F R O M M E E T I N G S

II INTERNATIONAL CONGRESS ON LIVER AND GASTROINTESTINAL DISEASE c/o Tufts University-New England Medical School of Medicine

Boston, 19th and 20th October, 1987

and

10TH COURSE OF LIVER DISEASES AND LIVER BIOPSY Firenze, 2nd-7th May, 1988

I n t he c o n t e x t o f t h e J o i n t V e n t u r e b e t w e e n Tufts U n i v e r s i t y - N e w E n g l a n d M e d i c a l C e n t r e o f B o s t o n a n d Istituto di Clinica Medica II o f t h e U n ive r s i t y o f F l o r e n c e , two i m p o r t a n t sc ien t i f i c u p d a t e m e e t i n g s h a v e b e e n h e l d , d e a l i n g with v a r i o u s a spec t s o f H e p a t o l o g y a n d G a s t r o e n t e r o l o g y . T h e f i rs t m e e t i n g t o o k p l a c e in B o s t o n o n t h e 19th a n d 20th O c t o b e r , 1987 a n d t h e s e c o n d , the 10th C o u r s e o f L ive r D i s e a s e s a n d L ive r Biopsy , was h e l d in F l o r e n c e b e t w e e n the 2 n d a n d 7th M a y 1988. T h e s e two m e e t i n g s h a v e b e e n m a d e p o s s i b l e t h a n k s to t he s p o n s o r s h i p o f Ministero della Pubblica Istruzione (Programma Nazionale Cirrosi Epatica) a n d Consiglio Nazionale delle Ricerche. T h e i m p o r t a n c e a n d top ica l i ty o f t he subjec ts s u m m a r i z e d h e r e m a k e th is col- l e c t i on u se fu l fo r a r a p i d u p d a t e in t he f i e ld o f l iver d i seases .

P A T H O G E N E T I C A N D C L I N I C A L ASPE CT S O F C H O L E S T A S I S

I. M. Arias: Department of Medicine, New England Medical Centre, Boston, MA, USA.

The pathobiology of intrahepatic cholestasis.

To pathologists, 'cholestasis' means canalicular bile plugs. To clinicians, it means increased se- rum alkaline phosphatase activity with normal or near normal SGOT and SGPT. To-physiologists, it means bile secretory failure which occurs at the level of the hepatocyte. Various drugs, chemi- cals, metabolites, viruses, metals, pigments, porphyrins, bile acids, and inheritable disorders pro- duce this syndrome in man and other animals. Advances in differential diagnosis by sonography, isotopic scans and magnetic resonance imaging have outpaced knowledge of pathobiology at the cellular and molecular levels. Recent studies employing isolated hepatocytes, vesicles derived from different domains of the plasma membrane, and photoaffinity probes for bile acids have provided new information into mechanisms and potential treatment of intrahepatic cholestasis. The following major advances will be reviewed: 1. because bile acids provide the driving force for bile secretion we will review their transport into and through the hepatocyte, identification of putative membrane and soluble transporters and the possible role of cytoskeletal elements in bile secretion and cholestasis; 2. bile acid secretion is driven by the hepatocyte's electrochemical gradient which is produced by transport ATPases which reside in the basal-lateral domain of the plasma membrane. These findings suggest that cholestasis may result from primary disorders in the basal-lateral domain which secondarily produce structural and functional canalicular defects which we recognize as intrahepatic cholestasis. Can cholestasis be the consequence of basal- lateral membrane defects? Several examples will be discussed; 3. plasma membrane fluidity is different in basal-lateral and canalicular portions and is a major contributor to reduced bile flow in experimental cholestasis produced by ethinyl estradiol or chlorpromazine. Correction of al- tered fluidity by several drugs, including S-adenosyl methionine, also restores membrane compo- sition and function and bile secretion; 4. immunocytochemical and intraceUular injection studies of isolated hepatocyte pairs or triplets suggest that the cytoskeleton plays a role in bile canalicu- lar contractility and bile flow. Calcium, anti-myosin light chain kinase and other substances have been injected into hepatocytes and their effect on bile canalicular contractility has been studied;

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5. bile canaliculi contain an abundan t Ca++-Mg ++ ATPase and 5' nucleotidase (AMPase) of un- known function. Recent evidence indicates that they are ectoenzymes, that is, thei r active sites face into the lumen of the bile canaliculus where they can degrade extracellular ATP to form adenosine. They are not transporters of ATPase which, in liver, is restricted to the basal-lateral domain. Bile canalicular membrane also contains a unidirectional t ranspor ter which returns adenosine from the canaliculus into the hepatocyte where it is reutilized. Extracellular ATP dam- ages hepatocyte structure and function. Is there extracellular ATP in normal bile canaliculi? Is its concentrat ion increased in cholestasis and thus does it play a role in pathogenesis? Hypoxia increases ATP breakdown and releases adenosine into the sinusoidal blood where it increases hepatic blood flow and oxygen delivery. Hypoxia also causes cholestasis. Is this related to in- creased extracellular ATP which exceeds the degradadve activity of the two ectoenzymes?

I. M. Arias: Depar tment of Medicine, New England Medical Centre, Boston, MA, USA.

Cellular polarity within the liver in relat ion to bile secretion.

Polarity of function within the liver has structural counterparts. The basic mechanisms which are responsible for functional polarity within the different cell types in the liver are be ing explored using modern cell an d molecular biologic techniques. Several levels of polarity were considered: 1. hepatic endothel ia l cells provide a barr ier to the entry of large molecules into Disse's spaces. The bar r ie r is manifested by numerous fenestrae which measure about 150 nm in diameter. Our studies indicate that these cytoplasmic holes have a cytoskeletal basis. The fenestrae are dynamic structures which can contract and relax when exposed to a variety of pharmacologic and physio- logic stimuli.. O the r studies reveal the mechanism of stimulus-response and the participation of specific kinases in the process; 2. hepatocytes have structural features in their plasma membrane which delineate a bile canalicular feature. Sealed plasma membrane vesicles have been obtained from the basal-lateral and canalicular domains of the plasma membrane and were used to local- ize the driving forces for various transport processes. Our studies indicate that the major driving force for bile secretion - - the sodium-potassium ATPase - - is in the basal-lateral domain and that most components in bile are transported by virtue of the electrochemical gradient which the pump generates within the cell. We propose that structural and functional features of chole- stasis due to drugs, such as ethinyl estradiol and others, result from disorders in the basaMateral domain of the plasma membrane; canalicular alterations are secondary to changes in intracellu- far pH, calcium concentration, etc.; 3. the bile canalicular domain contains an active calcium- magnesium ATPase and an AMPase, both of which are ectoenzymes and hydrolyze ATP in the bile canaliculus. We have identified a purine-specific unidirectional t ransport system in bile cana- liculi. Our observations suggest that extracellular ATP may be a factor in producing cholestasis. Hypoxia causes substantial release of adenosine across the sinusoidal plasma m e m b r a n e and possibly loss of ATP into the canaliculus.

A. M. Jezequel: Istituto di Semeiologia, Diagnosi e Terapia, UniversitY. di Ancona, Italy.

Ultrastructural aspects of cholestasis.

The alterations of the liver parenchyma associated with bile retention as detected by electron microscopy are numerous and may involve all cellular organelles. The pat tern of changes is often more diffuse and severe than suggested by examination of the tissue unde r light micro- scope. Constant lesions concern the 'bile secretory apparatus' of the hepatocytes involving the endoplasmic reticulum, the Golgi apparatus, the vesicular transport system and the plasma mem- b rane of the hepatocyte. Much emphasis has been placed in the past on changes o f the endo- plasmic reticulum with descriptions of hypertrophic smooth membranes of hepatocytes associat- ed with cholestasis. This has not been confirmed by morphometr ic studies conducted in man as well as in experimental models. The meaning of frequent mitochondrial changes including curling of cristae and increased surface density of the inne r mitochondrial membrane is still a matter of speculation. It is of interest that similar changes have been observed after cholic acid overload and in asymptomatic cholelithiasis. A role of mitochondria in the side-chain oxidation of cholesterol has been proposed, but recent observations on Zellweger's syndrome suggest that

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also peroxisomes have a role to play in bile acid synthesis. Altered microvilli with blebbing and shedding of the cytoplasm and distension of the canalicular wall are usually p rominen t features in cholestasis. The pericanalicular ectoplasm usually appears thickened due to abnormali t ies of the cytoskeleton and especially of the microfilaments leading to interference with the contractile component of the canalicular wall. Intracellular bile stasis is expressed morphologically ei ther as small lamellar residual bodies or as areas of confluent biliary necrosis. Extracellular bile stasis may appear as accumulation of particulate material in the canalicular lumen, or as plugs of electron dense material (thrombi) filling the lumen. Zonal differences may predispose to a major zone 3 distribution of canalicular precipitates. In any event, these alterations are not homoge- neously distributed and normal canaliculi rich in microvilli may be seen close to canaliculi with occluded lumen. Images of 'biliary regurgitation' indicating an inversed polarity of the liver cells may occur with presence of biliary material in intercellular spaces and in the Disse's spaces. This is not due to openings of junct ional complexes and passive transfer of particulate material from biliary spaces, since the tight junct ions are remarkably preserved even in cases of extreme dilata- tion of the canaliculi. No specific changes allow to discriminate between intra- and extrahepatic cholestasis.

E A. Berg: Medizinische Klinik, Tfibingen, West Germany.

Pathogenetic aspects in pr imary biliary cirrhosis and primary sclerosing cholangitis.

Primary biliary cirrhosis (PBC) is a disease of unknown etiology affecting the inter lobular and septal bile ducts. I f we suppose that an infectious agent may trigger the disease we would have to postulate that the agent may be presented to T cells on the surface of bile duct epithelial cells in association with bo th class I and II MHC antigens. Three different types of antigens may be exposed: a. the infectious agent itself; b. class II antigens plus the processed infectious agent; c. class I antigens plus a novel antigen which may be a new protein produced as a consequence of the infection. The focal nature of the disease fur ther implies that only some portal areas are initially infected and that at the beg inning of the disease a T cell-dependent cytotoxic reaction against single epithelial cells occurs giving rise to piecemeal necrosis. Spread of the antigen to other organs could account for the systemic nature of PBC observed in some instances. Thus, in salivary and lacrimal glands as well as in lung and pancreas, granulomatous reactions have also been found. Because of the observation that in PBC IgM globulins are preferentially produced, it seems not unlikely that at least some components of the infectious agent may be T cell-inde- penden t antigens which have the ability to trigger selectively-IgM-producing B cells. Indeed, dur ing the course of the disease a wide spectrum of different types of autoanfibodies and natural occurring antibodies can be detected in patients' sera reflecting also the strong immunogenic nature of the triggering agent. Mitochondrial antibodies are of great importance in the diagnosis of PBC especially when detected by ELISA and blotting, and the evaluation of the different subtypes such as anti-M2, anti-M4, anti-M8 and anti-M9 may help to predict the outcome of the disease. Primary sclerosing cholangitis (PSC) is also a chronic liver disease of unknown etiology which is characterized by fibrosing inf lammation of intra- and extrahepatic bile ducts. The dis- ease is often associated with chronic ulcerative colitis and affects young patients, most often men. A cholestatic biochemical profile can be seen with fluctuating levels of alkaline phosphatase and bilirubin. The aspartate aminotransferase levels are in most instances mildly elevated. No diag- nostic serological markers exist and PBC-specific anti-M2 antibodies are never associated with PSC. However, naturally occurring mitochondrial antibodies can be found in some patients indi- cating non-specific stimulation of the immune system. Histologically the initial stage of the dis- ease is characterized by increased connective tissue causing enlargement of the portal tracts. Spread into the periportal parenchyma and mild inflammatory changes may follow. In the ad- vanced stages fibrous septa and biliary cirrhosis are the predominant features. Immunological mechanisms may be of importance in the pathogenesis of PSC. An association with HLA-B8 and HLA-DR3 has been observed as well as increased levels of circulating immune complexes and increased complement metabolism indicating also an activation of the non-specific or amplifying immune system. Relating these features to fibrosis one could speculate that locally formed im- mune complexes could have stimulated macrophages which would then produce and release growth regulatory proteins for fibroblasts such as the platelet-derived growth factor and fibro- nectin.

