The protective effect of cyclosporine against cirrhotic alteration of the liver.

We have successfully applied the immunosuppressive drug cyclosporine in patients with primary biliary cirrhosis and in some patients with chronic active hepatitis. After several years of treatment, we have found a histologic remission tendency in cirrhotic alteration of some patients. This observation indicates that cyclosporine could have protective effects against fibrosis or cirrhotic alteration. To clarify this, we treated Sprague-Dawley rats in which the cirrhotic alteration of the liver was induced by injection of 0.5 ml/kg body weight carbon tetrachloride intramuscularly twice a week with cyclosporine (orally); the control animals were given saline solution instead of cyclosporine. After 6 weeks, we examined the liver histologically to determine the grade of fibrosis and cirrhosis and the grade of fatty degeneration; in group 2 we gave 1 mg/kg body weight cyclosporine daily, and in group 3 it was given every second day. We found excellent protective effects of cyclosporine against cirrhotic alteration in both groups compared with the control group. In group 3 only 25% of the animals showed grade 3 fibrosis and cirrhosis; however, the rate in the control animals (group 4) was 64.3%. In the daily application of cyclosporine (group 2) we found reduced effects of the drug compared with group 3. In group 1 we ordered 10 mg/kg body weight cyclosporine, which causes severe hepatotoxicity. In group 5, animals with hepatic damage from carbon tetrachloride were treated from the third week with cyclosporine. The effect of cyclosporine was not as beneficial compared with the groups in which we ordered cyclosporine from the first week. These results suggest excellent anticirrhotic effects of cyclosporine. This drug should be ordered as early as possible in the treatment of chronic hepatic damage and in an adequate minimal dosage.

[Successful treatment of primary biliary cirrhosis with cyclosporin]. / Erfolgreiche Behandlung der primären biliären Zirrhose mit Ciclosporin.

Four patients (aged 35, 52, 53 and 62 years) with primary biliary cirrhosis (raised levels of gamma-GT, alkaline phosphatase and immunoglobulins, as well as of antimitochondrial antibodies) were treated with cyclosporine and methylprednisolone. Different from the regimen for immunosuppressive treatment after organ transplantation, the dosage of cyclosporine was set not according to whole-blood levels but individualized to variations in serum transaminases. An initial dose of 2-2.5 mg/kg proved to be optimal. Higher doses caused a rise in liver enzymes or bilirubin, because of toxic effects produced by the cyclosporine, which is predominantly metabolized in the liver. Optimal initial doses correlated positively with serum IgG levels. In all patients there was an improvement not only in the abnormally high levels of liver enzymes but also in their general condition. There was a marked fall in antimitochondrial antibody titres and immunoglobulin values. These findings indicate that treatment of primary biliary cirrhosis with low doses of cyclosporine and methylprednisolone is effective and low in side effects and can arrest the progression of the disease even in an advanced stage.

Protective effect of aprotinin on ischemic hepatocellular damage.

During hepatic resection, occlusion of the hepatoduodenal ligament has been frequently applied to prevent intraoperative bleeding. To reduce hepatocellular ischemic damage in this procedure, we pretreated animals with Aprotinin. Three hours after an intravenous injection of 40,000 KIU Aprotinin in SD rats, we occluded the afferent hepatic vessels for 50-min and 60-min periods. 92% of occluded animals could sustain life after 60 min. Without premedication only 17 of 25 animals (68%) survived the 50-min occlusion, and 18 of 32 (56%) the 60-min occlusion. Biochemical analysis of sera was carried out 12 hr after a 40- and 60-min occlusion of the hepatoduodenal ligament with Aprotinin pretreatment. Furthermore we induced compensatory cirrhosis by application of CCL4 and biochemical analysis of sera was carried out after a 30-min occlusion. The elevation of SGOT and SGPT values was drastically reduced in the animals with Aprotinin medication in comparison with those without treatment. These observations suggest the highly protective effect of Aprotinin in the case of warm ischemic hepatic damage, especially in the cirrhotic liver. After pretreatment of LEW rats with Aprotinin (40,000 KIU i.v.), we perfused the livers with chilled Ringer solution containing 40,000 KIU Aprotinin/20 ml. We transplanted the livers orthotopically into LEW rats. With the application of Aprotinin liver preservation time increased to 10-15 hr. However, without Aprotinin the livers could be successfully preserved for only 4-6 hr. Our results indicated that premedication with high doses of Aprotinin provided highly protective effects against warm and cold ischemic damage of the liver.

