27, 32, 35, 36 We found MAC387 expression to be highest in patients transplanted find more sooner following acetaminophen ingestion, which could suggest that the influx of monocyte-derived macrophages to inflammatory foci occurs in the earlier phases of liver injury.14, 27 Experimental models demonstrate that the interaction between CCL2 and its receptor

CCR2 promotes efflux of CCR2-expressing monocytes from the bone marrow into the circulation.24, 37, 38 Our data demonstrate that despite reactive monocyte progenitor hematopoiesis and markedly elevated circulating CCL2 levels, there is a profound reduction in the absolute number of circulating monocytes that is proportional to the severity of acute liver injury (Figs. 1 and 2). This suggests that circulating monocytes are being recruited to the inflamed liver at a rate that exceeds bone marrow production resulting in a reduction in their numbers in the circulation. However, our data do not exclude the possibility that the depletion of circulating monocytes may also be attributed to apoptosis39 or recruitment to other tissues. Consistent with the previously published experimental APAP models12-14, 18 and human studies of AALF,25, 27 our data support the role of CCL2 in recruitment of circulating monocytes to the

liver during AALF. In Fig. 6, we show that necrotic liver tissue may act as a source of CCL2 secretion, as evidenced by the significantly elevated levels of monocyte chemoattractants selleck compound (CCL2, CCL3) in whole liver tissue, the chemokine gradient from necrotic to nonnecrotic tissue, and elevations in circulating levels of this chemoattractant. We also LY294002 solubility dmso found that all three circulating monocyte subsets express CCR2, suggesting that all three populations could be recruited to the inflamed liver. Our study, however, does not exclude the involvement of other chemokines in recruiting monocytes to the liver, and further studies are warranted to assess this. We observed

marked proliferation of the resident KC population within areas of necrosis (Fig. 4); this finding is in contrast to monocyte-derived infiltrating macrophages, where less than 1% were proliferating. Previous reports support the existence of two macrophage populations with distinct functional capabilities and self-renewal characteristics during steady state and inflammation. One population derived from circulating monocytes with little self-renewal potential is rapidly recruited to inflammatory sites, giving rise to the classical inflammatory macrophages that cause tissue destruction and necrosis.40 There is a second resident population with self-renewal capabilities that characterize later phases of inflammatory insult when tissue repair and regenerative responses prevail.7-10 Recently, the anti-inflammatory cytokine IL-4 has been shown to be a pivotal driver of macrophage self-renewal and tissue repair during experimental tissue injury.

We have observed that, as shown previously, 15, 16 the level of phospho-ERK at rest is higher in Pkd2KO cells, compared to controls, and, more impressive, is the difference between controls and Pkd2KO cells upon acute reduction in ER [Ca2+] by exposure to thapsigargin or TPEN (Figs. 4 and 5). The advantage of TPEN as a tool to acutely decrease ER [Ca2+] in this type of experiment is that, unlike thapsigargin or agonist that LEE011 cell line produce IP3, no rise in [Ca2+]c is generated, an event that may interfere with

the observed response. Of interest, unlike Pkd2KO cells, the small increase in phospho-ERK caused by ER Ca2+ depletion in controls is not accompanied by an increase in VEGF and HIF expression. The effect on ERK phosphorylation is clearly not secondary to an ER stress response caused by thapsigargin-induced depletion of ER Ca2+, because the

expression of three well-known markers of ER stress (e.g., BiP, ATF6, and PERK) after treatment with thapsigargin was not different between Pkd2KO selleck inhibitor and WT cell (see Supporting Fig. 4). Of note, ERK1/2 phosphorylation (at rest and after ER Ca2+ depletion) was PKA dependent, as shown by the inhibition by PKI. Hofer et al. have recently demonstrated that ER [Ca2+] levels regulate cAMP production through a STIM-1-dependent process. 20, 28 This mechanism, indeed, depends on the translocation of STIM1 and its ability to stimulate unknown plasma-membrane AC isoform(s). Our data show that, in Pkd2KO cells, see more not only depletion of ER Ca2+ with TPEN (or thapsigargin) increases cAMP production and PKA-dependent ERK1/2 phosphorylation, but also that PKA-dependent ERK1/2 activation is inhibited by antagonizing STIM-1 (with ML-9 and 2-APB 20) translocation. 20 The

