Infants with biliary atresia exhibit an altered amino acid profile in their newborn screening.

INTRODUCTION: Biliary atresia (BA) is a rare progressive neonatal cholangiopathy with unknown pathophysiology and time of onset. Newborn Screening (NBS) in Germany is routinely performed in the first days of life to identify rare congenital diseases utilizing dried blood spot (DBS) card analyses. Infants with biliary atresia (BA) are known to have altered amino acid profiles (AAP) at the time point of diagnosis, but it is unclear whether these alterations are present at the time point of NBS. OBJECTIVES: We aimed to analyze amino acid profiles in NBS-DBS of infants with Biliary Atresia. METHODS: Original NBS-DBS cards of 41 infants who were later on diagnosed with BA were retrospectively obtained. NBS-DBS cards from healthy newborns (n = 40) served as controls. In some BA infants (n = 14) a second DBS card was obtained at time of Kasai surgery. AAP in DBS cards were analyzed by targeted metabolomics. RESULTS: DBS metabolomics in the NBS of at that time point seemingly healthy infants later diagnosed with BA revealed significantly higher levels of Methionine (14.6 ± 8.6 µmol/l), Histidine (23.5 ± 50.3 µmol/l), Threonine (123.9 ± 72.8 µmol/l) and Arginine (14.1 ± 11.8 µmol/l) compared to healthy controls (Met: 8.1 ± 2.6 µmol/l, His: 18.6 ± 10.1 µmol/l, Thr: 98.1 ± 34.3 µmol/l, Arg: 9.3 ± 6.6 µmol/l). Methionine, Arginine and Histidine showed a further increase at time point of Kasai procedure. No correlation between amino acid levels and clinical course was observed. CONCLUSION: Our data demonstrate that BA patients exhibit an altered AAP within 72 h after birth, long before the infants become symptomatic. This supports the theory of a prenatal onset of the disease and, thus, the possibility of developing a sensitive and specific NBS. Methionine might be particularly relevant due to its involvement in glutathione metabolism. Further investigation of AAP in BA may help in understanding the underlying pathophysiology.

Amyloid precursor protein as a fibrosis marker in infants with biliary atresia.

BACKGROUND: Biliary atresia (BA) is a rare condition of unknown origin in newborns with jaundice. In BA bile ducts are non-functional, causing neonatal cholestasis and following liver fibrosis and failure. METHODS: This retrospective study included liver biopsies of 14 infants with BA aged [mean ± SD] 63 ± 23 days. Patients were grouped according to the clinical course (jaundice-free vs recurrent jaundice vs required liver transplantation or liver fibrosis (Ishak fibrosis score)) and followed for 1.61-5.64 years (mean 4.03). Transcriptome profiles were assessed using a panel of 768 fibrosis-specific genes, reanalyzed via qRT-PCR, and confirmed via immunostaining. Plasma from an additional 30 BA infants and 10 age-matched controls were used for amyloid precursor protein (APP) quantification by ELISA. RESULTS: Different clinical outcome groups showed a homogeneous mRNA expression. Altered amyloid-metabolism-related gene expression was found between cases with Ishak fibrosis score greater than 4. Immunostaining confirmed a distinct presence of APP in the livers of all BA subjects. APP plasma levels were higher in BA than in age-matched controls and correlated with the histological fibrosis grade. CONCLUSIONS: These results suggest that amyloidosis may contribute to BA and liver fibrosis, indicating that APP could serve as a potential liquid biomarker for these conditions. IMPACT: Biliary atresia patients with higher fibrosis scores according to Ishak have higher hepatic expression of amyloid-related genes while amyloid precursor protein accumulates in the liver and increases in the circulation. After a recent study revealed beta-amyloid deposition as a mechanism potentially involved in biliary atresia, we were able to correlate amyloid-metabolism-related transcript levels as well as amyloid precursor protein tissue and plasma levels with the degree of hepatic fibrosis. These findings suggest that amyloid precursor protein is a fibrosis marker in infants with biliary atresia, reinforcing the role of amyloid metabolism in the pathogenesis of this serious disease.

Alpha-1-antitrypsin improves anastomotic healing in intestinal epithelial cells model.