[Tuberculosis following liver transplantation]. / Tuberkulose nach Lebertransplantation.

Seven months after orthotopic liver transplantation because of terminal postnecrotic cirrhosis, a 55-year-old patient was found to have pulmonary tuberculosis with fever, cough and an infiltration in the left upper lobe. Sputum culture grew M. Tuberculosis. He received ethambutol (1.6 g/d) and isoniazid (400 mg three times weekly, after 1 1/2 months 200 mg thrice weekly). After eleven months the tuberculosis had healed with only minor residua. The function of the transplant was very good in the fourth year after the operation. The main side effect was a reversible rise in liver enzymes. If possible, patients should not be given hepatotoxic tuberculostatic agents after liver transplantation, in no circumstances rifampicin. Dosage should be adapted to the liver function so as to avoid damage to transplant function.

Significance of thymus differential ratio in immunologic status of graft tolerance.

LEW with BDE-heart graft received 0 (control), 15, or 40 mg cyclosporine (CsA)/kg b. wt. per day. On postoperative days 3, 5, 7, 10, and 14 in four animals each weight and cell count of thymus and spleen were determined, and thymus and spleen cell subpopulations were examined with monoclonal antibodies. The same tests were performed in FiS heart graft recipients without immunosuppression and ungrafted LEW which received 15 or 40 mg CsA. We expressed alterations in thymocyte subpopulations by using the differentiation ratio (DR), i.e., differentiated in % of all T-cells and by the ratio of helper to suppressor/cytotoxic T-cells (Th-Ts/c). In graft rejection the thymus showed no significant change in DR or Th-Ts/c. However, in the CsA-induced graft tolerance DR was elevated and at the same time Th-Ts/c declined, both showing maximum values on days 5 and 7 and a return to normal thereafter. FiS graft recipients exhibited similar thymus alterations as tolerant recipients, but less marked. In CsA-treated ungrafted LEW, elevation of DR was slight after 15 mg but very strong after 40 mg CsA (93% on day 7), and it did not return to normal in the latter group. Th-Ts/c was decreased in these ungrafted animals, but not as strongly as in tolerant graft recipients. Such thymus alterations were not observed in graft rejection. Spleen weights were strongly increased in graft rejection and unchanged in graft tolerance. Splenic Ts/c and Th-Ts/s were increased in CsA-treated tolerant recipients but not in graft rejection. We conclude that elevation of DR and decline of thymic Th-Ts/c in the initial postoperative phase are indicators of graft tolerance in organ recipients.

[Aspergillosis following liver transplantation as a hospital infection]. / Aspergillose nach Lebertransplantation als Hospitalismusinfektion.

In the first four weeks after a liver transplantation, there was an invasive aspergillosis with a lethal course in three out of five patients who were treated postoperatively in the same room. The clinical symptoms were very different. One patient was asymptomatic, and the diagnosis could only be made by autopsy. In another patient, pulmonary symptoms, and in the third patient, cerebral symptoms were the most prominent. In the two latter patients, the infection was demonstrated in the sputum and by bronchoalveolar lavage. The disease course was fulminant in all patients, and therapy was without success. Owing to this high incidence, mycological investigations were carried out on the ward. A flower bench in the hall beside the ward was probably the main focus of distribution. To avoid such nosocomial infections, foci of aspergillus distribution should if possible be removed from the surroundings of patients with weakened immune resistance.

Thymus alteration in hepatic regeneration.

