Sclerosing and obstructive cholangiopathy in biliary atresia: mechanisms and association with biliary innate immunity.

Biliary atresia (BA) is histologically characterized by a progressive, sclerosing cholangitis and the obstruction of extrahepatic bile ducts. In terms of the etiology and pathogenesis of BA, several viral infections consisting of dsRNA, including Reoviridae, have been implicated. Human biliary epithelial cells (BECs) possess an innate immune system consisting of Toll-like receptors (TLRs). BECs have negative regulatory mechanisms of TLR tolerance to avoid an excessive inflammatory response to lipopolysaccharide (LPS), a TLR4 ligand; however, they lack the tolerance to poly(I:C) (a synthetic analog of viral dsRNA), a TLR3 ligand. Treatment with poly(I:C) induces the expression of the apoptosis-inducer TNF-related apoptosis-inducing ligand (TRAIL), along with the antiviral molecule IFN-ß1, and reduces the viability of BECs by enhancing apoptosis. In response, surviving BECs increase their expression of various markers, including basic FGF [an epithelial-mesenchymal transition (EMT)-inducer], S100A4 (a mesenchymal marker), and Snail (a transcriptional factor), and decrease that of epithelial markers such as CK19 and E-cadherin before undergoing EMT. Extrahepatic bile ducts in BA infants frequently show a lack of epithelial markers and an aberrant expression of vimentin, in addition to the enhancement of TRAIL and apoptosis. dsRNA viruses may directly induce apoptosis and EMT in human BECs as a result of the biliary innate immune response, supporting the notion that Reoviridae infections may be directly associated with the pathogenesis of cholangiopathies in BA.

Dendritic Cells Regulate Treg-Th17 Axis in Obstructive Phase of Bile Duct Injury in Murine Biliary Atresia.

Several cell types are considered to be effector cells in bile duct injury in rhesus rotavirus (RRV)-induced experimental biliary atresia (BA). Here, we identified an increased T helper 17 (Th17) cell population in a BA mode. By depleting the Th17 cells, the BA symptoms (onset of jaundice, acholic stools and retarded growth) were attenuated and the survival rate was improved. Furthermore, we found that in mice with BA, the percentage of CD4+CD25highFoxp3+ T regulatory (Treg) cells decreased along with the increased percentage of Th17 cells. However, the absolute numbers of Treg and Th17 cells were both increased in liver of RRV-injected mice compared to saline-injected mice. The proportion of Th17 cells at 7 days post-infection was decreased if Treg cells isolated from normal adult mice, but not Treg cells from the livers of mice with BA, were intraperitoneally transferred on day 5 of life. In vitro experiments also showed that Treg cells from mice with BA had a diminished suppressive effect on Th17 cell generation. To determine the mechanisms, we investigated the production of cytokines in the liver. The level of IL-6, which has been shown to be abundantly secreted by activated dendritic cells (DCs), was remarkably elevated. Importantly, in a Treg/Th17 cell suppression assay, IL-6 was demonstrated to paralyze the Treg cells' suppressive effect on Th17 cells and eventually the unrestrained increase of Th17 cells contributed to bile duct injury. In conclusion, the DC-regulated Treg-Th17 axis, probably in conjunction with other effector T cells, aggravates progressive inflammatory injury at the time of ductal obstruction.

Liver radiofrequency ablation compromises the biological gut barrier.

AIM: Liver radiofrequency ablation (RFA) has been shown to disrupt the mechanical component of the gut barrier. The aim of the present study was to investigate the consequences of liver RFA on the biological gut barrier in terms of the effects of bile production rate and bowel inflammatory state on intestinal microflora balance. METHOD: A total of 25 New Zealand rabbits were assigned to five groups (n = 5 per group): group CBD: subjected to common bile duct (CBD) extracorporeal bypass; group CBD-RFA: subjected to CBD bypass plus one session of open liver RFA; group RFA: subjected to liver RFA; group sham: subjected to sham operation; and group TBD: subjected to total bile deviation (TBD). In groups CBD and CBD-RFA, bile production rate was assessed for 48 h. In groups sham and RFA, measurement of biliary glycine conjugates of cholic and deoxycholic acid levels, histopathologic examination of the non-ablated liver tissue, morphometric analysis, and histopathologic examination of the terminal ileum and microbiological analysis of fecal and tissue samples collected from the jejunum and the cecum (and in group TBD) were performed at 48 h post-operation. RESULTS: One session of liver RFA resulted in ablation of 18.7 ± 2.7% of liver weight. Following liver RFA, bile production rate was reduced, while the levels of biliary bile salts were not affected. There was mild injury of the non-ablated liver parenchyma, mild intestinal wall inflammation, intestinal mucosa atrophy, and intestinal microbial population overgrowth. CONCLUSION: Reduced in bile production and mild bowel inflammation secondary to liver RFA impaired the biological gut barrier as manifested by intestinal microflora imbalance.

