinical settings. Herein, by resorting to a murine model of PSC, we found that 3-IAld formulated for localized delivery in the gut alleviates hepatic inflammation and fibrosis by modulating the intestinal microbiota and activating the aryl hydrocarbon receptor-IL-22 axis to restore mucosal integrity. This study points to the therapeutic potential of 3-IAld in liver pathology.

Intestinal microbiota drives cholestasis-induced specific hepatic gene expression patterns

ABSTRACT Intestinal microbiota regulates multiple host metabolic and immunological processes. Consequently, any difference in its qualitative and quantitative composition is susceptible to exert significant effects, in particular along the gut-liver axis. Indeed, recent findings suggest that such changes modulate the severity and the evolution of a wide spectrum of hepatobiliary disorders. However, the mechanisms linking intestinal microbiota and the pathogenesis of liver disease remain largely unknown. In this work, we investigated how a distinct composition of the intestinal microbiota, in comparison with germ-free conditions, may lead to different outcomes in an experimental model of acute cholestasis. Acute cholestasis was induced in germ-free (GF) and altered Schaedler’s flora (ASF) colonized mice by common bile duct ligation (BDL). Studies were performed 5 days after BDL and hepatic histology, gene expression, inflammation, lipids metabolism, and mitochondrial functioning were evaluated in normal and cholestatic mice. Differences in plasma concentration of bile acids (BA) were evaluated by UHPLC-HRMS. The absence of intestinal microbiota was associated with significant aggravation of hepatic bile infarcts after BDL. At baseline, we found the absence of gut microbiota induced altered expression of genes involved in the metabolism of fatty and amino acids. In contrast, acute cholestasis induced altered expression of genes associated with extracellular matrix, cell cycle, autophagy, activation of MAPK, inflammation, metabolism of lipids, and mitochondrial functioning pathways. Ductular reactions, cell proliferation, deposition of collagen 1 and autophagy were increased in the presence of microbiota after BDL whereas GF mice were more susceptible to hepatic inflammation as evidenced by increased gene expression levels of osteopontin, interleukin (IL)-1β and activation of the ERK/MAPK pathway as compared to ASF colonized mice. Additonally, we found that the presence of microbiota provided partial protection to the mitochondrial functioning and impairment in the fatty acid metabolism after BDL. The concentration of the majority of BA markedly increased after BDL in both groups without remarkable differences according to the hygiene status of the mice. In conclusion, acute cholestasis induced more severe liver injury in GF mice compared to mice with limited intestinal bacterial colonization. This protective effect was associated with different hepatic gene expression profiles mostly related to tissue repair, metabolic and immune functions. Our findings suggest that microbial-induced differences may impact the course of cholestasis and modulate liver injury, offering a background for novel therapies based on the modulation of the intestinal microbiota.

Progressive dysfunction of collecting liver lymphatics during the development of extrahepatic cholestasis

