Core Hippo pathway components act as a brake on Yap and Taz in the development and maintenance of the biliary network.

The development of the biliary system is a complex yet poorly understood process, with relevance to multiple diseases, including biliary atresia, choledochal cysts and gallbladder agenesis. We present here a crucial role for Hippo-Yap/Taz signaling in this context. Analysis of sav1 mutant zebrafish revealed dysplastic morphology and expansion of both intrahepatic and extrahepatic biliary cells, and ultimately larval lethality. Biliary dysgenesis, but not larval lethality, is driven primarily by Yap signaling. Re-expression of Sav1 protein in sav1-/- hepatocytes is able to overcome these initial deficits and allows sav1-/- fish to survive, suggesting cell non-autonomous signaling from hepatocytes. Examination of sav1-/- rescued adults reveals loss of gallbladder and formation of dysplastic cell masses expressing biliary markers, suggesting roles for Hippo signaling in extrahepatic biliary carcinomas. Deletion of stk3 revealed that the phenotypes observed in sav1 mutant fish function primarily through canonical Hippo signaling and supports a role for phosphatase PP2A, but also suggests Sav1 has functions in addition to facilitating Stk3 activity. Overall, this study defines a role for Hippo-Yap signaling in the maintenance of both intra- and extrahepatic biliary ducts.

Developmental milestones in neonatal and juvenile C57Bl/6 mouse - Indications for the design of juvenile toxicity studies.

There is a growing demand for wild type mice and mouse models of disease that may be more representative of human conditions but there is little information on neonatal and juvenile mouse anatomy. This project produces sound and comprehensive histology background data on the developing neonatal mouse at different time points from Day 0 until Day 28. The work describes optimal methods for tissue harvesting, fixation and processing from the neonatal and juvenile mice which can be used in routine toxicology studies. A review of the available literature revealed inconsistencies in the developmental milestones reported in the mouse. Although it is true that the sequence of events during the development is virtually the same in mice and rats, important developmental milestones in the mouse often happen earlier than in the rat, and these species should not be used interchangeably.

Effects of Satureja khuzistanica essential oils in drinking water on mortality, production performance, water intake, and organ weights in broiler chickens reared under heat stress condition.

An experiment was conducted to examine the effects on mortality, production performance, water intake (WI), and organ weight of Satureja khuzistanica essential oil (SkEO) using 720 1-day-old Arian broiler chicks in a 42-day trial. Experimental treatments were addition of 0 (control(-)), 200, 300, 400, and 500 mg/L SkEO or 500 mg/L polysorbate 80 (control(+)) into drinking water. The birds were kept under natural ambient temperatures 4 to 6 °C above standard recommendation from days 22 to 42 of age. Addition of SkEO into drinking water at 200 and 500 mg/L decreased weight gain (P < 0.05) of the birds from days 29 to 35 of age with no differences in feed intake (FI) and feed conversion ratio (FCR) compared to control groups (P > 0.05). Supplementation of drinking water with 200, 300, 400, and 500 mg/L SkEO resulted in a 0.47, 4.40, 8.60, and 12.93% decrease in WI, respectively, from days 1 to 42 of age. The calculated European broiler index was greater for the birds received 400 mg/L of SkEO in their drinking water compared with that of the other birds (P < 0.05). Pancreas percentage was increased for the birds received 200 to 500 mg/L SkEO at days 21 and 42 of age compared with that of the control(-) birds (P < 0.05). The gall bladder weight was 17.56, 40.50, 12.16, and 38.73% greater for the birds received 200, 300, 400, and 500 mg/L SkEO compared with that of the control(-) birds, respectively. The results showed that an addition of 400 mg/L SkEO into drinking water for heat-stressed broiler chickens improves economic efficiency possibly by promoting digestion process, creating miniscule improvement in FCR and lowered mortality rate.

Non-linear increases in danazol exposure with dose in older vs. younger beagle dogs: the potential role of differences in bile salt concentration, thermodynamic activity, and formulation digestion.