Ethyl pyruvate prevents intestinal inflammatory response and oxidative stress in a rat model of extrahepatic cholestasis.

BACKGROUND: Ringer's ethyl pyruvate solution (REPS) has been shown to ameliorate liver injury in a murine model of extrahepatic cholestasis. The goal of the present investigation was to gain additional information about whether infusing REPS instead of Ringer's lactate solution (RLS) after inducing obstructive jaundice would be beneficial to intestinal barrier function, inflammatory response, and oxidative stress. METHODS: Male Sprague Dawley rats were divided into three groups: Group Sham (n=6), sham-treated controls; Group RLS (n=9), common bile duct ligation (CBDL) plus RLS; and Group REPS (n=9), CBDL plus REPS. On 14 d after BDL, the rats were sacrificed and intestinal permeability was analyzed. Ileal IL-6 and TNF-alpha levels, malondialdehyde (MDA), glutathione (GSH), myeloperoxidase (MPO), and NF-kappaB activity were determined. Histologic examination and apoptosis of ileum were also examined. RESULTS: Relative to sham-treated controls, CBDL in RLS-treated rats were associated with increased intestinal permeability to FITC-labeled dextran (4.51+/-0.85 versus 0.44+/-0.18, P<0.01), histopathologic damage and apoptosis (68.4+/-13.4 versus 6.7+/-1.9 pre-1000 villi cells, P<0.01). IL-6 and TNF-alpha level, MDA, MPO, and NF-kappaB activity in ileal tissues were also promoted, along with decreased GSH levels. Treatment with REPS significantly decreased intestinal permeability (3.37+/-0.71, P<0.01) and apoptosis (42.8+/-14.3 pre-1000 villi cells, P<0.01). Other changes were also significantly attenuated by treatment with REPS after CBDL. CONCLUSIONS: The present study demonstrates that administration of REPS, but not RLS, maintains intestinal barrier function and reduces intestinal oxidative damage, inflammatory response, and apoptosis in cholestatic rats. This effect of ethyl pyruvate may be useful for preventing intestinal injury in patients with biliary obstruction.

Invariant natural killer T cells suppress the neutrophil inflammatory response in a mouse model of cholestatic liver damage.

BACKGROUND & AIMS: NK1.1(+) TCRalphabeta(int) CD1-restricted T (NKT) cells are a unique subset of T lymphocytes that are believed to have an immunoregulatory role in a wide range of diseases. Most mouse NKT cells express a T-cell receptor that contains an invariant Valpha14Jalpha18 chain and recognizes antigenic glycolipids presented in association with major histocompatibility complex class Ib (CD1d) molecules. These invariant NKT (iNKT) cells have been implicated in cholestatic liver injury. METHODS: We examined the role of iNKT cells in liver injury associated with biliary obstruction in mice with ligations of the common bile duct. RESULTS: The number of activated iNKT cells increased markedly in the livers of mice following bile duct ligation. Plasma alanine aminotransferase levels, an indicator of liver injury, were significantly higher in iNKT cell-deficient (Jalpha18(-/-)) mice compared with wild-type mice following bile duct ligation. Photo image analysis of histologic sections confirmed that more damage was present in the livers of Jalpha18(-/-) mice; liver damage correlated with increases in keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2) production as well as neutrophil sequestration. Liver injury was significantly reduced in Jalpha18(-/-) mice treated with anti-KC and anti-MIP-2 or rendered neutrophil deficient before bile duct ligation. Similarly, Jalpha18(-/-) mice that were injected with iNKT cells before bile duct ligation exhibited significant decreases in neutrophil accumulation and liver damage. CONCLUSIONS: These data document the role of iNKT cells in suppressing the neutrophil proinflammatory response and neutrophil-dependent cholestatic liver damage.

