Lipid antigens in bile from patients with chronic liver diseases activate natural killer T cells.

Natural killer T (NKT) cells are an abundant subset of liver lymphocytes activated by lipid antigens presented on CD1d molecules that are expressed by cholangiocytes. We aimed to determine if bile from patients with chronic liver diseases contains antigenic lipids that can activate NKT cells. Using murine invariant (24.7, 24.8 and DN32.D3) and non-invariant (14S.6, 14S.7 and 14S.10) NKT hybridomas we investigated the presence of lipid antigens in bile collected from the gallbladder of patients undergoing liver transplantation due to end-stage liver disease. Biliary microbiota profiles were generated using 16S rRNA amplicon sequencing. We found that the patient bile samples contain antigens that activate both invariant and non-invariant NKT hybridomas (24.7, 24.8, DN32.D3, 14S.6, 14S.7 and 14S.10), as demonstrated by activation of at least one hybridoma by eight of 10 bile samples. Activation at high dilutions suggests that some antigens are highly potent. We used the non-invariant NKT hybridoma 14S.6 to screen 21 additional patient bile samples for NKT-reactivity and demonstrated that 12 of 21 bile samples resulted in activation, three of which gave a strong activation. Four of 12 activating bile samples contained microbial DNA. Our results reveal an immunological pathway that could be of critical importance in biliary immunology.

Validation of a cholangiographic prognostic model in primary sclerosing cholangitis.

BACKGROUND AND STUDY AIMS: We previously developed a prognostic model for primary sclerosing cholangitis (PSC), which was primarily based on a cholangiographic classification of the intra- and extrahepatic biliary tree lesions. The aim of the present study was to validate the performance of this model in an external cohort. PATIENTS AND METHODS: The validation dataset consisted of patients with PSC from a single referral center in Oslo, Norway. The patients' cholangiograms were scored according to the Amsterdam classification. We then examined whether adjusting the value of the original coefficients of the predictors or adding new predictors would improve the fit of the original model in the validation cohort. In addition, we evaluated calibration (closeness between observed and expected survival) and discrimination using the concordance index. RESULTS: A total of 111 patients (mean age 35 +/- 13 years; 76 % male) were included in the validation study. Baseline clinical characteristics were comparable between the two cohorts. None of the coefficients that were re-estimated in the validation cohort differed significantly from the values of the original model. Observed and expected survival curves were in close agreement across different risk groups. Discrimination of the original model was preserved in the validation cohort: the concordance index was the same in both cohorts. CONCLUSIONS: The prognostic model showed adequate performance in an independent series of patients. Therefore, we updated the model using the data from both cohorts to provide more robust estimates of transplant-free survival for individual patients. A nomogram was constructed, which can be used to predict medium- and long-term prognosis in individual patients with PSC.

Natural killer T cells mediate inflammation in the bile ducts.

Cholangiocytes function as antigen-presenting cells with CD1d-dependent activation of natural killer T (NKT) cells in vitro. NKT cells may act both pro- and anti-inflammatory in liver immunopathology. We explored this immune pathway and the antigen-presenting potential of NKT cells in the bile ducts by challenging wild-type and Cd1d-/- mice with intrabiliary injection of the NKT cell activating agent oxazolone. Pharmacological blocking of CD1d-mediated activation was performed with a monoclonal antibody. Intrabiliary oxazolone injection in wild-type mice caused acute cholangitis with significant weight loss, elevated serum levels of alanine transaminase, aspartate transaminase, alkaline phosphatase and bilirubin, increased histologic grade of cholangitis and number of T cells, macrophages, neutrophils and myofibroblasts per portal tract after 7 days. NKT cells were activated after intrabiliary injection of oxazolone with upregulation of activation markers. Cd1d-/- and wild-type mice pretreated with antibody blocking of CD1d were protected from disease. These findings implicate that cells in the bile ducts function as antigen-presenting cells in vivo and activate NKT cells in a CD1d-restricted manner. The elucidation of this biliary immune pathway opens up for potentially new therapeutic approaches for cholangiopathies.

Is a prolonged bleeding time associated with an increased risk of hemorrhage after liver biopsy?

