Neonatal Liver Research Articles

Organ-specific regulation of ATP7A abundance is coordinated with systemic copper homeostasis

Copper (Cu) is an essential cofactor for various enzymatic activities including mitochondrial electron transport, iron mobilization, and peptide hormone maturation. Consequently, Cu dysregulation is associated with fatal neonatal disease, liver and cardiac dysfunction, and anemia. While the Cu transporter ATP7A plays a major role in both intestinal Cu mobilization to the periphery and prevention of Cu over-accumulation, it is unclear how regulation of ATP7A contributes to Cu homeostasis in response to systemic Cu fluctuation. Here we show, using Cu-deficient mouse models, that steady-state levels of ATP7A are lower in peripheral tissues (including the heart, spleen, and liver) under Cu deficiency and that subcutaneous administration of Cu to these animals restore normal ATP7A levels in these tissues. Strikingly, ATP7A in the intestine is regulated in the opposite manner - low systemic Cu increases ATP7A while subcutaneous Cu administration decreases ATP7A suggesting that intestine-specific non-autonomous regulation of ATP7A abundance may serve as a key homeostatic control for Cu export into the circulation. Our results support a systemic model for how a single transporter can be inversely regulated in a tissue-specific manner to maintain organismal Cu homeostasis.

Multicystic dysplastic kidney: a new association of Wolcott-Rallison syndrome.

Wolcott–Rallison syndrome (WRS) is a rare autosomal recessive disorder due to mutations in the EIF2AK3 gene. It is characterized by permanent neonatal diabetes mellitus, skeletal dysplasia, liver impairment, neutropenia and renal dysfunction. Liver is the most commonly affected organ and liver failure is the commonest cause of death in this syndrome. The EIF2AK3 gene encodes a transmembrane protein PERK, which is important for the cellular response to endoplasmic reticulum (ER) stress. The absence of PERK activity reduces the ER’s abilities to deal with stress, leading to cell death by apoptosis. On acquiring febrile illness, affected patients suffer from liver injury, which may progress into liver failure and death. Renal involvement is less common and is mainly in the form of functional renal impairment at the advanced stage of the disease. Structural renal anomalies have not been reported in WRS. We report a 6-month-old girl who presented with neonatal diabetes on day 1 of life. Her genetic testing confirmed WRS due to missense mutation in the EIF2AK3 gene (c.2867G > A, p.Gly956Glu). Parents are first-degree cousins and both are heterozygous carriers to the mutation. 2 paternal uncles had the same mutation and died of liver disease at 1 and 14 years of age. Neither had a renal disease. She presented with hematuria during a febrile illness at the age of 5 months. Ultrasound scan showed right ectopic multicystic dysplastic kidney (MCDK). To the best of our knowledge, this is the first patient with WRS who is reported to have an MCDK disease.Learning points:Neonatal diabetes should be considered in babies presenting with early hyperglycemia particularly if there is a family history.Genetic diagnosis in neonatal diabetes enables disease confirmation, genetic counseling and anticipation of potential complications during concomitant situations such as acute illness, trauma or major surgery.There is lack of phenotype–genotype correlation in Wolcott–Rallison syndrome.Structural kidney abnormality, in our case MCDK, can be seen in WRS.

Enzyme replacement prevents neonatal death, liver damage, and osteoporosis in murine homocystinuria.

Classical homocystinuria (HCU) is an inborn error of sulfur amino acid metabolism caused by deficient activity of cystathionine β-synthase (CBS), resulting in an accumulation of homocysteine and a concomitant decrease of cystathionine and cysteine in blood and tissues. In mice, the complete lack of CBS is neonatally lethal. In this study, newborn CBS-knockout (KO) mice were treated with recombinant polyethyleneglycolylated human truncated CBS (PEG-CBS). Full survival of the treated KO mice, along with a positive impact on metabolite levels in plasma, liver, brain, and kidneys, was observed. The PEG-CBS treatment prevented an otherwise fatal liver disease characterized by steatosis, death of hepatocytes, and ultrastructural abnormalities of endoplasmic reticulum and mitochondria. Furthermore, treatment of the KO mice for 5 mo maintained the plasma metabolite balance and completely prevented osteoporosis and changes in body composition that characterize both the KO model and human patients. These findings argue that early treatment of patients with HCU with PEG-CBS may prevent clinical symptoms of the disease possibly without the need of dietary protein restriction.-Majtan, T., Hůlková, H., Park, I., Krijt, J., Kožich, V., Bublil, E. M., Kraus, J. P. Enzyme replacement prevents neonatal death, liver damage, and osteoporosis in murine homocystinuria.

