P. F. Heeckt

Human monocyte-derived neohepatocytes: a promising alternative to primary human hepatocytes for autologous cell therapy.

Background. There is growing interest in new therapeutic options for the treatment of end-stage liver diseases. In addition to mechanical devices supporting liver function, such as bioreactors, the transplantation of hepatocyte-like cells derived from (adult) stem cells offer great perspectives. We have generated hepatocyte-like (NeoHep) cells from terminally differentiated peripheral blood monocytes and, in this study, have evaluated these cells as a possible tool for autologous cell therapy. Methods. Peripheral blood monocytes were cultured under conditions that promote hepatocyte-like differentiation and were characterized for hepatocyte marker expression by reverse-transcriptase polymerase chain reaction, immunohistochemistry, and immunoblotting and for specific secretory and metabolic functions with the appropriate biochemical assays. Results. NeoHep cells resembled primary human hepatocytes with respect to morphology, expression of hepatocyte markers (albumin, cytochrome P450 isoenzymes, asialoglycoprotein receptor, coagulation factor VII), various secretory and metabolic functions (albumin secretion, urea production, lactate formation, and lactate dehydrogenase and aspartate transaminase release), and drug detoxification activities (phase I metabolization of ethoxycoumarin into 7OH-coumarin after stimulation with 3-methylcholanthren, induction of CYP3A4 activity, and phase II metabolization through UDP-glucuronidation of 4-methyl-umbelliferone). Conclusions. These data convincingly show that NeoHep cells display a phenotype and specific in vitro metabolic functions that are quantitatively and qualitatively comparable in part with those of primary human hepatocytes. These cells could thus be clinically applied in an autologous setting for the treatment of end-stage liver diseases or for improving liver function in patients who have undergone critical liver-mass resection.

Functional impairment of enteric smooth muscle and nerves caused by chronic intestinal allograft rejection regresses after FK506 rescue.

We have previously demonstrated that subclinical chronic rejection (CR) induces structural and functional alterations in enteric smooth muscle and nerves in a rat model of small intestinal transplantation. This study was designed to investigate the effect of prolonged FK506 rescue therapy on these sequelae of CR. Immunohistochemistry of BrdU-labeled muscle cells demonstrated that active proliferation of intestinal smooth muscle caused by CR was successfully aborted by FK506 rescue therapy after a period of 30 days (control = 0.14 +/- 0.09; CR = 30.4 +/- 1.73; rescue = 2.4 +/- 0.63 cells/jejunal cross-section, P < 0.01). However, FK506 did not reverse the already established increase in muscular thickness (control = 92 +/- 2.4; CR = 193 +/- 10.6; rescue = 188 +/- 8.1 microns) due to CR. Bethanechol stimulated circular muscle contractility was improved markedly with rescue therapy (maximal contractile force reached 39.5% of control values in CR grafts and 68.8% after rescue). Rescue therapy did not reverse the loss of NADPH-diaphorase positive myenteric ganglia (control = 37 +/- 1.4; CR = 28 +/- 2.9; rescue = 23 +/- 1.7 ganglia/cross-section). Despite the persistent loss of ganglia, inhibitory junction potentials (IJPs) improved significantly returning to control values with FK506 (control = 10 +/- 0.5; CR = 5 +/- 0.3; CR rescue = 10 +/- 0.7 mV; IJPs recorded at 1 pulse/150V/0.75 ms). Although structural changes in enteric smooth muscle and myenteric neurons induced by CR were not reversed, the progression of subclinical CR can be effectively curbed by FK506 rescue therapy. The improvement in muscular mechanics and inhibitory neural innervation is probably due to the cessation of infiltrating immunocytes and sprouting of remaining myenteric nerves.

Short-bowel syndrome - surgical treatment with long-term benefit?

Abstract Survival and management of patients with irreversible intestinal failure has been made possible by the development of total parenteral nutrition (TPN). Despite the progress of TPN severe and even fatal complications might occur. Different non-surgical and surgical therapies can be employed to either improve intestinal function or restore enteral autonomy to obviate the need for TPN. A comprehensive review of the pertinent literature on different treatment modalities for short-bowel syndrome and our own experience are presented to judge long-term benefit and make recommendations on up-to-date surgical management.

Heterotopic intestinal transplantation aggravates the insult of chronic rejection.

BACKGROUND Intestinal grafts are placed either heterotopically (out of continuity) or orthotopically (in continuity); the latter is believed to be advantageous, as intraluminal nutrients and intestinal secretions might modulate the intestinal immune status and possibly delay rejection. METHODS This study was designed to delineate the effects of heterotopic versus orthotopic allograft position on the morphology and function of intestinal smooth muscle in our rat model of chronic rejection. Syngeneic orthotopic grafts were evaluated to control for changes due to the transplantation process. RESULTS Histochemistry of the grafts muscularis externa showed a significant thickening due to hyperplasia and hypertrophy, which was most pronounced in heterotopic grafts (control = 92+/-2.4 microm, syngeneic grafts = 140+/-6.7 microm, orthotopic allografts = 278+/-26.6 microm, heterotopic allografts = 456+/-50 microm). In terms of function, muscle strips from allografts only generated 23% of the total bethanechol-induced contractile force in vitro compared to unoperated controls and syngeneic grafts. The mean resting membrane potential of control and isograft muscle cells was -69 +/- 0.9 mV with a slow-wave amplitude of 20+/-0.5 mV. Chronic rejection hyperpolarized the resting membrane potential of orthotopic allografts (-66 +/- 0.5 mV) and even more so of heterotopic allografts (-58 +/- 3.4 mV). Slow-wave amplitudes were decreased in orthotopic (14+/-0.9 mV) and nearly abolished in heterotopic allografts (2+/-1.2 mV). CONCLUSIONS Our data indicate that allografts in heterotopic position are most susceptible to the insult of chronic rejection exemplified by increased proliferative and hypertrophic transformation of intestinal smooth muscle and a marked decrease in mechanical and electrical activity.