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A. Gatta, R Angeli: Istituto di Medicina Clinica, UniversitS. di Padova, Italy.

Renal impairment in cholestasis.

Cholemic nephrosis is a very common complication in patients with obstructive jaundice. Gener- ally it has poor clinical relevance and benign evolution, but it may evolve to acute renal failure when a precipitating event, such as cholangitis, shock or biliary tract surgery, occurs in these patients. Cholemic nephrosis: the morphologic study of the kidney during cholestasis shows glo- merular lesions, particularly thickening of basal membrane, and regressive lesions of proximal tubular structures due to pigment deposition. These histological findings may be associated with clinically documentable alterations of renal function and both seem to be related to the degree and the duration of the jaundice. Involvement of kidney in obstructive jaundice may be clinically evidenced by casts and renal epithelial cells deeply bile-stained in urinary sediment, low molecu- lar weight proteinuria, and reduced urea clearance. Furthermore, reduced renal reabsorption of uric acid with consequent hypouricemia and renal tubular acidosis have been described in pa- tients with intrahepatic cholestasis. In jaundiced animals with chronic ligation of the common bile duct an increased tubular reabsorption of sodium and a reduced renal diluting capacity secondary either to increased ADH level or to a decreased delivery of sodium to the distal tubule have been observed. This impairment of renal sodium and water handling has not been de- scribed either in patients with extrahepatic cholestasis or in patients with primary biliary cirrho- sis. Patients with primary biliary cirrhosis, in contrast to patients with alcoholic or posthepatitic cirrhosis, do not show renal sodium or water retention except in the advanced phases of the dis- ease. Moreover, an exaggerated natriuretic response to extracellular volume expansion, due to a more pronounced inhibition of proximal sodium reabsorption, has been described in these pa- tients. A previous experimental study from our laboratory evidenced a significant correlation between the degree of renal histological lesions and urinary excretion of gamma-glutamyltrans- peptidase (GGT), an enzyme located in the brush border of renal tubular cells. Subsequently, in a clinical study we have observed that the increase of the urinary excretion of GGT precedes the detection of an impairment of renal function. Thus, the dosage of urinary GGT may be useful in the early diagnosis and in monitoring of cholemic nephrosis. The most relevant clinical finding concerning kidney function in cholestasis is however the great susceptibility of the kidney to hypovolemic and /o r septic shock and the consequent possible development of acute tubular necrosis. In the pathogenesis of this complication, besides the role of systemic and renal hemo- dynamics which will be discussed below, a possible direct effect of one or more bile compounds must be considered. Some in vitro and in vivo studies extensively documented the potential del- eterious effects of bilirubin and bile acids on the integrity and function of cells. Particularly it has been evidenced that conjugated bilirubin, at concentrations not exceeding those observed in blood during obstructive jaundice, may compromise cellular metabolism in an irreversible way when it is already impaired by anoxia. Moreover, an impairment in copper metabolism with copper deposition at a tubular level has been hypothesized to play a role in the pathogenesis of hyperuricuria and renal tubular acidosis in primary biliary cirrhosis. Acute renal failure during cholestasis: even if renal failure may develop in patients with uncomplicated obstructive jaundice, it is very common after surgery on the biliary tract, cholangitis or shock. The mean incidence of acute renal failure following surgery in patients with obstructive jaundice is 11-22% (range 3-50%). A number of factors including a hematocrit <30%, a total bilirubinemia >200 btmol/l and a malignant obstructive lesion are noted to predict the occurrence of acute renal failure in jaundiced patients following surgery. Patients with acute renal failure during extrahepatic chole- stasis show clinical and histopathological features of acute tubular necrosis. Only few cases of functional renal failure (hepatorenal syndrome) have been reported in patients with cholestasis even if it should be pointed out that a severe jaundice is present in most patients with hepatic cirrhosis who develop a hepatorenal syndrome. The prognosis of patients with obstructive jaun- dice complicated by an acute renal failure is poor. Acute renal failure in jaundiced patients after surgery for the relief of obstructive jaundice is associated with high mortality rate (25-80%), re- sulting the most frequent cause of death in these patients. Acute renal failure of patients with obstructive jaundice to renal ischemia is generally considered as a consequence of the alterations of systemic and renal hemodynamics related to cholestasis. Systemic hemodynamic disturbances include reduced cardiac output and peripheral vasodilation. The reduced cardiac output is sec- ondary to a negative chronotropic effect and to a possible negative inotropic effect of bile acids. The decrease in peripheral vascular resistance is secondary to a direct action of bile compounds on the autonomic nervous system or a direct effect on vascular smooth muscle. These hemody-

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namic disturbances are thought to be the principal determinants of hypotension and a reduced vascular response to vasopressor agents which can be frequently observed in jaundiced patients. But a contributory role of the reduction of volemia due to diuretic action of biliary salts should also be considered at least early in the course of cholemia. Renal hemodynamic alterations include a reduced total and cortical renal blood flow and an increased vascular response to vasopressor agents. They may be related to an enhanced adrenergic tone and to an increased level of plasma catecholamines secondary to the systemic hemodynamic disturbances, to the renal effects of circulating endotoxins or to biochemical alterations associated with cholestasis. Renal impairment in jaundiced patients is frequently associated with the presence of endotoxins in the peripheral blood. Beside the possibility of a Gram-negative infection, both an enhanced absorption of endotoxins from the gut as well as reduced Kupffer cell function may account for such a finding. Endotoxin may precipitate renal failure not only through a renal vasoconstriction and redistribution of the renal blood flow away from the cortex but also by promoting an intra- vascular coagulation. The histological finding of peritubular fibrin deposition in jaundiced pa- tients with acute renal failure seems to confirm this interpretation. Although no association has been found between the renal hemodynamic disturbances and plasma biochemical alterations in cholestasis, a possible pathogenetic role of hypercholesterolemia has been hypothesized in experimental studies.

H B V - A N D H D V - R E L A T E D H E P A T I T I S

B. Galanti, G. B. Gaeta, C. Gallo, G. Giusti: Cattedra di Virologia Clinica, UniversitY. di Palermo; Clinica delle Malattie Infettive, 1" Facolt/t di Medicina, Universit~ di Napoli; Cattedra di Statistica Sanitaria, 1" Facoltfi di Medicina, Universitfi di Napoli, Italy.

Epidemiology of HBV infection.

Type B hepatitis virus (HBV) can be transmitted only to man and to apes. Since the epidemiolog- ical role of apes is negligible, man with acute and particularly with chronic infection is the main virus reservoir. Progression to chronic infection is age-related. In fact, it is very common in neonatal infection, common in children and uncommon in adults. Chronic infection is fre- quent in immunocompromised hosts (hemodialysis, transplant recipients, etc.). HBV is present in blood, body secretions and excretions. The epidemiological role of blood, saliva, semen and vaginal secretion has been fully established. HBV is not detectable in feces because of its inacti- vation in the intestine. Early studies by McCallum in the 1940's demonstrated in type B hepatitis the high infectivity titre of serum by the parenteral route but not by the oral route. Also in the 1940's Bradley observed sexual transmission of type B hepatitis. More recently it has been shown that the infection can also be transmitted without sexual intercourse but with intimate contact, particularly in the family environment and in institutions. In acute infections viral replication is self-limiting and it stops within weeks or months. There is then e/anti-e seroconversion, disap- pearance of HBV-DNA and HBsAg/anti-HBs seroconversion. Conversely, in chronic infections viral replication is long-lasting and slowly tapering. After years there is e/anti-e seroconversion and integration of HBV-DNA in the DNA of the hepatocytes. There is no clearance of HBsAg. Both in healthy carriers and in patients suffering from chronic hepatitis the estimated rate of e/anti-e seroconversion is about 15% per year. In immunocompromised patients viral replication may last indefinitely. The infectivity titre of serum is very high in subjects with active viral replica- tion, and low or negligible after DNA integration. This may explain the wide range of infectious- ness of HBsAg carriers. The endemicity level of HBV can be evaluated on the basis of the preva- lence of HBsAg carriers and immune subjects. According to Maynard, the endemicity level of HBV infection is low when the carrier rate is less than 1% and the percentage of immune adults ranges between 4 and 10%. In countries with low endemicity levels, such as USA and Northern Europe, vertical transmission of infection is negligible and childhood infection uncommon. The

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endemicity level is defined intermediate when the prevalence of carriers ranges between 2 and 7% and when 20-55% of the adult population is immune. The intermediate endemicity level is characteristic of the Medi terranean basin where vertical transmission is present but uncommon, and chi ldhood infection is more common. A high endemicity level of HBV infection is present in wide geographical areas such as Equatorial Africa and Southeast Asia. In these areas both vertical transmission and chi ldhood infection are common. In vertical transmission, HBeAg- positive pregnant women play a very important role; in fact, up to 100% of newborn babies to HBeAg-positive mothers become infected. Horizontal transmission of HBV infection in child- hood implies intimate contact (e.g. eating from the same dish, dr inking from the same glass, sleeping in the same bed, shar ing spoons, combs, etc.). These intimate non-sexual contacts usual- ly occur in the family envi ronment and in institutions but not in days schools. In adult life HBV infection is frequently related to sexual activity and to drug abuse. Sometimes tat tooing or ear piercing are involved. Surgeons, physicians, dentists and nursing staff are at risk when their duties involve contact with blood. Iatrogenic transmission of HBV infection occurs where hygien- ic standards are poor. Post-transfusion type B hepatitis is now uncommon because of the high sensitivity of tests employed for the detection of HBsAg in blood donors. H B V infection in Italy: results of three wide recent epidemiological surveys carried out in Italy on navy recruits, preg- nan t women and blood donors show that the carr ier rate is 3-4% in Southern Italy and 1-2% in Nor thern Italy. But apart from the major difference between North and South, in all regions small scattered areas with low or high endemicity levels have been observed. A survey carried out on about 2,000 asymptomatic HBsAg carriers identified among blood donors showed that most of them had normal aminotransferase values (only 5.8% had abnormal ALT values). HBeAg was detected in 9.5% of carriers, while 68.7% of them were anti-HBe positive. T h e presence of anti-delta antibodies was 2.9%. Results of several seroepidemiological surveys studying the per- centage of immune subjects in different age groups allow us to estimate that the incidence of HBV infecti0n in Italy ranges from 100 up to 2,000 cases per 100,000 inhabitants per year. Most of these infections are asymptomatic and we estimate that in Italy less than 10% of HBV infec- tions cause acute symptomatic hepatitis. An exhaustive report gives us detailed information on HBV vertical transmission in Italy during the year 1986. The number of bir ths per year were about 300,000, both in Nor thern and in Southern Italy. Pregnant carriers were about 4,800 in the North and 8,600 in the South. The majority of newborn babies at risk received prophylaxis. Since several women escaped the HBsAg screening during their pregnancy, it has been estimated that in 1986 about 1,000 children still became infected (2/3 of them in the South) and about 25% of them became chronic carriers. Several epidemiological surveys have demonstra ted that since 1974 HBV has been responsible for about 50% of the cases of acute hepatitis in Italy. From 1972 a change in the epidemiological profile of acute type B hepatitis has been observed, the number of cases in children under 15 be ing greater in 1972 than in 1985. Drug addiction, uncommon in 1972, is now a p rominen t risk factor and as a consequence a great number of hepatitis cases can be observed in young adults. Moreover, in recent years the number of notified acute viral hepati- tis cases of all etiologies has dropped. Blood transfusion is no longer a significant risk factor for type B hepatitis. Surgery and dental care are implicated in the transmission of type B hepatitis. In areas of intermediate or high endemicity levels where HBV infection is common in children, we have a high number of chronic hepatitis cases. In a multicentre study we carried out in Italy on over 1,000 cases of CAH, we observed a mean prevalence of 60% of HBsAg-positive cases, but the prevalence was h igher (up to 90%) in children and teenagers.

C. Pourcel: Unit6 de Recombinaison et Expression Gtnt t ique , Institut Pasteur, Paris, France.

Synthesis in CHO cells of hepatitis B surface ant igen particles conta ining the pre-S2 region expression product.