amount of cAMP produced by a given cell results from the highly integrated function of several isoforms of ACs that respond to different stimuli and second messengers. 41 At least seven different ACs are expressed in cholangiocytes. 22 AC6 is localized also in the cilia and is involved in shear stress-induced signaling 19 and in the formation of gap junction and [Ca2+]c regulation in endothelial cells. 42 We, here, show that AC6 mediates most, if not all, SOcAMP in Pkd2KO cholangiocytes and in WT cholangiocytes after chronic ER Ca2+ depletion. On the contrary, the Ca2+ stimulated AC8 appears not to be involved in SO-cAMP response. Altogether, these data demonstrate that PC2 plays a key role in regulating Ca2+ and cAMP homeostasis in cholangiocytes.

Humans with chronic liver disease were also studied. Results: In healthy liver, hepatocytes strongly expressed D1 and stromal cells weakly expressed D3. During injury, hepatocyte expression of D1 decreased, while stromal expression of D3 increased, particularly in myofibroblasts. Repair-related changes in deiodinases were accompanied by reduced intrahepatic TH content and TH-regulated gene expression. Disrupting Hedgehog signaling in myofibroblasts reduced D3 and increased D1 expression, increased intrahepatic T4 concentration, and normalized TH-specific gene expression. Patients with advanced fibrosis

had less D1 and more D3 than patients with mild fibrosis. Their serum rT3 levels were also increased. Moreover, lower serum fT3/rT3 and fT4/rT3 distinguished advanced- from mild- fibrosis, even in individuals with similar serum levels of TSH and fT4. Conclusion: Hedgehog-dependent check details changes in liver stromal cells drive repair-related changes in hepatic deiodinase expression that promote intrahepatic hypothyroidism, thereby limiting exposure to T3, an important factor for hepatic differentiation. Changes in deiodinase expression correlate with reduced serum fT3/rT3 and fT4/rT3 ratios. Thus, increased serum rT3 may serve as a novel

biomarker of liver http://www.selleckchem.com/products/FK-506-(Tacrolimus).html disease severity in humans. Disclosures: Manal F. Abdelmalek – Consulting: Islet Sciences; Grant/Research Support: Mochida Pharmaceuticals, Gilead Sciences, NIH/NIDDK, Synageva, Genfit Pharmaceuticals Anna Mae Diehl – Consulting: Roche; Grant/Research Support: Gilead, Genfit The following people have nothing to disclose: Brittany Bohinc, Gregory A. Michelotti, Guanhua selleck chemicals Xie, Herbert Pang, Ayako Suzuki, Cynthia D. Guy, Dawn L. Piercy, Leandi Kruger, Marzena Swiderska-Syn, Mariana V. Machado, Thiago A. Pereira, Ann Marie Zavacki Background: Exosomes arise by inward budding of the limiting membranes of multivesicular

bodies which, upon fusion with the plasma membrane, result in their secretion and deposition into body fluids (e.g. blood, urine). Exosomes contain a complex mixture of microRNAs (miRs), mRNAs and proteins that reflect the transcriptional and translational status of the producer cell. Since this molecular payload is a “fingerprint” of the dynamic status of their producer cells, exosomes represent a potentially valuable resource for assessing liver disease or pathology. Our goal was to profile the microRNA content of serum exosomes in experimental liver fibrosis. Methods: PureExo Exosome Isolation Kits were used to isolate serum exosomes. MiR profiling was performed on exosomal RNA from 1ml of pooled serum (5 mice; 200μl/mouse) using a mouse miRnome miR PCR Array.

Humans with chronic liver disease were also studied. Results: In healthy liver, hepatocytes strongly expressed D1 and stromal cells weakly expressed D3. During injury, hepatocyte expression of D1 decreased, while stromal expression of D3 increased, particularly in myofibroblasts. Repair-related changes in deiodinases were accompanied by reduced intrahepatic TH content and TH-regulated gene expression. Disrupting Hedgehog signaling in myofibroblasts reduced D3 and increased D1 expression, increased intrahepatic T4 concentration, and normalized TH-specific gene expression. Patients with advanced fibrosis

