Heteropolymerization of S, I, and Z alpha1-antitrypsin and liver cirrhosis.

The association between Z alpha1-antitrypsin deficiency and juvenile cirrhosis is well-recognized, and there is now convincing evidence that the hepatic inclusions are the result of entangled polymers of mutant Z alpha1-antitrypsin. Four percent of the northern European Caucasian population are heterozygotes for the Z variant, but even more common is S alpha1-antitrypsin, which is found in up to 28% of southern Europeans. The S variant is known to have an increased susceptibility to polymerization, although this is marginal compared with the more conformationally unstable Z variant. There has been speculation that the two may interact to produce cirrhosis, but this has never been demonstrated experimentally. This hypothesis was raised again by the observation reported here of a mixed heterozygote for Z alpha1-antitrypsin and another conformationally unstable variant (I alpha1-antitrypsin; 39Arg-->Cys) identified in a 34-year-old man with cirrhosis related to alpha1-antitrypsin deficiency. The conformational stability of the I variant has been characterized, and we have used fluorescence resonance energy transfer to demonstrate the formation of heteropolymers between S and Z alpha1-antitrypsin. Taken together, these results indicate that not only may mixed variants form heteropolymers, but that this can causally lead to the development of cirrhosis.

Human blood group isoantigen expression on normal and malignant gastric epithelium studied with anti-A and anti-B monoclonal antibodies.

Variation in human blood group isoantigen expression on normal and malignant gastric epithelium was demonstrated with monoclonal antibodies to blood groups A and B in an indirect immunoperoxidase technique. The expected isoantigen expression was demonstrated on endoscopic biopsy specimens of normal gastric mucosa from 11 patients. Of 17 patients with gastric carcinoma (blood group A, 15; blood group AB, 2), complete loss of isoantigen expression was noted in 6 (35%). In these 6 patients, blood group isoantigen remained both in the adjacent uninvolved mucosa and at the margin of resection. The loss of isoantigen did not appear to be related to the degree of differentiation within the tumor, to the secretor status of the patient, or to the blood subgroup. Lymph node metastases reflected the isoantigen status of the primary tumor, being positive in 5 of 6 expression in all 17 patients or in an additional 15 patients studied with blood group O. These findings were discussed in the light of previously reported work on the localization of blood group isoantigens on malignant and nonmalignant gastric epithelium with the use of conventional antisera and a variety of immunohistologic techniques.

Antigen-specific immunosuppression induced by liver transplantation in the rat.

We have demonstrated that DA hearts grafted into PVG rats were completely protected from rejection by simultaneous liver transplantation from the same donor. In subsequent experiments PVG animals were given DA hearts followed 5 or 6 days later by livers from the same donor strain. Instead of the expected rapid rejection, all the grafts survived for at least 18 days, despite showing definite graft-swelling, a reliable clinical sign of early rejection, immediately prior to liver transplantation. In all 13 rats the heart size returned to normal and a strong beat returned within a few days. 6 animals survived indefinitely with healthy, beating heart grafts and the remaining 7 animals died of liver transplant rejection, but in these animals also the hearts were beating normally immediately prior to death. Histological examination of the hearts revealed no active rejection, but there was extensive myocardial scarring, compatible with resolution of a rejection reaction. It seems, therefore, that the liver grafts had entirely absorbed the vigorous immune response, terminating that which had already begun in the heart. This immunosuppressive effect was donor-specific and far more powerful than that of the immunosuppressive agent cyclosporine.

Orthotopic rat liver transplantation after long-term preservation by continuous perfusion with fluorocarbon emulsion.

Isolated rat livers have been stored for up to 25 hr using a continuous pulsatile perfusion technique with and without fluorocarbon emulsion oxygen carrier in the perfusate. After preservation, orthotopically isografted livers can maintain recipient animals in good health. The longest surviving animal is alive at 12 months. A prosthetic cuff technique for the portal vein anastomosis was specially developed for this experimental series. It has simplified vascular anastomosis after the portal vein cannulation that is required for machine preservation of the donor liver. We believe that the described method in the rat liver isograft model might be useful for studies of liver preservation.