The absence of a cell culture system capable of propagating HBV has greatly impeded the devel- opment of a vaccine against hepatitis B. The current vaccines consist of HBsAg 22-nm particles purified from the serum of HBV chronic carriers. Despite the proven efficacy and safety of the serum-derived vaccine, alternative methods of vaccine production are needed. This is because of limitations in the availability of h u m a n serum, the need of elaborate purification and inactivation procedures and the requirement of an innocuity test in chimpanzees. Plasma-derived vaccines are very expensive and their cost is beyond the possibilities of many countries which need mass

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infant vaccination programs. In this context, DNA recombinant technology is of primary impor- tance for developing new vaccines. I. Structure and antigenicity of the viral envelope: the HBV enve- lope contains proteins, carbohydrates and lipids. The carbohydrate residues are covalendy linked to the proteins and the proteins are anchored into a lipid bilayer. As is the rule for virus enve- lopes, the hydrophobic sequences of the proteins are intramembrane and the hydrophilic se- quences containing the carbohydrate are exposed outside and carry the antigenic determinants. The 22-nm particles contain two proteins, the major protein and the middle protein. The major protein is present in two forms, glycosylated (GP27S) and non-glycosylated (P24S). The middle protein exists in two glycosylated forms: the first (GP33S) contains one glycan whereas the second (GP36S) contains two glycosidic residues. The ratio of major protein to middle protein varies according to HBV multiplication. In the presence of virus multiplication, this ratio is around 2 whereas in the absence of virus multiplication, the middle protein represents only 1% of the total proteins. The major protein is encoded by the S gene and the middle protein by the pre-S2 plus the S gene. The middle protein contains an additional sequence of 55 amino acids at the N-terminus. The S gene translation product contains 2 hydrophilic sequences. The second one carries a glycan at position 145 and contains most or all of the antigenic determinants of HBsAg. Full antigenicity and immunogenicity of HBsAg need assembly of two protein molecules linked by disulphide bonds. HBsAg is a conformational antigen. Oligopeptides corresponding to anti- genic determinants induce a low immune response. The N-terminus of the middle protein is hydrophilic and carries a dominant epitope exposed outside. It contains a receptor for polymer- ized human serum albumin (pHSA) which plays a role in the attachment of the virus to the hepatocyte. Receptor activity is therefore absent or very low in HBsAg 22-nm particles of serum in the absence of virus multiplication. II. Structure of the recombinant plasmid: HBsAg is not as- sembled in bacteria. On the contrary, HBsAg particles can be obtained in eukaryotic cells, either in animal cells or yeast. Because full immunogenicity of HBsAg is highly dependent on its ter- tiary structure, an eukaryotic system is therefore needed for recombinant DNA vaccine. HBsAg particles produced in animal cells are homogeneous in size, whereas particles synthesized in yeast are heterogeneous. Principally, in animal cells, they are glycosylated and carbohydrates could influence the immune response. The structure of HBsAg particles synthesized in animal cells and especially the presentation of the HBsAg determinants could therefore be closer to that of virus particles of human origin, than particles produced in yeast. This could have consequen- ces on the quality of the immune response of the vaccine. Moreover, in animal cells, HBsAg particles are secreted without lysis of the cells. This facilitates the collection and purification of HBsAg. Because the appearance of antibodies to pHSA receptor is observed in the serum during recovery from hepatitis B, but never during evolution towards chronicity, this suggests that anti- bodies to pHSA receptor are important for viral clearance. We have therefore chosen to synthe- size 22-nm viral particles containing both the major and the middle proteins. The plasmid pSVS DHFR used to synthesize HBsAg particles carries two transcription units. The first consists of the 2.3-kb BgIII DNA fragment, including the S gene and the pre-S region, placed under the con- trol of the SV40 early promoter. The second unit consists of murine DHFR cDNA placed under the control of the MMTV-LTR promoter. CHO DHFR- cells were transfected with pSVS DHFR and clones resistant to methotrexate were selected. One clone (resistant to 50 nmol of methotre- xate) producing around 1 ~.g per 10 ~ cells and per day was chosen for HBsAg particle production. III. Structure and immunogenicity ofHBsAg 22-nm particles: HBsAg purified from cell culture super- natant of CHO cells consisted of 22-nm diameter particles with a density of 1.21 g /cm 3 in CsCI. The electrophoretic analysis of the proteins present in the particles showed that HBsAg particles contain both the major and the middle proteins. The major protein exists in two forms, glyco- sylated (GP28S) and non-glycosylated (P24S). The middle protein is glycosylated and represents about 30% of the total proteins. The particles contain both group and subtype HBsAg determi- nants. The presence of pHSA receptor activity was shown using hemagglutination test and solid- phase RIA. The immunogenicity was studied in mice. The particles elicit anti-HBs antibodies. The EDs0 was 0.04 [xg, identical to that obtained with particles of human origin. Moreover, the HBsAg particles were able to elicit antibodies to the pHSA receptor in mice. We compared the immune response to pre-S2 region encoded determinants in terms of immunogenicity, specific- ity, H-2 linked regulation and possible overlapping regulatory mechanisms. For this purpose, groups of mice from a panel of H-2 congenic strains were immunized intraperitoneally with 1 ~g of HBsAg 22-nm particles. Ten days after immunization, HBsAg responder mice (strain B10) con- tained IgG specific for the pre-S2 region encoded polypeptide but none for the S region encoded sequence. At 24 days, the response to the pre-S2 region was 25-fold greater than that to the S

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region. After secondary immunization, the response to the pre-S2 region remains greater. Im- munization of HBsAg total non-responder mice (strain B10S) elicits IgG response to the pre-S2 region 24 days after injection of viral particles. Furthermore, upon secondary immunization, antibodies to HBsAg were detected. Cumulatively these results indicate that the pre-S2 region is a superior immunogen to the S region with respect to the time of the primary response and the level of specific IgG. Furthermore, the pre-S2 region can have a positive influence on the S region response and circumvent non-responsiveness to the S region. These data have implications for the development of alternative vaccines. Inclusion of the pre-S2 region product in HBsAg 22-nm particles could augment the effectiveness of future recombinant hepatitis B vaccine.

C. Pourcel: Unit6 de Recombinaison et Expression G~n~tique, Institut Pasteur, Paris, France.

The use of transgenic mice containing HBV-DNA as a model for HBV infection: developmental and hormonal regulation of viral gene expression.

In the past four years transgenic mice have been widely used to study the specific expression of genes during development and in adult animals as well as the effect of the foreign gene product on the host. These mice also provide a unique opportunity to analyze the effect of host factors, such as hormones, on the.expression of the injected gene in physiological conditions. The lack of a laboratory animal 'naturally infectible by HBV led us to consider transgenic mice as a good alternative for analysis of HBV expression in vivo. By producing transgenic mice containing HBV-DNA sequences, we expected to reproduce some aspects of the chronic carrier state of HBV in human patients. Indeed the nature and cause of the chronic hepatitis due to persistence of viral DNA are still not clear as is the role of viral infection in the appearance of hepatoma. We also wished to clarify some aspects of the viral cycle such as its restricted host range and its specificity for the liver. We injected, in a first set of experiments, a recombinant plasmid contain- ing the HBV genome deleted of the core gene. This plasmid, PAC2, was shown to direct the expression of HBsAg in transfected mouse fibroblasts. We obtained 60 animals of which 6 were shown to contain plasmid sequences upon analysis of tail DNA by dot hybridization, and only 2 had HBsAg in the serum detected by RIA. Southern blot analysis of the genomic DNA showed that the 2 HBsAg-positive mice, males E l l and E36, had one copy of the plasmid integrated. Total RNA was extracted from different organs of male and female HBsAg-positive mice of the 2 strains and analyzed by Northern blot and hybridization to HBV. A 2.1-kb RNA species comigrat- ing with the mRNA encoding the major surface antigen polypeptide was detected only in the liver of all the mice. S1 mapping analysis showed that the 5' and 3' ends were at the correct position. This suggests that HBsAg expression is controlled specifically by liver factors interacting with viral sequences. It is supported by the discovery of a liver-specific enhancer element in the HBV genome. Serum HBsAg was titrated in males and females of F1 and F2 of E36 and in F2 of E l l mice. In the 2 strains, the HBsAg levels were, in most of the cases, 5 to 10 times higher in males than in females. Analysis of the liver RNA showed that the amount of 2.1-kb SmRNA paralleled the amount of serum HBsAg, suggesting that the regulation was at the mRNA level. These observations led us to hypothesize that HBV gene expression was regulated by both liver- specific factors and sex steroids. Therefore, we studied the time of appearance of HBsAg synthe- sis during development of transgenic mice and the differences in SmRNA and HBsAg produc- tion between males and females. Northern blot analysis showed that the synthesis of the 2.1-kb SmRNA started in the liver at day 15 of development together with the albumin mRNA. However, at that time, albumin mRNA was more efficiently expressed and it is only around birth that they reached the same level. During fetal life S gene expression increased, reaching a peak at birth, then decreased during the first week of life and increased again after sexual maturation more efficiently in males than in females. No difference between male and female liver SmRNA and HBsAg levels is seen during development and until puberty, probably because of the low levels of sex steroids a n d / o r receptors present at that time. We also analyzed the effect of steroid hormones by castrating mice and injecting different hormones. HBsAg production in castrated mice was reduced at least 10-fold compared to young mature animals. Injection of testosterone or estradiol into normal or castrated mice increased the production of HBsAg and SmRNA, thus confirming the positive regulation by sex hormones. However, HBsAg levels in females never reached those in males. This may be due to the so-called imprinting by testosterone in pre- and neonatal life. We also showed that injection of dexamethasone increased expression of HBsAg,

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which could explain the peak of HBsAg at birth. In most of our experiments the amount of hepatic rnRNA paralleled the serum HBsAg levels. After castration, the amount of SmRNA de- creased and increased again following injection of hormones. Based on other studies of genes regulated by steroids, we postulate that S gene expression is regulated at the trascriptional level. HBV-DNA carries in a very condensed form sequences that allow tissue specificity, developmen- tal and hormonal regulation. However, in the absence of hormonal stimulation by glucocorti- coids and /or sex steroids, S gene expression was lower than that of albumin which is constitu- tionally expressed at high level and is not affected by hormones. In humans, infection of infants or young children very often leads to a chronic carrier state without a difference between girls and boys. Our results suggest that boys do not have a higher risk of becoming chronic carriers until puberty. However, when adults are infected, 10% become chronic carriers with a male/ female ratio of 2. If viral antigens are produced at a higher rate in men, they may allow infected cells bearing these antigens to escape the host immune response and thus lead to chronic car- riage. After 2 years follow-up, mice of the 2 strains did not show any sign of liver pathology and anti-HBs antibodies were never produced. This confirms that HBsAg in itself is not toxic to the liver and suggests that the transgenic mice are immunotolerant to the antigen. In future experi- ments we wish to analyze the effect of other factors such as alcohol or chemical carcinogens in our transgenic mice as compared to normal mice.

A. L. Zignego, C. Brtchot: Unit~ de Recombinaison et Expression G6n~tique (INSERM U 163, CNRS UA 271), Institut Pasteur, Paris, France; Istituto di Clinica Medica II, Universit~t di Firenze, Italy; INSERM U 75, CHU Necker and Unit~ d'H~patologie, Htpital Laennec, Paris, France.

Hepatitis B virus and hepatocellular carcinoma.