LPS-binding protein mediates LPS-induced liver injury and mortality in the setting of biliary obstruction.

It is generally accepted that low levels of lipopolysaccharide (LPS)-binding protein (LBP) augment the cell's response to LPS, whereas high levels of LBP have been shown to inhibit cell responses to LPS. Clinical studies and in vitro work by our group have demonstrated that, in the setting of liver disease, increased or acute-phase levels of LBP may actually potentiate rather than inhibit an overwhelming proinflammatory response. Therefore, in the present studies we sought to determine the role of acute-phase LBP in mediating morbidity and mortality in animals challenged with LPS in the setting of biliary obstruction. Using LBP-deficient mice and LBP blockade in wild-type mice, we demonstrate that high levels of LBP are deleterious in the setting of cholestasis. Following biliary obstruction and intraperitoneal LPS challenge, hepatic injury, hepatic neutrophil infiltration, and mortality were significantly increased in animals with an intact LBP acute-phase response. Kupffer cell responses from these animals demonstrated a significant increase in several inflammatory mediators, and Kupffer cell-associated LBP appears to be responsible for these differences, at least in part. Our results indicate that the role of LBP signaling in inflammatory conditions is complex and heterogeneous, and elevated levels of LBP are not always protective. Increased LBP production in the setting of cholestatic liver disease appears to be deleterious and may represent a potential therapeutic target for preventing overwhelming inflammatory responses to LPS in this setting.

Mechanism of neutrophil accumulation in sinusoids after extrahepatic biliary obstruction.

BACKGROUND/AIMS: Polymorphonuclear neutrophil (PMN) infiltration represents a potential source of liver injury, but the precise mechanisms of PMN infiltration in cholestatic liver are not fully understood. METHODOLOGY: This study investigated hepatic expression of cytokine-induced neutrophil chemoattractant (CINC) 14 days after bile duct ligation, as well as the number of infiltrated PMNs in livers. Portal venous endotoxin levels were also evaluated. Furthermore, in vitro CINC production by isolated liver cells from obstructive jaundice (OJ) liver or sham-treated liver was evaluated after stimulation with tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta) or LPS. RESULTS: The number of infiltrated PMNs in sinusoids significantly increased in OJ liver, as compared to sham-treated liver. CINC mRNA expression was also increased in OJ liver. Immunohistochemical study revealed that the majority of the CINC-positive cells were hepatocytes. In vitro study proved that CINC production by isolated hepatocytes was markedly enhanced by IL-1beta stimulation in OJ liver. Furthermore, IL-1beta production by LPS-stimulated Kupffer cells isolated from OJ liver was significantly increased, compared to those from sham-treated liver. Portal venous endotoxin was detectable only in OJ rats. CONCLUSIONS: Excessive production of IL-1beta by activated Kupffer cells, as a result of portal endotoxemia, may play an important role for increased CINC release from hepatocytes in cholestatic liver, leading to PMN infiltration.

Hepatic macrophages promote the neutrophil-dependent resolution of fibrosis in repairing cholestatic rat livers.

BACKGROUND: Cholestatic liver injury from extrahepatic biliary obstruction is well characterized by inflammatory and fibrogenic mechanisms. Little is known, however, about mechanisms required to reverse injury and effect liver repair. We sought to determine the cellular and molecular requirements for repair after biliary decompression, focusing on the role of hepatic macrophages in regulating inflammation and matrix resolution. METHODS: Male Sprague-Dawley rats underwent bile duct obstruction for 7 days followed by ductular decompression. Rats were treated with gadolinium chloride (GdCl(3)) to deplete the macrophage populations 24 or 48 hours before decompression. Liver tissue obtained at the time of decompression or after 2 days of repair was processed for morphometric analysis, immunohistochemistry, quantitative RT-PCR and in situ hybridization. RESULTS: GdCl(3) treatment for either 24 or 48 hours before decompression reduced the numbers of ED2(+) Kupffer cells and ED1(+) inflammatory macrophages in obstructed livers; only 48 hours of pretreatment, however, reduced the neutrophil counts. Furthermore, 48-hour GdCl(3) pretreatment blocked matrix degradation. Quantitative polymerase chain reaction demonstrated decreased cytokine-induced neutrophil chemoattractant-1 (CINC-1; CXCL1) and intercellular adhesion molecule-1 mRNA expression after GdCl(3) treatment and the elimination of hepatic macrophages. Immunohistochemistry and in situ hybridization revealed that neutrophils and CINC-1 mRNA localize within regions of fibrotic activity during both injury and repair. CONCLUSION: We conclude that the macrophage population is not directly involved in fibrotic liver repair. Rather, hepatic macrophages regulate the influx of neutrophils, which may play a direct role in matrix degradation.