We performed 70% hepatectomy in LEW rats and examined immunologic alterations during hepatic regeneration; especially, thymus weight and cell count, T-cell subpopulations, differentiation ratio of thymocytes (DR) and ratio of T-helper to T-suppressor/cytotoxic cells (Th-Tsc). Strongest liver regeneration was observed on postoperative days 2-5 and it was completed on day 7. During hepatic regeneration a significant thymus atrophy in weight and cell count was found on day 3 and 5, it normalized from the 7th day on. T-cells were highly differentiated during liver regeneration with a DR of 35.7 +/- 2.5%, 64.0 +/- 4.4% and 38.8 +/- 3.0% on postoperative days 3, 5, and 7, respectively, and at the same time Th-Tsc ratio was reduced to 0.57 +/- 0.11, 0.38 +/- 0.04 and 0.57 +/- 0.05, respectively. DR and Th-Tsc ratio showed a trend to normalization from the 7th day on. No changes of thymus and T-cell subpopulations occurred in a sham-operated control group. Since we found such thymus alterations also in spontaneous or drug induced tolerant graft recipients, we conclude that the hepatic regenerative potential possesses a suppressive effect on immune responses.

Alteration of thymus and of immune regulatory-antifibroblast factors in acute and chronic hepatic damage.

We studied in this paper the behavior of immunosuppressive and fibroblast proliferation inhibitory factors in the acute, chronic damage and cirrhotic alteration of the liver. We induced in LEW-rats acute hepatic necrosis by i.v. application of dimethylnitrosamine (DMNA: 35 mg/kg b.wt.) and by i.m. injection of CCl4 (1 ml/kg b.wt., twice a week). After 2-4 weeks we found chronic hepatic damage and after 8-10 weeks liver cirrhosis. As a control, untreated animals were used. Sera and liver factors were prepared from the animals and used for inhibition tests of fibroblast proliferation and MLC reaction. Furthermore, cell count and cell subpopulation of the thymus were determined by monoclonal antibodies (W3/25, OX-8). LF of untreated and DMNA-treated animals exhibited very strong unspecific inhibition effects of fibroblast proliferation and allogenic stimulation. However, with progression of hepatic damage (chronic hepatitis and cirrhosis) both suppressive abilities were gradually reduced. Normal sera showed very slight inhibition of allogenic stimulation but sera of animals with acute hepatic damage showed very strong inhibition. In the 2 weeks of CCl4 treatment, their inhibitory abilities were more than 40%, and with progression of hepatic damage they were gradually reduced. Normal sera or sera of animals with chronic hepatic damage could not suppress the fibroblast proliferation; however, sera of acute hepatic damage inhibited it very strongly. With chronic hepatic damage, the thymus gradually atrophied and, after 10 weeks of CCl4 treatment, it had atrophied completely. Thymocyte differentiation was found only in animals with acute hepatic damage. This suggests that factors which were liberated from the damaged hepatocytes caused differentiation of the thymocytes.

[Problems in the reconstruction of bile flow in orthotopic liver transplantation]. / Probleme der Rekonstruktion des Gallenabflusses bei der orthotopen Lebertransplantation.

In hepatic transplantation complications of the biliary drainage were frequently observed. Ischemia of the extrahepatic bile duct which occurs for anatomical reasons can cause necrosis of the bile duct. The reconstruction of biliary drainage by biliodigestive anastomosis results in ascending infections of the graft. Biliary sludge could obstruct the intra- or extrahepatic bile duct. Recently, operation methods are mainly applied in which the function of Oddi's sphincter is preserved, i.e. choledocho-choledochostomy or gallbladder conduit method. If it is not possible to perform these methods the Roux-y-jejunum loop is used. Finally, an immediate operative revision of the biliary drainage is indicated if its complication is diagnosed.

Restoration of T cell responsiveness to interleukin-2 in recipients of pancreatic islet xenografts treated with cyclosporine.