Bleeding time determination is not advised as a general preoperative hemostasis screening test, but it might be useful in some patient groups. Patients referred for liver biopsy frequently have coagulation disturbances and are at risk of hemorrhage. In this prospective study 219 liver biopsies were carried out regardless of a prolonged bleeding time, but with minimum requirements for hemoglobin concentration, platelet count, and tests of the internal and external coagulation pathways. The bleeding time was prolonged in the case of 48 (22%) of the biopsies. Significant bleeding as defined by a hemoglobin decrease of > or =2.0 g/dl occurred in nine patients. Three of these patients were bone marrow transplanted. Patients with a prolonged bleeding time carried a five times higher risk of bleeding (odds ratio = 5.0; confidence interval = 1.1-21.8; p = 0.019). We conclude that the bleeding time may give additional information on the risk of bleeding in some patient groups undergoing liver biopsy.

Epidemiology of primary sclerosing cholangitis.

Many patients suffering from primary sclerosing cholangitis (PSC) have no symptoms--or rather unspecific symptoms. Most patients have a cholestatic biochemical profile, but a specific blood test for the diagnosis of PSC is lacking. The diagnostic test (endoscopic retrograde cholangiography (ERC)) is an invasive procedure with potential complications. Also, in some patients the diagnosis of PSC is not easy, even when ERC has been performed. Therefore true incidence and prevalence data on PSC are extremely few. Nevertheless, it seems well established that the epidemiology of PSC is not the same all over the world. PSC is most often seen in Northern Europe. In this part of the world PSC is also associated with inflammatory bowel disease in most cases--and in the Nordic Countries PSC has become the primary indication for hepatic transplantation.

Surgical treatment as a principle in patients with advanced abdominal carcinoid tumors.

Seventy-five patients with advanced abdominal carcinoid tumors (65 midgut, 10 others) have been examined retrospectively to evaluate the role of surgical treatment as a principle, irrespective of stage of disease. Eighteen of 52 patients (35%) exhibited the carcinoid syndrome. Two or more primaries were found in 39% of patients with midgut lesion, 81% of these patients had regional metastases, 5% of these patients had distant lymph node metastases, and 74% of the patients had liver secondaries. All patients underwent operation, an additional 34% of the patients had a further reoperation, 9% of the patients had a second reoperation, 3% of the patients had a third reoperation, and one patient (2%) had a fourth reoperation. Intraoperative debulking (liver excluded) was performed in 33% of the patients, and 48% of the patients had treatment (resection, hepatic artery ligation, embolization) directed at the liver. The postoperative mortality rate was 2% after the primary operation for midgut lesions. The median survival for midgut tumors was 92 months, compared to 40 months for other lesions (not significant). A significantly higher survival rate was revealed for those patients with midgut lesion who were undergoing intraabdominal debulking procedures (liver excluded); median survival was 139 months versus 69 months without debulking. For those patients with liver metastases, median survival after intervention was 216 months and 48 months without such treatment (p less than 0.001). It is concluded that resection of intraabdominal carcinoid tumor masses can be performed in a high proportion of patients. Despite the retrospective, uncontrolled nature of this study, the difference in survival probabilities in favor of aggressive surgical therapy is so marked that it is not unreasonable to conclude that surgery has played a role in prolonging life in these patients.

Involvement of guanylate cyclase and phosphodiesterases in the functional heterogeneity of human blood platelet subpopulations.

Cyclic nucleotides are involved in the regulation of platelet activation, shape change, and aggregation. In this study we have investigated the role of guanylate cyclase and phosphodiesterase in three functional heterogeneous human blood platelet subpopulations separated according to density. In low-density platelets aggregation was enhanced and inhibited less when cyclic GMP was increased by sodium nitroprusside, compared to high-density platelets. Low-density platelets possessed a lower basal level of cyclic GMP and exhibited a small increase in cyclic GMP after stimulation with sodium nitroprusside. Cyclic GMP-dependent phosphodiesterase activity was similar in high, low, and intermediate-density platelets. In contrast, the activity of the cyclic AMP-dependent phosphodiesterase was higher in low-density compared to high and intermediate-density platelets. These results suggest that regulation of cyclic GMP and cyclic AMP levels plays an important role in the functional heterogeneity of human blood platelets.