Liver-Directed Human Amniotic Epithelial Cell Transplantation Improves Systemic Disease Phenotype in Hurler Syndrome Mouse Model.

Mucopolysaccharidosis type 1 (MPS1) is an inherited lysosomal storage disorder caused by a deficiency in the glycosaminoglycan (GAG)‐degrading enzyme α‐l‐iduronidase (IDUA). In affected patients, the systemic accumulation of GAGs results in skeletal dysplasia, neurological degeneration, multiple organ dysfunction, and early death. Current therapies, including enzyme replacement and bone marrow transplant, improve life expectancy but the benefits to skeletal and neurological phenotypes are limited. In this study, we tested the therapeutic efficacy of liver‐directed transplantation of a placental stem cell, which possesses multilineage differentiation potential, low immunogenicity, and high lysosomal enzyme activity. Unfractionated human amniotic epithelial cells (hAECs) were transplanted directly into the liver of immunodeficient Idua knockout mouse neonates. The hAECs engraftment was immunohistochemically confirmed with anti‐human mitochondria staining. Enzyme activity assays indicated that hAECs transplantation restored IDUA function in the liver and significantly decreased urinary GAG excretion. Histochemical and micro‐computed tomography analyses revealed reduced GAG deposition in the phalanges joints and composition/morphology improvement of cranial and facial bones. Neurological assessment in the hAEC treated mice showed significant improvement of sensorimotor coordination in the hAEC treated mice compared to untreated mice. Results confirm that partial liver cell replacement with placental stem cells can provide long‐term (>20 weeks) and systemic restoration of enzyme function, and lead to significant phenotypic improvement in the MPS1 mouse model. This preclinical data indicate that liver‐directed placental stem cell transplantation may improve skeletal and neurological phenotypes of MPS1 patients. Stem Cells Translational Medicine 2017;6:1583–1594

Radiation Exposure Enhances Hepatocyte Proliferation in Neonatal Mice but not in Adult Mice.

There is a natural tendency to expect that irradiation of an infant organ prior to development-related expansion will result in a higher risk of developing cancer than that of fully-developed adult tissue, and this has generally been observed. However, if tissues also vary in their initial responses to radiation depending on age, the interplay between tissue- and age-dependent risk would potentially be quite complex. We have previously shown opposing age-dependent induction of apoptosis for the intestinal epithelium and hematopoietic cells in mice, but such data are not yet available for the liver. Here, we have examined markers of DNA damage, initiation of DNA damage responses, cell cycle arrest, apoptosis and proliferation, as well as gene expression, in the B6C3F1 mouse liver over the hours and days after irradiation of mice at 1 or 7 weeks of age. We found that induction and resolution of radiation-induced DNA damage is not accompanied by significant changes in these cellular end points in the adult liver, while in infant hepatocytes modest induction of p53 accumulation and p21-mediated cell cycle arrest in a small fraction of damaged cells was overshadowed by a further stimulation of proliferation over the relatively high levels already found in the neonatal liver. We observed distinct expression of genes that regulate cell division between the ages, which may contribute to the differential responses. These data suggest that the growth factor signaling environment of the infant liver may mediate radiation-induced proliferation and increased liver cancer risk after irradiation during early life.

TARGETED INVESTIGATION OF NOVEL MESENCHYMAL STEM CELL BIOMARKERS OF BRONCHOPULMONARY DYSPLASIA IN PREMATURELY BORN INFANTS