Lymphatics are necessary anatomical structures of body systems to maintain tissue fluid and molecular homeostasis and are tightly involved in immune reactions of the body. Transport function of lymphatics is altered during inflammation, which impacts the pathogenesis of many diseases. Despite their importance in liver function, the functional characteristics of liver lymphatic vessels (LLVs) have not been evaluated. In this study we, for the first time, measured the function of LLVs in normal and cholestatic rats following extrahepatic bile duct ligation (BDL). In this study parameters of lymphatic contractility/pumping of isolated, cannulated and pressurized segments of LLVs were determined in male 5 moth old Sprague‐Dawley rats. The following animal groups were used: control, 1, 3 and 7 days, after BDL (BDL1, BDL3 and BDL7) and sham control 3 days after surgery. Segments of LLVs located in close proximity to the liver were dissected for the study. Lymphatic contractility parameters were measured and analyzed in isolated, cannulated and pressurized LLV segments under standard transmural pressures and imposed flow ranges in all groups. Healthy LLVs with outer diameter of ~500±50 μm have significant lymphatic tone (7–9%), moderate contraction amplitude (10–25%), high contraction frequency (14–25 contractions/min) demonstrating therefore strong pumping (4–7 volumes/min). Contractility of LLVs from sham and control group were not significantly different. BDL induced specific changes in tone and phasic contractions of LLVs. BDL caused an increase in vessel size (up to 900±100 μm of outer diameter in BDL7 group), progressive lowering of lymphatic tone over period of time after BDL (up to ~2–3‐fold lower lymphatic tone in BDL7 group). Effects of BDL on lymphatic contraction amplitude were as follows: BDL1‐LLVs contraction amplitude was ~2–2.5 times higher than control, BDL3– back to control values, BDL7– amplitude was 0 (a complete cessation of phasic contractions). At the same time contraction frequency in BDL1 and BDL3 LLVs was ~2–2.2 fold lower than control with no contractions in BDL7 group. Cumulatively, we observed a progressive decline in LLVs' pumping in BDL1 and BDL3 groups with complete lymphatic pump failure in BDL7 group. Additionally, LLVs possess comparatively light flow‐dependent responses, which were ablated by BDL. The data indicate that over the time course of cholestasis, the transport capabilities of LLVs are progressively diminished introducing severe, harmful disturbances in fluid/macromolecular homeostasis of the cholestatic liver, which could alter proper trafficking of immune cells and promote liver inflammation. Further research on underlying mechanisms of liver lymphatic dysfunction will shed light on the pathogenesis of cholestatic liver disease and may reveal novel therapeutic approaches.Support or Funding Information1U01HL123420 (DZ, AG); DK110035 (SG, GA)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cholestasis Reveals Severe Cortisol Deficiency in Neonatal Pituitary Stalk Interruption Syndrome.

ObjectivesCholestasis has been reported during the course of congenital hypothalamic-pituitary deficiency, but crucial information is lacking regarding both its origin and prognosis. We aimed to characterize the course of cholestasis and factors contributing to it in patients with deficiency due to pituitary stalk interruption syndrome (PSIS).MethodsWe conducted a retrospective single-center, case-cohort study including 16 patients with PSIS diagnosed before one year of age. We collected clinical and biological parameters from medical records and compared the characteristics of the endocrine syndrome in PSIS patients with and without cholestasis.Results5/16 patients had cholestasis, all with a neonatal onset and multiple hypothalamic-pituitary deficiency. Patients with cholestasis presented with lower Apgar score and higher rate of ophthalmic malformations: 3/5 vs 1/11, p = 0.03 and 5/5 vs 4/11, p = 0.02, respectively. The plasma cortisol level was strongly decreased in patients with cholestasis: 12.4 ng/mL (8–15 ng/mL) vs 79.4 ng/mL (10–210 ng/mL), p = 0.04. Cholestasis resolved within 9 months following hormone supplementation. No development of chronic liver disease was observed during a median follow-up of 9.4 years (range, 1.3–13.3 years).ConclusionsCholestasis is a frequent symptom at presentation of PSIS during the neonatal period that may help earlier diagnosis and that indicates a profound cortisol deficiency.

Percutaneous treatment of biliary complications in pediatric patients after liver transplantation.

Evaluation of the efficiency and safety of the percutaneous treatment of biliary complications in pediatric liver transplant recipients. We conducted a retrospective analysis of children who underwent biliary percutaneous interventions after pediatric liver transplantation (PLT) over a 4-year period. Kind of biliary complication, interval between liver transplantation and intervention, status of the vessels, procedural interventional management, technical and clinical success, course of cholestasis, PTBD-related complications and patient survival were analyzed. 23 percutaneous transhepatic biliary drainages (PTBD) were placed in 16 children due to 18 biliary complications. The drains were customized individually by shortening and cutting additional holes. PTBD placement was performed with technical and clinical success in all children. 4 children received PTBD to bridge the time to retransplantation and surgical revision. One child received PTBD for successful treatment of anastomotic leakage. Long-term dilation of biliary stenoses was performed in 13 children using PTBD. One of these 13 patients showed recurrent stenosis during a median follow-up of 295 days. Bilirubin values decreased significantly after PTBD placement for biliary stenosis. One patient suffered from bacteremia after PTBD replacement. PTBD treatment for biliary complications after PLT is effective and safe.