had less D1 and more D3 than patients with mild fibrosis. Their serum rT3 levels were also increased. Moreover, lower serum fT3/rT3 and fT4/rT3 distinguished advanced- from mild- fibrosis, even in individuals with similar serum levels of TSH and fT4. Conclusion: Hedgehog-dependent TSA HDAC solubility dmso changes in liver stromal cells drive repair-related changes in hepatic deiodinase expression that promote intrahepatic hypothyroidism, thereby limiting exposure to T3, an important factor for hepatic differentiation. Changes in deiodinase expression correlate with reduced serum fT3/rT3 and fT4/rT3 ratios. Thus, increased serum rT3 may serve as a novel

biomarker of liver Sirolimus disease severity in humans. Disclosures: Manal F. Abdelmalek – Consulting: Islet Sciences; Grant/Research Support: Mochida Pharmaceuticals, Gilead Sciences, NIH/NIDDK, Synageva, Genfit Pharmaceuticals Anna Mae Diehl – Consulting: Roche; Grant/Research Support: Gilead, Genfit The following people have nothing to disclose: Brittany Bohinc, Gregory A. Michelotti, Guanhua selleck chemicals llc Xie, Herbert Pang, Ayako Suzuki, Cynthia D. Guy, Dawn L. Piercy, Leandi Kruger, Marzena Swiderska-Syn, Mariana V. Machado, Thiago A. Pereira, Ann Marie Zavacki Background: Exosomes arise by inward budding of the limiting membranes of multivesicular

bodies which, upon fusion with the plasma membrane, result in their secretion and deposition into body fluids (e.g. blood, urine). Exosomes contain a complex mixture of microRNAs (miRs), mRNAs and proteins that reflect the transcriptional and translational status of the producer cell. Since this molecular payload is a “fingerprint” of the dynamic status of their producer cells, exosomes represent a potentially valuable resource for assessing liver disease or pathology. Our goal was to profile the microRNA content of serum exosomes in experimental liver fibrosis. Methods: PureExo Exosome Isolation Kits were used to isolate serum exosomes. MiR profiling was performed on exosomal RNA from 1ml of pooled serum (5 mice; 200μl/mouse) using a mouse miRnome miR PCR Array.

After optimization of the membrane-engineering process, the virus detection limit for TMV

and CLRV with the bacteria-based biosensor system was 1 pg/ml, representing a 1000-fold improvement over currently available methods. Although the novel biosensor is still in its proof-of-concept stage of development, its sensitivity and speed (assay time: 60–100 s) could make it a very promising tool for high throughput, field-based virus screening. “
“To clarify the phytoplasma associated with Huanglongbing (HLB), a detection survey of phytoplasma in field citrus trees was performed using the standardized nested PCR assay with primer set P1/16S-Sr and R16F2n/R16R2. The HLB-diseased learn more citrus trees with typical www.selleckchem.com/products/NVP-AUY922.html HLB symptoms showed a high detection of 89.7% (322/359) of HLB-Las, while a low detection of phytoplasma at 1.1% (4/359) was examined in an HLB-affected Wentan pummelo (Citrus grandis) tree (1/63) and Tahiti lime (C. latifolia) trees (3/53) that were co-infected with HLB-Las. The phytoplasma alone was also detected

in a healthy Wentan pummelo tree (1/60) at a low incidence total of 0.3% (1/347). Healthy citrus plants were inoculated with the citrus phytoplasma (WP-DL) by graft inoculation with phytoplasma-infected pummelo scions. Positive detections of phytoplasma were monitored only in the Wentan pummelo plant 4 months and 3.5 years after inoculation, and no symptoms developed. The citrus phytoplasma infected and persistently

survived in a low titre and at a very uneven distribution in citrus plants. Peanut witches’ broom (PnWB) phytoplasma (16SrII-A) and periwinkle leaf yellowing (PLY) phytoplasma belonging to the aster yellows group (16SrI-B) maintained in periwinkle plants were inoculated into healthy citrus plants by dodder transmission. The PnWB phytoplasma showed infection through positive detection of the nested PCR assay in citrus click here plants and persistently survived without symptom expression up to 4 years after inoculation. Positive detections of the phytoplasma were found in a low titre and several incidences in the other inoculated citrus plants including Ponkan mandarin, Liucheng sweet orange, Eureka lemon and Hirami lemon. None of the phytoplasma-infected citrus plants developed symptoms. Furthermore, artificial inoculation of PLY phytoplasma (16SrI-B) into the healthy citrus plants demonstrated no infection. The citrus symptomless phytoplasma was identified to belong to the PnWB phytoplasma group (16SrII-A). “
“This study investigated whether foliar sprays of potassium silicate (KSi), sodium molybdate (NaMo) or a combination of both (KSi + NaMo), with or without the fungicide azoxystrobin (Azox), could reduce anthracnose symptoms, improve photosynthesis and increase yield.