PURPOSE: To explore the possibility that age-related changes in physiology may result in differences in drug bioavailability after oral administration of lipid based formulations of danazol. METHODS: Danazol absorption from lipid formulations with increasing drug load was examined in younger (9 months) and older (8 years) beagles. Age related changes to hepatic function were assessed via changes to systemic clearance and serum bile acid concentrations. Changes to lipolytic enzyme activity and intestinal bile salt concentration were evaluated using in vitro lipolysis. RESULTS: Drug exposure increased linearly with dose in younger animals. In older animals, bioavailability increased with increasing dose to a tipping point, beyond which bioavailability reduced (consistent with initiation of precipitation). No differences in hepatic function were apparent across cohorts. Changes to enzyme concentrations in lipolysis studies had little impact on drug precipitation/solubilisation. In contrast, higher bile salt concentrations better supported supersaturation at higher drug loads. CONCLUSIONS: Differences in animal cohort can have a significant impact on drug absorption from lipid based formulation. For danazol, bioavailability was enhanced under some circumstances in older animals. In vitro experiments suggest that this was unlikely to reflect changes to metabolism or lipolysis, but might be explained by increases in luminal bile salt/phospholipid concentrations in older animals.

Comparative pathology and ecological implications of two myxosporean parasites in native Australian frogs and the invasive cane toad.

Myxosporean parasites Cystodiscus axonis and C. australis are pathogens of native and exotic Australian frog species. The pathology and ecological outcomes of infection with these parasites were investigated in this study. Gliosis was correlated to Cystodiscus axonis plasmodia in the brains of (9/60) tadpoles and (3/9) adult endangered Green and golden bell frogs using ordinal regression. Severe host reactions to C. axonis (haemorrhage, necrosis, and vasulitis) were observed in the brains of threatened Southern bell frogs (8/8), critically endangered Booroolong frogs (15/44) and Yellow spotted bell frogs (3/3). Severe brain lesions were associated with behavioural changes, neurological dysfunction, and spontaneous death. Both C. axonis and C. australis develop in the bile ducts of tadpoles, the plasmodia were significantly associated with biliary hyperplasia, inflammation and the loss of hepatocytes in (34/72) Green and golden bell frog tadpoles using ordinal regression. These lesions were so severe that in some cases 70% of the total liver was diseased. Normal liver function in tadpoles is necessary for metamorphosis, metabolism, and immune function. We postulate that this extensive liver damage would have significant host health impacts. Severe hepatic myxosporidiosis was more prevalent in tadpoles examined in autumn and winter (overwintered), suggestive of delayed metamorphosis in infected tadpoles, which would have serious flow-on effects in small populations. We compared the sensitivity of histopathology and species-specific PCR in the detection of C. australis and C. axonis. PCR was determined to be the most sensitive method (detection limit 1 myxospore equivalent of ribosomal DNA). Histology, however, had the advantage of assessing the impact of the parasite on the host. It was concluded that these parasites have the potential for significant ecological impacts, because of their high prevalence of infection and their ability to cause disease in some frogs.

Gerentological studies on the gross and histomorphology of the vesicular gland of gaddi goat (Copra hircus) / Estudios gerontológicos en anatomía e histomorfología de la glándula vesicular de cabra gaddi (Cabra hircus)

The study was conducted on the vesicular glands of 30 entire pure male Gaddi goats from birth to 5yrs and 6 months of age, divided into three equal size groups of 10 animals in each viz; Pre-pubertal (lday old to <18 months of age), Pubertal (18 months to < 5years of age) and Post-pubertal (>5 years) age groups. In new born kid the glands were small, white cord like. It became "?" shaped at 6 months and "S" shaped at 12 months of age. A significant growth in size and weight of gland occurred at 6 month, then up to 12 month and beyond it grew slowly. The secretory acini of the lobules were lined by pseudostratified ciliated columnar epithelium which contained A-tall columnar, B-basal and C-narrow columnar type of cells. The number per unit area and size of the gland increased with age significantly from birth in the prepubertal animals and up to puberty. In post-pubertal animals it did not grow significantly, rather the connective tissue elements were increased in the capsule (adventitia). The intralobular connective tissue however, decreased at the expense of growth of acini at all ages.