The chronic hepatitis B virus (HBV) infection is the most important factor involved in the patho- genesis of hepatocellular carcinoma (HCC). This was first suggested by epidemiological studies showing a close association between chronic HBV carrier state and HCC. Results of molecular biology studies investigating the presence, state and structure of HBV genome (HBV-DNA) in infected cells suggest a possible role of HBV in hepatocarcinogenesis and observations of animal models of infection further support this. However, the precise role of HBV in liver cancer as well as the respective importance of cofactors remain, at present, unclear. Structure of hepatitis B virus DNA: the genome of the HBV is a small, 3,000-3,300 nucleotide-long circular DNA molecule with a single-stranded region of variable length. The maintenance of the circular structure is ensured by a base-pairing of the five prime ends of the two strands. At both sides of the cohesive ends, there is an eleven or twelve base-pair direct repeat which plays a role in HBV replication. The two copies are termed DR1 and DR2. The genome contains four open reading frames termed S/pre-S, C, P and X, all of which are located on the same DNA strand (minus strand DNA). The S/pre-S region codes for the proteins of the viral envelope and the C gene codes for the core proteins. Overlapping all the other coding regions is a large open reading frame named P which is believed to encode the viral polymerase. The function of the X gene is unknown. The B virus is part of the hepadnavirus group which also includes the woodchuck hepatitis virus (WHV), the beechey ground squirrel hepatitis virus (GSHV), the Pekin duck hepatitis B virus (DHBV) and a recently proposed member, the tree squirrel hepatitis B virus (THBV). There are notable genetic and biological analogies between these viruses. All lead to persistent viral infection and a rela- tionship between woodchuck virus chronic infection and hepatocellular carcinoma has been clearly established. HCC has also been reported in ground squirrels and Pekin ducks chronically infected with GSHV and DHBVI It is noteworthy that the HBV and other hepadnaviruses share certain important features with retroviruses which are implicated in several forms of animal and human tumors. For example, they utilize a unique mechanism for genome replication by reverse transcription of RNA and share considerable genome homologies. It has also been suggested that they evolve from a common ancestor. Moreover, there are also some important differences such as the fact that the hepadnaviruses do not integrate into cellular DNA as a necessary step in their replication cycle. Southern blot analysis of HBV-DNA: using the Southern blot technique with the cloned HBV-DNA as a probe, it is possible to define the state of HBV in the liver cells of patients with different liver pathologies. Integration of viral DNA in liver cells has been shown in most of the HBsAg-positive tumors analyzed with this technique and also in cell lines derived from them with aspects suggesting mono- or oligoclonal proliferation of the cells and DNA

339

patterns varying from one tumor to another. Integrated HBV-DNA has also been demonst ra ted in the non-tumorous parts of liver with HCC. T he restriction DNA patterns relative to tumorous and non- tumorous tissue from the same livers are often different but may share some common bands, especially when early tumors are analyzed. Integrated HBV-DNA has also been found in the liver of HBV chronic carriers without apparent tumor. As in the case of integrat ion observed in non-tumorous parts of liver with HCC, the precise organization of the viral DNA is still not clear. Both integration at multiple sites and clonal proliferation of cells have been described. At the acute stage of HBV infection it is also possible to detect viral integration. This usually occurs in multiple sites of genomic DNA and in only a few of the infected cells. HBV integrat ion has also been detected in non-hepat ic cells of various origins; mononuc lea r cells, where viral se- quences in both integrated and free form are described, are particularly interesting. It has been suggested that the infection of these hematic cells may be important in de termining some immu- nological changes found in viral hepatitis and that this may represent an extrahepatic reserve of the virus. It is not impossible that this mononuc lear cell colonization may also in some way interfere with the antitumoral response of the host. To summarize at this stage: integrated se- quences have been shown to be present in most of the HBsAg-positive tumors analyzed and it appears that, at least in some patients, integration may precede overt development of HCC. This can occur in some chronically and also acutely infected patients. Moreover, it is a p h e n o m e n o n which is often only present in a low percentage of the infected cells, that probably might not always be necessary for main tenance of a tumorous phenotype and which is not necessary for the viral replication such as it is, for instance, for the retroviruses. One can speculate that inte- gration might be related to the cellular DNA replication as in the case of liver cell regenerat ion and that expansion of some of these infected cells would be due to viral gene expression. In- deed, it has been hypothesized that a lack of HBcAg and HBeAg expression would allow some infected cells with integrated HBV-DNA and HBsAg synthesis to escape the immune response. Cloning and characterization of integrated HBV and adjacent DNA sequences: the Southern blot anal- ysis has been completed by the cloning of the DNA sequences to define their structure more accurately. Using this approach the nucleotide sequence of the viral and adjacent DNA has been analyzed together with the genetic rearrangements secondary to HBV integration. The organi- zation of the integrated HBV-DNA is complex and there is no unique feature common to all tumors. It still appears that the function between the viral and cellular DNA sequences is fre- quently located in the cohesive ends of the HBV genome. With regard to cellular DNA also, there are no constant situations. However, interesting studies performed on h u m a n and animal HCC suggest that chromosomal rearrangements at the site of HBV integration might be one of the mechanisms involved in HBV carcinogenesis. Taken together, the results shown suggested that the complex situation of a chronic hepatitis virus infection may lead to several different modes of cellular transformation. The same cellular genes may become inappropriately ex- pressed as a direct consequence of the viral integration in their vicinity and proto-oncogenes may become activated by chromosomal breaks that have occurred on a background of cellular regenerat ion in a chronically infected liver. Finally, al though sequences similar to known viral oncogenes have not been found in the HBV genome, it is not possible to exclude the existence of viral t ransforming genes. For example, it has been suggested but not so far proved, that the X gene is in some way involved in cell transformation. HBsAg-negative HCC: it is noteworthy that in studies done in France, viral DNA was detected, al though generally in a small amount, in liver cells of most of the individuals investigated with HBsAg-negative HCC and associated cirrhosis. In addition, the study of alcoholics showed that those without HCC had a much lower rate of HBV positivity and evidence has been obtained for the presence of HBV-DNA in some patients with cirrhosis due to genetic hemochromatosis and HCC. These findings suggest tha t the effect of the virus in the development of HBsAg-negafive liver cancers might differ from that present in HBsAg-posifive cases where the majority of the cells contain HBV-DNA and it is possible that interactions between HBV infection, cirrhosis, iron excess a n d / o r alcohol may be involved in the pathogenesis of the HBsAg-negafive HCC. Furthermore, the use of more accurate techniques such as molecular hybridization analysis, radioimmunoassays based on particular monoclonal anti-HBs antibodies and transmission experiments in chimpanzees suggest that in some HBsAg- negative sera, regular HBV particles are present, whereas in other sera HBV variants may be found. Conclusions: all the studies carried out so far confirm the strong association between HBV and HCC and suggest the possibility of a direct role for HBV-DNA integration in the host ge- nome. The HBV could behave as an initiating event or as a promoting factor which occurs after initiation of liver cells by o ther factors.

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L. Cantley: Tufts University School of Medicine, Boston, MA, USA.

Oncogenes and carcinogenic mechanisms.

Genes whose mutation or overproduction result in cell transformation and tumor production have been classified as proto-oncogenes. In recent years research on endogenous proto-onco- genes and virus-encoded oncogenes has indicated that many of these genes encode for growth factors or growth factor receptors. Other oncogenes appear to encode for mutated forms of pro- teins which are involved in transducing signals from growth factor receptors to the nucleus. The realization that cell transformation involves the same biochemical mechanisms as normal cell growth, but with loss of the normal restraint, has provided new insight into the mechanisms of cell proliferation. An understanding of these mechanisms is of particular interest in tissues such as the liver where both regeneration and tumor formation are of considerable clinical interest. Many of the protein products of oncogenes are enzymes with protein-tyrosine kinase activity. The relevant targets of these protein kinases are not known but are thought to be involved in propagating the growth signal to the nucleus. We have been investigating targets of three differ- ent tyrosine kinases which are involved in growth regulation: the pp60 v'~ oncogene product, the polyoma middle t/pp60 '-'= complex, and the platelet-derived growth factor receptor. The first two proteins are responsible for tumor formation by the rous sarcoma virus and polyoma virus and the third is the receptor for the simian sarcoma virus-encoded transforming protein, v-sis. All three tyrosine kinases phosphorylate an 85,000 Da protein when activated in fibroblasts. Phos- phorylation of this pi-otein at a tyrosine residue correlates very well with transforming capability in a series of mutants of potyoma virus middle t gene. In addition, we have found a novel lipid kinase activity which copurifies with the 85,000 Da protein through a series of immunoprecipita- tion and chromatography steps. This lipid kinase exclusively phosphorylates phosphatidylinositol and will not phosphorylate phosphatidylglycerol or diacylglycerol. In addition, unlike the major phosphatidylinositol kinase in red cells, liver and fibroblasts which phosphorylate the 4' position on the inositol ring, this enzyme phosphorylates the 3' position. Cells transformed with polyoma virus have elevated levels of phosphatidylinositol-3'-phosphate (PI-3-P) compared to non-trans- formed fihroblasts and red ceils have no detectable PI-3-P. This previously unreported lipid is not an intermediate in the inositol-l,4,5-triphosphate signaling pathway but appears to be a novel phosphatidylinositol pathway involved in growth regulation. We propose that this novel PI kinase is activated when phosphorylated by certain oncogene-encoded tyrosine kinases and that the PI-3-P produced by this enzyme is critical in growth regulation.

M. Rizzetto: Divisione di Gastroenterologia, Ospedale S. Giovanni Battista, Torino, Italy.

Clinical and epidemiological aspects of 8 virus infection.

The present conception of hepatitis 8 virus (HDV) is that of a defective or incomplete virus of very reduced genome dimensions, which do not enable it to replicate autonomously. Nonethe- less, it is able to replicate and cause liver damage when drawn into the orbit of the hepatitis B virus. The concept of the HDV as a subviral entity which behaves as a satellite of a mature and competent virus (B virus) has no equivalent in human virology but can be found in the vegetable world. Examples are viriodes, minute defective entities which have similar structure and dimen- sion to HDV. Among the viriodes there are so-called RNA satellites which, like the HDV, are singularly inert but become virulent when drawn into the orbit of another mature vegetable virus capable of autonomous replication. Evidently, the mature vegetable virus provides the satellite with a necessary function for its activation, just as the HBV provides the HDV with a biological aid necessary for infection and replication. The interviral symbiosis between HBV and HDV takes place in a chimeric virion represented by a spherical particle covered by HBsAg, in which the elements of HDV are enclosed, i.e. the RNA genome (of about 1,700 base) and the 8 antigen which is the specific protein of the genome. The HDV borrows the capsule from the HBV which is required to protect its own identity and thus it becomes able to infect in the same way as the HBV and transmits itself through the vectors and modes of the HBV. The parenteral pathway is the most efficient in the transmission of HDV, as demonstrated by the diffusion of hepatitis D in drug addicts. The risks of post-transfusional 8 hepatitis during transfusions with blood checked for HBsAg are very low given that the presence of HBsAg excludes the virus D carrier from donating blood. The risk of infection from virus 8 therefore increases with overcrowding and poor hygiene, as demonstrated by the high incidence of HDV in communities at a social disad-

841

vantage in tropical areas. In general, the geographical incidence of HDV correlates with that of HBV and increases with its density. The relationship between HDV and HBV is very high in the western part of the Amazon basin (100%) and varies between 20 and 80% in subtropical areas, where HBV is endemic. Pockets of infection have been localized in the Mediterranean basin, in Arab countries and Asian Russia. Because HDV is defective, it can only develop in subjects with HBsAg but, unlike virus B infection (which can also be non-pathogenic), HDV is highly patho- genic. Its infection worsens the course of the underlying virus B hepatitis, either adding further viral damage or causing it ex novo in healthy HBV carriers. Both the individuals who are suscepti- ble to HBV and the HBsAg carriers are sensitive to HDV, but the help given by virus B occurs in different ways. In healthy subjects infected simultaneously by HBV and HDV, it is the concomi- tant expression of HBV which enables the expression of HDV. In HBsAg carriers, however, the defective virus finds the substrate required for its replication in the preexisting B infection. The clinical pictures which derive from coinfection are similar to those of acute hepatitis B. Superinfection can cause acute forms of hepatitis, but more usually the disease becomes chronic with all the long-term consequences. In asymptomatic HBsAg carriers, superinfection may pre- sent as a hepatitis which simulates acute virus B hepatitis. In these subjects, the disease is often serious and superinfection is largely responsible for the fulminant hepatitis found in the World. The forms of HDV superinfection which become chronic determine progressive hepatitis. There is a significant correlation between chronic HDV infection and the serious forms of liver disease. Conventional therapeutic attempts have been disappointing. Preliminary studies with interferon have shown that this '.drug' is active in inhibiting viral replication and in the improvement of the liver disease, but the results have proved only transitory.