After optimization of the membrane-engineering process, the virus detection limit for TMV

and CLRV with the bacteria-based biosensor system was 1 pg/ml, representing a 1000-fold improvement over currently available methods. Although the novel biosensor is still in its proof-of-concept stage of development, its sensitivity and speed (assay time: 60–100 s) could make it a very promising tool for high throughput, field-based virus screening. “
“To clarify the phytoplasma associated with Huanglongbing (HLB), a detection survey of phytoplasma in field citrus trees was performed using the standardized nested PCR assay with primer set P1/16S-Sr and R16F2n/R16R2. The HLB-diseased this website citrus trees with typical Protein Tyrosine Kinase inhibitor HLB symptoms showed a high detection of 89.7% (322/359) of HLB-Las, while a low detection of phytoplasma at 1.1% (4/359) was examined in an HLB-affected Wentan pummelo (Citrus grandis) tree (1/63) and Tahiti lime (C. latifolia) trees (3/53) that were co-infected with HLB-Las. The phytoplasma alone was also detected

in a healthy Wentan pummelo tree (1/60) at a low incidence total of 0.3% (1/347). Healthy citrus plants were inoculated with the citrus phytoplasma (WP-DL) by graft inoculation with phytoplasma-infected pummelo scions. Positive detections of phytoplasma were monitored only in the Wentan pummelo plant 4 months and 3.5 years after inoculation, and no symptoms developed. The citrus phytoplasma infected and persistently

survived in a low titre and at a very uneven distribution in citrus plants. Peanut witches’ broom (PnWB) phytoplasma (16SrII-A) and periwinkle leaf yellowing (PLY) phytoplasma belonging to the aster yellows group (16SrI-B) maintained in periwinkle plants were inoculated into healthy citrus plants by dodder transmission. The PnWB phytoplasma showed infection through positive detection of the nested PCR assay in citrus selleckchem plants and persistently survived without symptom expression up to 4 years after inoculation. Positive detections of the phytoplasma were found in a low titre and several incidences in the other inoculated citrus plants including Ponkan mandarin, Liucheng sweet orange, Eureka lemon and Hirami lemon. None of the phytoplasma-infected citrus plants developed symptoms. Furthermore, artificial inoculation of PLY phytoplasma (16SrI-B) into the healthy citrus plants demonstrated no infection. The citrus symptomless phytoplasma was identified to belong to the PnWB phytoplasma group (16SrII-A). “
“This study investigated whether foliar sprays of potassium silicate (KSi), sodium molybdate (NaMo) or a combination of both (KSi + NaMo), with or without the fungicide azoxystrobin (Azox), could reduce anthracnose symptoms, improve photosynthesis and increase yield.

Several studies have demonstrated sinusoidal endothelial dysfunction at the liver microcirculation in chronic liver diseases, especially Selleckchem LBH589 in cirrhosis,13-16 but also in the early stages of nonalcoholic fatty liver disease (NAFLD).17, 18 In addition, we have recently demonstrated in patients with cirrhosis that bacterial translocation further worsens liver endothelial dysfunction.19 A large corpus of data shows that endothelial dysfunction may be ameliorated by statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors).20-22 This occurs also in cirrhosis and portal hypertension.23-25 Experimental models,2 several observational studies in humans,26

and a meta-analysis27 suggest

that statins might improve vascular inflammation and microvascular dysfunction in sepsis. Pirfenidone order However, the potential of these drugs for preventing endotoxin-induced liver vascular abnormalities has never been explored. This study aimed at evaluating the changes in liver microcirculation induced by LPS, and whether the administration of simvastatin might prevent liver microvascular dysfunction in a rat model of endotoxemia. eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; lipopolysaccharide (LPS); MOF, multiorgan failure; NAFLD, nonalcoholic fatty liver disease; PPP, portal perfusion pressure. Male Wistar rats, weighing 275-300 g, were caged in pairs on a 12:12-hour light-dark cycle, in a temperature- and humidity-controlled environment. The animals were kept in environmentally controlled animal facilities at the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS). All experiments were approved by the Laboratory Animal Care and Use Committee of the University of Barcelona and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, NIH Publication 86-23, revised 1996). The effects this website of LPS (5 mg/kg intraperitoneally) as compared with vehicle administration were evaluated 6 hours and 24 hours after the injection. Subsequent studies were performed at