PURPOSE: Intestinal anastomosis is a routine procedure in pediatric surgery, with leakage being a significant complication. Human alpha1-antitrypsin (AAT), whose physiological serum concentrations range from 0.9-2.0 mg/ml, is known to accelerate wound healing and stimulate the expression of cell proliferation-related genes. We hypothesized that AAT might enhance anastomotic healing. METHODS: In a monolayer of non-tumorigenic HIEC-6 epithelial cells derived from fetal intestine a scratch was created. Standard medium without (control) or with AAT (0.5 and 1 mg/ml) was added. Cells were observed using a Life-Cell Imaging System. Cell proliferation was assessed, and the expression of proliferation-related genes was measured by qRT-PCR. RESULTS: In the presence of AAT, the scratch closed significantly faster. Cells treated with 1 mg/ml AAT showed 53% repopulation after 8 h and 97% after 18 h, while control cells showed 24% and 60% repopulation, respectively (p < 0.02). The treatment with AAT induced HIEC-6-cell proliferation and significantly increased the mRNA-expression of CDKN1A, CDKN2A, ANGPTL4, WNT3 and COL3A1 genes. AAT did not change the mRNA-expression of CXCL8 but decreased levels of IL-8 as compared to controls. CONCLUSION: At physiological concentrations AAT accelerates the confluence of intestinal cells and increases cell proliferation. The local administration of AAT may bear therapeutic potential to improve anastomotic healing.

Pleural CD14+ monocytes/macrophages of healthy adolescents show a high expression of metallothionein family genes.

Nowadays laparoscopic interventions enable the collection of resident macrophage populations out of the human cavities. We employed this technique to isolate pleural monocytes/macrophages from healthy young adults who underwent a correction of pectus excavatum. High quality CD14+ monocytes/macrophages (plMo/Mφ) were used for RNA-sequencing (RNA-seq) in comparison with human monocyte-derived macrophages (MDM) natural (MDM-0) or IL-4-polarized (MDM-IL4). Transcriptome analysis revealed 7166 and 7076 differentially expressed genes (DEGs) in plMo/Mφ relative to natural MDM-0 and polarized MDM-IL4, respectively. The gene set enrichment analysis, which was used to compare RNA-seq data from plMo/Mφ with single-cell (scRNA-seq) data online from human bronchial lavage macrophages, showed that plMo/Mφs are characterized by a high expression of genes belonging to the metallothionein (MT) family, and that the expression of these genes is significantly higher in plMo/Mφ than in MDM-0 or MDM-IL4. Our results provide additional insights on high MTs-expressing macrophage subsets, which seem to be present not only in bronchial lavage of healthy adults or in pleural exudates of lung cancer patients but also in pleural fluid of healthy young adults. Macrophage subsets expressing high MTs may have specific roles in lung defense, repair, and homeostasis, and require further investigations.

Growth Factors Assessed during Kasai Procedure in Liver and Serum Are Not Predictive for the Postoperative Liver Deterioration in Infants with Biliary Atresia.

BACKGROUND: Biliary atresia (BA) is a neonatal cholangiopathy characterized by progressive destruction of the biliary system resulting in liver cirrhosis. Residual bile drainage can temporarily be achieved through Kasai portoenterostomy (KPE) and some children show long-term survival with their native liver. However, most children eventually require liver transplantation (LTX). As several growth factors (GF) and chemokines have been shown to promote fibrogenesis in the liver, we assessed whether GF are predictive for the course of disease. MATERIAL AND METHODS: Liver and sera samples were collected from 49 infants with BA during KPE. Levels of 13 different GF were measured by multiplex immunoassay. Patient outcomes were stratified into favorable (bilirubin < 20 µmol/L at 2-year follow-up) and unfavorable (LTX). GF levels were compared between groups by a t-test, correlation coefficients were calculated, and principal component analyses performed. RESULTS: Twenty-two patients showed a favorable and 27 an unfavorable disease course. No relation of GF and outcome could be established. In both groups, high levels of SDF-1alpha/CXCL12 (1473.0 ± 497.5 pg/mL), FGF2 (301.2 ± 207.8 pg/mL), and VEGF-a (209.0 ± 146.4 pg/mL) levels were measured within the liver, followed (in descending order) by PDGF-bb, LIF, GM-CSF, BDNF, VEGF-d, beta-NGF, IL-7, SCF, PIGF-1, and EGF. Serum marker levels showed much higher mean variation compared to hepatic values and no correlation to the protein microenvironment in the liver. CONCLUSIONS: Our study demonstrates high amounts of GF in livers from infants with BA at KPE, but no correlation to the outcome or serum values could be established. Our data suggest that local or systemic GF levels are unsuitable for prediction of the disease course. Collectively, we conclude that in BA the degree of proliferative activity caused by GF is a dismissible factor for the further course of disease.