Decreased mucosal IgA levels in ileum of patients with chronic ulcerative colitis.

Patients with chronic ulcerative colitis (CUC) are known to have decreased spontaneous IgA secretion by colonic mononuclear cells. The aim of this study was to determine whether a similar alteration exists in the apparently healthy ileum of patients with CUC. The concentration of IgA was measured in the supernatant from homogenized mucosal ileal biopsies using a sandwich-type ELISA. The concentration of IgA was significantly (P=0.025) decreased in the ileum of patients with CUC (N=24) in comparison to normal ileum (N=10). The number of mucosal IgA-containing mononuclear cells (MNC) was also determined using an avidin-biotin-immunoperoxidase technique on paraffin-embedded ileal sections. Although reduced, the number of positive cells and their distribution was not significantly different in the ileum of patients with CUC (N=20) when compared to normal ileum (N=10). We suggest that decreased mucosal IgA levels are a panintestinal condition in CUC and that this is a primary alteration rather than a secondary response to the inflammatory process. Considering the role of IgA, we propose that decreased mucosal IgA levels in CUC may predispose to the disease by a reduction of the immune-mediated exclusion mechanism and/or by an impairment of the down-regulation of the inflammatory response.

In situ Hybridisierung und RT-PCR zeigen eine erhöhte Freisetzung von IFN- γ in jejunaler Muskularis und Mukosa bei chronischer Abstoßung nach Dünndarmtransplantation

Chronische Abstosungsreaktionen stellen eine Hauptursache von Organdysfunktion nach Transplantation dar. Wir haben kurzlich gezeigt, das es wahrend chronischer Abstosungsreaktionen nach Dunndarmtransplantation zu deutlichen strukturellen Veranderungen innerhalb des Transplantates mit Schadigung vor allem in der intestinalen Muskulatur und den intrinsischen Nervenzellen sowie einer erhohten Freisetzung von MHC Klasse I und II-Molekulen kommt [1]. Es ist bekannt, das Interferon gamma (IFN-γ) ursachlich bei der Freisetzung von MHC Klasse I und II-Molekulen beteiligt und daruber hinaus ein potenter Makrophagen-stimulierender Faktor ist [2, 3]. Ziel dieser Studie war es, die erhohte Freisetzung von IFN-γ und die Aktivierung von Makrophagen unter chronischen Abstosungsbedingungen zu untersuchen und IFN-γ produzierende Zellen zu lokalisieren.

Niedermolekulares Heparin vermindert die Folgen chronischer Abstoßung an der intrinsischen Innervation allogener Dünndarmtransplantate

Wir haben kurzlich im Rattenmodell gezeigt, das bereits subklinische chronische Abstosungsreaktionen intestinaler Transplantate eine signifikante Destruktion der intrinsischen Nervenzellen mit Abnahme der neuromuskularen Transmission verursachen und damit zu Motilitatsstorungen fuhren. Chronische Abstosung erzeugt zudem eine Hyperplasie und Hypertrophie der intestinalen Muskulatur, sowie Schadigung der mechanischen Kontraktionskraft glatter Muskelzellen [1, 2]. Heparin vermag die Proliferation glatter vaskularer Muskelzellen und atherosklerotische Gefasveranderungen in Transplantaten zu hemmen [3,4]. Diese Eigenschaften konnten eine Minderung der negativen Effekte chronischer intestinaler Abstosung bewirken. In der vorliegenden Studie haben wir daher den Einflus niedermolekularen Heparins (Logiparin, Novo Nordisk A/S, Danemark) auf die morphologischen und funktionellen Veranderungen intestinaler Nerven und Muskulatur wahrend subklinischer chronischer Abstosung untersucht.

Einfluß der Dünndarmtransplantation und chronischen Abstoßung auf die Morphologie und Funktion der intestinalen Muskulatur

Die Dunndarmtransplantation ist seit Einfuhrung effektiver Immunsuppressiva klinisch moglich geworden und gewinnt zunehmend als definitive Therapie des Kurzdarmsyndroms an Bedeutung. Mit der erfolgreichen Unterdruckung akuter Abstosung durch Cyclosporin (CsA) und FK 506 kommt es zu langzeitigem Transplantatuberleben. Damit treten chronische Abstosung und zunehmende gastrointestinale Motilitatsstorungen des Transplantats in den Vordergrund [1]. Die vorliegende Studie wurde durchgefuhrt, um die bisher unbekannten Auswirkungen der Dunndarmtransplantation, ge¬folgt von chronischer Abstosung, auf die Morphologie und Funktion der intestinalen Muskulatur zu untersuchen.