24 hours in rats treated with simvastatin (25 mg/kg, orally), given 3 and 23 hours after LPS/saline challenge, and in rats treated with simvastatin (25 mg/kg/24 hours, orally) from 3 days before LPS/saline injection (the last dose 1 hour before the hemodynamic study). After LPS/saline injection livers were isolated and perfused with Krebs buffer in a recirculation fashion with a total volume of 100 mL at a constant flow rate of 35 mL/min. An ultrasonic transit-time flow probe (model T201; Transonic Systems, Ithaca, NY) and a pressure transducer (Edwards Lifesciences, Irvine, CA) were placed on line, immediately ahead of the portal inlet cannula, to continuously monitor portal flow and perfusion pressure.

Several studies have demonstrated sinusoidal endothelial dysfunction at the liver microcirculation in chronic liver diseases, especially MLN2238 cell line in cirrhosis,13-16 but also in the early stages of nonalcoholic fatty liver disease (NAFLD).17, 18 In addition, we have recently demonstrated in patients with cirrhosis that bacterial translocation further worsens liver endothelial dysfunction.19 A large corpus of data shows that endothelial dysfunction may be ameliorated by statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors).20-22 This occurs also in cirrhosis and portal hypertension.23-25 Experimental models,2 several observational studies in humans,26

and a meta-analysis27 suggest

that statins might improve vascular inflammation and microvascular dysfunction in sepsis. IDH inhibitor drugs However, the potential of these drugs for preventing endotoxin-induced liver vascular abnormalities has never been explored. This study aimed at evaluating the changes in liver microcirculation induced by LPS, and whether the administration of simvastatin might prevent liver microvascular dysfunction in a rat model of endotoxemia. eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; lipopolysaccharide (LPS); MOF, multiorgan failure; NAFLD, nonalcoholic fatty liver disease; PPP, portal perfusion pressure. Male Wistar rats, weighing 275-300 g, were caged in pairs on a 12:12-hour light-dark cycle, in a temperature- and humidity-controlled environment. The animals were kept in environmentally controlled animal facilities at the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS). All experiments were approved by the Laboratory Animal Care and Use Committee of the University of Barcelona and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, NIH Publication 86-23, revised 1996). The effects selleck chemical of LPS (5 mg/kg intraperitoneally) as compared with vehicle administration were evaluated 6 hours and 24 hours after the injection. Subsequent studies were performed at

24 hours in rats treated with simvastatin (25 mg/kg, orally), given 3 and 23 hours after LPS/saline challenge, and in rats treated with simvastatin (25 mg/kg/24 hours, orally) from 3 days before LPS/saline injection (the last dose 1 hour before the hemodynamic study). After LPS/saline injection livers were isolated and perfused with Krebs buffer in a recirculation fashion with a total volume of 100 mL at a constant flow rate of 35 mL/min. An ultrasonic transit-time flow probe (model T201; Transonic Systems, Ithaca, NY) and a pressure transducer (Edwards Lifesciences, Irvine, CA) were placed on line, immediately ahead of the portal inlet cannula, to continuously monitor portal flow and perfusion pressure.