TRAIL mediates liver injury by the innate immune system in the bile duct-ligated mouse.

UNLABELLED: The contribution of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a death ligand expressed by cells of the innate immune system, to cholestatic liver injury has not been explored. Our aim was to ascertain if TRAIL contributes to liver injury in the bile duct-ligated (BDL) mouse. C57/BL6 wild-type (wt), TRAIL heterozygote (TRAIL(+/-)), and TRAIL knockout (TRAIL(-/-)) mice were used for these studies. Liver injury and fibrosis were examined 7 and 14 days after BDL, respectively. Hepatic TRAIL messenger RNA (mRNA) was 6-fold greater in BDL animals versus sham-operated wt animals (P < 0.01). The increased hepatic TRAIL expression was accompanied by an increase in liver accumulation of natural killer 1.1 (NK 1.1)-positive NK and natural killer T (NKT) cells, the predominant cell types expressing TRAIL. Depletion of NK 1.1-positive cells reduced hepatic TRAIL mRNA expression and serum alanine aminotransferase (ALT) values. Consistent with a role for NK/NKT cells in this model of liver injury, stress ligands necessary for their recognition of target cells were also up-regulated in hepatocytes following BDL. Compared to sham-operated wt mice, BDL mice displayed a 13-fold increase in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and an 11-fold increase in caspase 3/7-positive hepatocytes (P < 0.01). The number of TUNEL and caspase 3/7-positive cells was reduced by >80% in BDL TRAIL knockout animals (P < 0.05). Likewise, liver histology, number of bile infarcts, serum ALT values, hepatic fibrosis, and animal survival were also improved in BDL TRAIL(-/-) animals as compared to wt animals. CONCLUSION: These observations support a pivotal role for TRAIL in cholestatic liver injury mediated by NK 1.1-positive NK/NKT cells.

[Autoimmune pancreatitis with normal IgG4-Levels: 4 case reports and review of the literature]. / Autoimmune Pankreatitis bei normalem IgG4: 4 Fallberichte und Literaturübersicht.

We report four cases of autoimmune pancreatitis in an 18-, a 22- and a 26-year-old male patient and a 20-year-old female patient. The 20-year-old female patient was admitted to the hospital with upper abdominal pain and jaundice, the 18-year-old patient with recurrent acute pancreatitis and cholestasis, the 26-year-old patient with right upper abdominal pain for four weeks and laboratory findings suggesting an acute pancreatitis. The 22-year-old patient presented with painless jaundice. EUS-guided fine needle aspiration was performed in all patients. The cytological findings and the EUS were decisive for the diagnosis of autoimmune pancreatitis in all four cases. In contrast, no patient showed elevated IgG4, or antibodies for carboanhydrase-II, for lactoferrin, or rheumatoid factor, serum markers reported to be positive in autoimmune pancreatitis. All patients were treated successfully with steroids, one patient relapsed after discontinuing the steroid medication and required renewed therapy. These case reports demonstrate that autoimmune pancreatitis should be considered in the differential diagnosis in cases of pancreatitis and/or jaundice also in western countries. As demonstrated, the diagnosis should not be based solely on the elevation of IgG4 or autoantibodies.

[Autoimmune pancreatitis--a surgical disease?]. / Die autoimmune Pankreatitis--eine chirurgische Krankheit?