Increased serum bile acids after extrahepatic biliary obstruction causes leakiness to the blood brain barrier via the disruption of tight junctions

The brain is protected by the blood brain barrier (BBB), which becomes leaky during obstructive cholestasis via an unknown mechanism. A common feature of cholestasis is increased serum bile acids, which can signal via the membrane bound receptor, TGR5. Our aim was to determine the effects of bile acids in the permeability of the BBB during the course of cholestasis. After bile duct ligation (BDL) in the rat, serum bile acid concentrations increased which was paralleled by increased BBB permeability as shown by an Evan's blue exclusion assay. In vitro, there was increased permeability of a monolayer of rat brain microvessel endothelial cells (RBMEC) after treatment with serum from BDL animals compared to controls and also after treatment with cholic acid (CA), deoxycholate (DCA) and chenodeoxycholate (CDCA), but not after ursodeoxycholate (UDCA). In parallel, the TGR5 agonist betulinic acid also increased permeability in vitro. Analysis of RBMECs treated with CA, CDCA, DCA, and betulinic acid all exhibited a disorganized cytoskeleton (demonstrated by F‐Actin staining) and an altered distribution of the tight junction proteins ZO‐1 and ZO‐2. Our data suggests that the increased circulating serum bile acids contribute to the increased permeability of the BBB seen during obstructive cholestasis. This is possibly via a mechanism involving TGR5 activation and subsequent disruption of the tight junctions.

Cholestasis in neonatal intensive care unit: incidence, aetiology and management

Prevalence, aetiology, management and outcome of cholestasis were evaluated in infants admitted to neonatal intensive care unit (NICU). Medical records of all infants admitted to two Italian level III NICUs from January 2005 to August 2007 were retrospectively reviewed. The role of ursodeoxycholic acid (UDCA) therapy was also investigated. Twenty-seven of 1289 enrolled infants developed cholestasis. In 25 infants, cholestasis had a multifactorial basis, while in two, no aetiology was found. UDCA did not significantly affect clinical and biochemical course of cholestasis. During a period of 12 months, eight cholestatic infants died, one underwent liver transplantation and 18 fully recovered. Infants admitted in NICU have a rate of cholestasis higher than that reported in the general population of live births; in most cases, cholestasis is associated to multiple risk factors and shows a favourable outcome. UDCA does not seem to affect clinical course of cholestasis in this setting.

Concurrent Course of Transient Neonatal Diabetes with Cholestasis and Paucity of Interlobular Bile Ducts: A Case Report

We report for the first time a patient with both transient neonatal diabetes mellitus (TNDM) and idiopathic neonatal cholestasis, with both features resolving over a similar time course. Cholestasis was due to paucity of interlobular bile ducts (PILBD). Genetic analysis was consistent with a uniparental disomy of chromosome 6. Paucity of interlobular bile ducts is common in Alagille syndrome but also occurs by unknown mechanisms in a wide spectrum of other diseases. We propose a shared explanation for this patient's TNDM and PILBD mediated by the noted chromosomal abnormality. We suggest that hepatobiliary function be evaluated in patients with TNDM to determine the prevalence and course of cholestasis of the disease.

High Rates of Mortality and Morbidity Occur in Infants With Parenteral Nutrition–Associated Cholestasis

Extremely few data are available about the natural history of parenteral nutrition (PN)-associated cholestasis. The authors evaluated a cohort of infants at a large center to determine the outcome of PN-associated cholestasis in infants with some gastrointestinal function. The authors reviewed the records of all infants admitted to a level 3 neonatal intensive care unit over a 16-month period who had the diagnosis of PN-associated cholestasis. Records were reviewed in these infants for course of cholestasis, laboratory values, outcome, and infection rate. Sixty-six patients were admitted who met the study criteria. There were 10 deaths and 1 referral for liver transplant (Death/TPlant) (17%) in the first year of life. All Death/TPlant infants had at least 1 positive blood culture after the onset of cholestasis. Maximum conjugated bilirubin (MaxCB) in Death/TPlant infants was 15.7 +/- 2.2 (SEM) compared to 8.4 +/- 1.0 mg/dL in babies who recovered. Of 21 infants with a MaxCB > or =10.0, Death/TPlant occurred in 8/21 (38%). Of 40 babies with positive blood cultures, 11 were in the Death/TPlant group vs no deaths among the 25 without positive blood cultures. Average time to resolution from the MaxCB to a CB <2.0 mg/dL was 66 +/- 7 days (n = 49). Infants with PN-associated cholestasis have high rates of mortality despite the presence of some gastrointestinal function. These data support further evaluation and the development of novel forms of therapy for babies with parenteral-associated CB > or =2 mg/dL with emphasis on interventions for infants with a CB >10 mg/dL.

