Cross-species transplantation tolerance: rat bone marrow-derived cells can contribute to the ligand for negative selection of mouse T cell receptor V beta in chimeras tolerant to xenogeneic antigens (mouse + rat----mouse).

Mixed xenogeneic bone marrow reconstitution (mouse + rat----mouse) results in stable mixed lymphopoietic chimerism (1-48% rat), long-term survival, and the induction of stable functional donor-specific transplantation tolerance to xenoantigens in vivo. To examine the role of negative selection of potentially xenoreactive T lymphocytes during tolerance induction across a species barrier, mixed xenogeneic chimeras (mouse + rat----mouse) were prepared and analyzed using a mixture of mouse and rat bone marrow cells for relative T cell receptor (TCR)-V beta expression on mouse T cells. In mixed xenogeneic chimeras (B10 mouse + rat----B10 mouse), T cell maturation proceeded normally in the presence of rat bone marrow-derived elements, and functional donor-specific tolerance to rat xenoantigens was present when assessed by mixed lymphocyte reactivity in vitro. V beta 5, which is expressed at high (undeleted) levels in normal B10 mice, was consistently deleted in B10 recipients of Wistar Furth (WF), but not F344 rat bone marrow, whereas the coadministration of either F344 rat or WF rat bone marrow with B10 mouse bone marrow cells resulted in a significant decrease in expression of TCR-V beta 11. Taken together, these data demonstrate for the first time that rat bone marrow-derived cells can contribute in a strain-specific manner to the ligand for negative selection of specific mouse TCR-V beta during tolerance induction across a species barrier.

Nitric oxide production in host-versus-graft and graft-versus-host reactions in the rat.

The present study was designed to determine whether .N = O produced in vivo during the rejection of histoincompatible tissues might permit serum NO2-/NO3- levels to serve as markers of a rejection reaction. Rat syngeneic and allogeneic liver, heart, bone marrow/spleen cell, small bowel, skin, and sponge matrix grafts were performed and the stable end-products of .N = O, NO2-/NO3-, were serially assayed in the serum of the grafted animals. A significant rise of serum NO2-/NO3- levels in the allografted animals preceded the onset of clinical signs of rejection or graft-versus-host disease, with the exception of the skin and sponge matrix graft models, where elevated serum NO2-/NO3- levels were never observed. In all transplant models, normal serum NO2-/NO3- levels were observed at all times in animals that received syngeneic grafts. Furthermore, treatment of allograft recipients with the immunosuppressive agents FK 506 or cyclosporine A inhibited .N = O production. Determination of serum creatinine levels demonstrated that the elevated serum NO2-/NO3- levels were not caused by kidney dysfunction. Serum NO2-/NO3- levels might be useful early serum markers of the initiation of a rejection reaction or graft-versus-host disease when functional markers of graft dysfunction are not apparent.

[Role of stem cells in adult hepatic regeneration]. / Die neuen Wege des Prometheus: Adulte Leberstammzellen.

Central to the successful surgically treatment numerous liver diseases is the ability of the organ to regenerate. The understanding of the process of self-renewal has both changed and progressed over the last few decades. For many years, the assumption was that the liver regenerates primarily through the division of mature liver cells. However, over the last few years there has been increasing evidence of the participation of stem cells. Intrahepatic stem cells, so-called oval cells, are activated under conditions of severe or chronic liver disease and originate from the canals of Hering. In addition, extrahepatic stem cells may migrate from the bone marrow into the liver when the regenerative capacity of the liver itself is depleted. It is not yet fully clear how the different stem cell populations interact with both each other and the mature liver cell population to achieve homeostatic cell and differentiation equilibrium in the diseased and/or regenerating organ. In any case, the outstanding growth potential of liver stem cells may become a clinically viable option in the field of cell transplantation.

Prevention of graft-versus-host disease following allogeneic bone marrow transplantation in rats using FK506.

FK506 and cyclosporine were used for the prevention of acute graft-versus-host disease. Acute GVHD was induced in Lewis rats by total-body irradiation and subsequent reconstitution with allogeneic (ACI) bone marrow and spleen cells (BMTx). GVHD was assessed by both clinical and histologic parameters during the experiment duration of 60 days, and longer for selected animals. All untreated BM recipients died within 26 days from severe acute GVHD. GVHD was prevented with CsA during the period of immunosuppressive therapy, but it appeared within a few days afterward. FK506-treated BM recipients were also protected, but they had a markedly prolonged GVHD-free period after therapy was discontinued. Most such animals eventually developed GVHD but with notable exceptions. Maintenance therapy with doses of FK506 as low as 0.1 mg/kg every other day (1/20 of daily induction dose) was infallible insurance against delayed GVHD. The relevance of these findings to GVHD caused by lymphoid-containing solid organs such as the intestine was discussed.