The immunosuppressive mechanism of cyclosporine (CsA) was studied using pancreatic islet xenotransplantation. A dose of 40 mg/kg/day CsA significantly prolonged survival of hamster islet grafts in BDE rats to 14.0 +/- 7.1 days compared with 2.0 +/- 0.9 days in controls (P less than 0.01), and antihamster lymphocytotoxic antibody was not detected in recipient sera even after rejection. Responses of spleen cells to pokeweed mitogen (PWM) and to phytohemagglutinin (PHA) were inhibited at least 21 days after transplantation in recipients treated with CsA. The addition of exogenous interleukin-2 (IL-2) to spleen cell cultures on day 7 had no effect on the impaired response to PHA. At the time of graft rejection (day 14), the T cell response to PHA recovered with the addition of IL-2. However, significant changes were not observed in the ratio of spleen cell subpopulations, which were studied with monoclonal antibodies of W3/ 13, OX-12, W3/25, and OX-8. From these results we conclude that CsA can significantly prolong islet xenograft survival in closely related species, and inhibit the production of humoral antibodies and T cell responses. At the time of graft rejection, recipient T cell responsiveness to IL-2 was restored. This suggest that islet xenograft rejection is caused by the recovery of cellular immunity in recipients treated with CsA.

Immune regulatory effect of hepatic factor associated with thymus alteration.

This study was carried out to clarify the mechanism of the immune regulatory effect of factors which were liberated from the ischemic damaged liver. By occlusion of the hepatic vessels (hepatic artery and portal vein) for 40 min daily during 5 days to induce the ischemic damage of the liver, reduced thymus weight (50 +/- 5 mg; control, 274 +/- 23 mg) and cell count (0.7 +/- 0.3 X 10(7); control, 3.5 +/- 0.3 X 10(8] and complete differentiation of thymocytes were observed, i.e., helper cells reacting to monoclonal antibody W3/25 were 34 +/- 8% and suppressor/cytotoxic cells to OX-8, 49 +/- 5% (in control W3/25:89 +/- 1%, OX-8:89 +/- 1%). These quantitative and qualitative changes of thymocytes were correspondent to those of animals treated with 40 mg CsA/kg per day for 5 days; however, medication with 10 mg prednisolone/day 5 times could not induce any alteration of thymocyte subpopulation (W3/25:89 +/- 1%, OX-8:87 +/- 1%) although the weight and cell count decreased to 92 +/- 8 mg and 4.1 +/- 0.6 X 10(7), respectively. Furthermore, 5 days after liver allotransplantation (BDE to LEW), the weight and cell count of the thymus were extremely reduced (58 +/- 6 mg, 2.7 +/- 0.2 X 10(7], and thymocyte differentiation was observed (W3/25:56.6%, OX-8:61 +/- 11%). On the other hand, in heart transplantation the atrophy of the thymus was not so strong (105 +/- 28 mg, 1.3 +/- 0.6 X 10(8], and there was no change in the subpopulation (W3/25:89 +/- 2%, OX-8:88 +/- 1%).(ABSTRACT TRUNCATED AT 250 WORDS)

Successful treatment of hepatic coma by a new artificial liver device in the pig.

In this study, we tested a new artificial liver device using liver pieces in 8-h hemoperfusion of comatous porcine blood and compared two alternative tissue preparations. Acute hepatic coma in the pigs was induced by complete devascularization of the liver. The animals were killed in stage IV coma (15-25 h after the operation), and 1 l blood was perfused over 200 g fresh or DMSO-preserved liver cubes. After the devascularization GOT, GPT, GLDH, AP, LDH, SDH, bilirubin, free fatty acid, and bile acid levels in serum increased progressively. Ammonia concentrations underwent a rapid increase in the first 9 h of coma development from 126.0 +/- 9.9 to 321.9 +/- 62.2 mumol/l. Most of the amino acids in serum were elevated and molar ratio of BCAA/AAA declined from 3.87 +/- 0.79 to 0.92 +/- 0.24. In the course of hemoperfusion ammonia was removed from the perfusate to 71% of the initial values using fresh and to 39% using preserved tissue. Correspondingly, there was an increase in urea concentrations. Amino acid metabolism was ameliorated during the perfusion; Fischer's quotient increased from 0.91 +/- 0.15 to 1.38 +/- 0.14 (fresh liver) and from 0.89 +/- 0.14 to 2.11 +/- 0.44 (preserved liver); neuroexcitatory amino acids Asp and Glu were markedly elevated. Energy charge of the liver cells increased and reached levels exceeding 0.5 in both experimental groups, a balanced energy metabolism was maintained and suggests active metabolization by the liver pieces. In comparison with fresh tissue, preserved liver cubes proved effective. We consider our artificial liver device capable of temporary hepatic support in acute necrosis of the liver and suppose that its efficiency can be potentiated by combining this system with other procedures.