[41, 42] Next, screening studies by Sveger and Eriksson documented that 15%-20% of infants with α-1-AT deficiency (PiZZ) present with neonatal cholestasis.[43] In Cincinnati we were greatly aided by our colleague Kevin Bove, a pediatric pathologist, who developed an interest and expertise in interpretation of biopsy findings from children with a variety of hepatobiliary disorders.[9, 44] It became clear that if we were to study

diseases such as neonatal cholestasis we needed to understand the normal physiologic events occurring at this stage of liver development. A series of adaptations must occur during transition of the infant to extrauterine life; specifically, the liver of a newborn must conform to the unique metabolic demands that result from discontinuation of the bidirectional exchange of nutrients through the placenta and the biotransformation mechanisms shared with the mother.[31] These RO4929097 maturational changes as the transition is made from an intrauterine existence to independent life occur predominantly through enzyme induction triggered by substrate and hormonal input. The efficiency with which these anatomic and physiologic adaptations

are established determines the ability of the newborn to cope with a new environment.[31, 45, 46] Historically, there are dramatic examples of inefficiency of hepatic metabolic and excretory function in early life, most notably “physiologic jaundice” (unconjugated hyperbilirubinemia characteristic of the newborn). We therefore were not surprised to discover an analogous phase, which we termed “physiologic cholestasis.” this website We documented that in newborns there is a cholestatic phase of liver development, manifest by delayed hepatic clearance of endogenous and exogenous compounds.[45-47] The morphological and functional differences that characterize the

neonatal versus the mature liver are responsible not only for a decrease in bile flow but also the production of abnormal bile acids. This renders the developing liver uniquely vulnerable to exogenous insults such as E. coli sepsis with endotoxemia, the intravenous administration of amino acids during total parenteral nutritional support, and hypoxia/hypoperfusion.[44, 48, 49] Good fortune once again intervened—my first fellow in Pediatric Gastroenterology learn more at CCHMC was Fred Suchy, who enthusiastically joined me for studies further delineating normal and abnormal hepatobiliary function in neonates. We were able to document that multiple steps in the enterohepatic circulation were reduced in early life, evidenced by elevated serum bile acid levels, reduced intraluminal bile acid concentrations, and reduced hepatocellular transport (uptake and excretion) of bile acids. Another striking feature of “physiologic cholestasis” was the presence of a large proportion of “atypical” bile acids (yet typical for the developmental phase) that are not found in adult human bile.

[41, 42] Next, screening studies by Sveger and Eriksson documented that 15%-20% of infants with α-1-AT deficiency (PiZZ) present with neonatal cholestasis.[43] In Cincinnati we were greatly aided by our colleague Kevin Bove, a pediatric pathologist, who developed an interest and expertise in interpretation of biopsy findings from children with a variety of hepatobiliary disorders.[9, 44] It became clear that if we were to study

diseases such as neonatal cholestasis we needed to understand the normal physiologic events occurring at this stage of liver development. A series of adaptations must occur during transition of the infant to extrauterine life; specifically, the liver of a newborn must conform to the unique metabolic demands that result from discontinuation of the bidirectional exchange of nutrients through the placenta and the biotransformation mechanisms shared with the mother.[31] These buy Rucaparib maturational changes as the transition is made from an intrauterine existence to independent life occur predominantly through enzyme induction triggered by substrate and hormonal input. The efficiency with which these anatomic and physiologic adaptations

are established determines the ability of the newborn to cope with a new environment.[31, 45, 46] Historically, there are dramatic examples of inefficiency of hepatic metabolic and excretory function in early life, most notably “physiologic jaundice” (unconjugated hyperbilirubinemia characteristic of the newborn). We therefore were not surprised to discover an analogous phase, which we termed “physiologic cholestasis.” Ruxolitinib in vivo We documented that in newborns there is a cholestatic phase of liver development, manifest by delayed hepatic clearance of endogenous and exogenous compounds.[45-47] The morphological and functional differences that characterize the

neonatal versus the mature liver are responsible not only for a decrease in bile flow but also the production of abnormal bile acids. This renders the developing liver uniquely vulnerable to exogenous insults such as E. coli sepsis with endotoxemia, the intravenous administration of amino acids during total parenteral nutritional support, and hypoxia/hypoperfusion.[44, 48, 49] Good fortune once again intervened—my first fellow in Pediatric Gastroenterology check details at CCHMC was Fred Suchy, who enthusiastically joined me for studies further delineating normal and abnormal hepatobiliary function in neonates. We were able to document that multiple steps in the enterohepatic circulation were reduced in early life, evidenced by elevated serum bile acid levels, reduced intraluminal bile acid concentrations, and reduced hepatocellular transport (uptake and excretion) of bile acids. Another striking feature of “physiologic cholestasis” was the presence of a large proportion of “atypical” bile acids (yet typical for the developmental phase) that are not found in adult human bile.