Phenotypic Switch of Human Peritoneal Macrophages during Childhood.

INTRODUCTION: Human peritoneal macrophages are resident in the abdominal cavity where they support the specific microenvironmental regulation. We have previously observed a phenotypic switch of murine macrophages during infancy that was associated with a functional development. To investigate the age related changes in human peritoneal macrophages, we analyzed peritoneal macrophages of children undergoing laparoscopic procedures. MATERIALS AND METHODS: Immunologically healthy children who received minimally invasive surgery in our department were included in this study. In all cases, the written consent was obtained. At the beginning of laparoscopy, physiologic NaCl-solution was instilled and manually removed through the umbilical trocar to gain macrophages. Lavage cells were processed for flow cytometry analysis. CD14+ myeloid cells were monitored for specific lineage marker expression. RESULTS: A total of 21 donors (age: 7 days-18 years) were included and divided into three groups. In all age groups, 97% of myeloid cells expressed CD11b. 70% of these expressed CD14. Three subsets of CD14 cells were detected on the basis of CD14/CD16 expression (CD14 + CD16dim, CD14 + CD16inter, and CD14 + CD16high). In neonates, >80% belonged to the CD14 + CD16high subset, reducing to 30% in adolescents. In none of the cases, the M2 markers CD23 and CD25 were expressed. CONCLUSION: This is the first study showing that lineage marker expression of peritoneal macrophages in neonates differs from that in adults. The knowledge about neonatal tissue resident macrophages might help to understand their complex interaction and to use specific macrophage properties for therapeutic approaches.

Synthetic retinoid AM80 inhibits IL-17 production of gamma delta T cells and ameliorates biliary atresia in mice.

BACKGROUND & AIMS: Recent evidence suggests that Interleukin (IL)-17-producing gamma delta ( γδ ) T cells are the dominant pathogenic cellular component in designated autoimmune or inflammatory diseases, including biliary atresia (BA). We have previously demonstrated that retinoids effectively suppress T-helper cell (Th) 17 differentiation. METHODS: Here, we established an in vitro system, enabling investigations of the effect of AM80 on the IL-17 production of γδ T cells. Additionally, we tested the therapeutic effect of AM80 in the Rotavirus-induced mouse model of BA. Co-incubation of γδ T cells with IL-23 and anti-CD28 mAb proved most effective in inducing an IL-17 response in vitro. The effect of AM80 on human CCR6+CD26+ V δ 2 cells was assessed by flow cytometry. RESULTS: AM80 efficiently reduced IL-17 production by murine γδ T cells and the expression of the master transcription factor Retinoid-Orphan-Receptor- γ t (ROR γτ ) in a dose-dependent manner. The fraction of human CCR6+CD26+ V δ 2 cells was significantly reduced by co-incubation with AM80. Moreover, AM80 also inhibited IL-17 production by liver-infiltrating γδ T cells isolated from animals suffering from BA. Intraperitoneal treatment with AM80 ameliorated BA-associated inflammation. However, AM80 treatment was not sufficient to control disease progression in the murine model, despite reduced inflammatory activity in the animals. CONCLUSIONS: Retinoids are very efficient in down-regulating IL-17 production by γδ T cells in vitro and, to a lesser extent, in the BA mouse model. However, retinoids do not suffice for the control of disease progression. Thus, our data suggest that IL-17 is not the only factor contributing to the pathogenesis of BA. LAY SUMMARY: Biliary atresia (BA) is a rare disease which affects infants, causing progressive liver failure in most children, and is the most common indication for paediatric liver transplantation. We have previously demonstrated that IL-17, produced by γδ T cells, contributes to hepatic inflammation in the murine model of BA and is increased in the livers of infants suffering from the disease. In the study at hand, we demonstrate that treatment with AM80, a synthetic retinoid with superior pharmacological properties, effectively inhibits the IL-17 production of gamma delta T cells without generating systemic immunosuppression. Although all-trans retinoic acid (ATRA) has been demonstrated to suppress differentiation of IL-17-producing conventional T-helper cells (Th17) in vitro, the therapeutic application of ATRA in vivo is limited by the compound's potential side effects caused by its instability and lack of receptor specificity. Our study is the first to show that AM80 suppresses the IL-17 production of γδ T cells in a very efficient manner and that hepatic inflammation is ameliorated in mice suffering from BA. However, AM80 treatment does not suffice to block the disease progression. We conclude that factors other than IL-17 drive the progressive inflammation in BA. The addition of retinoids to the treatment regime of children suffering from BA might decrease the disease burden; however, further research is needed to clarify the pathomechanism and possible therapeutic interventions in humans.

