Staffan Meurling

The instant blood-mediated inflammatory reaction characterized in hepatocyte transplantation.

Background. Hepatocyte transplantation (HcTx) has proven to be a safe procedure, although the functional results have been unsatisfactory, probably due to insufficient engraftment or a loss of transplanted mass or function. In this study, we investigate whether hepatocytes in contact with blood induce an inflammatory reaction leading to, similar to what happens in clinical islet transplantation, an instant blood-mediated inflammatory reaction (IBMIR) resulting in an early loss of transplanted cells. Methods. By using an experimental model that mimics the portal vein blood flow, we could study different parameters reflecting the effects on the innate immunity elicited by hepatocytes in contact with ABO-matched human blood. Results. We report that all aspects of the IBMIR such as platelet and granulocyte consumption, coagulation, and complement activation were demonstrated. Addition of various specific inhibitors of coagulation allowed us to clearly delineate the various stages of the hepatocyte-triggered IBMIR and show that the reaction was triggered by tissue factor. Analysis of a case of clinical HcTx showed that hepatocyte-induced IBMIR also occurs in vivo. Both the inflammatory and the coagulation aspects were controlled by low-molecular-weight dextran sulfate. Conclusion. Isolated hepatocytes in contact with blood induce the IBMIR in vitro, and there are indications that these events are also relevant in vivo. According to these findings, HcTx would benefit from controlling a wider range of signals from the innate immune system.

Fetal rat hepatocytes: isolation, characterization, and transplantation in the Nagase analbuminemic rats.

BACKGROUND In contrast to adult hepatocytes, fetal hepatocytes (FH) are thought to be highly proliferative, less immunogenic, and resistant to cryopreservation and ischemic injury. These qualities could enhance FH engraftment, proliferation, and gene transfer requiring active DNA synthesis. METHODS Rat FH were obtained using the nonperfusion collagenase/DNase digestion method. Free and cultured cells were studied using electron microscopy, fluorescence-activated cell sorting, and Northern analysis using alpha-fetoprotein and albumin as markers of hepatocyte lineage. DNA synthetic activity was measured in quiescent and mitogen-stimulated fetal and adult hepatocytes by [3H]thymidine incorporation. Susceptibility of cultured FH to retrovirally mediated gene transfer was studied using an amphotropic retroviral vector carrying the Escherichia coli lac-Z gene. Nagase analbuminemic rats were used as recipients to study the effects of intraportal FH transplantation. Analysis of serum albumin was carried out by enzyme-linked immunosorbent assay. RESULTS In fetal liver, 87+/-2% of the cells showed morphological and molecular features of hepatocytes. DNA synthetic activity in nonstimulated cultured FH was 10 times greater than the maximal hepatocyte growth factor-driven response in adult rat hepatocytes. A total of 5-15% FH stained positive for X-gal; results of transduction in adult hepatocyte cultures were negative. In Nagase analbuminemic rat recipients, FH produced significant amounts of albumin only when a hepatic regenerative stimulus was applied. Immunohistochemistry confirmed presence of albumin-positive hepatocytes. CONCLUSIONS Fetal rat liver from the late gestation period is highly enriched with hepatocyte progenitors. They are highly proliferative and susceptible to retroviral transduction and can engraft and function in the adult rat liver if transplanted under a hepatic regenerative stimulus.

Intestinal ischemia measured by intraluminal microdialysis

Abstract Objective. To evaluate the possibility of detecting intestinal ischemia by intraluminal microdialysis and comparing the ileum and colon. Methods. The studies were performed on male Sprague-Dawley rats. In the first part of the study, microdialysis catheters were placed in the sigmoid part of the colon and in the subcutaneous adipose tissue. In the second part of the study, microdialysis catheters were placed in the lumen of the ileum and the colon. The infrarenal aorta was clamped proximal to the cranial mesenteric artery. Microdialysate levels of glucose, lactate, pyruvate and glycerol were measured. Intestinal specimens were removed at the end of the ischemic period for microscopic evaluation. Results. Intraluminal microdialysis could detect early signs of ischemic injury in the ileum, as well as in the colon, with a marked increase of lactate, lactate/pyruvate ratio and glycerol. The increased levels of intraluminal glycerol showed a positive correlation to prolonged ischemia and to higher degrees of intestinal damage. Conclusion. Intraluminal measurement of glycerol is a good marker for intestinal ischemia. Intraluminal microdialysis in the colon is easily accessible through the rectum, and may prove to be a valuable clinical tool for diagnosing intestinal ischemia.