Secondary Osteoporosis in Liver Transplant Recipients: a Longitudinal Study in Patients With and Without Cholestatic Liver Disease.

BACKGROUND: Metabolic bone disease is one of the major long-term complications in liver transplant recipients, but it remains unclear which patients are at highest risk for developing severe bone disease following transplantation. METHODS: A total of 46 consecutive, adult patients with chronic liver disease accepted for a liver transplantation waiting list were prospectively included in the study. The patients were classified into two groups: group A-chronic cholestatic liver disease (n = 28), and group B-chronic non-cholestatic liver disease (n = 18). Bone mineral density (BMD) was measured at acceptance for the waiting list and at 3, 12 and 36 months following transplantation. Markers of bone turnover (serum-bone specific alkaline phosphatases (bALP), s-osteocalcin, s-l-collagen-C-terminal telopeptide (1-CTP) and urine N-terminal telopeptides u-Ntx) were measured at acceptance and at 3, 6, 12, 24 and 36 months following transplantation. BMD and markers of bone turnover were compared with similar values in a matched control group of 42 healthy individuals. RESULTS: BMD decreased significantly during the early post-transplantation period (median bone loss femoral neck (FN) 3 months post-transplant 8.5%). BMD levels declined slightly from 3 to 12 months following transplantation and increased thereafter. The relative bone loss was greatest among group B patients (relative bone loss FN 3 months post-transplant: group A, 8% versus group B, 13%; P = 0.04). At 36 months, 8/17 group A and 2/9 group B patients had BMD levels that exceeded the pretransplant levels (P = 0.12). The early bone loss was positively correlated with an increase in resorption markers (s-1-CTP and u-Ntx). Group B had higher levels of both s-1-CTP and u-Ntx at 3 and 6 months post-transplant than group A patients (P = 0.03). Bone formation markers increased slowly from 6 months post-transplant and onwards. Relative bone loss was positively correlated to total glucocorticoid dose during the first 3 months post-transplant. There were no differences in BMD between patients receiving tacrolimus versus those receiving'cyclosporin A. CONCLUSION: Bone loss following liver transplantation is considerable in patients with both cholestatic and non-cholestatic liver disease, the first group has the poorest starting-point while the latter group has the greatest bone loss following transplantation. Bone loss is closely correlated with biochemical markers of bone resorption and total dose of glucocorticoids given post-transplant.

Detection of antineutrophil cytoplasmic antibodies in primary sclerosing cholangitis: a comparison of the alkaline phosphatase and immunofluorescent techniques.