Constitutive TNF-α signaling in neonates is essential for the development of tissue-resident leukocyte profiles at barrier sites.

Newborn infants have a high disposition to develop systemic inflammatory response syndromes (SIRSs) upon inflammatory or infectious challenges. Moreover, there is a considerable trafficking of hematopoietic cells to tissues already under noninflammatory conditions. These age-specific characteristics suggest a hitherto unappreciated crucial role of the vascular endothelium during the neonatal period. Here, we demonstrate that healthy neonates showed already strong endothelial baseline activation, which was mediated by a constitutively increased production of TNF-α. In mice, pharmacological inhibition of TNF-α directly after birth prevented subsequent fatal SIRS but completely abrogated the recruitment of leukocytes to sites of infection. Importantly, in healthy neonates, blocking TNF-α at birth disrupted the physiologic leukocyte trafficking, which resulted in persistently altered leukocyte profiles at barrier sites. Collectively, these data suggest that constitutive TNF-α-mediated sterile endothelial activation in newborn infants contributes to the increased risk of developing SIRS but is needed to ensure the postnatal recruitment of leukocytes to organs and interfaces.-Bickes, M. S., Pirr, S., Heinemann, A. S., Fehlhaber, B., Halle, S., Völlger, L., Willers, M., Richter, M., Böhne, C., Albrecht, M., Langer, M., Pfeifer, S., Jonigk, D., Vieten, G., Ure, B., von Kaisenberg, C., Förster, R., von Köckritz-Blickwede, M., Hansen, G., Viemann, D. Constitutive TNF-α signaling in neonates is essential for the development of tissue-resident leukocyte profiles at barrier sites.

Hepatocyte growth factor levels in livers and serum at Kasai-portoenterostomy are not predictive of clinical outcome in infants with biliary atresia.

Biliary atresia (BA) is characterized by progressive destruction of the biliary system leading to liver fibrosis and deterioration of liver function. Serum hepatocyte growth factor (HGF) has been shown to be increased in cirrhotic diseases including BA. The aim of this study was to investigate the prognostic value of HGF levels in sera and liver tissue for the further disease course. A total of 49 serum and liver samples from infants with BA were acquired during Kasai-portoenterostomy (KPE) and analyzed by multiplex immunoassay including HGF, as marker of liver regeneration, and Interleukin 6 (IL-6) as a marker of inflammation. Both mediators showed no correlation with the outcome defined as favorable (survival with native liver (SNL)) or, in contrast, rapid deterioration of liver function requiring transplantation. Our data suggest that the degree of liver regeneration indicated by high levels of HGF within the liver is a dismissible factor in the post-KPE disease course.

Impact of Capnoperitoneum on Renal Perfusion and Urine Production in Infant and Adolescent Pigs: Crystalloid versus Colloid Fluid Resuscitation.

BACKGROUND: Infants are likely to develop anuria during laparoscopy which is uncommon in older patients. The reason for this susceptibility remains unknown. We compared the impact of CO2 pneumoperitoneum on renal perfusion and urine production in piglets compared with adolescent pigs. We furthermore investigated the effects of different resuscitation strategies. MATERIALS AND METHODS: Male piglets (n = 21) were divided into four groups: (a) infant controls (n = 5), (b) infants with crystalloid restitution (n = 6), (c) infants with colloidal restitution (n = 5), and (d) adolescents with crystalloid restitution (n = 5). Animals were ventilated, the central vessels and ureters were cannulated, and the animals were subjected to a 3-hour, 10 mm Hg CO2 pneumoperitoneum followed by 2-hour resuscitation. Renal perfusion was assessed by fluorescent microspheres and the rate of urine flow was measured. RESULTS: Urine production significantly decreased after insufflation only in the infant crystalloid and adolescent group, but not in controls or infants treated with colloids. In the infant crystalloid group, urine production remained at levels below 20% of baseline throughout the experiment. In this group, the renal perfusion dropped significantly after the beginning of the capnoperitoneum and remained significantly reduced throughout the experiment. CONCLUSION: Our data indicates that capnoperitoneum impairs renal perfusion and urine production in infants. In moderate-pressure capnoperitoneum, this effect cannot be compensated by application of crystalloids but with colloids.