Outcome and management in infants with esophageal atresia – A single centre observational study

BACKGROUND/PURPOSE A successful outcome in the repair of esophageal atresia (EA) is associated with a high quality pediatric surgical centre, however there are several controversies regarding the optimal management. The aim of this study was to investigate the outcome and management EA in a single pediatric surgical centre. METHODS Medical records of infants with repaired EA from 1994 to 2013 were reviewed. RESULTS 129 infants were included. Median follow-up was 5.3 (range 0.1-21) years. Overall survival was 94.6%, incidences of anastomotic leakage 7.0%, recurrent fistula 4.6% and anastomotic stricture 53.5% (36.2% within first year). In long gap EA (n=13), delayed primary anastomosis was performed in 9 (69.2%), gastric tube in 3 (23.1%) and gastric transposition in one (7.7%) infants. The incidences of anastomotic leakage and stricture in long gap EA were, 23.1% and 69.2%, respectively. Peroperative tracheobronchoscopy and postoperative esophagography were implemented as a routine during the study-period, but chest drains were routinely abandoned. CONCLUSION The outcome in this study is fully comparable with recent international reports showing a low mortality but a significant morbidity, especially considering anastomotic strictures and LGEA. Multicenter EA registry with long-term follow up may help to establish best management of EA.

Control of IBMIR Induced by Fresh and Cryopreserved Hepatocytes by Low Molecular Weight Dextran Sulfate versus Heparin

Rapid destruction of hepatocytes after hepatocyte transplantation has hampered the application of this procedure clinically. The instant blood-mediated inflammatory reaction (IBMIR) is a plausible underlying cause for this cell loss. The present study was designed to evaluate the capacity of low molecular weight dextran sulfate (LMW-DS) to control these initial reactions from the innate immune system. Fresh and cryopreserved hepatocytes were tested in an in vitro whole-blood model using ABO-compatible blood. The ability to elicit IBMIR and the capacity of LMW-DS (100 μg/ml) to attenuate the degree of activation of the cascade systems were monitored. The effect was also compared to conventional anticoagulant therapy using unfractionated heparin (1 IU/ml). Both fresh and freeze–thawed hepatocytes elicited IBMIR to the same extent. LMW-DS reduced the platelet loss and maintained the cell counts at the same degree as unfractionated heparin, but controlled the coagulation and complement systems significantly more efficiently than heparin. LMW-DS also attenuated the IBMIR elicited by freeze–thawed cells. Therefore, LMW-DS inhibits the cascade systems and maintains the cell counts in blood triggered by both fresh and cryopreserved hepatocytes in direct contact with ABO-matched blood. LMW-DS at a previously used and clinically applicable concentration (100 μg/ml) inhibits IBMIR in vitro and is therefore a potential IBMIR inhibitor in hepatocyte transplantation.

Bacterial translocation from defunctionalized rat small bowel

Abstract Bacterial translocation from the intestine may cause severe infectious complications in a number of clinical situations, including the short bowel syndrome and after small bowel transplantation. The aim of the present study was to develop a simplified model for the study of bacterial translocation from a defunctionalized intestine. An ileal segment from untreated or cyclosporine-treated rats was exteriorized as a Thiry-Vella loop. After 1, 3, or 7 days, bacterial translocation and distribution of immunocompetent cells were assessed. The data obtained were compared with data from animal subjected to intestinal transplantation. Translocation to the mesenteric lymph nodes was detected in 60% of the Thiry-Vella loop animals on day 1, in 100% on day 3, and in 83% on dy 7; concomitantly, the number of macrophages and T-cells in the mesenteric lymph nodes increased from day 1 until day 7. The degree of bacterial translocation on day 3 and 7 in animals with a Thiry-Vella loop was comparable with that observed 7 days after intestinal transplantation. Furthermore, treatment with cyclosporine A enhanced the number of translocating bateria. In the model presented here bacterial translocation occurs from the small bowel to the mesenteric lymph nodes. The model offers possibilities to study the mechanisms and immunological phenomena associated with microbial translocation.