Short-term myeloid reconstitution following TBI is not adversely affected by doses of FK506 that abrogate lethal GVHD.

Studies were undertaken to determine whether the doses of FK506 that are effective for acute GVHD prophylaxis following lethal irradiation and bone marrow transplantation (BMT) would also suppress myeloid cell reconstitution. FK506 (3 mg/kg/day) abrogated acute lethal graft versus host disease (GVHD) in lethally irradiated C57BL/10SnJ (H-2b) recipient mice given histoincompatible BM plus spleen cells from B10.BR (H-2k) donors and this dose was used in all of the studies. Endogenous and exogenous myeloid repopulation was studied in mice given daily injections of either FK506, an equivalent amount of carrier solvent or no treatment throughout the interval between total body irradiation (TBI) and the day of assay. Repopulation was studied after 400 or 500 cGy TBI (endogenous) and after 950 cGy TBI plus injection with syngeneic BM (exogenous). No consistent adverse effects of FK506 were seen during either exogenous or endogenous recovery. Parameters studied included hematocrit (Hct), WBC count, cells per humerus, spleen weight, splenic colony-forming units, % spleen or BM 59Fe uptake and colony forming cells per humerus. Similarly, when lethally irradiated secondary recipients were reconstituted with BM from FK506 treated primary recipients (lethal irradiation plus exogenous BM), no consistent effects were observed. These data suggest that FK506 given to prevent GVHD would not compromise the myeloid recovery that is critical for survival in the interval of time following shortly after BMT.

FK 506 reverses acute graft-versus-host disease after allogeneic bone marrow transplantation in rats.

Severe graft-versus-host disease was induced by transplantation of ACI rat bone marrow and spleen cells into irradiated Lewis rat recipients. Treatment with FK 506 or cyclosporine A (CsA) was started after clinical and histologic evidence of acute GVHD was present. A 14-day course of FK 506 at 1.0 mg/kg/day could rescue 100% of the animals suffering from GVHD. In contrast only one half of the animals treated with CsA at a high dose of 25 mg/kg/day recovered. After cessation of immunosuppressive therapy, FK 506-treated animals displayed a marked prolonged disease-free interval as compared to CsA-treated bone marrow recipients. Recurrence of the disease in these animals could be prevented when FK 506 treatment was continued after the induction period with a low maintenance dose of 0.1 mg/kg/day every other day.

Allogeneic hepatocyte transplantation using FK 506.

Hepatocyte transplantation is an intriguing alternative to orthotopic liver transplantation. While engraftment of syngeneic hepatocytes can be achieved with relative ease, engraftment of allogeneic hepatocytes has been far more complicated. We used FK 506 (Tacrolimus), a novel and highly efficient immunosuppressant, which has been reported to augment liver regeneration in rats. Recipients of isolated syngeneic (LEW) and allogeneic (Wistar F.) rat hepatocytes (major histocompatibility barrier) recieved different immunosuppressive regiments with FK 506 or Cyclosporine A (CsA). Mature syngeneic hepatocytes could be retrieved up to post op day 300 with the lowest number of hepatocytes on post op day 20. Following allogeneic transplantation, no mature hepatocytes could be identified after post op day 10, though ductular like structures within the spleen were found in FK 506 but not CsA-treated animals. The epithelial cells of ductular like structures exhibit cytological features of CK-19 positive cells. Our results suggest that under CsA or FK 506 immunosuppression long-term survival of mature allogeneic hepatocytes within the spleen cannot be achieved across a major histocompatibility barrier though FK 506 allows engraftment of allogeneic donor type ductular cells.

Selective intraportal transplantation of DiI-marked isolated rat hepatocytes.