Long-term outcome and necessity of liver transplantation in infants with biliary atresia are independent of cytokine milieu in native liver and serum.

PURPOSE: Biliary atresia (BA) is a rare disease of unknown pathogenesis in infants characterized by an inflammatory, progressive destruction of the biliary system and deterioration of liver function. The standard treatment for BA is a Kasai-hepatoportoenterostomy (KPE). However, liver transplantation (LTX) becomes necessary in about 50-80% of cases. Therefore, some authors advocate for primary LTX in BA, but this would require early markers to predict which children would benefit from KPE or to show rapid progression to liver cirrhosis (RLC) instead. METHODS: Snap-frozen liver biopsies and sera samples of 57 infants with BA were collected during KPE. Clinical and follow-up data were assessed via the biliary atresia and related diseases registry (BARD-online.com). Protein-levels of 25 pro- and anti-inflammatory mediators of 49 infants were assessed via multiplex protein-immunoassay and analyzed by t-test as well as multidimensional principal component analysis. RESULTS: 22 different immunomodulatory mediators were detectable in livers of children with BA, while serum protein levels were very low to undetectable. Following KPE, 33 BA patients showed RLC that required early LTX, while 24 had favorable course of disease with long-term survival with native liver (SNL). There were no significant differences between RLC and SNL in terms of local (from liver samples) nor systemic (from sera) immunomodulatory mediators. Protein levels were much lower in sera than in livers without statistical correlation. CONCLUSION: Our data suggest that local or systemic immunomodulatory mediators are unsuitable for predicting the disease course of BA. Thus, no deduction for optimal treatment strategy can be drawn. Collectively, we conclude that in BA, the degree of inflammation and protein microenvironment in the liver at the time-point of KPE are dismissible factors for the future course of disease.

Dextran sodium sulfate (DSS) induces necrotizing enterocolitis-like lesions in neonatal mice.

BACKGROUND: Necrotizing enterocolitis (NEC) is an inflammatory bowel disease of preterm human newborns with yet unresolved etiology. An established neonatal murine model for NEC employs oral administration of lipopolysaccharides (LPS) combined with hypoxia/hypothermia. In adult mice, feeding dextran sodium sulfate (DSS) represents a well-established model for experimental inflammatory bowel disease. Here we investigated the effect of DSS administration on the neonatal murine intestine in comparison with the established NEC model. METHODS: 3-day-old C57BL/6J mice were either fed formula containing DSS or LPS. LPS treated animals were additionally stressed by hypoxia/hypothermia twice daily. After 72 h, mice were euthanized, their intestinal tissue harvested and analyzed by histology, qRT-PCR and flow cytometry. For comparison, adult C57BL/6J mice were fed with DSS for 8 days and examined likewise. Untreated, age matched animals served as controls. RESULTS: Adult mice treated with DSS exhibited colonic inflammation with significantly increased Cxcl2 mRNA expression. In contrast, tissue inflammation in neonatal mice treated with DSS or LPS plus hypoxia/hypothermia was present in colon and small intestine as well. Comparative analysis of neonatal mice revealed a significantly increased lesion size and intestinal Cxcl2 mRNA expression after DSS exposure. Whereas LPS administration mainly induced local neutrophil recruitment, DSS treated animals displayed increased monocytes/macrophages infiltration. CONCLUSIONS: Our study demonstrates the potential of DSS to induce NEC-like lesions accompanied by a significant humoral and cellular immune response in the small and large intestine of neonatal mice. The new model therefore represents a good alternative to LPS plus hypoxia/hypothermia administration requiring no additional physical stress.

Conventional alpha beta (αß) T cells do not contribute to acute intestinal ischemia-reperfusion injury in mice.