Se realizó un estudio de las glándulas vesiculares de 30 cabras Gaddi, macho, totalmente puras desde el nacimiento hasta los 5 años y 6 meses de edad, divididas en tres grupos de 10 animales cada uno, pre-puberal (día 1 de edad a <18 meses de edad), pubertad (18 meses a <5 años de edad) y post-puberal (> 5 años). En el recién nacido las glándulas eran pequeñas, como un cordón blanco. Se presentó en forma de "?" a los 6 meses y de "S" a los 12 meses de edad. Un importante crecimiento en tamaño y peso de la glándula se produjo a los 6 meses, luego de 12 meses y más, creció lentamente. Los acinos secretores de los lóbulos se alinearon como epitelio columnar ciliado pseudoestratificado, los cuales contienen células de tipo A-columnar alto, B-basal y C-columnar estrecho. El número por unidad de área y el tamaño de la glándula aumentó significativamente con la edad desde su nacimiento en los animales prepuberes y hasta la pubertad. En animales post-puberales no creció significativamente, y los elementos del tejido conectivo se incrementaron en la cápsula (adventicia). Sin embargo, el tejido conectivo intralobular, disminuyó a expensas del crecimiento de los acinos, en todas las edades.

Developmental changes in Ca2+ homeostasis and contractility in gallbladder smooth muscle.

Relatively little is known about the contribution of Ca(2+)-dependent and -independent mechanisms in the contractility of neonatal gastrointestinal smooth muscle. We therefore studied Ca(2+) homeostasis and Ca(2+) sensitization mechanisms in 10-day-old and adult guinea pig gallbladder smooth muscle to elucidate developmental changes in these processes. Gallbladder contractility was evaluated by isometrical tension recordings from strips, intracellular Ca(2+) concentration was estimated by epifluorescence microscopy of fura-2-loaded isolated cells, and protein expression and phosphorylation were assessed by Western blot analysis. The neonatal gallbladder contracted significantly less to CCK than adult tissue, but this correlated with an increased Ca(2+) mobilization, suggesting immaturity of Ca(2+) sensitization mechanisms. The enhanced Ca(2+) release in the newborn gallbladder was the result of the increase in the size of the releasable Ca(2+) pool. Moreover, in neonatal smooth muscle cells, neither the plasma membrane Ca(2+) pump nor the Na(+)/Ca(2+) exchanger collaborate in the extrusion of Ca(2+). In contrast, in these cells, there is an increase in phospholamban phosphorylation, which could drive to an overactivity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase pump. The reduced Ca(2+) sensitivity in neonatal tissues was demonstrated by the lack of effect to Y-27362, an inhibitor of Rho kinase (ROCK), and GF-109203X, an inhibitor of PKC, on agonist-induced contraction. In addition, the neonatal gallbladder showed lower levels of RhoA, ROCK, PKC, and two effectors [C-kinase-dependent inhibitor of 17 kDa (CPI-17) and myosin phosphatase targetting 1 (MYPT1)] as well as an absence of CPI-17 and MYPT1 phosphorylation in response to agonists. In conclusion, our results indicate that the main mechanisms involved in smooth muscle contractility are under developmental regulation.

Cholesterol gallstone formation in overweight mice establishes that obesity per se is not linked directly to cholelithiasis risk.

The relationship between obesity and cholesterol cholelithiasis is not well understood at physiologic or genetic levels. To clarify whether obesity per se leads to increased prevalence of cholelithiasis, we examined cholesterol gallstone susceptibility in three polygenic (KK/H1J, NON/LtJ, NOD/LtJ) and five monogenic [carboxypeptidase E (Cpe (fat)), agouti yellow (A(y)), tubby (tub), leptin (Lep(ob)), leptin receptor (Lepr (db))] murine models of obesity during ingestion of a lithogenic diet containing dairy fat, cholesterol, and cholic acid. At 8 weeks on the diet, one strain of polygenic obese mice was resistant whereas the others revealed low or intermediate prevalence rates of cholelithiasis. Monogenic obese mice showed distinct patterns with either high or low gallstone prevalence rates depending upon the mutation. Dysfunction of the leptin axis, as evidenced by the Lep(ob) and the Lepr (db) mutations, markedly reduced gallstone formation in a genetically susceptible background strain, indicating that in mice with this genetic background, physiologic leptin homeostasis is a requisite for cholesterol cholelithogenesis. In contrast, the Cpe (fat) mutation enhanced the prevalence of cholelithiasis markedly when compared with the background strain. Since CPE converts many prohormones to hormones, a deficiency of biologically active cholecystokinin is a likely contributor to enhanced susceptibility to cholelithiasis through compromising gallbladder contractility and small intestinal motility. Because some murine models of obesity increased, whereas others decreased cholesterol gallstone susceptibility, we establish that cholesterol cholelithiasis in mice is not simply a secondary consequence of obesity per se. Rather, specific genes and distinct pathophysiological pathways are responsible for the shared susceptibility to both of these common diseases.