BACKGROUND: The reported prevalence of antineutrophil cytoplasmic antibodies (ANCA) in primary sclerosing cholangitis (PSC) varies considerably (26-85%). Part of this may reflect methodological differences but part may reflect the differences in the patient groups analysed. To resolve this issue we compared the sensitivity and specificity of the immunoalkaline phosphatase (IALP) and immunofluorescence (IF) techniques in four different populations. METHOD: Sera from four centres were tested blind on alcohol-fixed neutrophils using both techniques. PATIENTS: USA: 14 PSC, 14 primary biliary cirrhosis (PBC); Sweden: 32 PSC, 3 autoimmune hepatitis (AIH), 14 PBC, 11 chronic liver disease; Norway: 32 PSC, 14 AIH, 13 PBC, 1 hepatitis C. Italy: 8 PSC, 14 PBC, 8 viral hepatitis. Thirty-six normal healthy volunteers from Oxford, together with positive and negative controls, were also tested. RESULTS: The healthy controls were all ANCA negative. The diagnostic sensitivity and specificity, respectively, of ANCA for PSC using the IALP technique for the different test sera were: USA 71% and 93%, Sweden 66% and 96%, Norway 69% and 46%, Italy 50% and 95%. The diagnostic sensitivity and specificity, respectively, of the IF technique on the same sera were: USA 50% and 86%, Sweden 56% and 86%, Norway 47% and 61%, Italy 50% and 91%. Overall, combining all four groups, detection of ANCA using the IALP technique gave a diagnostic sensitivity of 66% with a specificity of 74% for PSC. In contrast, the IF technique gave an overall diagnostic of only 51% (P = 0.044, compared with IALP) with a specificity of 73%. Although overall the IALP technique was more sensitive than IF, the differences in sensitivity and specificity between the two techniques did not reach statistical significance for any individual group. Furthermore, the small differences in sensitivity between the four groups using either technique were not significant. However, the IALP technique had greater specificity in the US, Swedish and Italian groups compared with the Norwegian group (P < 0.05) whereas no statistically significant differences in specificity were noted between the groups using the IF technique. CONCLUSION: This study shows that the IALP method of ANCA detection is at least as sensitive as IF for the serological diagnosis of PSC. Indeed, combining data from all four centres, the IALP technique was significantly more sensitive than IF. We therefore recommend the use of the IALP technique, which is also easier to interpret and does not require the use of a specialist fluorescent microscope. The lack of a wide variation in sensitivity between IALP and IF for any individual patient group reported in this study suggests that the previously reported regional differences in ANCA prevalence in PSC of between 26% and 85% may be patient, related, rather than due to ethnic or methodological differences in ANCA detection, perhaps reflecting possible disease heterogeneity within PSC, or case selection bias. Further studies are needed to investigate this intriguing possibility. Such differences, if confirmed, will need to be taken into account when assessing the use of ANCA as a serological marker of PSC.

Diminution of postprandial release of pancreatic polypeptide in myotonic dystrophy.

The meal-stimulated release of pancreatic polypeptide (PP), gastrin, somatostatin and glucagon was studied in nine patients with myotonic dystrophy (MD) and in 11 healthy controls. PP-release was significantly reduced in MD compared to controls. This reduction may be related to the abnormal gut motility demonstrated in MD. The release of gastrin, somatostatin and glucagon was not significantly different in the two groups.

Etiology and pathogenesis in primary sclerosing cholangitis.

The etiology and pathogenesis of the inflammatory and fibrotic bile duct lesions characteristic of primary sclerosing cholangitis (PSC) is unknown, but several lines of evidence support the contention that genetic and immunologic factors are involved. There is an association with human leukocyte antigens (HLA) with an increased frequency of DR3, DR6, and DR2 positive haplotypes. DRB3*0101(DR52a) is the most strongly associated allele in some studies, but the HLA gene conferring the primary HLA associated susceptibility to PSC remains to be established. There is an aberrant expression of HLA class II antigens (DR and DP) on bile duct epithelial cells, with the potential to present antigens to the surrounding T-lymphocytes. A defective suppressor T-cell function has been suggested in some studies. The patients may have elevated levels of circulating immune complexes, immunoglobulins, and non-organ-specific autoantibodies. Antibodies to perinuclear antigens (pANCA) are present in about 80% of cases. Increased metabolism of complement C3, reduced clearance of immune complexes, and increased concentration of biliary immune complexes have been found. The strong association between PSC and ulcerative colitis (UC) has not been explained. The detection of circulating IgG antibodies against a specific epitope shared by epithelial cells in the bile ducts and colon in about two-thirds of PSC patients may be of importance. Portal bacteremia secondary to a diseased bowel may possibly contribute to development of liver disease in UC. Viral infections and toxic and ischemic factors have also been implicated in the pathogenesis of PSC. In conclusion, PSC seems to occur in genetically predisposed individuals, mediated by immunologic mechanisms. The primary event triggering the disease development is, however, unknown.

The effect of trimipramine, cimetidine and atropine on gastric secretion.

A comparison was made of increasing doses of trimipramine (0.05-0.40 mg/kg/h), atropine (7-28 microgram/kg/h) and cimetidine (0.30-2.40 mg/kg/h) on the gastric secretion stimulated by 3 microgram/kg/h of histamine dihydrochloride as continuous infusion, each dose step lasting 30 minutes. In 9 healthy subjects it was found that trimipramine had no significant effect on the output of acid. The largest dose of atropine caused a reduction by 77%, and cimetidine a reduction of 89% during the last 15 minutes portions. The study suggests that the healing of peptic ulcer by trimipramine is not linked to the effect on the histamine-stimulated gastric acid secretion.