PURPOSE: Ischemia-reperfusion injury (IRI) is associated with significant patient mortality and morbidity. The complex cascade of IRI is incompletely understood, but inflammation is known to be a key mediator. In addition to the predominant innate immune responses, previous research has also indicated that αß T cells contribute to IRI in various organ models. The aim of this study was to clarify the role αß T cells play in IRI to the gut. METHODS: Adult wild-type (WT) and αß T cell-deficient mice were subjected to acute intestinal IRI with 30min ischemia followed by 4h reperfusion. The gene expression of pro-inflammatory cytokines was measured by qPCR, and the influx of leukocyte subpopulations in the gut was assessed via flow cytometry and histology. Pro-inflammatory cytokines in the serum were measured, and transaminases were assessed as an indicator of distant organ IRI. RESULTS: Intestinal IRI led to an increased expression of pro-inflammatory cytokines in the gut tissue and an influx of leukocytes that predominantly consisted of neutrophils and macrophages. Furthermore, intestinal IRI increased serum IL-6, TNF-α, and ALT/AST levels. The αß T cell-deficient mice did not exhibit a more significant increase in pro-inflammatory cytokines in the gut or serum following IR than the WT mice. There was also no difference between WT- and αß T cell-deficient mice in terms of neutrophil infiltration or macrophage activation. Furthermore, the increase in transaminases was equal in both groups indicating that the level of distant organ injury was comparable. CONCLUSION: An increasing body of evidence demonstrates that αß T cells play a key role in IRI. In the gut, however, αß T cells are not pivotal in the first hours following acute IRI as deficiency does not impact cytokine production, neutrophil recruitment, macrophage activation, or distant organ injury. Thus, αß T cells may be considered innocent bystanders during the acute phase of intestinal IRI.

IL-17A blockade or deficiency does not affect progressive renal fibrosis following renal ischaemia reperfusion injury in mice.

OBJECTIVES: IL-17A contributes to acute kidney injury and fibrosis. Therefore, we asked whether IL-17A deficiency or treatment with a IL-17A blocking antibody impacts severe renal ischaemia reperfusion injury (IRI) and the progression to chronic kidney disease (CKD). METHODS: IL-17A-deficient and wild-type (WT) mice underwent transient unilateral renal pedicle clamping for 45 min to induce IRI and subsequent renal fibrosis. Furthermore, a neutralizing anti-IL-17A antibody (mAb) was injected into WT mice before induction of renal IRI intravenously. On days 1, 7 and 21, inflammation, fibrosis, leukocyte infiltration and pro-inflammatory and pro-fibrotic cytokine expression were assessed in kidneys using histology, qPCR and flow cytometry. KEY FINDINGS: IL-17A was significantly increased after renal IRI in WT kidneys. Levels of pro-inflammatory (MCP-1) cytokine and pro-fibrotic (collagen 1α1, fibronectin) transcripts were similar in the experimental groups studied. IL-17A deficiency had no effect on renal T-cell influx or the number, inflammatory phenotype, or spatial distribution of macrophages. Similarly, administration of an IL-17A blocking antibody did not attenuate inflammation. CONCLUSIONS: Despite the effects of IL-17 in other inflammation models, neither genetic IL-17A deficiency nor treatment with an IL-17A blocking antibody attenuated IRI and progression to CKD. We conclude that in severe renal IRI IL-17A is not crucially involved in disease progression.

The viral dsRNA analogue poly (I:C) induces necrotizing enterocolitis in neonatal mice.

BACKGROUND: Necrotizing enterocolitis (NEC) is a life-threatening gastrointestinal disease in premature infants with high mortality and morbidity with uncertain pathogenesis. Recent research focused on the role of intraluminal bacteria and lipopolysaccharide (LPS). However, an additional role of viral agents in the pathogenesis of NEC has recently been postulated. We assessed the role of polyinosinic:polycytidylic acid (pIC) mimicking viral dsRNA in contributing to the development of NEC in neonatal mice. METHODS: Four-d-old C57BL/6J pups were stressed by asphyxia and hypothermia twice daily. Animals were either fed by formula only (FO), formula containing LPS or pIC. After 72 h, mice were euthanized, intestines harvested, and the severity of NEC was assessed. RESULTS: Breastfed mice showed no evidence of NEC. Very mild NEC-like lesions were observed in mice fed by FO. Supplementation of LPS or pIC to the formula led to increased intestinal tissue damage and inflammation compared with FO in a similar manner. CONCLUSION: Our study demonstrates the ability of viral factors to induce NEC in neonatal mice even in the absence of LPS. Furthermore, we present a new mouse model of pIC-induced NEC which may be used to obtain further mechanistic insights in the pathogenesis of this disease.