The term autoimmune pancreatitis (AIP) describes a nonalcoholic, chronic lymphoplasmocytic pancreatitis. The lymphoplasmocytic infiltration is characterized by periductal localization of predominantly CD4-positive T cells, fibrosis, and acinar atrophy, frequently resulting in stenosis of the main pancreatic and distal common bile ducts. Imaging studies often reveal a diffuse narrowing of the pancreatic main duct and swelling of the pancreatic head wrongly suggesting the presence of a malignant tumor. Clinical signs include mild abdominal pain, jaundice, recurrent episodes of acute pancreatitis, and even new-onset diabetes mellitus. Additionally, AIP can be associated with other autoimmune diseases such as Sjögren's syndrome, primary sclerosing cholangitis, chronic inflammatory bowel diseases, and retroperitoneal fibrosis. Serological markers include autoantibodies and increased levels of gamma globulin and especially IgG4. Steroids seem to be effective in improving clinical symptoms as well as in the resolution of pancreatic and bile duct narrowing. This distinguishes AIP from other forms of pancreatitis and from pancreatic neoplasms. Further studies of the underlying pathophysiologic mechanisms, prognosis, and new diagnostic tools are needed to provide adequate and effective treatment in the future. In this article, we summarize the current knowledge about AIP and present 17 cases that underwent surgical resection at our institution from 2003 to 2004.

Biliary pancreatitis-associated ascitic fluid activates the production of tumor necrosis factor-alpha in acinar cells.

OBJECTIVE: Acute pancreatitis is associated with increased cytokine release from different cell sources. We have investigated the ability of acinar cells, in comparison with inflammatory peripheral blood cells, to produce tumor necrosis factor (TNF)-alpha in response to pancreatitis-associated ascitic fluid (PAAF). DESIGN: Controlled, randomized animal study. SETTING: University research laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Flow cytometry using phycoerythrin-labeled monoclonal anti-TNF-alpha antiserum. MEASUREMENTS AND MAIN RESULTS: PAAF (20%, v:v) obtained from rats with acute pancreatitis induced by bile-pancreatic duct obstruction significantly increased TNF-alpha production in acinar cells, as measured by flow cytometry using phycoerythrin-labeled monoclonal anti-TNF-alpha antiserum. Neither heating of PAAF nor the addition of soybean trypsin inhibitor or neutralizing amounts of anti-TNF-alpha monoclonal antiserum reduced the acinar cell TNF-alpha production. Monocytes and lymphocytes did not produce TNF-alpha in response to PAAF. Likewise, the typical monocyte and lymphocyte stimulating factors-lipopolysaccharide (10 microg/microL) and phorbol 12-myristate 13-acetate (250 ng/mL) plus ionomycin (1 microg/mL), respectively-were not able to produce TNF-alpha in acinar cells. By comparison of the two acinar cell populations differentiated by flow cytometry, R2 cells (with higher forward scatter values) showed a greater ability to produce TNF-alpha in response to PAAF than R1 cells. Acinar cell nuclear factor-kappaB was activated, but TNF-alpha production was not totally inhibited in presence of N-acetyl cysteine (30, 100 mM). CONCLUSIONS: The production of TNF-alpha from different cell sources is selectively activated. PAAF may be involved in the pathophysiology of acute pancreatitis by TNF-alpha production in acinar cells through mechanisms partially mediated by nuclear factor-kappaB activation. PAAF components, such as TNF-alpha or trypsin, are not responsible for acinar cell activation. TNF-alpha was induced by heat-resistant PAAF factors, displaying acinar cells with higher forward scatter (R2) a greater ability to increase the TNF-alpha production than R1 cells.

Reduced oncotic necrosis in Fas receptor-deficient C57BL/6J-lpr mice after bile duct ligation.