Abstracts Abstract 47 Table 1 EGA (wks) All BPD, n (%) BPD after correction, n (%) BPD or death, n (%) BPD or death after correction, n (%) Decrease in BPD rate* *P<0.02. Conclusions BPD is common in high risk preterm infants. Using the standard definition, high altitude is associated with significantly increased rates of BPD. At our altitude, the rate of BPD was two-fold lower following FiO2 correc- tion for altitude. Further diagnostic criteria to adjust for altitude seems warranted. neonatal characteristics. Half of the samples have been ana- lyzed. Processing of the remaining samples and collection of outcomes data is ongoing. Standard curves were used from the pilot study. Approximately half of the subjects have developed BPD and demonstrated low baseline Csf1 levels and rising TGF-B levels post-ventilation. The subjects without BPD had stable Spp1, Csf1, and TGF-B levels. Conclusions Levels of Spp1, Csf1, and TGF-B are asso- ciated with BPD. Further data collection is underway to reach study power. Statistical analysis will follow comple- tion of sample processing. Larger multi-center studies are needed to confirm this association, which will guide tar- geted therapy against BPD. NEONATAL HEPATIC COX2 EXPRESSION IS MEDIATED VIA AN NFκB-DEPENDENT MECHANISM FOLLOWING INFLAMMATORY STRESS S McKenna, BE Butler, CJ Wright. University of Colorado, Aurora, CO 10.1136/jim-d-15-00013.49 TARGETED INVESTIGATION OF NOVEL MESENCHYMAL STEM CELL BIOMARKERS OF BRONCHOPULMONARY DYSPLASIA IN PREMATURELY BORN INFANTS RG LaGrandeur, 1 F Bany-Mohammed, 1 C UY, 1 DM Cooper, 2 M Aslam 1 . 1 University of California Irvine Medical Center, Orange, CA; University of California Irvine Medical Center, Orange, CA 10.1136/jim-d-15-00013.48 Purpose of Study Bronchopulmonary dysplasia (BPD) is a chronic disease of preterm infants caused by oxygen tox- icity, inflammation, and ventilator use leading to arrested alveolar development. Current therapies lack effectiveness and cause undesirable side effects. Our work has shown mesenchymal stem cell conditioned-media to have protect- ive effects in mouse BPD models. Analysis identified Osteopontin (Spp1) and Macrophage colony stimulating factor 1 (Csf1) as key factors to suppress the TGF-B surge in the lungs, leading to protection against BPD. Our pilot study has shown it to be feasible to quantify Spp1, Csf1, and TGF-B in the tracheal aspirate fluid (TAF) of preterm infants and generated standard curves. Our aim is to deter- mine the association between Spp1 and Csf1 and BPD by quantifying these markers in the TAF of preterm infants. Methods Used Infants under 32 weeks gestational age intubated within 24 hours of life were enrolled into the UCI IRB-approved study. Those with neuromuscular or congenital anomalies or pulmonary hemorrhage were excluded. The 1st TAF sample was obtained at intubation, before surfactant dosing. The 2nd was obtained at extuba- tion or the 4th day if still intubated. Spp1, Csf1, TGF-B, and IgA levels were analyzed using ELISA. IgA was used as control to correct for TAF volume. Infants were followed prospectively for outcomes data including the development of BPD. Summary of Results 21 infants were enrolled and TAF obtained. Subjects were similar in their maternal and J Investig Med 2016;64:135–345 Purpose of Study Patent Ductus Arteriosus(PDA) occurs commonly in preterm neonates and is associated with numerous severe co-morbidities. Exposure to chorioamnio- nitis and early-onset sepsis are risk factors for PDA; add- itionally, prostaglandin byproducts are elevated in the serum of infants with PDA. However, the mechanisms linking inflammatory stress and PDA are unknown. COX2 is an inducible enzyme responsible for prostaglandin pro- duction. The cellular source and transcriptional regulation of COX2 following inflammatory stress in neonates are unknown. We hypothesize that the transcription factor NFκB regulates inflammatory-stress induced COX2 expres- sion in neonates. Methods Used Fetal (E15, E19) wild type mice were exposed to intrauterine LPS (250 mg); neonatal(P0) and adult wild type mice were exposed to IP LPS (5 or 50 mg/g). COX2 expression was assessed by qPCR. The time course of LPS-induced hepatic NFκB activation was determined using Western blot to assess NFκB inhibitory protein degradation and NFκB subunit nuclear transloca- tion. COX2 expression in isolated neonatal and adult hepatic macrophages and adult livers following clodronate-induced macrophage ablation was assessed by qPCR. COX2 expression in cultured macrophages and in neonatal livers following NFκB inhibition was assessed by qPCR. All experiments were performed in triplicate. Summary of Results Hepatic COX2 expression was induced in fetal, neonatal, and adult mice following LPS exposure. COX2 expression was enriched in isolated neo- natal and adult hepatic macrophages and attenuated in adult livers following macrophage ablation by clodronate. Cultured macrophages demonstrated LPS-induced NFκB activation and COX2 expression and attenuated COX2 expression following NFκB inhibition. Neonatal and adult mice demonstrated hepatic LPS-induced NFκB activation. Pharmacologic NFκB inhibition in neonatal mice attenu- ated hepatic LPS-induced COX2 expression.