Interleukin 17, Produced by γδ T Cells, Contributes to Hepatic Inflammation in a Mouse Model of Biliary Atresia and Is Increased in Livers of Patients.

BACKGROUND & AIMS: Biliary atresia (BA) is a rare disease in infants, with unknown mechanisms of pathogenesis. It is characterized by hepatobiliary inflammatory, progressive destruction of the biliary system leading to liver fibrosis, and deterioration of liver function. Interleukin (IL) 17A promotes inflammatory and autoimmune processes. We studied the role of IL17A and cells that produce this cytokine in a mouse model of BA and in hepatic biopsy samples from infants with BA. METHODS: We obtained peripheral blood and liver tissue specimens from 20 patients with BA, collected at the time of Kasai portoenterostomy, along with liver biopsies from infants without BA (controls). The tissue samples were analyzed by reverse transcription quantitative polymerase chain reaction (PCR), in situ PCR, and flow cytometry analyses. BA was induced in balb/cAnNCrl mice by rhesus rotavirus infection; uninfected mice were used as controls. Liver tissues were collected from mice and analyzed histologically and by reverse transcriptase PCR; leukocytes were isolated, stimulated, and analyzed by flow cytometry and PCR analyses. Some mice were given 3 intraperitoneal injections of a monoclonal antibody against IL17 or an isotype antibody (control). RESULTS: Livers from rhesus rota virus-infected mice with BA had 7-fold more Il17a messenger RNA than control mice (P = .02). γδ T cells were the exclusive source of IL17; no T-helper 17 cells were detected in livers of mice with BA. The increased number of IL17a-positive γδ T cells liver tissues of mice with BA was associated with increased levels of IL17A, IL17F, retinoid-orphan-receptor C, C-C chemokine receptor 6, and the IL23 receptor. Mice that were developing BA and given antibodies against IL17 had lower levels of liver inflammation and mean serum levels of bilirubin than mice receiving control antibodies (191 µmol/L vs 78 µmol/L, P = .002). Liver tissues from patients with BA had 4.6-fold higher levels of IL17 messenger RNA than control liver tissues (P = .02). CONCLUSIONS: In livers of mice with BA, γδ T cells produce IL17, which is required for inflammation and destruction of the biliary system. IL17 is up-regulated in liver tissues from patients with BA, compared with controls, and might serve as a therapeutic target.

Distinct phenotypic features of neonatal murine macrophages.

Neonates rely on their innate immune system. Resident tissue macrophages are considered to be initiators and regulators of the innate immune response and thus, appear to be especially important to neonates. We hypothesized that the phenotype and function of neonatal tissue macrophages differ from their adult counterparts. Peritoneal macrophages from neonatal (<24 h) and adult (6 weeks old) C57BL/6J mice were isolated and analyzed by high-content chipcytometry. After stimulation for 6 h with LPS (0, 1, 10, 100 ng/mL), macrophage transcriptome was analyzed by microarray and cytokine release was measured using multiparametric bead assays. Antigen presenting capacity was compared by T-cell stimulation assays. We observed that neonatal murine peritoneal macrophages are characterized by selective lack of expression of F4/80, MHC class II, and costimulatory molecules (CD80, CD86). Furthermore, we found differences in the transcriptome between neonatal and adult macrophages, unstimulated and after LPS stimulation. Although neonatal macrophages showed a significantly increased secretion of proinflammatory cytokines upon LPS stimulation, their potential to induce T-cell proliferation was significantly reduced. In conclusion, we observed a distinct phenotype of the neonatal macrophage population. The specific functions of this macrophage population could help to understand the excessive inflammatory reactions observed in the very young.

Increased inflammatory reaction to intestinal ischemia-reperfusion in neonatal versus adult mice.