[Prolongation of ischemic tolerance time of donor livers by alkaline preservation solutions]. / Verlängerung der Ischämietoleranzzeit der Spenderleber durch alkalinisierte Konservierungslösungen.

Intracellular enzyme activities can be greatly influenced by alterations of pH, and non-physiologic pH may inhibit cell metabolism. The study was undertaken to examine the influence of pH values in preservation solution on ischemic tolerance time of the liver. BDE rat livers were used. Livers were preserved for 20 min or 2 h in warm ischemia after an initial perfusion with Ringer's solution at pH 9.0, 7.4, and 6.0. The values of total adenine nucleotide (TAN) and energy reserve (ER) in the livers were determined at the end of the preservation. After 20 min of warm ischemia, TAN values at pH 9.0 and 7.4 fell to 2.727 +/- 0.255 and 2.410 +/- 0.164 mumol/g, respectively (normal values: 3.414 +/- 0.270 mumol/g) and ER values to 0.786 +/- 0.186 mumol/g at pH 9.0 and to 0.446 +/- 0.095 mumol/g at pH 7.4 (normal values: 2.962 +/- 0.214 mumol/g). A similar trend was also observed after 2 h of warm ischemia. The preservation with a solution at pH 6.0 did not present any difference as compared to that at pH 7.4. Four-hour preservation in cold ischemia with Ringer's solution at pH 9.0 rendered higher values of TAN (2.635 +/- 0.085 mumol/g) and ER (0.336 +/- 0.026 mumol/g) than those in preservation at pH 7.4. No significant difference between TAN and ER values was found when 4-h preservation at pH 7.4 and 6.0 was compared. In another group an intermittent liver perfusion at 1-h interval was performed with chilled Ringer's solution; afterwards GOT, GPT, beta-glucuronidase, and acid phosphatase values in the effluents were evaluated. All of these enzymes showed higher concentration in the effluent with solution at pH 7.4 than that at 9.0. These results suggested that better intracellular energy reserve and organ viability can be maintained by preservation with alkaline solution. Furthermore, ER values seemed to be an excellent indicator of the organ viability during preservation. These were also confirmed by orthotopic hepatic transplantation in pigs. Livers were successfully preserved with alkaline Ringer's solution for up to 12 h. However, without change of pH, livers could not be preserved for more than 4.5 h.

Comparison of a charcoal sorbent fiber with commercial charcoals for hemoperfusion.

A charcoal sorbent fiber (Enka, F.R.G.), was assessed for impurities, surface area, and adsorptive properties of its native charcoal, and compared with other uncoated activated charcoals. In vivo and in vitro hemocompatibility of the fiber were assessed as well as the adsorptive properties for endogenous toxins. The charcoal of the fiber had few impurities and a high surface area of 1,200 m2/g charcoal. For measuring the adsorptive speeds, 2 g of the uncoated charcoals were milled and screened to a particle size of 150-250 microns (Enka; 30-40 microns) and then mixed with the solutions of the individual solutes. The charcoal types of Enka, used in the charcoal sorbent fiber, and of Sutcliffe Speakman, used in the acrylic hydrogel coated charcoal, exhibited the highest adsorptive rates for bromthalein (middle molecular weight marker) and inulin (high molecular weight marker). No hematological differences among the various charcoals were found during the in vivo hemoperfusions. In the in vitro hemoperfusions with heparinized fresh blood, the fibers showed the lowest loss of leucocytes and thrombocytes. In the in vitro evaluation of the absorbents for hepatic support, the charcoal fiber and the petroleum pitch charcoal of Asahi had the best adsorptive properties for substances in the low molecular weight range.