Developmental differences between newborn and adult mice in response to romiplostim

Thrombocytopenia is frequent among sick neonates. While most cases are transient, some neonates experience prolonged and severe thrombocytopenia. These infants often pose diagnostic and therapeutic challenges, and may receive large numbers of platelet transfusions. Romiplostim (ROM) is a thrombopoietin (TPO)-receptor-agonist approved for treatment of adults with chronic immune thrombocytopenia (ITP). The immature platelet fraction (IPF) is a novel measure of newly produced platelets, which could aid with the diagnostic evaluation of thrombocytopenic neonates. This study had the following two objectives: (1) compare the response of newborn and adult mice to escalating doses of ROM in vivo and (2) assess the correlation between IPF and megakaryocyte (MK) mass in newborn and adult treated and untreated mice. In the first set of studies, newborn (day 1) and adult mice received a single subcutaneous (SC) dose of ROM ranging from 0 to 300 ng/g, and platelet counts were followed every other day for 14 days. Both sets of mice responded with dose-dependent platelet and IPF increases, peaking on days 5–7 post-treatment, but neonates had a blunted response (2.1-fold compared to 4.2-fold maximal increase in platelet counts, respectively). On day 5 post-treatment with 300 ng/g ROM, MKs in the bone marrow (BM) and spleen of adult mice were significantly increased in numbers and size (p < 0.0001 for both) compared to controls. MKs in the spleen and BM (but not liver) of treated neonates also increased in number, but not in size. The immature platelet count (IPC, calculated as IPF x platelet count) was highly correlated with the MK number and size in neonatal and adult BM and spleen, but not neonatal liver. The lack of response of neonatal liver MKs was not due to a cell-intrinsic reduced responsiveness to TPO, since neonatal liver progenitors were more sensitive to murine TPO (mTPO) in vitro than adult BM progenitor. In vivo treatment of newborn mice with high mTPO doses or with higher doses of ROM (900 ng/g) resulted in peak platelet counts approaching 3-fold of controls. Taken together, our data indicate that newborn mice are less responsive to ROM than adult mice in vivo, due to a combination of likely pharmacokinetic differences and developmental differences in the response of MKs to thrombopoietic stimulation, evidenced by neonatal MKs increasing in numbers but not in size. PK/PD studies in human infants treated with ROM are warranted.

Presenting Features and Prognosis of Ischemic and Nonischemic Neonatal Liver Failure.

To describe the epidemiological features, clinical characteristics and outcomes of neonates diagnosed with liver failure, as well as determine prognostic factors. Cohort study conducted at a single tertiary referral and university-affiliated pediatric center. Hospital records of all neonates diagnosed with liver failure between January 2003 and December 2015 were retrospectively reviewed, and data on clinical and laboratory findings, treatment, and outcomes were collected. Survival analysis (Kaplan-Meier) and Cox regression were performed to identify prognostic factors at diagnosis. Liver failure diagnosis was established using the pediatric acute liver failure study group's diagnostic criteria for every patient with coagulopathy and biochemical pattern of liver disease. Forty-five patients were included. In our series, most cases were secondary to ischemia (28.9%). Other causes were neonatal hemochromatosis (17.8%), viral infections (13.3%), and inborn errors of metabolism (13.3%). A total 55.6% (25/45) of the patients died (median age: 16 days; range 1-235 days). Alanine aminotransferase (ALT) at diagnosis was associated with higher mortality or the need for liver transplantation on day 21 after diagnosis (P = .006). For every 500 IU/L increase in ALT serum levels, the mortality/liver transplantation rate increased 1.3 times (hazard ratio 95% confidence interval: 1.1-1.6). Although ischemic neonatal acute liver failure presents with higher ALT levels, these cases appear to have better outcomes. Higher international normalized ratio tended to increase mortality/transplantation (hazard ratio 1.02; 95% confidence interval 0.91-1.2). Neonatal liver failure should perhaps be considered in the differential diagnoses of any coagulopathy. ALT and international normalized ratio levels at diagnosis could predict prognosis in the short term. Ischemic liver failure appears to have a better prognosis.

Hepatic fibrosis scores and serum biomarkers in pediatric hepatology.

Hepatic fibrosis scores and serum biomarkers in pediatric hepatology.