AIM: Neonate and preterm patients are threatened by exaggerated inflammation of the gut. This study tests the hypothesis that the neonatal gut is prone to inflammation, by comparing the inflammatory reaction of neonatal and adult murine intestine to ischemia and reperfusion. METHODS: Neonatal (4 days, n=36) and adult (4 weeks, n=12) C57BL/6J mice were randomly divided between ischemia-reperfusion (IR) and untreated controls (Con). In IR animal's intestinal ischemia was induced by clamping the superior mesenteric artery (30 minutes) followed by reperfusion (4 hours). After the experiment, RNA was extracted from the small intestines and the expression of the chemokines CXCL1/KC and CXCL2/MIP-2 were determined by quantitative real-time reverse transcription-polymerase chain reaction. Flow cytometry was used to analyze neutrophil influx (Live+ Ly-6G+ ) in isolated cell populations. RESULTS: We observed a strong increase in all measured proinflammatory endpoints after IR in both adult and neonatal mice. However, the inflammatory reaction was significantly stronger in neonatal murine intestines, with a significantly higher increase in CXCL1/KC expression and neutrophil accumulation as compared with adults (p<0.05). CONCLUSION: The intestines of neonatal mice reacted with an increased inflammatory response to the ischemic insult. This increased susceptibility could help to explain the exaggerated inflammation seen in diseases such as necrotizing enterocolitis.

Carbon dioxide modifies the morphology and function of mesothelial cells and facilitates transepithelial neuroblastoma cell migration.

BACKGROUND: The response of mesothelial cells to surgical trauma and bacterial contamination is poorly defined. We have recently shown that CO(2) pneumoperitoneum increases systemic metastasis of neuroblastoma cells in a murine model. Thus, we hypothesized that CO(2) alters the morphology and function of mesothelial cells and facilitates transmesothelial tumor cell migration. MATERIALS AND METHODS: Murine mesothelial cells were exposed to 100% CO(2) and 5% CO(2) as control. Scanning electron microscopy (SEM) investigations, as well as LPS-induced granulocyte-colony stimulating factor (G-CSF) production and mitochondrial activity (MTT assay) were measured. Transmesothelial migration of neuroblastoma cells (Neuro2a) was determined using a transwell chamber system. RESULTS: CO(2) incubation was associated with a significant destruction of the microvillar formation in SEM. Migration studies showed that the barrier function of the mesothelial monolayer decreased. A significantly increased migration of neuroblastoma cells was identified after 100% CO(2) exposure (P < 0.05). Although the conversion of MTT as an indicator of mitochondrial activity was only slightly and not significantly reduced after CO(2) incubation, the release of G-CSF induced by LPS was completely blocked during the incubation with 100% CO(2) (P < 0.05). The capacity of G-CSF release recovered after the incubation. CONCLUSION: We observed that peritoneal mesothelial cells lose their typical cell morphology by CO(2) incubation, which is accompanied by facilitated migration of neuroblastoma cells. Moreover, the synthesis of immunological factors is blocked, but this effect is not long lasting. These mechanisms may explain an increased metastasis rate of neuroblastoma cells after CO(2) pneumoperitoneum, which was recently observed in a murine model.

Obesity and the associated mediators leptin, estrogen and IGF-I enhance the cell proliferation and early tumorigenesis of breast cancer cells.

Breast cancer continues to be a major cause of cancer deaths in women. Estrogen, which is also produced by the adipose tissue, is held responsible for the elevated risk of breast cancer in obese women. However, the adipose tissue secrets hormones and adipokines such as leptin and IGF-I and these substances could also contribute to an increased breast cancer risk for obese women. In this study, the impact of obesity on cell proliferation was investigated. The carcinogen 7, 12, dimethylbenz[a]anthracene (DMBA) was administered to normal weight and diet-induced obese female Sprague-Dawley rats. Cell proliferation was evaluated by immunohistological staining of BrdU-incorporation. In the mammary glands and inguinal lymphatic nodes of the obese rats, cell proliferation was significantly increased, indicating a significant influence of obesity on breast cancer. Effects of leptin, estrogen, and IGF-I on the proliferation of MCF-7 cells in vitro were assessed using an MTT assay. Cell culture experiments demonstrated a mitogenic role of these three mediators on cell proliferation. Our data demonstrate a stimulative effect of substances produced by the adipose tissue on breast cancer. Body weight specific cell proliferation suggests that obesity-related adipokines and mediators enhance cell proliferation and increase the risk for breast cancer.