Ultrasonographic study on the growth and dimensions of healthy children and adults organs.

We measured by ultrasound the spleen, the kidneys, the pancreas in 323 children (age 0-12), the thyroid in 60 children of same age range, and all these organs in 180 adults (in adults we measured additionally the testis, the gallbladder and the choledochus). Children and adults were all healthy, without evidence of pathologies potentially involving these organs. The children have been selected looking at clinical and hematological parameters, while the adults have been selected among a military population, that is the most significant sample of healthy young adults. We measured the length, the transversal diameter and the thickness of the spleen, kidneys, pancreas, testis and thyroid, the diameter of the choledochus and the maximum length of the gallbladder (pre and post stimulus). We found a good correlation between age and dimensions for pancreas, kidneys and spleen in children, representing the progressive growth of these organs. Our data represent an assessment of the normal dimensions of these organs in vivo by means of ultrasound, and therefore they are an useful tool to discriminate pathologically enlarged or reduced organs, both in children and in adults.

Effects of epidermal growth factor on neonatal pancreatic growth in the guinea pig.

CONCLUSION: EGF and/or transforming growth factor-alpha (TGF-alpha) are likely to be important in the rapid pancreatic growth that occurs in the neonatal guinea pig. BACKGROUND: Rapid pancreatic growth is observed during the neonatal period in the guinea pig. The growth factors that are involved are not known but may include members of the EGF family. METHODS: Mini-osmotic pumps were implanted on the day of birth for continuous infusion of EGF (30 microgram/d). Pancreatic DNA, RNA, and protein contents were determined at 4 and 15 d, along with wet weights of the pancreas, duodenum, jejunoileum, colon, and gallbladder. Pancreatic EGF and TGF-alpha concentrations were measured in adult controls, in control neonates at 1, 4, 8, and 15 d, and also at 4 and 15 in guinea pigs receiving either EGF or the cholecystokinin receptor antagonist devazepide (25 nmol/kg/h). RESULTS: EGF infusion significantly increased the weight of the stomach and duodenum at 4 d and all the gastrointestinal organs, including the pancreas, at 15 d. Exogenous EGF increased pancreatic DNA, RNA, and protein content at 4 and 15 d. Endogenous EGF and TGF-alpha concentrations in the pancreas were significantly higher at birth than in adults (P < 0.001) and P < 0.01, respectively) and declined during the first 2 wk postpartum. At 15 d, EGF concentrations remained significantly higher than adult levels (P < 0.01), but TGF-alpha concentrations had declined to adult values. Infusion of EGF decreased concentrations of endogenous EGF in the pancreas at 4 and 15 d (both P < 0.05) and decreased TGF-alpha concentrations at 4 d (P < 0.001). Devazepide infusion caused a significant decrease in endogenous pancreatic EGF concentrations at 15 d (P < 0.05).

Effects of epidermal growth factor, cholecystokinin, and secretin on growth of the alimentary tract in the neonatal guinea pig.

Gut hormones and growth factors are likely to be involved in the functional changes and substantial growth of the alimentary tract that occurs during early neonatal life. The present experiments investigated the effects of continuous subcutaneous infusion of cholecystokinin (CCK), secretin, and epidermal growth factor (EGF) in neonatal guinea pigs for 4 or 15 days. At doses of 20, 100 or 500 pmol/kg/h, CCK and secretin had no effect on alimentary tract organs except for an increase in pancreatic weight at 4 days with the highest dose of CCK and an increase in stomach weight at 4 days with the highest dose of secretin. In contrast, EGF showed dose-dependent and time-dependent effects on all the alimentary tract organs when infused at 70, 210 and 630 pmol/kg/h. These results suggest that EGF, but not CCK or secretin, is likely to be an important trophic factor during the neonatal period.

A developmental study of the localization of NADPH-diaphorase in the ganglionated plexus of the guinea-pig gallbladder.