Hypothermic preservation of the rat liver assessed by orthotopic transplantation. II. Evaluation of citrate solutions.

Rat livers were flushed with isotonic citrate solution (IC), hypertonic citrate solution (HC), or Collins' solution (C2), and were stored at 0 degree C for 8-16 hr. Following 8-hr preservation, the number of animals surviving for one month was greatest with IC grafts (6/8) and least with C2 grafts (2/7). There was no significant difference between the two citrate groups. Following 12-hr preservation, the IC group was superior to the HC group (3/6 vs. 1/6). Following 16-hr preservation, all four animals in each citrate group died within 24 hr. The ability of livers to produce bile was greater in the citrate groups, compared with the C2 group. A bile flow rate less than 0.3 microliter/min/g liver, 15 min after implantation, appeared to predict subsequent poor survival. There were no significant histological differences between biopsies taken from each group at the end of the transplant operation. Biochemical measures of liver function were best in animals that had received IC grafts, and functional damage after preservation was consistently greater in the C2 group than the citrate groups. The best results were obtained with grafts preserved with IC solution.

Successful 48-hour preservation of the rat liver by continuous hypothermic perfusion with haemaccel-isotonic citrate solution.

Eleven rat livers were stored at 7 degrees C for 24 or 48 hr using a continuous nonpulsatile perfusion method. The perfusate was based on the isotonic citrate preservation solution but contained, in addition, gelatin polypeptides (Haemaccel, Hoechst) and fluorocarbon emulsion (FC-43, Green Cross Corp.). Isologous livers were orthotopically transplanted after preservation, and long-term survival was 3/6 following 24 hr preservation and 4/5 following 48 hr preservation. All the biopsies taken immediately after revascularization were histologically normal. The seven surviving animals were killed at two months and histology showed biliary obstruction, but in all cases the hepatocytes appeared to be well preserved. These late histological findings are common in any use of this transplantation model and are believed to be associated with the difficulty of obtaining a satisfactory anastomosis of the bile duct. The perfusate described here is capable of providing reliable 48 hr preservation of the rat liver.

Damage to the biliary tract during preservation.

Biliary complications following liver transplantation continue to present major difficulties. Damage to the biliary tract has been reported after preservation of the liver before transplantation. Studies in the rabbit are reported that confirm the extensive biliary autolysis during preservation. This damage is directly related to the presence of bile in the biliary tract and can be reduced by the washout of bile and biliary perfusion.

The possibility of resuscitating livers after warm ischemic injury.

The number of clinical liver transplants that can be performed is limited by the availability of suitable donor organs. If it were possible to harvest and use livers after cardiac arrest, the supply could be improved. The mechanisms of damage in warm ischemia are not yet well understood and the consequences of transplanting a liver that is unable to provide immediate life-support are unacceptable. This study aims to identify areas for more detailed study in an attempt to improve the quality of livers harvested after significant warm ischemia, and to select acceptable organs for transplantation. Porcine livers were subjected to 75 min of warm ischemia and then perfused at 37 degrees C for 3 hr, during which period biochemical monitoring was carried out. At the end of the perfusion, histological and transmission electron microscopical studies were made. Large amounts of the intracellular enzymes ALT, AST, and LDH were released into the perfusate during the first 30 min of perfusion, but this--and the further amounts released during the subsequent 2.5 hr--was influenced by the composition of the perfusate. The inclusion of the substrates fructose and oleate, plus amino acids, substantially reduced this release and also improved the ability of the livers to metabolize ammonia. Oxygen free-radical scavengers had a significant, but smaller, beneficial effect. Electron microscopy confirmed the value of perfusion in improving cell morphology, and the additional value of including metabolic substrates. This study shows that hepatocellular structure and function can be improved by appropriate perfusion methods that also provide a simple means of monitoring some important functions. Both metabolic support and neutralization of oxygen free-radical action have a role to play in this approach to rendering ischemically injured livers acceptable for clinical use.