Natural Course of Cholestasis in Neonates on Extracorporeal Membrane Oxygenation (ECMO): 10-Year Experience at a Single Institution

The study objectives were to confirm the incidence of cholestasis and determine factors that contribute to its development and the natural course of cholestasis in neonates treated with extracorporeal membrane oxygenation (ECMO). This was a retrospective chart review including all patients receiving ECMO between 1995 and 2005 at Kosair Children's Hospital. Neonates were grouped as having cholestasis or no cholestasis. A total of 211 patients underwent ECMO between 1995 and 2005. Thirty patients (14%) developed cholestasis. There was no difference in demographics or diagnoses between patients developing cholestasis on ECMO vs. those who did not. The development of cholestasis on ECMO was related to run time, 148 hours +/- 111 (no cholestasis group) vs. 252.8 hours +/- 187 (cholestasis group), p < 0.001 respectively. The majority of patients received veno-arterial ECMO, but there was no difference in cholestasis related to type of ECMO support. Direct hyperbilirubinemia and elevation of hepatic enzymes resolved in all but one patient by the time of discharge. Our incidence of cholestasis (14%) on ECMO is lower than previously reported series. Longer duration of ECMO and other complications on ECMO (renal, infectious, and metabolic) are more likely in the patients developing cholestasis. Follow-up showed resolution of cholestasis in all patients without hepatic sequelae.

Glucagon-Like Peptide-1 and Its Receptor Agonist Exendin-4 Modulate Cholangiocyte Adaptive Response to Cholestasis

Cholangiopathies are characterized by progressive dysregulation of the balance between proliferation and death of cholangiocytes. In the course of cholestasis, cholangiocytes undergo a neuroendocrine transdifferentiation and their biology is regulated by neuroendocrine hormones. Glucagon-like peptide-1 (GLP-1), secreted by neuroendocrine cells, sustains beta-cell survival in experimental diabetes and induces the neuroendocrine transdifferentiation of pancreatic ductal cells. GLP-1 receptor (GLP-1R) selective agonist exendin-4 is used in humans as a novel therapeutic tool for diabetes. The aim of this study was to define if GLP-1 modulates cholangiocyte biologic response to cholestasis. Expression of GLP-1R in cholangiocytes was determined. Effects on cholangiocyte proliferation of the in vitro and in vivo exposure to GLP-1 or exendin-4, together with the intracellular signals, were then studied. Synthesis of GLP-1 by cholangiocytes and the effects of GLP-1R blockage on their growth were also determined. Cholangiocytes express the GLP-1 receptor, which is up-regulated in the course of cholestasis. GLP-1 and exendin-4 increase cholangiocyte growth both in vitro and in vivo. The GLP-1R signal is mediated by the phosphatidyl-inositol-3-kinase, cAMP/Protein Kinase A, and Ca(2+)-CamKIIalpha but not by the ERK1/2 and PKCalpha pathways. Proliferating cholangiocytes synthesize GLP-1: neutralization of its action by GLP-1R antagonist blunts cholangiocyte response to cholestasis. GLP-1 is required for the cholangiocyte adaptive response to cholestasis. Cholangiocytes are susceptible to the activation of GLP-1R and respond with increased proliferation and functional activity. Exendin-4 availability for employment in humans and these data may open novel perspectives for the medical treatment of cholangiopathies.