H E P A T I C A N D E X T R A H E P A T I C F U N C T I O N A L A L T E R A T I O N S

I N C H R O N I C L I V E R D I S E A S E

R. Mazzanti, S. Moscarella, E Gentilini: Istituto di Clinica Medica II, Universit/t di Firenze, Italy.

Alcoholic liver disease and lipid peroxidation.

Among the various mechanisms that have been proposed to explain ethanol hepatotoxicity, a particular feature of oxidative stress is the peroxidation of membrane phospholipids. Lipid per- oxidation is a chain reaction of oxidative steps which lead to the decomposition of polyunsatu- rated fatty acids in the phospholipids of cellular membranes with the formation of intermediate products such as conjugated dienes and end products such as lipid hydroperoxides, aldehydes, hydrocarbons and others. These products of lipid peroxidation are toxic and oxidants them- selves. Moreover, they are important because they can be used to measure lipid peroxidation. Once lipid peroxidation is set in motion, profound alterations of the structure and function of the cellular membranes result, which derive from changes in membrane fluidity and modifica- tion of protein-lipid interactions. Several defence lines against lipid peroxidation and oxidative damage in general are present in the cytosol as well as in the membranes, such as GSH peroxi- dases, SOD, vitamins E, A, and ascorbic acid. Therefore, we can imagine that, in vivo, pro-oxidant and anti-oxidant factors are in equilibrium in normal conditions. Coming to the possible involve- ment of lipid peroxidation in alcoholic liver disease (ALD), it has been suggested that increased lipid peroxidation is responsible for the initiation of alcoholic liver damage. In fact, it has been shown that lipid peroxidation increases after acute and chronic alcohol intoxication in experi- mental conditions and in different animals. Since the first demonstration of increased hepatic lipid peroxides, by Di Luzio and Kalish in 1966, after an acute intoxication with ethanol in rats in vivo, many other researchers have reported results which showed increased lipid peroxidation by alcohol also using tools other than malonaldehyde (MDA) determination for detecting lipid per- oxidation. Although some controversial reports have also been published on this matter, on the basis of the main reports it is possible to ask: 1. By which mechanism can ethanol metabolism stimulate lipid peroxidation? 2. Does lipid peroxidation play a pathogenic role in ALD in man? Regarding the first point, the large production of oxygen-derived free radicals, the increase in

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the liver of iron concentrat ion and the decrease in hepatic antioxidant factors may play an important role. It is known that e thanol is mainly oxidized in the liver by alcohol dehydrogenase (ADH), which is sited in the cytosol and needs NAD § as a cofactor. In the case of a large alcohol intake, other metabolic pathways can act in metabolizing ethanol, i.e. the microsomal ethanol oxidizing system (MEOS), which requires NADPH and cytochrome P~0 for this oxidation, the catalase, which uses hydrogen peroxide, NADPH oxidases that produce the hydrogen peroxide necessary for catalase activity, and OH. free radicals. M1 these oxidative pathways for ethanol produce acetaldehyde, a reactive compound that is fur ther oxidized to acetate by acetaldehyde dehydrogenase (ALDH) hepatic activity which occurs mosdy in mi tochondr ia and, to a lesser extent, in the cytosol. This enzyme also needs NAD § as a cofactor, and in recent years its activity has been found decreased in alcoholics suggesting that this enzyme impairment may be of great importance in causing an increase in hepatic acetaldehyde concentration. Acetaldehyde, how- ever, can be metabolized in vivo also by the action of aldehyde oxidase and xanthine-oxidase which lead to the production of oxygen-derived free radicals (O~-. and H20~). Free radicals are also formed during e thanol oxidation by MEOS and by catalase. The possibility that an excess of oxygen-derived free radicals are generated in the liver during ethanol metabolism is very impor- tant because they can react with the hepatic iron which is frequendy found in increased quanti- ties in alcoholics. We have recently confirmed previous works suggesting that chronic alcohol consumption increases the intestinal absorption of iron, at least in rats. I ron is a powerful cata- lyst of lipid peroxidation which promotes the formation of the most reactive free radical, OH.. The production of large quantities of free radicals and acetaldehyde can contr ibute to the de- crease in the cellular content of glutathione (GSH), probably the main cytosolic antioxidant factor. This latter p h e n o m e n o n , which may play a critical role in permitt ing an increase in lipid peroxidation, can be caused by several mechanisms in acute and chronic alcohol intoxication. In fact, e thanol metabolism may affect the GSH turnover in the liver, due to the fact that excess of acetaldehyde can b ind covalently with GSH, free radicals increase the oxidation of GSH to GSSG which is excreted into the bile canaliculi, acetaldehyde can increase the efflux of GSH from the liver into the plasma compartment, and ethanol and acetaldehyde can inhibit the synthesis of new GSH. In chronic alcohol abuse these possibilities can act synergically with alcohol-associat- ed malnutri t ion which reduces the availability of GSH precursors, and other cellular antioxidants such as vitamins E and A. Moreover, malnutri t ion can affect the activity of detoxifying enzymes such as SOD, glutathione reductase and peroxidase and alcohol-metabolizing enzymes such as ADH and ALDH. The decrease in cellular GSH content below cridcal values has been suggested to be a crucial factor in the inhibition of Ca ++ transport at different cellular levels. According to this hypothesis, this p h e n o m e n o n could be particularly important because it seems that the in- tegrity of the cytoskeleton, and the life of the cell, depend on the ability to regulate the Ca +* transport. Thus, on the basis of the knowledge of its metabolism, it is possible to postulate that e thanol can en hance lipid peroxidation, as experimental findings had already suggested. Com- ing to the second point, is it possible that lipid peroxidation is involved in the pathogenesis of ALD in man? It must be said that the reports concerning lipid peroxidation in alcoholics are few and, moreover, without clearcut evidence for the pathogenic role of lipid peroxidation in ALl). Suematsu and coworkers, in 1981, found that alcoholics with ALD had a h igher MDA both in serum and in the liver in comparison to controls without liver disease. Two years later, Shaw and coworkers published a paper where they reported the results obtained in alcoholic and non- alcoholic liver patients which showed that in the former group the hepatic and serum concentra- tions of conjugated dienes were greater than in the latter. We have also found that in alcoholic liver cirrhosis pen tane expiration increased. These studies therefore showed that an increased lipid peroxidation occurs in alcoholic liver damage also in man. Accordingly, a reduction of GSH was shown in alcoholics with ALl) compared to controls a n d / o r patients with chronic liver dis- ease (CLD) by other authors. In partial agreement with these results, we found that the reduction of liver content in GSH in alcoholics with ALD appeared related to the severity of the liver damage. This led us to perform a study in which lipid peroxidation was compared in alcoholics affected by chronic alcoholic liver disease (CALl)) and patients with CLD. All patients were matched for age, sex, nutrit ion and, in particular, liver function tests. The results showed that MDA concentrat ion in liver homogenates and in serum was similar in the two groups. These findings were supported by the fact that also GSH liver concentration was similar in these two groups of liver patients. Interestingly, mitochondrial hepatic ALDH, which metabolizes acetal- dehyde and MDA, showed a higher activity in CALD than CLD patients, whereas the cytosolic enzyme did not show significant difference:

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patients n ~ liver MDA serum MDA liver GSH low-Km ALDH high-Kin ALDH (nmol/mg protein) (nmol/ml) (nmol/mg protein) (nmol/mg protein/rain)

CALD 24 1.0 + 0.3 1.9 + 0.1 24.9 + 2.2 21.2 + 2.8 67.5 + 6.4 CLD 14 0.9 -4- 0.2 1.8 • 0.1 22.9 4- 4.7 10.7 • 1.7 59.6 • 11.2 p value ns ns ns <0.01 ns

Whether the higher mALDH activity could be responsible for the lack of difference in lipid peroxidafion between alcoholic and non-alcoholic liver patients, or whether these findings mean that lipid peroxidation can be a consequence of the liver damage itself, is not yet determined. In conclusion, lipid peroxidafion is a logical mechanism to explain the toxicity associated with large and chronic ingestion of alcoholic beverages. Acute and chronic alcohol abuse can induce lipid peroxidation in particular conditions (fasting, anfioxidant depletion) both in animals and in man. Nevertheless, the role of lipid peroxidation in the pathogenesis of ALD remains to be definitely proved and other possible free radical damages on different cellular constituents such as DNA and proteins must be considered in the pathogenesis of alcoholic liver disease.

G. Buzzelli, C. Smorlesi, P. Dattolo, G. P. Focardi: Istituto di Clinica Medica II, Universit~ di Firenze, Italy.

Impaired glucose tolerance in chronic liver disease.

Impaired gluizose tolerance and overt diabetes are frequently encountered in patients affected by chronic liver disease (CLD), though with different prevalence according to various authors. In- deed, the occurrence of diabetes ranges from 5 to 30% of cases with chronic hepatitis and 7 to 75% among cirrhofics, whereas data concerning an impaired glucose tolerance are 5 to 45% and 6 to 55%, respectively. Such disagreement could be mainly due to different stimuli and variable criteria adopted to characterize the altered glucose metabolism, as well as heterogeneity in grouping patients according to the incidence of familial diabetes and age (personal data are summarized in the following table):

family history of diabetes age (years)

negative (%) positive (%) 20-49 (%) over 50 (%)

normal glucose tolerance 86 (65.6) 8 (24.2) 4l (69.5) 53 (50.5)

impaired glucose tolerance 18 (13.8) 4 (12.1) 6 (10.2) 16 (15.2)

diabetes 27 (20.6) 21 (63.7) 12 (20.3) 36 (34.3)

total 131 33 59 105

However, the prevalence of glucose intolerance and diabetes is significantly correlated to the severity of liver involvement. Glucose intolerance observed in CLD patients is characterized by hyperinsulinemia with insulin resistance and hyperglycemia following both overnight fasting and oral glucose load. Hyperglycemia is mainly due to impaired hepatic and peripheral uptake of glucose. In the course of cirrhosis the portal blood supply to the liver is progressively bypassed into the systemic circulation, owing to intra- and extrahepatic shunt opening and to reduced liver cell mass, with subsequent hyperglycemia. This is further enhanced by a defective glucose-phos- phorylating enzyme activity recently observed in the hepatocytes of cirrhotics with glucose intol- erance. Moreover, insulin resistance seems to play a major role in determining the impaired glucose tolerance at a peripheral level. The presence of hyperinsulinemia with insulin resistance in CLD patients with intolerance to glucose is based on a decreased hepatic clearance because of liver structural and vascular modifications occurring during cirrhosis rather than on pancreatic p-cell overproduction. Studies on simultaneous determinations of both insulin and C peptide also

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confirmed the former hypothesis. There has been much controversy concerning the pathogene- sis of insulin resistance, which seems to be related to insulin receptor status in some studies and to various defects at pre- and postreceptor sites in others. Growth hormone (GH), glucagon and free fatty acids (FFA) have been constantly found elevated in CLD patients with glucose intoler- ance. This could account for a prereceptor mechanism played by such insulin antagonist fac- tors in inducing insulin resistance and hyperglycemia. Growth hormone may produce insulin resistance by: 1. reducing tissue glucose uptake, 2. increasing FFA release from adipose tissue, 3. counteracting insulin for intracellular glucose oxidation, and 4. opposing receptor binding to insulin. Glucagon is thought to carry out its activity by inducing: 1. hyperglycemia via glycogen- olysis and gluconeogenesis within the liver, 2. lipid degradation via FFA release from adipose tissue, 3. insulin and GH release, and 4. opposition to insulin activity at a postreceptor site. The free fatty acids are known to interfere with peripheral glucose uptake and degradation. Some authors have reported decreased insulin receptors in cirrhotics with glucose intolerance. How- ever, such a finding seems to be due to long-lasting receptor exposure to abnormally high insulin levels (down regulation phenomenon), rather than representing the primary reason for insulin resistance. An important mechanism in the pathogenesis of the insulin resistance of CLD pa- tients can be reasonably attributed to postreceptor alterations, recently observed at a hepatic level, mainly consisting of impaired enzyme activities (glucokinase and glucose-6-phosphatase). So far, the pathogenesis of glucose intolerance observed in the course of CLD remains only partially clarified, despite being extensively investigated. The impaired hepatic uptake of glucose is still the only ascertained pathogenetic mechanism.