Serum alpha-glutathione S-transferase--a sensitive marker of hepatocellular damage associated with acute liver allograft rejection.

The wide hepatic distribution, high cytosolic concentration, and short in vivo plasma half-life of serum alpha-glutathione s-transferase are properties which may make monitoring this enzyme more clinically useful than conventional biochemical liver function tests as a marker of hepatocellular damage associated with acute liver allograft rejection. In a prospective longitudinal study of 58 liver transplants in 45 patients, serum alpha-glutathione S-transferase concentrations rose significantly more consistently and more rapidly than conventional liver function tests in association with acute rejection. However, a rise in alpha-glutathione S-transferase was less specific for rejection than conventional liver function tests although none of the tests had a positive predictive value for rejection of greater than 32%. Compatible with the particularly short in vivo plasma half-life of this enzyme, alpha-glutathione S-transferase concentrations fell to or toward normal more rapidly than conventional liver function test measurements following uncomplicated transplantation as well as during high-dose steroid treatment of rejection. This may be valuable, both in improving the resolution of biochemical changes associated with early rejection episodes and in determining when treatment of rejection has been successful. Further studies are warranted, however, to assess whether the fall in GST during rejection treatment does genuinely reflect the histological resolution of rejection.

Preservation of the canine liver for 24-48 hours using simple cold storage with UW solution.

The results of a series of 29 orthotopic liver transplants in the dog are described. The livers were preserved in a new cold storage fluid, UW solution, and were successfully transplanted after periods of storage of 24, 30, 36, and 48 hr. All six animals transplanted after 24 hr survived beyond 5 days after transplantation and had excellent graft function. Four of six survived for at least 5 days after 30 hr of cold storage, and five of five after 36 hr. Five of six consecutive dogs that received transplants that had been cold-stored for 48 hr survived for 5 or more days. This solution represents a substantial advance over all existing cold storage solutions for liver preservation.

An association between cytomegalovirus infection and chronic rejection after liver transplantation.

BACKGROUND: Previous studies suggest a link between cytomegalovirus (CMV) infection and chronic rejection. Since these studies, more sophisticated diagnostic methods with high sensitivity and specificity for CMV have been developed and effective therapy/prophylaxis for CMV is now available. We sought CMV prospectively by polymerase chain reaction of serum and urine and by conventional methods in a group of 33 patients undergoing 57 transplants during 1993 or 1994, selected from a larger series. There were 13 grafts lost to chronic rejection. The remaining 44 grafts that did not develop chronic rejection served as controls and comprised 15 successful primary grafts, 15 second transplants, 8 third transplants, and 6 primary grafts that were lost for reasons other than chronic rejection. RESULTS: The combination donor CMV antibody negative with recipient antibody positive and the duration of CMV infection >30 days were associated with an increased relative risk of chronic rejection. In contrast, the presence of CMV infection alone, symptomatic CMV infection, the detection of CMV by PCR of serum or urine, and the peak/cumulative viral load were not predictive. CMV infection occurred earlier in those undergoing a second transplant for chronic rejection than for those undergoing a second transplant for other reasons. In addition, a human leukocyte antigen B mismatch was associated with prolonged CMV infection. CONCLUSION: These data are consistent with the hypothesis that prolonged subclinical cytomegalovirus infection is associated with an increased risk of chronic rejection.

CsA levels in the early posttransplant period--predictive of chronic rejection in liver transplantation?