Endogenous Opioids Modulate the Growth of the Biliary Tree in the Course of Cholestasis

There is poor knowledge on the factors that modulate the growth of cholangiocytes, the epithelial cell target of cholangiopathies, which are diseases leading to progressive loss of bile ducts and liver failure. Endogenous opioids are known to modulate cell growth. In the course of cholestasis, the opioidergic system is hyperactive, and in cholangiocytes a higher expression of opioid peptide messenger RNA has been described. This study aimed to verify if such events affect the cholangiocyte proliferative response to cholestasis. The presence of the delta opioid receptor (OR), muOR, and kappaOR was evaluated. The effects on cholangiocyte proliferation of the in vitro and in vivo exposure to their selective agonists, together with the intracellular signals, were then studied. The effects of the OR antagonist naloxone on cell growth were also tested both in vivo and in vitro. Cholangiocytes express all 3 receptors studied. deltaOR activation strongly diminished the proliferative and functional response of cholangiocytes to cholestasis, whereas muOR resulted in a slight increase in cell growth. The deltaOR signal is mediated by the IP3/CamKIIalpha/PKCalpha pathway, which inhibits the cAMP/PKA/ERK1/2/AKT cascade. In contrast, muOR activation stimulates the cAMP/PKA/ERK1/2/AKT cascade but does not affect the IP3/CamKIIalpha/PKCalpha pathway. The blockage of endogenous opioid peptides by naloxone further enhanced cholangiocyte growth both in vivo and in vitro. The increase in opioid peptide synthesis in the course of cholestasis aims to limit the excessive growth of the biliary tree in the course of cholestasis by the interaction with the deltaOR expressed by cholangiocytes.

Nervous and Neuroendocrine regulation of the pathophysiology of cholestasis and of biliary carcinogenesis

Cholangiocytes, the epithelial cells lining the biliary ducts, are the target cells in several liver diseases. Cholangiopathies and cholangiocarcinoma generate interest in many scientists since the genesis. The developing mechanisms, and the therapeutic tools of these diseases are still undefined. Several studies demonstrate that many hormones, neuropeptides and neurotransmitters regulate malignant and non-malignant cholangiocyte pathophysiology in the course of chronic biliary diseases. The aim of this review is to present the findings of several studies published in the recent years that contributed to clarifying the role of nervous and neuroendocrine regulation of the pathophysiologic events associated with cholestasis and cholangiocarcinoma development. This manuscript is organized into two parts. The first part offers an overview of the innervation of the liver and the origin of neuroendocrine hormones, neurotransmitters and neuropeptides affecting cholangiocyte function and metabolism. The first section also reviews the effects played by several neuroendocrine hormones and nervous system on cholangiocyte growth, survival and functional activity in the course of cholestasis. In the second section, we summarize the results of some studies describing the role of nervous system and neuroendocrine hormones in the regulation of malignant cholangiocyte growth.

Risk factors of cholestasis in very low-birth-weight infants.

To evaluate the incidence, clinical course, and possible risk factors of cholestasis in very low-birth-weight infants. A retrospective study of 143 very low-birth-weight infants was performed. Cholestasis was defined as direct-reacting bilirubin > 2 mg/dL for more than 14 days. The clinical course of cholestasis was described, and perinatal risk factors were evaluated for associations with the development and severity of cholestasis. Cholestasis was present in 31 infants (21.7%). The mean (SD) age of onset was 30.3(15.3) days after birth or 26.0 (15.6) days after receiving parenteral nutrition, and the mean (SD) duration was 77.1 (33.8) days. In half of the cholestatic infants, bilirubin continued to rise after discontinuing parenteral nutrition. One infant developed signs of liver cirrhosis and died, two infants died with progressive cholestasis, while the other 28 patients recovered. Analysis of risk factors revealed that birthweight and duration of fasting significantly correlated with the development of cholestasis, and that sepsis significantly influenced the severity of cholestasis. Cholestasis is a common complication of extreme prematurity. The clinical course seems benign but long-term sequelae are unknown. Immature liver function and absence of stimuli for intestinal motility and hormonal secretion predispose to decreased bile flow, while sepsis further impairs hepatic ductular secretion and aggravates cholestasis.