The Impact of Early Postnatal Vitamin A Supplementation on Childhood Obesity Risk ‐ A Study in Rat Model

Childhood obesity is a serious medical concern because it is associated with many immediate and long‐term health consequences, including diabetes, hypertension, and cardiovascular disease. However, studies are limited about the effects of early nutritional supplementation on later health outcomes. The objective of the study was to investigate whether early postnatal vitamin A (VA) supplementation alters metabolic functions related to childhood obesity risk later in life, using a rat model. We hypothesized that early‐life VA supplementation in rats is associated with altered expression of genes related to insulin resistance and thermogenesis at neonatal period, with carry‐over effects at prepubescent age. In addition, previously in our laboratory, we showed that the early postnatal treatment with VARA, a retinoid combination of VA and 10% retinoic acid (RA), significantly decreases the expression of retinol‐binding protein 4 (Rbp4) in the liver of neonates as well as adult rats and reduced liver Rbp4 expression is associated with improved glucose tolerance. A defined high‐fat VA‐marginal (HFVAM) diet, which is modified to contain 45 kcal‐% fat and a marginal level of VA (0.35 μg of retinol as retinyl palmitate per gram diet), was fed to Sprague‐Dawley dams from pregnancy until the pups were euthanized at postnatal day (P) 12 or 5 weeks of age. Three doses of VA, VARA, or canola oil as control, were adjusted based on body weight (6 mg VA/kg of body weight) and administered orally to the pups on P0/1, P4, and P10. Plasma and tissues, including liver and adipose tissues, were collected on P12 and 5 weeks of age. Body composition of adolescent rats (5 weeks old) was measured by dual‐energy X‐ray absorptiometry (DEXA) to determine the effects of early VA supplementation on adiposity in prepubertal rats. Total retinoid concentrations in the plasma and tissues were determined by ultra performance liquid chromatography (UPLC). Data were compared to age‐ and sex‐matched normal chow‐fed non‐obese rats as references. There was no significant difference in body weight change between treatment groups, indicating similar growth patterns and successful randomization among pups. The adolescent rats fed a HFVAM diet had significantly higher body fat (%), as compared to rats fed a normal chow diet, but there was no difference in body fat between sexes in each treatment group. The VA‐supplemented HFVAM diet‐fed adolescent rats had significant higher serum total retinol concentration, as compared to normal chow‐fed rats. Both neonatal and adolescent VA‐supplemented rats had increased hepatic retinoid storage, as compared to the oil‐dosed group, suggesting the possibility of “carry‐over” effects of early postnatal VA‐supplementation on metabolic changes later in life. However, no difference was observed in the brown adipose tissue retinoid concentrations between treatment groups in adolescent rats. The relationship between early‐life VA supplementation and metabolic functions related to obesity risk remains to be determined by gene expression analysis.Support or Funding InformationThe project was supported by the Pennsylvania State University Childhood Obesity Training Program funded by USDA National Institute for Food and Agriculture Grant #2011‐67001‐30117 Program A2121.

Neonatal acute liver failure: a diagnosis challenge.

Neonatal acute liver failure is a rare, very severe disease with a high rate of mortality. It is clinically and etiologically different from acute liver failure seen in older children and adults. Coagulopathy with an international normalized ratio ≥ 3 is the critical parameter that defines it. The most common causes are fetal alloimmune hepatitis, previously called neonatal hemochromatosis, viral infections, metabolic disorders, and hemophagocytic lymphohistiocytosis. There is a group of treatable diseases that require a very early diagnosis for the prescription of an adequate treatment. Patients should be immediately referred to a specialized facility where pediatric liver transplantation is available to implement such therapeutic alternative, if indicated.

Conditional postnatal deletion of the neonatal murine hepatic circadian gene, Npas2, alters the gut microbiome following restricted feeding