Dopaminergic and adrenergic influence on gastric acid and pepsin secretion stimulated by food. The role of vagal innervation.

Dopaminergic and adrenergic mechanisms were studied in a dog model which made possible physiological stimulation by food and comparison of vagally innervated and denervated acid and pepsin response at the same time. Five dogs were equipped with two pouches separated from the stomach, and stimulation was done by a standard meal - a mixture of liver, heart and bonemeal , 10 g/kg. The meal was combined with different doses of dopamine 0.1 - 2.0 - 20.0 - 40.0 micrograms/kg/min and with single doses of phentolamine, propranolol and sulpiride separately and combined with dopamine. Acid and pepsin secretion were inhibited in dose-response manner by dopamine in innervated mucosa, but all other effects of the compounds were different in parietal cells and chief cells and in vagally innervated and denervated mucosa.

Incidence of malignancies of the Billroth II operated stomach. A prospective follow-up.

In 1976 endoscopy with at least 20 biopsies and cytology were performed in 108 patients 20-25 years after partial gastrectomy (Billroth II). In one patient advanced carcinoma and in three cases severe dysplasia or carcinoma in situ, were found. At the follow-study in 1979, 7 patients had died of causes other than gastric carcinoma. A re-examination with endoscopy, biopsies and cytology were performed in fifty-eight patients. The present study did not show a progress of dysplasia in the gastric remnant during the three years of follow-up. The observations may suggest that re-examinations with gastroscopy and multiple biopsies every 3-5 years may be satisfactory in detecting carcinoma of the gastric remnant.

The effect of gastric bypass operation on glucose tolerance in obesity.

A series of variables involved in glucose handling were monitored before and after gastric bypass operation for morbid obesity. Blood glucose, insulin, C-peptide, gastric inhibitory polypeptide (GIP), pancreatic polypeptide (PP), and gastrin were measured basally and after an oral glucose load. Blood glucose, insulin, C-peptide, and PP were also measured after an intravenous glucose load. Adrenocortical function was evaluated by measuring plasma cortisol and urinary excretion of 17-hydroxy-corticosteroids and 17-ketosteroids. Nine subjects were examined before and 3 and 12 months after operation. Glucose tolerance improved postoperatively concomitant with decreased basal levels of C-peptide and insulin, increased hepatic insulin extraction, and evidence of reduced adrenocortical function. Parallel with reduced insulin resistance, support for an increase in both insulin secretion and removal was obtained postoperatively. It is concluded that the considerable endocrine abnormalities seen in morbid obesity can be normalized after gastric bypass operation and weight reduction.

Follow-up and prospective studies of the classification of liver disease.

Two hundred patients with different liver diseases were observed during a period of 6-8 years. The diagnosis at the first hospitalization was based on morphological criteria (and, in some cases, additional clinical information). In 162 of the cases an initial 'specific' diagnosis could be made. By the time of the follow-up study the diagnosis was confirmed in 73% of them. In 22 of 38 patients who were initially unclassifiable, the diagnosis was made definite by the follow-up study. Eighty-five patients were hospitalized for re-examination 6-8 years after the initial study. Several of the liver diseases initially had quite typical patterns of clinical chemical data. Allocation by discriminant analysis was therefore in good agreement with the morphological classification. The follow-up study showed that several patients with initially atypical patterns of clinical chemical results had their diagnosis changed. In 35 patients with the final diagnosis of chronic active hepatitis (CAH) or primary biliary cirrhosis (PBC), laboratory data from the last hospitalization were used for discriminant analysis with teaching data from the initial study. Ninety-seven per cent were correctly allocated, and we conclude that these patients retain recognizable patterns of laboratory results for several years, even when given immunosuppressive treatment. The potential clinical usefulness of discriminant analysis of laboratory data for differential diagnosis was evaluated by a prospective study of 65 patients with the morphological diagnosis of CAH or PBC. Correspondence with the morphological classification system was found in almost 90% of the cases.