Carbohydrate metabolism in the course of intrahepatic cholestasis in pregnancy

Glucose metabolism was evaluated in pregnant women with clinically and biochemically demonstrated intrahepatic cholestasis. Laboratory investigations included measurements of serum glucose concentrations on fasting and 2 hours after breakfast, the glucose tolerance test (100 gm oral glucose load), and 24-hour glycemia profile. All patients were admitted to the II Department of Obstetrics and Gynecology, Institute of Obstetrics and Gynecology of the Medical School in Warsaw, Poland. None of the patients exhibited manifest diabetes mellitus or had any clinical history suggestive of previous diabetes. The serum samples collected 2 hours after breakfast demonstrated higher glucose concentrations in women with intrahepatic cholestasis when compared with healthy control subjects. The glucose tolerance tests demonstrated consistently higher concentrations of glucose in blood serum samples after loading in the study group. The 24-hour glycemia profile showed greater glucose concentrations in serum samples collected 2 hours after breakfast and after supper. These results suggest that in the course of cholestasis in pregnancy, visible changes occur in the carbohydrate metabolism of the pregnant woman.

Sulphated lithocholic acid conjugates in serum from children with hepatic and intestinal diseases.

Sulphated lithocholic acid conjugates (SGLC) were measured in the sera of 268 children with various hepatic and intestinal disorders. Two groups were distinguished: (I) SGLC concentration less than or equal to 1.2 mumol/l, n = 198, and (II) SGLC concentration greater than 1.2 mumol/l, n = 70. In 28 patients of the latter group the SGLC concentration was less than 25% of the concentration of glycocholic acid (GC) in the same serum sample. This group (IIA) consisted predominantly of patients with cholestasis, as characterized by high serum bile acid levels and deviating liver function tests. The rest of the group (IIB), with SGLC levels exceeding 25% of the GC concentration and relatively low serum bile acid concentrations, showed no clear cholestatic symptoms. A postprandial increase in serum SGLC (delta SGLC) greater than 1.0 mumol/l was found in only 1 of 32 patients of group I (3%), in 1 of 6 patients of group IIA (17%), but in 9 of 11 patients of group IIB (81%). delta SGLC did not correlate with delta GC in the same test, which indicated that a general hepatic bile acid clearance defect was not responsible. In two patients with intermittent cholestasis, the distinct postprandial rise in serum SGLC that was always found during anicteric periods could be prevented by adding cholestyramine to the test meal. We conclude that elevated serum concentrations of SGLC develop during the course of cholestasis but may also be caused by influx of this bile acid from the intestine. Because of its hepatotoxic properties, SGLC may be involved in the initiation or perpetuation of specific cholestatic phenomena.

Microsomal drug metabolism during α-naphthylisothiocyanate-induced cholestasis

Impaired microsomal drug metabolism has been previously reported after α-naphthylisothiocyanate (ANIT) administration to rats. Our aim was to study the time course of changes in microsomal metabolism in relation to the time course of cholestasis produced by oral administration of a single 200-mg/kg dose of ANIT and to determine whether hypoactivity and hypertrophy of the smooth endoplasmic reticulum (SER) precedes the onset of cholestasis. During the first 12 hr after ANIT, bile flow remained unchanged, hexobarbital sleeping time was prolonged, and in vitro oxidation of aminopyrine and aniline was impaired in 10,000 g supernatants of liver homogenates from ANIT-treated rats, but not in 105,000 g microsomes from the same animals. Washout of inhibitory concentrations of ANIT or its metabolites during the preparation of the microsomes probably explains the discrepancy between the two in vitro preparations used to assess SER metabolic activity. After the onset of cholestasis drug oxidation by 105,000 g microsomes was also impaired and remained so until normal bile flow was reestablished. Decreased microsomal cytochrome P-450 paralleled the changes in (105,000 g) microsomal activity, but at no time was microsomal protein yield per gram of liver significantly altered. The data suggest that pre-cholestatic impairment of microsomal drug metabolism was not associated with a hypertrophic, hypoactive SER. Subsequent reversible SER hypoactivity was the result of bile stasis. Elevation of plasma alanine aminotransaminase provided an early index of ANIT hepatotoxicity, while elevation of plasma bilirubin and 5′-nucleotidase was associated with cholestasis and correlated with the diminution and recovery of bile flow.