The increasing success of clinical liver transplantation has brought rejection to the forefront as a cause of morbidity and graft loss. The relationship of immunosuppressive drug doses and levels to acute and chronic rejection remains a matter of debate. The effect of blood CsA levels and drug doses on the incidence of acute and chronic rejection and the impact of acute rejection episodes on the occurrence of chronic rejection were studied in 146 grafts in 132 patients. These patients were transplanted in the 4-year period from June 1989 using CsA-based immunosuppression (CsA, azathioprine, prednisolone). Liver grafts in patients maintained on median CsA levels (whole blood, trough level) of > or = 175 micrograms/L in the first 28 days posttransplant had a significantly lower incidence of chronic rejection (2 out of 49 vs. 22 out of 97; P = 0.002). There was no significant difference in incidence of graft loss due to fatal sepsis (6% vs. 5%) or nephrotoxicity between the high and low CsA level groups. The overall graft loss rate was lower in the higher CsA level group (22% vs. 37%). The total doses of the individual drugs did not correlate with the incidence of acute or chronic rejection. Although the occurrence of acute rejection itself did not determine later chronic rejection, late occurrence (P < 0.00001) and multiple episodes (two or more; P = 0.0002) of acute rejection were significant risk factors for the occurrence of chronic rejection. We conclude that to minimize graft loss to rejection, CsA levels should be maintained at greater than 175 micrograms/L in the early posttransplant period, and late and recurrent episodes of acute rejection should be prevented.

Randomized trial to evaluate the clinical benefits of serum alpha-glutathione S-transferase concentration monitoring after liver transplantation.

BACKGROUND: An increase in serum alpha-glutathione S-transferase concentration (GST) has been shown to be a more sensitive and specific marker of hepatocellular damage than equivalent increases in transaminase activities. A randomized clinical trial of 60 liver transplants in 49 patients was carried out to assess the clinical benefits of GST monitoring as a supplementary test to routine liver function tests during the first 3 postoperative months after liver transplantation. METHODS: Mortality and morbidity were compared in graft recipients who had their GST reported daily to the ward (reporting group) and graft recipients who did not. RESULTS: The 3-month survival rate was significantly greater in the reporting group (P=0.033) and the risk of graft loss was halved (relative hazard ratio=0.50; P=0.29). The reporting group also had significantly more patients who spent less than 3 weeks in the hospital throughout the follow-up period (P=0.036). In addition, the reporting group experienced a lower frequency of biopsies per graft (P=0.038), less severe rejection (P=0.015), and a lower incidence of infection episodes per graft (P=0.03). GST increased by >50% above the upper limit of the reference range at a median of 1 day before the equivalent change in alanine transaminase in association with allograft rejection in the combined groups (95% confidence interval=1 to 2 days) but was lower on the day of diagnosis of rejection in the reporting group (P=0.02). This is compatible with the earlier diagnosis of rejection in the reporting group. CONCLUSIONS: We conclude that the monitoring of GST may improve patient care, reducing both mortality and morbidity.

Asymptomatic arteritis of the uterine cervix.

A necrotizing arteritis in the uterine cervix is described as an incidental finding in surgical material from 10 patients. The histological features are compared with those seen in polyarteritis nodosa. Subintimal hyaline deposition and a relative paucity of neutrophil and eosinophil polymorphs characterized the lesion but the histological appearance was never sufficiently specific to exclude confidently the possibility of polyarteritis nodosa. None of the patients had evidence of multisystem disease either at the time of operation or at subsequent follow-up assessment.

Metastasizing ependymoma of the cauda equina.

A case of metastasizing ependymoma is described, only the second in the British literature. The tumour, which arose in the cauda equina, spread to humerus and pleura over a period of 32 years. It is postulated that, in common with most of the previously reported cases, dissemination was a direct consequence of either surgical intervention or radiotherapy since neither the primary tumour nor its metastases shows any of the conventional morphological criteria of malignancy. The literature of similar cases is briefly reviewed.

Neutropenic enterocolitis due to Clostridium septicum infection.

Three cases of neutropenic enterocolitis are described. This unusual condition occurs almost exclusively in neutropenic patients and has a fulminating course which is almost invariably fatal without surgical intervention. The lesion is centered on the caecum and hitherto its pathogenesis has been unclear, partly because most studies have been performed on material obtained at necropsy. In these three patients, all of whom were treated surgically, Clostridium septicum was identified by specific immunofluorescence in the bowel wall of the resected specimens. Two patients also had C septicum septicaemia. Various forms of mucosal damage can be identified which predispose towards invasion of the bowel wall by this organism. These cases provide further confirmation of a primary role for C septicum in the pathogenesis of neutropenic enterocolitis.