Transplantation of isolated hepatocytes is a promising alternative to orthotopic liver transplantation in experimental animal models with acute hepatic failure and hereditary enzyme defects. Conventional light microscopy identification of hepatocytes within recipient livers has been limited due to the inability to distinguish between donor and recipient liver cells. In this study, we labeled hepatocytes intracellularly with the fluorescent dye DiI-18 prior to selective intraportal or intrasplenic transplantation. Syngeneic LEW rat hepatocytes were isolated and 2 x 10(7) fluorescence-labeled cells were transplanted by intraportal infusion selectively into 2/3 of the recipient liver lobules to avoid lethal portal hypertension. Rats were sacrificed on postop days 1, 3, 5, 10, 20, and 40. Histological examination was performed using light and fluorescence microscopy counterstained by light green dye. The quantity of transplanted hepatocytes residing within the recipient liver was determined by FACS analysis after enzymatic digestion of the recipient liver lobules. Engrafted hepatocytes were identified in the periportal regions of transplanted liver lobules. The stained hepatocytes were retrieved up to 20 days postop using fluorescent microscopy. Using FACS analysis the number of labeled hepatocytes was found to diminish over time following transplantation from 2.1% on postop day 1 to 0.5% on day 10. Labeled hepatocytes transplanted into the spleen were retrieved in clusters up to 20 days postop (the last day of observation). Furthermore, the migration of labeled hepatocytes from spleen to liver parenchyma was observed following intrasplenic transplantation. However, after selective intraportal transplantation, only fluorescent debris was found in splenic and pulmonary tissue upon examination of various organs. This article describes the method of fluorescent labeling of rat hepatocytes and reports the feasibility and limitations of using DiI-18 as a marker.

Induction of donor-specific transplantation tolerance to skin and cardiac allografts using mixed chimerism in (A + B-->A) in rats.

Mixed allogeneic chimerism (A + B-->A) was induced in rats by reconstitution of lethally irradiated LEW recipients with a mixture of T-cell depleted (TCD) syngeneic and TCD allogeneic ACI bone marrow. Thirty-seven percent of animals repopulated as stable mixed lymphopoietic chimeras, while the remainder had no detectable allogeneic chimerism. When evaluated for evidence of donor-specific transplantation tolerance, only those recipients with detectable allogeneic lymphoid chimerism exhibited acceptance of donor-specific skin and cardiac allografts. Despite transplantation over a major histocompatibility complex (MHC)- and minor-disparate barrier, animals accepted donor-specific ACI skin and primarily vascularized cardiac allografts permanently, while rejecting third party Brown Norway (BN) grafts. The tolerance induced was also donor-specific in vitro as evidenced by specific hyporeactivity to the allogeneic donor lymphoid elements, yet normal reactivity to MHC-disparate third party rat lymphoid cells. This model for mixed chimerism in the rat will be advantageous to investigate specific transplantation tolerance to primarily vascularized solid organ grafts that can be performed with relative ease in the rat, but not in the mouse, and may provide a method to study the potential existence of organ- or tissue-specific alloantigens in primarily vascularized solid organ allografts.

Induction of chronic graft-versus-host disease in a rat model after transplantation of sensitized small bowel allografts.

The recent success in controlling acute rejection in clinical small bowel transplantation has resulted in a number of patients with functioning grafts and an occasional occurrence of graft-versus-host disease (GVHD). To better understand this complication following small bowel transplantation, a model of chronic GVHD was developed, using the Brown Norway-->Lewis rat strain combination. When the Lewis recipients were immunocompromised at the time of transplantation and received a graft specifically sensitized against Lewis, fatal GVHD developed in 3 of 5 animals. Serial histologic evaluation and determination of donor major histocompatibility complex (MHC) class I antigens were used to delineate the course of GVHD. Although the histologic results were inconsistent, with the exception of the animals developing fatal GVHD, the detection of donor MHC antigens correlated well with the development of GVHD. Determination of donor MHC class I antigens may serve as useful indicators for the development of GVHD.

Laparoscopic fundoplication.

BACKGROUND: The effectiveness of laparoscopic Nissen fundoplication (LNF) was assessed in patients with chronic gastroesophageal reflux disease (GERD) using pH study and different quality-of-life indexes. We correlated both types of data and hypothesised that improvement in quality of life following LNF does not necessarily correlate with improvement in pH values. METHODS: Seventy patients presenting with typical symptoms of GERD (14 with Barrett's esophagus) underwent LNF between May 1997 and December 2000. All patients were evaluated both prior to and 3 months after surgery using 24-h pH study, endoscopy, and a validated quality-of-life questionnaire. RESULTS: Following LNF, reflux was reduced to normal in all but six patients. Howevers despite persistent reflux, the Gastrointestinal Quality of Life Index (GQLI), of these six patients improved postoperatively from 79.5 +/- 12.2 to 111.7 +/- 8.3. These results correlate with those of patients who had normal postoperative pH studies-namely, 88.5 +/- 19.3 to 112 +/- 16.7. There was no difference in quality-of-life improvement between patients with Barrett's esophagus and those without it. CONCLUSION: There is only a weak correlation between quality-of-life assessment and pH study. Because the patient's quality of life is likely to improve following LNF, an objective means parameter of assessing the effectiveness of antireflux surgery, such as pH study or endoscopy, is recommended.

Mesh penetration of the sigmoid colon following a transabdominal preperitoneal hernia repair.