G. Laffi, E. Meacci, F. Marra, P. Gentilini: Istituto di Clinica Medica II, Universit~ di Firenze, Italy.

Platelet function in fiver cirrhosis.

An impairment of hemostasis frequendy occurs in patients with liver disease. Quantitative and qualitative alterations of platelets are involved in this phenomenon. The quantitative alteration is represented by a reduction in platelet number, whereas qualitative abnormalities consist of im- pairment of platelet aggregation. Circulating platelets are significandy reduced in patients with cirrhosis and mild impairment of liver function. Severe thrombocytopenia with a platelet count in the range of 50,000-100,000 is usual in advanced liver cirrhosis. Splenomegaly secondary to portal hypertension is considered the main cause of thrombocytopenia. Studies using 5~C-labelled platelets show an increased splenic platelet pooling in cirrhotic patients with thrombocytopenia. Platelet life span is normal or slightly reduced, megakaryocyte content in the bone marrow is normal or increased and the total body platelet mass is maintained. Redistribution of platelets from the peripheral circulation in the presence of maintained platelet production, normal total body platelet mass and nearly normal platelet life span appears to be adequate to explain the thrombocytopenia in such patients. Other causes of thrombocytopenia in cirrhotics should be considered. Increased platelet consumption at the sites of incompletely endothelized endovascu- lar surfaces in necrosing-regenerating liver tissue can be a consistent cause of reduced platelet survival. Agglutinins directed against platelets have been described in cirrhotics, but it has not been established how much they contribute to the development of thrombocytopenia. Deficiency of folic acid can contribute to thrombocytopenia. Alcohol is per s e a cause of thrombocytopenia, decreasing the life span of platelets and reducing platelet production from megakaryocytes. It is difficult to estimate the contribution of thrombocytopenia to the bleeding tendency found in liver disease. No clear relationship has been found between platelet count and abnormal bleed- ing in patients with cirrhosis. Occurrence of less hemostatically effective platelets with reduced volume has been recognized in cirrhotics. Platelet aggregation in response to ADP and adrenalin is reduced in cirrhotics and this defect is related to the severity of the disease, but not to its etiology. No clinical correlation has been found between impaired platelet aggregation and oc- currence of hemorrhage in these patients. In a recent study, we evaluated the aggregating re- sponse to collagen and to different doses of adrenalin, ADP and arachidonic acid in healthy subjects and in two groups of cirrhotics. Cirrhotics were divided into two groups, moderate and severe, respectively, according to hepatocellular impairment, as judged on a point system for different parameters. Platelet aggregation in response to collagen and arachidonic acid was sig- nificantly reduced in both groups of patients and the defect was more pronounced but not significant in patients with severe liver impairment. It is still under discussion whether the defect

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in platelet function is due to a primary defect in platelets themselves or to abnormal factors contained in the plasma of cirrhotics. The abnormal presence of fibrin monomer has been considered a possible cause of reduced platelet aggregation. Subsequent studies have not con- firmed any significant correlation between the presence of fibrinogen degradation products in the serum of cirrhotics and reduced platelet aggregation. Crossover studies realized by adding platelet-poor plasma from patients to control platelets and vice versa, failed to show a significant effect of plasma on platelet aggregation. We recently performed an extensive study of aggrega- tion on washed platelets in cirrhotic patients with severe liver disease (Child's C class). Aggrega- tion studies on washed platelets allowed us to exclude any interference related to defective plas- matic factor. A significant reduction of platelet aggregation was observed in cirrhotic patients with advanced disease, thus strongly suggesting the hypothesis that this event is presumably related to an intraplatelet defect. The possible pathophysiological mechanism involved in such a defect is still under evaluation. An impairment of arachidonic acid metabolism has been suggest- ed; in fact, a reduced platelet TxA~ synthesis was observed in these patients, and the occurrence of this defect is related to defective platelet aggregation. The reduced platelet TxA~ synthesis is not due to a reduced stimulus (i.e., a reduction of endogenous thrombin) or to a reduced Tx synthase activity, since we have previously demonstrated that added endogenous thrombin does not result in an improved TxA~ synthesis and that platelet PG~ production is also significantly reduced, thus pointing to a reduced arachidonic acid availability a n d / o r more complex defect. The reduced platelet aggregation and TxP h synthesis in response to increasing concentration of arachidonic acid seems to rule out the possibility that the observed abnormalities might be due to a reduced substrate for cyclooxygenase activity. The possibility however that exogenous as well as endogenous arachidonic acid is unavailable within the platelets cannot be excluded from these results. Another possible mechanism involved in defective platelet aggregation observed in cir- rhotic patients is related to an impairment of the transmembrane signalling mechanism. This mechanism was studied in cirrhotic patients by prelabelling platelets with [1-'4C] arachidonic acid and then measuring the production of phosphatidic acid, arachidonic acid and TxA~ which are related to sequential activation of phospholipase C, Ph and cyclooxygenase. These studies confirmed that patients with severe liver disease (Child's C class) have a reduced synthesis of all these compounds during platelet aggregation, thus suggesting an impairment of the transmem- brahe signalling mechanism. Changes in lipid composition and the consequent reduced fluidity of platelet plasma membrane observed in cirrhotic patients may be responsible for impairment of platelet response to agonists. In conclusion, severe thrombocytopenia and defective platelet aggregation are usual complications of advanced stages of cirrhosis. T h e main cause of thrombo- cytopenia is related to splenomegaly secondary to portal hypertension. Further causes involved in this phenomenon are increased platelet consumption, the presence of agglutinins, folic acid deficiency and alcohol abuse. An impairment of the platelet signalling mechanism probably related to a reduced fluidity of platelet membrane seems to be the main event involved in the defective platelet aggregation observed in these patients.

T. B. Reynolds: Department of Medicine, University of Southern California, Los Angeles, CA, USA.

Pathogenetic factors and medical treatment of portal hypertension.

Pathogenetic factors in portal hypertension: though the liver appears to be a relatively solid organ, about a litre of portal venous blood flows through it each minute at a low pressure head (4-6 mmHg) indicative of the liver's low resistance to blood flow. The litre of portal blood joins at the sinusoidal level with about 500 ml of arterial blood that has been reduced to a similarly low pressure by a high resistance in the arteriolar bed. Via a unique arrangement of sinusoids with markedly fenestrated walls, the combined portal and arterial blood streams make extensive con- tact with the microvilli of the hepatocytes in the Disse's spaces to enable the liver to perform its numerous metabolic tasks. The major reason for portal hypertension is increased mechanical resistance to blood flow through the liver due to architectural disturbance. Intrasinusoidal colla- gen deposition in the perivenular area in alcoholic liver injury, portal zone fibrosis in chronic hepatitis, and nodular regeneration with compression of portal and terminal hepatic veins in cirrhosis are all mechanisms for distortion of the microvasculature. There are other reasons for increased resistance in the large portal and hepatic venous channels as well (see the following table):

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A. Increased resistance to blood flow

I. Complete or partial inferior vena caval obstruction, above the hepatic vein orifices, by a web lesion;

2. main hepatic vein occlusion (thrombus, tumor, idiopathic sclerosis);

3. hepatic venular occlusion (veno-occlusive disease, radiation, chemotherapeutic agents);

4. hepatic parenchymal disease (alcoholic liver disease, chronic hepatitis, cirrhosis of various causes, sarcoidosis, nodular regenerative hyperplasia, congenital hepatic fibrosis, metastatic tumor);

5. portal venular occlusion (schistosomiasis, tumor emboli);

6. main portal vein occlusion (thrombosis, tumor).

B. Increased splanchnic inflow

7. Splanchnic arteriovenous fistula.

C. Uncertain cause

8. Idiopathic portal hypertension;

9. hematologic portal hypertension (myelofibrosis, chronic leukemia, osteopetrosis, Gaucher's disease, etc.).

With increasing resistance in the portal stream, main portal vein pressure rises from its normal low level of 4-6 "mmHg to a plateau of 25-30 mmHg. By the time portal pressure reaches this level, extensive collateral circulation has developed that usually prevents the pressure from rising higher. A second potential contribution to portal hypertension that has been difficult to assess is increased inflow into the portal vein by way of the splanchnic and splenic arteries. This appears to be the primary cause of portal hypertension with splanchnic arteriovenous (A-V) fistula and with 'big spleen' hematological disorders, even though increased vascular resistance ultimately develops in the hepatic microcirculation in these conditions. Increased inflow may be important in idiopathic portal hypertension, though recent evidence favors intrahepatic portal venous oc- clusion as the cause. Studies with microspheres in animal models of portal hypertension suggest that increased portal inflow, perhaps as a reflex response to decreased hepatic portal perfusion, might play a part in all portal hypertension. Medical treatment of portal hypertension: when esopha- geal varices are present in a patient with chronic liver disease, there is advanced pathology in the liver that is, to a large extent, irreversible; it is unreasonable to expect any type of treatment to be highly successful or to greatly prolong life. Alcoholic liver disease offers the greatest potential for treatment since there is at least a possibility of partial reversal of the underlying liver pathology with abstinence from alcohol. While admitting that none are spectacularly successful, there are now a number of potential treatments available for patients who have survived an episode of bleeding from esophageal varices. Some of these newer treatments might even be applied pro- phylactically, before the first episode of variceal bleeding, if it was possible to predict when bleeding was likely to occur. Factors that might determine the propensity of varices to bleed include: 1. damage to the mucosa overlying the varices from acid reflux; 2. size and degree of protrusion of the varices; 3. portal vein pressure and 4. pressure in the varices themselves. Two studies show an absence of mucosal damage in specimens of the lower esophagus obtained at surgery during variceal bleeding. There is general agreement that large varices are more likely to bleed than small ones. There is disagreement about the importance of portal pressure level in predicting variceal bleeding: most data, including our own, do not show a relationship between portal pressure and propensity :o bleeding in patients who already have varices present. The only published study on intravariceal pressure did not show a close relationship between pres- sure and bleeding. Propranolol was proposed as medical therapy for portal hypertension by Le- brec and colleagues from France. They found consistent lowering of wedged hepatic vein pres- sure after propranolol averaging 23% and this effect continued for as long as the medication was given (up to 9 months). Cardiac output fell to about the same degree as wedged hepatic vein pressure, on the average, but there was no significant correlation between the two measurements in individual patients. Hepatic blood flow fell in their patients, but to a lesser degree than wedged hepatic vein pressure. The presumed mechanism of action of propranolol in lowering