We have recently shown in both non-human primates and in rodents that fetal and neonatal hepatic expression of the circadian transcription factor, Npas2, is modulated by a high fat maternal diet and plays a critical role in establishing life-long metabolic homeostasis. Similarly, we and others have also established the importance of the maternal and early postnatal diet on establishment of the early gut microbiome. We hypothesized that altered circadian gene expression solely in the neonatal liver would result in gut microbiome dysbiosis, especially with diet-induced metabolic stress (ie, restricted feeding). Using a murine model in which we conditionally knock out Npas2 in the neonatal liver, we aimed to determine the role of the circadian machinery in gut dysbiosis with restricted feeding. We collected fecal samples from liver Npas2 conditional knockout (n= 11) and wild-type (n= 13) reproductive-aged mice before (study day 0) and after the restricted feeding study (study day 17). Extracted DNA was sequenced using the MiSeq Illumina platform using primers specific for the V4 region of the 16S ribosomal DNA gene. The resulting sequences were quality filtered, aligned, and assigned taxonomy. Principal coordinate analysis was performed on unweighted and weighted UniFrac distances between samples with a permutation analysis of variance to assess clustering significance between groups. Microbial taxa that significantly differ between groups of interest was determined using linear discriminate analysis effect size and randomForrest. Principal coordinate analysis performed on weighted UniFrac distances between male conditional knockout and wild-type cohorts revealed that the gut microbiome of the mice did not differ by genotype at the start of the restricted feeding study but did differ by virtue of genotype at the end of the study (P= .001). Moreover, these differences could be at least partially attributed to restricted feeding-associated alterations in relative abundance of the Bacteroides genus, which has been implicated as crucial to establishing a healthy gut microbiome early in development. Here we have provided an initial key insight into the interplay between neonatal establishment of the peripheral circadian clock in the liver and the ability of the gut microbiome to respond to dietary and metabolic stress. Because Npas2 expression in the liver is a target of maternal high-fat diet-induced metabolic perturbations during fetal development, we speculate that these findings have potential implications in the long-term metabolic health of their offspring.

Cystathionine beta-synthase deficiency alters hepatic phospholipid and choline metabolism: Post-translational repression of phosphatidylethanolamine N-methyltransferase is a consequence rather than a cause of liver injury in homocystinuria.

Classical homocystinuria (HCU) due to inactivating mutation of cystathionine β-synthase (CBS) is a poorly understood life-threatening inborn error of sulfur metabolism. A previously described cbs-/- mouse model exhibits a semi-lethal phenotype due to neonatal liver failure. The transgenic HO mouse model of HCU exhibits only mild liver injury and recapitulates multiple aspects of the disease as it occurs in humans. Disruption of the methionine cycle in HCU has the potential to impact multiple aspect of phospholipid (PL) metabolism by disruption of both the Kennedy pathway and phosphatidylethanolamine N-methyltransferase (PEMT) mediated synthesis of phosphatidylcholine (PC). Comparative metabolomic analysis of HO mouse liver revealed decreased levels of choline, and choline phosphate indicating disruption of the Kennedy pathway. Alterations in the relative levels of multiple species of PL included significant increases in PL degradation products consistent with enhanced membrane PL turnover. A significant decrease in PC containing 20:4n6 which primarily formed by the methylation of phosphatidylethanolamine to PC was consistent with decreased flux through PEMT. Hepatic expression of PEMT in both the cbs-/- and HO models is post-translationally repressed with decreased levels of PEMT protein and activity that inversely-correlates with the scale of liver injury. Failure to induce further repression of PEMT in HO mice by increased homocysteine, methionine and S-adenosylhomocysteine or depletion of glutathione combined with examination of multiple homocysteine-independent models of liver injury indicated that repression of PEMT in HCU is a consequence rather than a cause of liver injury. Collectively, our data show significant alteration of a broad range of hepatic PL and choline metabolism in HCU with the potential to contribute to multiple aspects of pathogenesis in this disease.

Chronic Intermittent Hypoxia Causes Lipid Peroxidation and Altered Phase 1 Drug Metabolizing Enzymes in the Neonatal Rat Liver

Critically ill preterm neonates requiring oxygen therapy often experience frequent apneas with intermittent hypoxia (IH). IH-induced oxidative stress causes lipid peroxidation, which targets the liver and contributes to toxic drug reactions. We tested the hypothesis that incremental IH episodes induce oxidative damage in the neonatal liver and alter the expression of genes that regulate drug metabolism. Newborn rats were exposed to increasing IH episodes (12% O2) during hyperoxia (50% O2), or placed in room air (RA) until postnatal day 21 (P21) for recovery from IH (IHR). RA littermates served as controls, and pups exposed to 50% O2 served as hyperoxia controls. Hepatic histopathology, biomarkers of oxidative stress and oxidative DNA damage, antioxidants, and expression of genes that regulate drug metabolism were assessed. Oxidative stress and DNA damage, evidenced by 8-isoprostaglandin F2α (8-isoPGF2α) and 8-hydroxy-2'-deoxyguanosine (8-OH-dG), respectively, increased as a function of IH episodes, and was associated with decreased superoxide dismutase (SOD) and increased catalase activities. Pathological changes including cellular swelling, steatosis, necrosis, and focal sinusoid congestion were seen in IH, but not in IHR. Similarly, IH was associated with upregulation of several genes involved in DNA repair, which were downregulated during IHR. Of the genes involved in drug metabolism, aldehyde dehydrogenases (involved in lipid peroxidation) and cytochrome P450 (CYP) genes of the 2C family (involved in oxidative stress) were robustly upregulated both in IH and in IHR. Hepatic oxidative stress and lipid peroxidation occurring in response to chronic IH have implications for preterm infants, and may explain, in part, the pharmacokinetic variations and drug toxicities in this vulnerable population.