The laparascopic transabdominal preperitoneal (TAPP) repair of an inguinal hernia is an established technique associated with notably low rates of recurrence and complication. Inguinal pain and anal bleeding following a TAPP procedure may result from the penetration of the repair mesh into the sigmoid colon. In this case report, we discuss this particular complication following the TAPP procedure. Subsequently, we describe the diagnostics as well as the surgical treatment necessary.

Paralysis of the femoral nerve following totally extraperitoneal laparascopic inguinal hernia repair.

Totally extraperitoneal preparation (TEP) of an inguinal hernia is an established method of treating inguinal hernias associated with an acceptable complication rate (2-12%) and low rate of recurrence (0-3%). This is the first reported case of sensorimotor paralysis of the femoral nerve following the complete endoscopic mesh treatment of a primary inguinal hernia to the left side. Following a discussion of the necessary diagnostic and therapeutic steps, traumatic postsurgical paralysis of the nerve as well as spontaneous paralysis of the femoral nerve are discussed. The prognosis is positive given the lack of macroscopic evidence of any direct damage to the nerve.

A peritoneal cavity chamber for intravital microscopy of the liver under conditions of pneumoperitoneum.

BACKGROUND: Intravital microscopy allows direct visualization of the hepatic microvasculature. We report on a novel application of this technique using a chamber model that simulates the conditions of pneumoperitoneum. METHODS: For this purpose, we designed a peritoneal cavity chamber for rats. In the present study, we evaluated the technical procedure without any induction of increased intraabdominal pressure to assess undisturbed hepatic microcirculation. Intravital microscopy of the liver was performed in 12 rats. Animals that underwent the same operative procedure without the chamber served as controls (n = 12). RESULTS: Hepatic sinusoidal perfusion rate, leukocyte-endothelial cell interaction, and bile flow showed no significant differences between the groups. Operating time was longer in the chamber group. CONCLUSION: The peritoneal cavity chamber is an attractive approach for the study of hepatic microvascular, cellular, and molecular mechanisms that are important to our understanding of the potential harmful effects of laparoscopy on hepatic circulation and liver function.

Microcirculation and excretory function of the liver under conditions of carbon dioxide pneumoperitoneum.

BACKGROUND: To date, the effects of increased abdominal pressure, as given during carbon dioxide (CO(2)) pneumoperitoneum, on hepatic microcirculation and biliary excretion are unknown. METHODS: Using a custom-made peritoneal cavity chamber, we performed intravital microscopy of the left liver lobe under conditions of CO(2) pneumoperitoneum in a rat model. In addition, biliary excretion was assessed. RESULTS: The establishment of a CO(2) pneumoperitoneum of 4 or 8 mmHg resulted in sinusoidal perfusion failure that was more pronounced in the periportal regions than in the midzonal and pericentral regions of the liver acinus. Biliary excretion was considerably reduced at an intraabdominal pressure of 8 mmHg. Leukocyte-endothelial cell interactions increased significantly in both hepatic sinusoids and postsinusoidal venules. CONCLUSION: Alterations in hepatic microcirculation and liver function must be taken into consideration in any kind of laparoscopic surgery and may be of particular clinical relevance in patients with liver pathology.

Elevated expression of hormone-regulated rat hepatocyte functions in a new serum-free hepatocyte-stromal cell coculture model.

The specific performance of the adult hepatic parenchymal cell is maintained and controlled by factors deriving from the stromal bed; the chemical nature of these factors is unknown. This study aimed to develop a serum-free hierarchical hepatocyte-nonparenchymal (stromal) cell coculture system. Hepatic stromal cells proliferated on crosslinked collagen in serum-free medium with epidermal growth factor, basic fibroblast growth factor, and hepatocyte-conditioned medium; cell type composition changed during the 2-wk culture period. During the first wk, the culture consisted of proliferating sinusoidal endothelial cells with well-preserved sieve plates, proliferating hepatic stellate cells, and partially activated Kupffer cells. The number of endothelial cells declined thereafter; stellate cells and Kupffer cells became the prominent cell types after 8 d. Hepatocytes were seeded onto stromal cells precultured for 4-14 d; they adhered to stellate and Kupffer cells, but spared the islands of endothelial cells. Stellate cells spread out on top of the hepatocytes; Kupffer cell extensions established multiple contacts to hepatocytes and stellate cells. Hepatocyte viability was maintained by coculture; the positive influence of stromal cell signals on hepatocyte differentiation became evident after 48 h; a strong improvement of cell responsiveness toward hormones could be observed in cocultured hepatocytes. Hierarchial hepatocyte coculture enhanced the glucagon-dependent increases in phosphoenolpyruvate carboxykinase activity and messenger ribonucleic acid (mRNA) content three- and twofold, respectively; glucagon-activated urea production was elevated twofold. Coculturing also stimulated glycogen deposition; basal synthesis was increased by 30% and the responsiveness toward insulin and glucose was elevated by 100 and 55%, respectively. The insulin-dependent rise in the glucokinase mRNA content was increased twofold in cocultured hepatocytes. It can be concluded that long-term signals from stromal cells maintain hepatocyte differentiation. This coculture model should, therefore, provide the technical basis for the investigation of stroma-derived differentiation factors.