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portal venous pressure is lowering of cardiac output and blockade of [3-adrenergic vasodilatory 'tone' in the splanchnic bed. Selective ~3-blockers such as metoprolol and atenolol appear to have less capacity to lower wedged hepatic vein pressure in relationship to their cardiac output-lower- ing action. Spectacular results in the prevention of recurrent upper gastrointestinal bleeding in patients with cirrhosis and varices were reported in a controlled trial by Lebrec and colleagues. During a 2-year trial period, 79% of patients with propranolol were free of rebleeding compared to 32% treated with placebo. Patients enrolled in the trial all had alcoholic liver disease and were in relatively good condition, without ascites or jaundice. Our experience with propranolol at USC has been less hopeful. In 19 patients, mean net portal pressure was lowered from 15.3 to 12.7 mmHg by doses of propranolol titrated to lower heart rate by 25%; only 9 of the 19 patients had a fall in portal pressure of more than 2 mmHg. Four additional randomized controlled trials of ~3-blocker therapy to prevent recurrent variceal bleeding have now been reported. Three involv- ing a total of 226 patients failed to show any benefit from ~3-blockers, while one involving 24 patients did show a significanfly lower rebleeding rate while taking Nadolol. It is difficult to reconcile these divergent findings. The positive trials could be mirages or the negative results could be due to insufficient numbers of cases, poor compliance with treatment or variable effect of ~-blockers on portal pressure. The status of propranolol treatment remains uncertain at this point in time. Several other drugs have been shown to lower portal pressure experimentally (isosorbide dinitrate, oral nitroglycerine, ketanserin, verapamil); extensive clinical trials have not been done with these agents. A recent multicentre trial in France enrolled patients with esopha- geal varices that had ~aot yet bled and treated them with propranolol or placebo. The incidence of bleeding was lowei" and survival higher in the patients treated with propranolol. Propranolol has few serious side-effects in patients with liver disease so this is an attractive method of treat- ment pending further studies to confirm Pascal's results. Endoscopic sclerotherapy is another alter- native to shunt surgery with more promise than propranolol. Technical proficiency has in- creased rapidly since the reintroduction of sclerotherapy by Terblanche and associates in 1979. Most centres now use a flexible endoscope without general anesthesia. Morbidity from the proce- dure is low. The ideal sclerosant and technique are not yet established. In a controlled trial from King's College Hospital in London, survival after one year was significantly improved by sclero- therapy. At Los Angeles County-USC Medical Centre we conducted a similar controlled trial using a mixture of 3.0% sodium tetradecyl and 50% glucose injected directly into the varices. With 110 patients in our trial, there were significantly fewer episodes of variceal bleeding among treated patients, fewer episodes of bleeding per patient-month of follow-up and fewer patients with bleeding episodes. Complications include esophageal ulcers, substernal pain, transient fever and sepsis. Though pulmonary complications have not been clinically noted, there can be em- bolization of sclerosing material to the lungs as indicated by lung scan after incorporation of radioiodinated macroaggregated albumin into the injected material. All trials have had a number of sclerotherapy failures with recurrence of variceal bleeding after multiple injections. It is uncer- tain whether the appearance of gastric varices is hastened by sclerotherapy; they are difficult to inject and can be the site of fatal bleeding. Randomized controlled trials comparing chronic sclerotherapy and shunt surgery have been done or are under way in at least three centres. Cello et al. concluded that the treatments were equally effective in the acute treatment of bleeding. The results in the trial by Warren et al. favour sclerotherapy providing that distal splenorenal shunt is done as a 'rescue' in sclerotherapy failures. In a third trial at USC involving 55 patients, the results are inconclusive at three years. Pending further data the most reasonable approach to esophageal varices appears to be sclerotherapy with surgical shunt reserved for sclerotherapy failures. At what point to call sclerotherapy a failure can be a difficult clinical decision. The next few years should show us the relative merits of pharmacological therapy and sclerotherapy and allow their comparison with surgical treatment in controlled trials. However, no treatment is likely to be highly successful. Major effort should be directed toward prevention of cirrhosis and toward hepatic transplantation when advanced disease develops.

V. Arroyo: Clfnico I Provincial, Barcelona, Spain.

Water and sodium retention in cirrhosis.

The clinical course of patients with cirrhosis of the liver is frequently complicated by abnormali- ties of renal function that play a major role in the pathogenesis of ascites and contribute to the poor prognosis of these patients. Sodium retention is the most common renal abnormality in

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cirrhosis. It is constantly present in patients in whom ascites is developing but not in those with compensated cirrhosis. Initially, sodium retention in cirrhosis was considered to be due to a plasma volume contraction secondary to ascites formation. At present, however, there is general agreement that this abnormality is a cause and not a consequence of the accumulation of fluid within the peritoneal cavity. Sodium retention in cirrhosis usually occurs in the setting of a normal GFR, indicating that the predominant mechanism of this abnormality is an increased tubular reabsorption. Cirrhotic patients with ascites also demonstrate an impaired renal ability to excrete free water. In many patients this disorder can only be detected by measuring free water clearance after a water overload. In other cirrhotics, however, the abnormality is so marked that they are unable to eliminate their regular water intake. These latter patients develop severe hy- ponatremia and hypo-osmolality. Approximately 35% of cirrhotic patients with ascites admitted to the hospital have a significant dilutional hyponatremia (serum sodium concentration lower than 130 mEq/1). There is strong evidence suggesting that sodium and water retention in cirrho- sis is related to changes in endogenous neurohumoral systems. The renin-angiotensin-aldo- sterone system, which stimulates sodium reabsorption in the distal nephron, and the sympathetic nervous system, which enhances sodium reabsorption in the proximal tubule, loop of Henle and distal and collecting tubules, are activated in most cirrhotics with ascites and correlate closely with the degree of sodium retention. In addition, the release of natriuretic substances may be reduced in these patients. Sodium retention in cirrhosis is, therefore, a multifactorial phenome- non that takes place along the entire nephron. The cardiac release and plasma levels of the atrial natriuretic peptide, a powerful natriuretic substance synthesized by the atrial myocytes, are increased in cirrhotics with ascites. This peptide, therefore, is apparently not involved in the pathogenesis of sodium retention in these patients. Baseline plasma concentration of antidiuret- ic hormone (ADH) is elevated in cirrhotics with ascites, particularly in patients with hyponatre- mia and hypo-osmolality, and does not decrease after the administration of a water overload. In addition, the impairment of free water excretion in these patients correlates closely with plasma ADH concentration. Finally, the renal synthesis of prostaglandins (as estimated by the urinary excretion of these compounds), which are powerful inhibitors of the hydro-osmotic effect of ADH, is increased in cirrhotics with preserved renal ability to excrete free water and markedly reduced in patients with dilutional hyponatremia. Therefore, water retention in cirrhosis is likely to be due to the simultaneous occurrence of a non-osmotic hypersecretion of ADH and an im- paired tubular synthesis of prostaglandins. The increased activity of the renin-angiotensin and sympathetic nervous systems and the non-osmotic hypersecretion of ADH in cirrhosis are proba- bly due to a circulatory disturbance characterized by an inappropriately dilated arterial vascular bed relative to the available circulating blood volume (effective hypovolemia). This assumption is based on the following data. Patients with cirrhosis and ascites are usually hypotensive despite their having an increased plasma volume and cardiac index. This hemodynamic profile indicates the existence of a reduced peripheral resistance secondary to arteriolar vasodilation. Angioten- sin-II blockade in these patients is associated with a further reduction of arterial pressure and peripheral resistance, indicating that the circulatory disturbance would be even more intense if endogenous vasoconstrictors were not acting in the peripheral vasculature. The expansion of the circulating blood volume, either by head-out water immersion or by the insertion of a LeVeen shunt, produces a marked suppression of plasma renin, norepinephrine and ADH concentra- tions in cirrhotics with ascites. Finally, early investigations in alcoholic patients with chronic liver diseases and recent studies in experimental animals have shown that portal hypertension, by an unknown mechanism, induces an intense and generalized splanchnic arteriolar vasodilation. The following hypothesis may explain the pathogenesis of sodium and water retention in cirrho- sis. Portal hypertension produces splanchnic vasodilation that would tend to reduce the arterial pressure. Automatically, there is stimulation of the sympathetic nervous system activity, renin release, and antidiuretic hormone secretion that would return the arterial pressure to normal or near-normal levels. These factors maintain blood pressure by increasing the arterial vascular resistance and also by promoting sodium and water retention that expands the blood volume. In patients with moderate portal hypertension, the maintenance of arterial pressure would occur mainly through the expansion of the circulating blood volume, filling the dilated vascular bed, suppressing the signals that stimulate vasoactive systems, and normalizing the renal excretion of sodium and water. This sequence of events probably explains what occurs in cirrhotic patients without ascites, who have moderate portal hypertension, increased plasma volume and cardiac index, reduced peripheral resistance, normal concentrations of renin, norepinephrine, and anti- diuretic hormone, and preserved renal ability to excrete sodium and free water. When portal

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hypertension becomes more severe, however, the renal retention of fluid would not be able to correct the circulatory disturbances, probably because the splanchnic vasodilation would be more intense and, more importantly, because almost all of the fluid retained in the kidney would extravasate within the peritoneal cavity leading to ascites formation. In these patients, therefore, sympathetic nervous system activity, renin release, and antidiuretic hormone secretion would remain increased to maintain the arterial blood pressure, thus perpetuating sodium and water retention and ascites formation.

G. T. Haupert, Jr.: Department of Medicine, Harvard Medical School, Cambridge, MA, USA.

Atrial and hypothalamic natriuretic factors, identification and mechanisms.

The regulation of fluid and electrolyte homeostasis by the kidney is accomplished by a complex interaction of neural, hemodynamic, hormonal and epithelial transport processes. Alterations in the balance of these processes lead to the disruption of normal electrolyte and water homeosta- sis, including the accumulation of edema fluid, which contributes to the morbidity of patients with liver, renal and cardiac dysfunction. Although much is known about hemodynamic, hor- monal and tubular transport processes occurring in the kidney, a comprehensive explanation of the regulation of renal Na * excretion would now appear to require an understanding of the roles of two 'new' endogerious factors which are felt to play a key role in the regulation of water and electrolyte balance. The atrial natriuretic peptides (ANP) have been shown to exert potent effects on glomerular filtration in vivo, and to alter sodium transport via the voltage-dependent Na + channel in cultured renal epithelial cells. A second compound, whose structure is distinct from the atrial peptides but as yet incompletely characterized, has been partially purified from blood, urine, whole brain and hypothalamus. This substance has some of the biological properties of the cardiac glycosides and exerts its effects on sodium and potassium transport by inhibition of the sodium, potassium, adenosine triphosphatase (Na, K-ATPase), the enzymatic expression of the Na + pump. We have previously isolated and partially purified such a substance from bovine hypothalamus. This low molecular weight, non-peptidic compound has been shown to be a high- affinity, specific, reversible inhibitor of the purified Na,K-ATPase, with a mechanism of action similar to but not identical to that of the cardiac glycosides. This hypothalamic factor (HF) has also been found to be a potent regulator of the Na § pump in renal tubular epithelial cells, with binding and dissociation reactions consistent with physiologic regulation in vivo. Since ANP in- hibits with high affinity an amiloride-sensitive Na § channel in these same renal epithelial cells and since ANP is secreted from hypothalamus, the putative source of the Na,K-ATPase inhibitor, we have recendy explored the possibility that the two factors, ANP and HF, are interrelated in physiology. A method was developed to study the production of the Na,K-ATPase inhibitor in vitro using slices of rat brain incubated in culture medium. Supernatants from the brain incu- bates were purified using the same methods for purification of HF from bovine hypothalamus. The purified product was effective in inhibiting both Na § pump activity in human erythrocytes and purified canine renal Na,K-ATPase in vitro. Elution patterns from the chromatographies were identical to those of HF extracted from bovine hypothalamus, suggesting that the same substance is being recovered from both sources. The pattern of release of the Na,K-ATPase inhibitor from rat brain incubates was consistent with a secretory process. ANP injected intrave- nously, or included in the in vitro incubation of brain tissue, decreased the release of the Na, K- ATPase inhibitor from brain by 74% and 42%, respectively. Inhibitory effects of ANP were dose- dependent both in vivo and in vitro, and the threshold effect for inhibition in the in vitro studies occurred at 10 -~ M ANP, which is very close to the physiologic range for ANP binding to specific membrane receptors. Control experiments using the neuropeptide arginine vasopressin show- ed no effect either in vivo or in vitro, on release of the Na, K-ATPase inhibitor, suggesting that the effects o f ANP may be specific. These studies indicate that ANP is capable of regulating the release from brain of a Na, K-ATPase inhibitor with similar chromatographic characteristics to the one previously obtained from extraction of bovine hypothalamus, and raise the possibility that the two are interrelated in the regulation of fluid and electrolyte balance. Final structural characterization of the endogenous Na,K-ATPase inhibitor, with development of a radioimmu- noassay to detect its presence in body fluids, and further understanding of the interaction of the two natriuretic compounds, should provide important new insights into the pathogenesis of fluid and electrolyte abnormalities associated with cirrhosis, nephrosis and congestive cardiac failure.

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A. L. Gerbes: Medizinische Klinik II, Ludwig Maximilian Universit~it, Munich, West Germany.

Atrial natr iuret ic factor and sodium re ten t ion of cirrhosis.