A histochemical investigation of age-related changes that occur with respect to the localization of NADPH-diaphorase in the ganglionated plexus of the guinea-pig gallbladder was carried out. In all age groups examined (embryonic stages day 34 and 52, 2 to 4-day old, 6-month old and 2-year old), the mean percentage of NADPH-diaphorase-positive neurons per ganglion was obtained by taking the number of neurons that were immunoreactive to protein gene product 9.5 (a general neuronal marker) as 100%. In addition, the possible co-existence of NADPH-diaphorase and nitric oxide synthase in the ganglionated plexus of 2 to 4-day old and 6-month old guinea-pig gallbladder was investigated. NADPH-diaphorase was not present in the ganglionated plexus of the gallbladder at embryonic day 34. At embryonic day 52, all the protein gene product 9.5-immunoreactive neurons showed positive staining to NADPH-diaphorase; this dropped to a minimum at 2-4 days (26.7%), rose slightly at 6 months (33.6%), and finally returned close to the 100% value at 2 years. In the gallbladders of 2-year old guinea-pigs, some (3 out of 10) ganglia were devoid of protein gene product 9.5-immunoreactive neurons, but NADPH-diaphorase-stained granules were found within the ganglia. However, all those neurons that were immunopositive to protein gene product 9.5 also expressed NADPH-diaphorase. Moreover, NADPH-diaphorase-positive neurons in the gallbladder of 2 to 4-day-old and 6-month-old guinea-pigs were found to express nitric oxide synthase.

Age-related changes in gallbladder contractility and cytoplasmic Ca2+ concentration in the guinea pig.

Gallbladder (GB) motility diminishes with aging. This study was performed to characterize mechanisms that are involved in changes in GB contractility that occur during aging. Cytoplasmic Ca2+ concentrations ([Ca2+]i) and the contractile force of guinea pig GB muscle strips were simultaneously measured using fura-2 and force-displacement transducers. The binding ability of the Ca2+ channel antagonist and GB muscle compliance were also examined. The COOH-terminal octapeptide of cholecystokinin (CCK-8) evoked a dose-dependent increase in force and [Ca2+]i. Changes of [Ca2+]i and contractile force of muscle strips in response to CCK-8 were significantly greater in young (2 mo old) compared with mature and aged (12 and 24 mo old) guinea pigs (changes in [Ca2+]i, ED50: 46.1 nM at 2 mo, 6.1 microM at 12 mo, and 2.8 mM at 24 mo; changes of contractile force, ED50: 24.8 microM at 2 mo, 2.1 mM at 12 mo, and 357 mM at 24 mo). However, the magnitude of the contraction at each percent change in [Ca2+]i was actually similar in young and aged guinea pigs. In a Ca(2+)-free buffer, the responses of [Ca2+]i and force to CCK-8 in both young and aged GB muscles decreased, but those were still dose and age dependent. Binding ability of the Ca2+ channel antagonist did not differ in the young and aged groups, but the compliance of the GB muscle strip decreased with aging. These results suggest that both a reduced mobilization of intracellular Ca2+ and a decreased muscle compliance are responsible, at least in part, for age-related reduced contraction of guinea pig GB in response to CCK.

[Three dimensional growth of the extrahepatic bile ducts--surgical inferences]. / Körpermassebezogenes Wachstum der extrahepatischen Gallenwege--chirurgische Schlussfolgerungen.

Allometric growth of length and increasing of lumen area of the extrahepatic bile ducts were examined morphometrically in 50 human cases at the age of 28 gestational weeks up to 19 years. We calculated the flow resistance in relation to body weight with the help of the principle of Hagen-Poisoille. Principle ways to better bile flow in cases of non-correctable biliary atresia or bile duct hypoplasia are derived.

Gap junctions and zonulae occludentes of hepatocytes during biliary atresia in the lamprey.