[Bile duct lesions in liver trauma]. / Gallenwegsläsionen bei Lebertrauma.

Injuries of the biliary tract remain an important concomitant lesion following liver trauma. Intra- and extrahepatic bile duct injuries differ substantially in etiology and choice of surgical procedure. Intrahepatic bile duct injuries gain clinical relevance postoperatively following primary surgical liver treatment. Characteristic consequences of intrahepatic bile duct injuries are: infected necrosis, abscess, bilious fistula, bilhemia, hemobilia and biliary duct stricture. In contrast, concomitant organ or vessel injury are of primary importance in extrahepatic biliary duct injury. Lesions of the distal biliary duct, always associated with duodenal or pancreatic injury, remains a particular thrill in general surgery. The approach to surgical treatment in case of biliary duct injury and its consequences are outlined and discussed in detail.

[Hepatocellular transplantation--from the beginning to clinical application]. / Hepatocelluläre Transplantation--von den Anfängen bis zum klinischen Einsatz.

Within the past 30 years hepatocyte transplantation has been developed in rodents for the cure of acute and chronic liver failure. Organ shortage and the enormous costs of orthotopic liver transplantation have promoted intensive research in transplantation of isolated hepatocytes. Transplanted syngeneic hepatocytes survive indefinitively in rodents, allowing correction of various inherited liver enzyme defects or enhancing recipient survival in experimentally induced acute liver failure. The clinical application of hepatocyte transplantation has been recently demonstrated in a young patient suffering from Crigler-Najjar syndrome type I, with successful long-term survival of allogeneic hepatocytes. This review reports the historical development and the published data on experimental and clinical hepatocyte transplantation.

[Mesenteric ischemia. Is diagnostic laparoscopy of value?]. / Mesenteriale Ischämie. Hat die diagnostische Laparoskopie einen Stellenwert?

In the past decade laparoscopic surgery replaced many open operations in general surgery. Apart from therapeutic uses in cholecystectomy, appendectomy, hernia surgery, gastric fundoplication, and increasingly also large intestine surgery, it is indicated diagnostically first of all for unclear abdominal findings and for staging of intra-abdominal malignancies. To date laparoscopy has been used occasionally for diagnosis and therapy of mesenteric ischemia. Patients suffering from mesenteric ischemia are usually old and have comorbid conditions. Quick diagnosis and therapy are necessary due to the pathogenesis of the disease. The low rate of morbidity as well as the easy availability of laparoscopy in principle favor the employment of laparoscopy also for mesenteric ischemia. Against the background of increasing experience in the area of laparoscopic surgery, this study gives an overview of the present value of laparoscopy for mesenteric ischemia.

[Teaching and learning in surgery--The Gottingen curriculum]. / Lehren und Lernen in der Chirurgie--das Göttinger Curriculum.

The study of medicine in Germany is in need of reform. Oversubscribed courses, the lack of practical reference, scarce patient contact and cancelled lessons define the educational landscape for many students. Since at present we cannot carry out global reforms, the quality of medical education is highly dependent on the commitment of those responsible in the individual institution. The aim of the revised surgical curriculum in Göttingen is to demonstrate how medical education with a high didactic level including multimedia teaching can be realised despite large student numbers. Duties both in patient care and education are coordinated through integrated organisation. Educational content and structure are made transparent by online guidebooks. In the surgical examination course and the practical course in surgery, patient-oriented tuition takes place consistently in small groups. The Teaching Studio provides additional aids such as interactive CD-ROMs, online research, textbooks, ultrasound equipment, examination and suturing dummies. Curriculum-relevant information, including the weekly case examples and instruction guides for clinical practical experience, is presented on an Internet website. Constant evaluation of the curriculum not only documents student motivation, but also serves to improve the educational concepts on a continuous basis. On completion of the practical course in surgery, 65% of participants maintain that they have reached the defined learning objectives "well" or "very well" (prior to reform only 17%). In the summer semester 2000, 46% of students gave top marks in the practical course in surgery with respect to tuition in patient contact. The results of the evaluations verify the positive feedback from the student body.