Neutrophils aggravate cholestatic liver injury after bile duct ligation (BDL). Recently, it was suggested that hepatocellular apoptosis might be critical for liver injury in this model. To test the hypothesis that apoptosis could be a signal for neutrophil extravasation and injury, we assessed parameters of apoptosis and inflammation after BDL using 2 different approaches: (1) wild-type and Fas receptor-deficient lpr mice of the C57BL/6J or C3H/HeJ strains, and (2) treatment with the pancaspase inhibitor z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk)in C3HeB/FeJ mice. After BDL for 3 days, total cell death was estimated to be between 10% and 50% of all cells evaluated. However, less than 0.1% of hepatocytes showed apoptotic morphology in all 3 strains. Processing of procaspase-3, caspase-3 enzyme activities, and immunohistochemical staining for cytokeratin 18 cleavage products indicated no activation of caspases. Real-time reverse-transcriptase polymerase chain reaction analysis revealed increased expression of many inflammatory mediators but no effect on proapoptotic genes. More than 50% of all accumulated neutrophils were extravasated and colocalized with foci of oncotic hepatocytes and chlorotyrosine adducts. z-VAD-fmk treatment had no effect on apoptosis or liver injury after BDL but eliminated apoptosis after galactosamine/endotoxin in C3HeB/FeJ mice. In Fas receptor-deficient lpr mice (C57BL/6J), expression of inflammatory mediators, neutrophil accumulation and extravasation, chlorotyrosine adduct formation, and liver injury were reduced. This protection was not observed in lpr mice of the endotoxin-resistant C3H/HeJ strain. In conclusion, liver injury (oncotic necrosis) after BDL correlated with the severity of the inflammatory response. The minimal amount of apoptosis had no effect on inflammation or on the overall injury.

Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice.

Obstruction of the common bile duct in a variety of clinical settings leads to cholestatic liver injury. An important aspect of this injury is hepatic inflammation, with neutrophils as the prominent cell type involved. However, the pathophysiologic role of the infiltrating neutrophils during cholestatic liver injury remains unclear. Therefore, we tested the hypothesis that neutrophils contribute to the overall pathophysiology by using bile duct-ligated (BDL) wild-type animals and mice deficient in the beta(2) integrin CD18. In wild-type animals, neutrophils were activated systemically as indicated by the increased expression of Mac-1 (CD11b/CD18) and L-selectin shedding 3 days after BDL. Histologic evaluation (48 +/- 10% necrosis) and plasma transaminase levels showed severe liver injury. Compared with sham-operated controls (< 10 neutrophils per 20 high-power fields), large numbers of neutrophils were present in livers of BDL mice (425 +/- 64). About 60% of these neutrophils had extravasated into the parenchyma. In addition, a substantial number of extravasated neutrophils were found in the portal tract. In contrast, Mac-1 was not up-regulated and plasma transaminase activities and the area of necrosis (21 +/- 9%) were significantly reduced in CD18-deficient animals. These mice had overall 62% less neutrophils in the liver. In particular, extravasation from sinusoids and portal venules (PV) was reduced by 91% and 47%, respectively. Immunohistochemical staining for chlorotyrosine, a marker of neutrophil-derived oxidant stress, was observed in the parenchyma of BDL wild-type but not CD18-deficient mice. In conclusion, neutrophils aggravated acute cholestatic liver injury after BDL. This inflammatory injury involves CD18-dependent extravasation of neutrophils from sinusoids and reactive oxygen formation.

The anti-inflammatory effects of circulating fatty acids in obstructive jaundice: similarities with pregnancy-induced immunosuppression.

Rheumatoid arthritis (RA) is ameliorated during both obstructive jaundice and pregnancy. Previous studies of polymorphonuclear leukocyte (PMN) function during pregnancy have shown reductions in the stimulated release of arachidonic acid (AA) and leukotriene B4 (LTB4), and lower NADPH oxidase activity. These changes may account for the amelioration of RA. The cause of this reduction in PMN function appears to be a progressive change in circulating fatty acids (FA), with a reduction in polyunsaturated FA, predominantly AA. The NADPH oxidase responsible for the respiratory burst has a direct requirement for polyunsaturated FA, particularly AA. We investigated whether the same changes in PMN function and FA, occur during obstructive jaundice. Patients with biliary obstructions were investigated before and after surgical correction (n=14). Obstructive jaundice caused significant changes in the proportions of serum and cellular FA. There was a striking reduction in polyunsaturated FA, particularly AA (48% in serum, p<0.001; 42% in PMNs, p<0.001) and an increase in mono-unsaturated oleic acid (24% in serum, p<0.001; 15% in PMNs, p<0.005). Similar changes occurred in mononuclear cell FA. Jaundice also caused a significant reduction in PMN function. Respiratory burst activity was reduced by between 32% and 38% in response to physiological and non-physiological stimuli, and there were similar significant reductions in the release of AA and LTB4. These changes in stimulated PMN function were evident whether or not the cells were first primed with tumour necrosis factor alpha (TNFalpha). Incubation of PMNs from healthy donors in pooled serum from patients with obstructive jaundice caused a reduction of 32% in cellular AA and 38% in NADPH oxidase activity. These findings support the idea that circulating FA can regulate PMN inflammatory responsiveness. The FA-induced attenuation in PMN activity in both jaundice and pregnancy may explain their ameliorating effects upon RA.