Gap junctions and zonulae occludentes of hepatocytes were examined in thin sections and freeze-fracture replicas from livers of larval and juvenile adult lampreys and during the phase of metamorphosis when bile ducts and bile canaliculi disappear (biliary atresia). Larvae possess zonulae occludentes at the canaliculi which are composed of one to five (mean = 2.81) junctional strands that provide a bile-blood barrier. Morphometry demonstrates that during biliary atresia the decreases in number of junctional strands and apico-basal depth of the zonulae occludentes are accompanied by an increase in the frequency of gaps or interruptions in the strands and in a breakdown of the bile-blood barrier. The zonulae occludentes completely disappear during metamorphosis and are not found in the adult liver. Gap junctions of the larval liver occupy 1% of the surface of the plasma membrane and have a mean area of 0.167 micron 2 but, following an initial decline in these parameters during early biliary atresia, they rise sharply in later stages of metamorphosis and in adults are 3.2% and 0.502 micron 2, respectively. The events of alteration in junctional morphology during lamprey biliary atresia is in many ways comparable to the changes in gap junctions and zonulae occludentes during experimental and pathological intra- and extrahepatic cholestasis in mammals.

Modulation of hepatic biotransformation and biliary excretion of bile acid by age and sinusoidal bile acid load.

Pericentral hepatocytes excrete bile acids more slowly and biotransform them more than periportal cells. This may reflect adaptation to low pericentral bile acid concentration or may be intrinsic. We studied two models in which pericentral bile acid concentrations are high: the 72-h choledocho-caval shunt (CCS) rat and the 3- to 4-wk-old rat. Livers were perfused forward or backward to assess periportal or pericentral hepatocyte function. Taurodeoxycholate (TDC) was infused at 32 nmol X min-1 X g liver-1, and a bolus of [3H]TDC was given to assess metabolism and excretion of bile acids. In CCS livers perfused backward, pericentral cells resembled periportal cells of controls in that time to excrete 50% of administered [3H]TDC (t50) was reduced by two-thirds and [3H]TDC biotransformation was reduced by about half. In young livers t50 was half that of adult livers when perfused backward. Biotransformation, however, was not reduced. Young livers biotransformed more than adult controls for any given residence time of bile acid in the liver. We conclude that the difference between pericentral and periportal cells as regards bile acid processing is adaptive. Livers from young rats biotransform more bile acid than those from controls under similar conditions.

[The development of the guinea pig gallbladder epithelial cells. I. Light microscopical and carbohydrate-histochemical investigations (author's transl)]. / Zur Entwicklung des Gallenblasenepithels des Meershweinchens. I. Färberisch-lichtmikroskopische und kohlenhydrat-histochemische Untersuchungen

The development of the guinea pig gallbladder epithelium was studied from the 19th day of intrauterine life to the 31st postnatal day by means of histological and histochemical staining reactions. At first, the epithelium is a columnar pseudostratified one. Its transformation into a simple columnar eptihelium is terminated by the 31st day of the intrauterine life. Then the epithelial cells become more columnar and their nuclei acquire a basal position. Somewhat later the epithelium invaginates the underlying mesenchyme. Up to the 57th day the epithelium contains much glycogen. Neutral and carboxylated mucosubstances are demonstrable after the 30th day. From the 48th day onwards sulphated mucosubstances can be visualized in some cells in the depth of the invaginations and from the 51st day in the epithelial cells of the gallbladder. "Light" mucoid cells appear first in the epithelium of day 58. After the 6th postnatal day the "light" cells are rarely seen in the invaginations. The development of the gallbladder epithelium is completed about the 10th postnatal day. The epithelial mucosubstances of the gallbladder of the adult animal could be classified as GC- mucins and S-mucinsA, 1.0 MgCl2.

Alterations in biliary lipids of mice during dehydrocholic acid feeding.

Mice were fed a lithogenic diet consisting of Purina chow and 0.5% dehydrocholic acid (DHA group). Controls received Purina chow. Every 2 wk for 20 wk animals were killed, and biliary phospholipid, cholesterol, and bile salt concentrations were determined, as well as the extent of gallstone formation. With time there was a gradual, significant decline in the concentration and the relative composition of phospholipid in both groups compared with initial values. There was a significant increase in biliary cholesterol concentration and relative amount in the DHA group compared with the control. No significant differences were found in the relative amounts of bile salt or phospholipid between the two groups. Feeding DHA resulted in an increased concentration of bile salts and the sum of measured lipid compared with controls. After 8 wk, gallstones were found in approximately 60% of autopsied animals and correlated with increased cholesterol concentration. Our data support the hypothesis that there is a component of cholesterol secretion that may not be bile salt- or phospholipid-dependent. Our data also suggest that biliary phospholipid secretion decreases with age.