Possible use of the growing liver biological set for hepatic recovery after toxic damage (an experimental study)

The lack of acceptable pharmacological approaches for restoration of the injured liver is associated with complex of mechanisms involved in hepatic regeneration and with difficulty of the target selection. The aim of this research was to study the hepatoprotective function of the extract from both the growing and regenerating liver containing a natural set of factors crucial for the hepatic restoration. Extracts from both regenerating liver of rats after 70% hepatic resection and the growing liver of neonatal pigs were obtained using own original technique. The set of resultant extracts was named as the hepatic regeneration set (HRS). HRS fractionation was carried out using the Toyopearl HW-50S sorbent. The efficiency of HRS and its fractions was estimated using a model of the mouse liver thioacetamide injury and monitoring hepatic enzyme activity in blood serum. The activities of AST and ALT in intact animals were 50 U/l and 80 U/l, respectively; after thioacetamide administration they increased to 2059±212 U/l and 4280±440 E/l, respectively (p<0.05). Treatment of injured animals with HRS from the rat regenerating liver resulted in a significant decrease of transaminase activities to 924±148 U/l (AST; p<0.05) and 1633±308 U/l (ALT; p<0.05). A similar effect was observed after treatment with HRS from the neonatal pig liver: the AST decreased to 937±138 U/l (p<0.05), while ALT activity decreased to 1710±237 U/l (p<0.05). HRs fractionation resulted in identification two active fractions characterized by much higher (8-29) hepatotropic effect that that of the whole extract. These fractions contained peptide/protein components with the range of molecular mass of 3-60 kDa (fraction 1) and 3-25 kDa (fraction 2a). Fraction 1 also contained some polynucleotides in fraction 1. Subsequent studies of these fractions exceeding the hepatotropic effect of original HRS is clearly needed to identify their individual components by immunochromatography methods, ELISA, MRM mass spectrometry and quantitative PCR.

Neonatal liver abscess with impending rupture presenting as abdominal wall swelling: A rare case

Neonatal liver abscess is a rare entity, and till date, fewer than 100 cases have been reported in the literature. In general, they occur in preterm infants with certain risk factors like umbilical vein catheterization. Treatment consists of aspiration, drainage of the abscess with antibiotics. We are reporting here a case of the liver abscess with impending rupture in a 1-month-old male child.

TNF-α and Interleukin-6 Levels in Clinical Early Onset Neonatal Sepsis Toward Acute Liver Injury

TNF-α and IL-6 level have an important role in acute liver injury during early onset neonatal sepsis. This study aims to investigate the correlation of TNF-α and IL-6 levels in neonates with clinical early onset neonatal sepsis toward the occurrence of acute liver injury 44 neonates with clinical early onset neonatal sepsis based on SIRS criteria and Rodwell hematologic scored ≥3 were included in this study. Acute liver injury is defined as elevated of AST, ALT, or AST: ALT ratio &lt;1. TNF-α and IL-6 levels were measured using ELISA methods. This study showed that TNF-α correlated significantly with AST level (p&lt;0,001, r=0,570), ALT level (p&lt;0,001, r=0,554), and AST: ALT ratio (p&lt;0,001, r=0,652). This study also showed that IL-6 correlated significantly with AST level (p&lt;0,001, r=0,523), ALT level (p&lt;0,001, r=0,482), AST: ALT ratio (p&lt;0,001, r=0,603). Regression test using backward methods showed that TNF-α influence acute liver injury (indicated by AST, ALT, and AST: ALT ratio) more than IL-6. We concluded that TNF-α and IL-6 level in clinical early onset neonatal sepsis correlated with acute liver injury, whereas early onset neonatal sepsis was correlated with acute liver injury.Keywords: Acute liver injury, clinical early neonatal sepsis, IL-6 level, TNF-α level