Deficiency of the expression of CD45RA isoform of CD45 common leukocyte antigen in CD4+ T lymphocytes in children with infantile cholestasis.

The immunological background of the pathological changes that appear in infantile cholestasis (infections, inflammatory process in the liver) is largely unknown. With the use of double color flow cytometry, we assessed the distribution of functionally different lymphocyte subpopulations in the peripheral blood of 29 infants with extra and intra-hepatic cholestasis (12 and 17 patients, respectively), aged from 1 to 8.6 months. Control group consisted of 15 age-matched, healthy infants. We examined: (1) the expression of CD3, CD4, CD8, CD19 lymphocyte surface receptors; and (2) the distribution of lymphocyte subsets with distinctive surface Ag characteristics of 'naive' (CD45RA+) and 'memory' (CD45RO+) cells in both CD4+ and CD8+ cell populations. The surface markers expression was evaluated in terms of percentage of positive cells and receptor density. The following changes in the expression of lymphocyte surface markers are described: (1) a decrease in the percentage of total CD3+, CD4+ cells but normal percentage of CD8+ cells and elevated proportion of CD19+ B cells; (2) a reduction of the proportion of 'naive' CD4+ lymphocytes but normal percentage of 'naive' CD8+ as well as 'memory' CD4+ and CD8+ cell subsets; (3) a decrease in density of CD3, CD4+, CD8 receptors, and D45RA isoform in a subset of 'naive' CD4+ cells. We conclude that deficiency of 'naive' CD4+ T cell subset which possess important effector and immunoregulatory functions, and low expression of certain lymphocyte receptors known to be engaged in T cell activation, possibly reflect a defect of cell mediated immunity that may account for viral and bacterial infections, often observed in infants with cholestasis.

Cytokine characteristics of jaundice in mouse liver.

OBJECTIVE: The aim of this study is to clarify the perioperative cytokine changes and their mechanism in jaundiced liver. MATERIALS AND METHODS: Obstructive jaundice was induced using a common bile duct ligation (CBDL) and a two-thirds hepatectomy (HEP) was performed in six- to seven-week-old male C3H/HeN mice. When hepatectomy was added to CBDL, it was carried out 2 to 5 days after CBDL. The serum interleukin 6 (IL-6) levels and heat shock protein (HSP)-70 expression were evaluated. One mg per mouse of methylprednisolone (MPL) was intraperitonealy administered in some mice of CBDL+HEP group. RESULTS: The post-hepatectomy IL-6 values at 2 and 3 days after CBDL were significantly lower than those in the HEP group, while those at 5 days after CBDL were significantly higher than those in HEP group. The serum IL-6 value of the steroid group was significantly lower than that of non-steroid group in HEP group. However, no steroid effects were recognized on post-hepatectomy IL-6 values at 3 days after CBDL, steroid inhibited post-hepatectomy IL-6 production at 5 days after CBDL. No expression of HSP70 protein was observed in the control group, but HSP70 protein was expressed in both the hepatocytes and Kupffer cells 3 days after CBDL, then at 5 days after CBDL, no HSP70 protein was expressed in the Kupffer cells. CONCLUSIONS: In the early period of obstructive jaundice, the IL-6 level after hepatectomy did not increase in comparison to HEP group, and steroid had no effect on IL-6 level. According to the progression of obstructive jaundice, the IL-6 level after hepatectomy increased to a higher level than before, and the effect of MPL was restored. HSP70 is thus suggested to have an important role in cytokine production.

Effects of vitamin E on neutrophil phagocytosis during experimental obstructive jaundice.