Chronic coronary periarteritis in two patients with chronic periaortitis.

Two cases of chronic coronary periarteritis associated with chronic periaortitis are described. In one, the chronic periaortitis took the form of perianeurysmal fibrosis around a thoracic atherosclerotic aneurysm; in the other it resembled idiopathic retroperitoneal fibrosis. The findings in the two subjects support the unifying concept of chronic periaortitis, to include conditions previously known as idiopathic retroperitoneal or mediastinal fibrosis, perianeurysmal retroperitoneal fibrosis and inflammatory aneurysms of the aorta. The observations are also compatible with the view that the disease is due to hypersensitivity to atheroma.

Expression of c-myc oncogene in coeliac disease.

A monoclonal antibody, produced by peptide immunisation was used to detect the distribution of p62c/myc by immunohistology in normal and coeliac small intestinal mucosa. The effect of gluten in four treated coeliac patients was investigated by taking serial jejunal biopsy specimens for six hours after a 10 g oral gluten challenge. There was a progressive increase in p62c/myc staining intensity in the villus enterocytes extending to the crypts, which accompanied the classical morphological changes occurring in the mucosa.

Abnormal distribution of c-myc oncogene product in familial adenomatous polyposis.

Monoclonal antibodies raised by synthetic peptide immunisation were used to determine the distribution of the protein product of the c-myc gene by immunocytochemical staining of archival wax embedded material from patients with familial adenomatous polyposis. Polyps from 18 cases of familial adenomatous polyposis, 10 of whom had developed malignant change, and 30 normal control colonic biopsy specimens were examined. A consistent staining pattern was observed in normal mucosa; nuclear staining in the basal proliferative zone; mixed nuclear and cytoplasmic staining in the maturation zone; and cytoplasmic localisation in the surface mature zone. In contrast, the polyps and carcinomata showed a mixed pattern of cytoplasmic and nuclear localisation in the basal proliferative zone with nuclear persistence throughout the crypts to the surface mature zone. This abnormal distribution of the c-myc oncogene product may have a role in the evolution of polyps and their subsequent malignant transformation into familial adenomatous polyposis.

Fibrosis and other histological features in chronic hepatitis C virus infection: a statistical model.

AIMS: To study the inter-relation between hepatic fibrosis and other histological features of chronic hepatitis C virus (HCV) infection. METHODS: Liver biopsy specimens from 200 consecutive patients with chronic HCV infection were graded and staged separately for necro-inflammatory activity and for fibrosis. The interaction between fibrosis and other histological features was evaluated by univariate and multivariate analysis, followed by hierarchical log linear modelling. RESULTS: The most striking feature was the presence of portal tract inflammation in 177 (89%) of 200 samples. Lymphoid aggregates/follicles were observed either alone or as part of the general inflammatory infiltration of the portal tracts in 120 (60%) of 200 samples. Fatty change (macro- and microvesicular steatosis) was observed in 76 (38%) samples: mild to moderate in 60 (30%) and diffuse in 16 (8%). Bile duct damage was found in 30 (15%) of 200 specimens. Lobular activity was found in 154 (77%) of 200 samples and was significant in 44; piecemeal necrosis was present in 79 (40%). Thirty one (16%) patients had stage 0 liver fibrosis, 27 (14%) had stage 1, 69 (35%) had stage 2, 43 (22%) had stage 3, 16 (8%) had stage 4, and 12 (6%) had stage 5. On log linear analysis, piecemeal necrosis, lobular inflammation and steatosis were linked directly with fibrosis. Portal tract inflammation was linked directly and indirectly via piecemeal necrosis and lobular inflammation with fibrosis. The presence of lymphoid aggregates was associated with bile duct damage. CONCLUSIONS: Portal tract inflammation with lymphoid aggregates or follicles, together with fatty change, bile duct damage and/or lobular activity, are characteristic of chronic HCV infection, confirming previous reports. Piecemeal necrosis, lobular inflammation, portal inflammation, and steatosis are linked directly with fibrosis in this statistical model, suggesting a close inter-relation in the development of fibrosis/cirrhosis.