Recellularization via the bile duct supports functional allogenic and xenogenic cell growth on a decellularized rat liver scaffold

ABSTRACTRecent years have seen a proliferation of methods leading to successful organ decellularization. In this experiment we examine the feasibility of a decellularized liver construct to support growth of functional multilineage cells. Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds. Initially, we recellularized liver scaffolds using a human tumor cell line (HepG2, introduced via the bile duct). Subsequent studies were performed using either human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein) or rat neonatal cell slurry (introduced via the bile duct). Bio-chambers were used to circulate oxygenated growth medium via the portal vein at 37C for 5-7 days. Human HepG2 cells grew readily on the scaffold (n = 20). HepG2 cells co-cultured with HUVECs demonstrated viable human endothelial lining with concurrent hepatocyte growth (n = 10). In the series of neonatal cell slurry infusion (n = 10), distinct foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. The presence of cholangiocytes was verified by CK-7 positivity. Quantitative albumin measurement from the grafts showed increasing albumin levels after seven days of perfusion. Graft albumin production was higher than that observed in traditional cell culture. This data shows that rat liver scaffolds support human cell ingrowth. The scaffold likewise supported the engraftment and survival of neonatal rat liver cell slurry. Recellularization of liver scaffolds thus presents a promising model for functional liver engineering.

Pathophysiology, prevention, treatment, and outcomes of intestinal failure-associated liver disease.

Intestinal failure-associated liver disease (IFALD) remains a serious problem in the treatment of infants with nutritional problems and short bowel syndrome. A review of the recent literature from 2010 to 2016, concentrating on articles related to the pathophysiology of IFALD and to outcomes of novel nutritional and pharmacological therapies for neonatal cholestasis in the post-surgical neonate. The pathophysiology of IFALD relates to an increase sensitivity of the neonatal liver to cholestasis in the non-fed state; prolonged cholestasis almost inevitably results in liver damage which will progress from fibrosis to cirrhosis. Clinically discerned risk factors include premature birth, inflammation, sepsis, disruption of the enterohepatic circulation by creation of a proximal stoma, and the duration and type of parenteral nutritional support. Within the hepatocyte, the regulatory enzyme farsanoid receptor X (FXR) appears to play a pivotal role in the development of cholestasis. Recent studies have shown that its activity is suppressed by sepsis, and by plant phytosterols found in soy-based lipid preparations. This paradigm is reflected in the emerging consensus for the care of post-surgical neonates, which is based around a multi-disciplinary team approach. Using an algorithm-driven approach, an appropriate balance between caloric support and prevention of IFALD can be achieved. Further prospective studies are required to further refine the optimal sequence of use of these therapies and the long-term effects on neurological development and hepatic function. However, with optimal care, the number of IF patients progressing to end-stage liver disease because of IFALD should be very low.

Potential risks of maternal administration of Mucophylline on the pups of albino rats during lactation

The present study was undertaken to evaluate the potential risks of the mucolytic and broncholytic drug, Theophylline derivatives (Mucophylline) maternally administered on the pups. The nursing rats orally administered from 1st postpartum day (PPD) to 21th PPD with two different doses 30.83mg/kg (low dose) and 66.61mg/kg (Human equivalent dose (HED)). On the 21th PPD, the postnatal developmental signs, skeletal malformation and the histopathology of neonatal liver, kidney and brain were examined. Our results showed that Mucophylline induced a significant reduction in the neonatal weight and length, delayed, weak and incomplete ossification, wavy ribs and the neonatal liver revealed histopathological changes, pyknotic hepatocytes, cytoplasmic vacuolization, dilated sinusoid and necrotic area. Kidney revealed alternation changes, enlargement of the glomerulus, renal tubules degeneration and lymphatic infiltration. Brain (cerebral cortex and cerebellum) showed neurodegenerative changes, vacuolization of neuropil, congested and dilated blood vessel and dark stain neurons. Our results showed that the activities of non-enzymatic (GSH) and enzymatic (GST, CAT) antioxidants were insignificantly decrease in both neonatal brain and liver tissues of rats administered with 30.83mg/kg and 61.66mg/kg of Mucophylline and insignificant increase in MDA levels in both neonatal brain and liver tissues. However, significant reduction (P≤0.05) in the content of GR was recorded in neonatal brain tissue of rats administered with 30.83mg/kg and 61.66mg/kg of Mucophylline during lactation period in comparison with control. These support and proof the potential risks of the maternal administration of Mucophylline on pups.