BACKGROUNDS/AIMS: Depression of non-specific immunity is one of the systemic complications of biliary obstruction. Vitamin E, which decreases during prolonged obstructive jaundice, may be beneficial to diseased function of neutrophils. In this study we want to investigate changes in neutrophil phagocytosis and the effect that vitamin E supplementation has on this function METHODOLOGY: Rats were divided into 5 groups as follows: the control group and 4 other groups that underwent double ligation and division of the common bile duct. Two of these 4 groups (Group 3 and 5) received vitamin E during the experiment. Alkaline phosphatase, aspartate aminotransferase, bilirubin serum levels, white blood cell count and neutrophil phagocytosis index were determined for group 2 and 3 at the end of the 15 days and for group 1, 4 and 5 at the end of the 21 days. RESULTS: There was a significant increase in white blood cell counts and biochemical parameters in group 2, 3, 4, and 5 (P < 0.05). Neutrophil phagocytosis index significantly increased 15 days after bile duct ligation (P < 0.001) (Group 2) and significantly decreased 21 days after bile duct ligation (P < 0.001) (Group 4). Neutrophil phagocytosis index in vitamin E pretreated groups were significantly decreased at the end of the 15 days (P < 0.001) (Group 3) and increased at the end of the 21 days (P < 0.001) (Group 5). CONCLUSIONS: Finally, If vitamin E is administered for further days and weeks of prolonged jaundiced, neutrophil phagocytosis index improves.

Progress in developing animal models for biliary atresia.

Animal models for extrahepatic biliary atresia (EHBA) have failed to simulate the course of the disease. Until now only a few aspects of the entity could be investigated and no model was helpful in discovering the etiology of EHBA. Following the suspicion of a viral and hepatotropic infection, investigations in an infectious mouse model were continued. The results of previous and topical studies are summarized here. Infection of newborn Balb/c-mice with rhesus rotavirus (RRV) leads to cholestasis in 85% of the animals followed by a lethality of 90%. Preparation and histomorphological investigation of liver and ligamentum duodenale reveal EHBA of varying extent. Clinical course and morphological findings in mice are very similar to EHBA in newborn children and the results are presented in a chronological table. Hepatobiliary morbidity and lethality after RRV infection is higher in Balb/c-mice than in other mouse strains. This observation supports the suspicion that immunocompetence might be a determining factor in the etiology of EHBA. Initial therapeutic trials were made using this model by treating infected newborn mice with interferon-alpha (IFN). The prophylactic application of IFN protects the infected mice from cholestatic symptoms and appears to induce partial immunity. Their descendants are protected against the hepatotropic effect of RRV infection. Infected animals presenting with clinical signs of cholestasis can be treated successfully by IFN-therapy for one week. In the presented animal model. EHBA can be better induced and simulated than by any other method. As a first trial, a non-surgical and more etiologically orientated therapeutic method is tested in this model.

Conclusive evidence of endotoxaemia in biliary obstruction.

BACKGROUND: Endotoxaemia is implicated in the pathophysiology of obstructive jaundice. The EndoCab enzyme linked immunosorbent assay (ELISA) is a novel assay which measures endogenous antibody (IgG) to the inner core region of circulating endotoxins (ACGA). AIMS: To investigate the significance of endotoxaemia in biliary obstruction using the EndoCab assay and assess the specificity of the humoral response to endotoxin compared with an exogenous antigenic challenge (tetanus toxoid, TT). METHODS: Three groups of adult male Wistar rats were studied: no operation, sham operation, and bile duct ligation for 21 days (BDL). In the second study, rats rats received prior immunisation with TT. RESULTS: In the preliminary experiment, plasma ACGA was significantly increased in the BDL group (306.6 (18.3)% versus 119.9 (6.7)% and 105.2 (4.6)% in the sham and no operation groups, respectively; p < 0.001). Although the mean endotoxin concentration in the BDL group was greater than that in the control groups this was not significant. There was a strong positive correlation between ACGA and endotoxin concentrations (p = 0.0021). In the second study mean ACGA after 21 days of BDL was significantly elevated (267.1 (31.2)% versus 101.6 (21.2)% at baseline, p < 0.0001). ACGA was unaffected in the other two groups. TT antibody concentrations fell in all three groups; only in the BDL group was the fall significant (97.6 (5.3)% versus 78.8 (4.2)% at baseline, p < 0.05). CONCLUSIONS: The specific rise in ACGA supports the hypothesis that endotoxin has an integral role in the pathophysiology of obstructive jaundice. The production of anticore glycolipid antibodies specifically reflects systemic endotoxaemia in this model. The EndoCab assay provides a novel, sensitive, and specific method for endotoxin detection.