Concepts of the pathophysiology of renal sodium retent ion and ascites formation in cirrhosis of the liver have become rather complex. There is increasing evidence that activation of the renin- aldosterone system is just one of several factors involved in the impaired volume regulation of cirrhosis. For many years, a deficiency of a putative natriuretic ho rmone in cirrhosis has been postulated, but never satisfactorily demonstrated. Thus, with the discovery of the well-defined atrial natriuretic factor (ANF) there was considerable interest in the role of this novel volume- regulating peptide ho r m one in cirrhosis. For a bet ter unders tanding of the possible implications of ANF some of its salient features will be shortly displayed, such as release upon appropriate stimuli, b inding to specific receptors, action on kidney, vascular smooth muscles, adrenal gland and central nervous system, relationship with other hormonal systems and inhibi t ion of its ac- tion by antisera to ANF. The first communicat ion on ANF plasma levels in patients with cirrhosis showed that there is no absolute deficiency of ANF in plasma as had been postulated for the putative natriuretic hormone. These findings have been confirmed by several groups, report ing ANF plasma concentrat ions in patients with cirrhosis and ascites equal to or h igher than normal. However, the immunoreactive ANF in plasma of patients with cirrhosis might comprise h igher molecular weight forms with reduced biological activity, as has been suggested in patients with congestive hear t failure. By analysis of irANF with high performance gel permeat ion chromato- graphy and reverse phase chromatography, we found no evidence for major structural differ- ences of circulating ANF in patients with cirrhosis. The reactivity of the ANF system to volume stimulation might provide information superior to that derived from determinat ion of basal plasma levels. Furthermore, it has been well described that ANF counteracts the renin-aldoster- one system in various ways. Therefore, we investigated the response of ANF and renin-aldosterone to acute volume stimulation and the relationship of these hormones to natriuresis. As a tool to induce central hypervolemia with atrial distension, prompting natriuresis and diuresis, head-out water immersion (WI) in a thermoneutral bath was used. After 1-h immersion, ANF plasma concentrat ions had doubled in healthy subjects as well as in patients with cirrhosis but without ascites. ANF increase in patients with cirrhosis and ascites, however, was significantly blunted. Plasma renin activity (PRA) and plasma aldosterone concentrat ion (PAC) were elevated in cir- rhotic patients, especially in those with ascites. Following immersion, PRA and PAC were reduced similarly in controls, cirrhotics without and cirrhotics with ascites. WI induced a more pronounc- ed natriuresis in controls than in cirrhotic patients. Neither ANF nor PRA or PAC correlated to basal or immersion-stimulated natriuresis. In an attempt to characterize the counteract ion of ANF and PAC, ratios of ANF over PAC (ANF/PAC) were calculated. These ANF/PAC ratios were found to be significantly correlated to basal and immersion-stimulated natriuresis in cirrhotic patients, and particularly closely in those with ascites. Even though correlations do not necessari- ly imply a cause-effect relationship, our data suggest that the relationship of ANF with renin- aldosterone influences sodium regulation in patients with cirrhosis. Renal response to ANF was tested by intravenous administration of synthetic ANF. Natriuretic effects of bolus ANF adminis- tration and of ANF infusion for 30 min were found to be blunted in cirrhotic patients with effects varying in a wide range. Thus, while reports on ANF in cirrhosis are still controversial in many aspects, an impairment of ANF release and renal response to ANF may be postulated and ANF, in relationship with renin-aldosterone, may play a role in sodium retention of cirrhotics.

P. Gentilini: Istituto di Clinica Medica II, Universifft di Firenze, Italy.

Vasoactive factors in the pathogenesis of the hepatorenal syndrome.

During the course of acute and chronic liver disease, renal function is frequently and often progressively impaired. T he pathogenesis of renal failure during acute liver disease is not yet clear, but is l inked to toxic factors (acid hydrolase, mercaptans, radical functions). Its evaluation is difficult due to the impossibility of accurately studying this type of patient. Vice versa, in the chronic form and particularly in cirrhosis, the pathogenetic mechanisms of renal functional impairment (RFI) have been more carefully studied for longer periods of time. At present, the greatest importance tends to be given to the imbalance between circulating plasma volume and the

351

vascular bed, which becomes much larger due to a pe rmanen t per ipheral and splanchnic vasodi- lation. This makes the circulating volume 'ineffective' in correctly stimulating the baroreceptors situated in the arterial compartment. Consequently, through an abnormal and persistent stimula- tion of the sympathetic nervous system, there is a release of vasoconstricting factors (catecholamines) which seem to be the main determinants of the compensatory per ipheral vasoconstriction and renal cortex vasoconstriction. This type of hemodynamic situation also brings about an abnor- mal activation of the renin-angiotensin-aldosterone system, with increased product ion and probably increased activity of angiotensin II in the kidney, in advanced stages resembling a state of shock. Added to these two vasoactive factors are other factors in the kidney with an autacoid action, such as thromboxane and probably the leukotrienes and platelet-activatingfactor (PAF). For a long time, a moderate RFI with decrease of renal clearances of more than 50% of the i r theoretical value is found during the course of the disease in about 15-20% of all patients with chronic liver disease. The incidence progressively increases as the ascites becomes more tense and refractory until the terminal phases are reached, when significant decreases in renal function are found in more than 50% of patients. This is more frequent in older patients. The true hepatorenal syndrome (HRS) is found in only about 5% of ascitic patients and in this case is characterized by a marked oliguria with practically absent sodiuria, a urinary osmolarity which is greater than plasmatic, and rapid and progressive increase of azotemia and plasma creatinine levels. In these stages, the long-lasting compensatory action at a renal level by stimulation of protective systems, particularly by the hyperproduction of vasodilatingprostaglandins (PGF n and PGI~) and by the activation of the bradykinin kinin system, is exhausted. Consequently, the balance between vasodilating and vaso- constricting substances is progressively weighted in favour of the latter with consequent more marked renal hypoxia and lack of correct cortical function, whereas the medullary function of concentrat ion remains preserved and paradoxically increased. The p h e n o m e n o n is aggravated by the opening of arteriovenous shunts which al though compensatory at first, subsequently deviate most of the arterial blood from the cortex to the medulla or directly towards the systemic circula- tion. Such a modified hemodynamic situation is in continuous precarious equil ibrium and is subject to sudden worsening, as may happen for example with the administrat ion of non-steroi- dal anti-inflammatory drugs (NSAID) or because of other situations which can decrease the circulating volume. In the former case, renal cyclooxygenase is blocked with decreased PG pro- duction and rapid deficit of glomerular filtration rate. In other cases, bleeding, diarrhea, inco- ercible vomiting and too drastic diuretic treatment cause a sudden decrease of circulating vol- ume, provoking a fur ther renal ischemia. In fact, in these cases there is an increasing stimulation of the RAA system with more intense renal vasoconstriction and a rapid and progressive in- crease in azotemia and creatininemia. These conditions justify the term prerenal azotemia orpseudo- hepatorenal syndrome for which the most effective treatment is plasma re-expansion. This proce- dure enables pseutlo-hepatorenaI syndrome to be distinguished from the true hepa torena l syn- drome and is useful because it blocks, at least in part, the production of strongly vasoconstrictive endogenous substances. Atrial natriuretic factor (ANF) is a hormone regulating plasma volume and is produced and released from atrial ceils following an increase in central b lood volume. It is capable of causing peripheral vasodilation and an intense renal excretion of sodium and water. This factor in some cases is increased during cirrhosis, but reports in the literature are conflicting. Another factor with intense diuretic activation, but with a probable per iphera l vaso- constrictive action, is the hypothalamicfactor (HF), a molecule which, unlike ANF, is small, non- peptidic and able to act on membrane Na§247 with a similar mechanism to that of cardiac glycosides. Its release by the hypothalamus is controlled by ANF which acts with a negative feedback mechanism. The two factors are in continuous intercorrelation with each o ther and an imbalance could be of great importance in the pathogenesis of water and sodium retent ion in the cirrhotic patient. Completely separate from these is the antidiuretic hormone (ADH), which is directly affected by a lowering of volemia and plasma osmolarity and is therefore constantly raised during cirrhosis, especially when ascites is evident. The presence of endotoxins in the blood may be of importance in the release of vasoconstricting autacoid substances in the kidney. Endotoxins seem to be capable of inducing a notable production of PAF from the glomerular mesangial cells. From a therapeutic point of view, bear ing in mind the imbalances of the renal vasoactive systems, the following are necessary: 1. continuous plasma re-expansion; 2. if neces- sary, insertion of a LeVeen shunt (new type of catheter); 3. prohibition of NSAID; 4. administration of drugs which, al though still experimental, are able to block the production of vasoconstrictive substances at the level of the kidney, or else their interaction with membrane receptors. At pre- sent, studies are being carried out on selective receptor inhibitors for both th romboxane and PAF.

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L. Varticovski: Department of Medicine, New England Medical Centre, Boston, MA, USA.

Mechanisms of phospholipase A~ regulation and availability of arachidonic acid.

Phospholipase A~ (PLA~) is a specific enzyme which catalyzes the breakdown of membrane phos- pholipids to arachidonic acid. This product is the precursor of a variety of biologically active molecules, such as prostaglandins and leukotrienes. The availability of arachidonic acid determines the extent and severity of inflammatory processes. Because glycerol-sn-2 in cell phospholipids is enrich- ed in arachidonic acid, it has been proposed that phospholipid hydrolysis leading to liberation of arachidonate is regulated by phospholipase A (PLA). Theoretically, two kinds of PLA could be involved: PLA~ which releases arachidonic acid directly, or PLA~ which acts in conjunction with lysophospholipase. The products of PLA~ (1-acyl-lysophospholipids) are detected in platelets and other cells during activation; however, 2-acyl-lysophospholipids have not been found during cell activation. These observations suggest that PLA 2 plays an important role in cell activation. Arachidonate release can also occur through phospholipid hydrolysis by PLC in conjunction with diglyceride lipases which are widely distributed. This process has been examined specifically for phospha- tidylinositol-specific PLC. The reaction products, inositol phosphate and diacylglycerol, appear as soon as 5 sec after appropriate stimuli in platelets and all other mammalian cells thus far exam- ined. The relative contribution of PLA~ and PLC in arachidonic acid release remains to be deter- mined in each system. PLA 2 is a Ca++-dependent, small, water-soluble enzyme that hydrolyzes the fatty acid ester bond at the sn-2 position of 1,2-diacyl-sn-phosphoglycerides. PLA~ are abundant in nature, specificalIy in pancreatic secretion and animal venoms. Over 40 phospholipases have been sequenced and can be separated into two groups whose difference resides in the presence of a disulfide bridge in pancreatic PLA~ which belongs to the first group. No functional differ- ences have been seen between these two groups of enzymes. All reported enzymes are secretory PLA~. There is recent evidence that intracellular PLA~ may have different properties. Availability of free arachidonic acid is a key event in production of eicosanoids and in mediating inflamma- tory response. It has been known for a long time that glucocorticoids have anti-inflammatory activity. Hirata and Axelrod observed that glucocorticoids induce synthesis of a protein which inhibits PLA~. A family of glucocorticoid-induced PLA~ inhibitors, named lipocortins, has been identified. Lipocortins are specific PLA~ inhibitors which bind to phospholipids in Ca§ - dent fashion. At the same time that PLA~ inhibitory activity was characterized, lipocortins 1 and 2 were isolated and sequenced as substrates for protein tyrosine kinases. As the sequences became available, it was realized that these are the same proteins. Subsequently, lipocortins 1 and 2 were also found to be excellent substrates for protein kinases A and C. Phosphorylation affects the affinity of lipocortins for Ca ++ and phospholipids. The functional consequences of phosphoryl- ation of lipocortins on PLA~. inhibitory activity remain to be determined. Regulation of PLAz by lipocortins, which are induced by glucocorticoids, may play an important role in controlling inflammation in liver and other tissues.

N. Villari: Dipartimento di Fisiopatologia Clinica - Uniter di Radiodiagnostica, UniversitS. di Firenze, Italy.

Modern diagnostic imaging techniques in the study of hepatocellular carcinoma.

Echography is undoubtedly the technique of choice in the study of focal lesions of the liver. In particular, it is apt for detection and evaluation of hepatocellular carcinomas. Computed tomo- graphy (CT) is therefore used only for a further diagnostic assessment of the hepatic lesions, which are always studied beforehand with echography. Only occasionally, CT may detect initial hepatic tumors during upper abdominal examinations performed for other reasons. In this case, it is possible to demonstrate limited hepatocellular carcinomas that otherwise, when they are eventually studied with CT, are rather extensive or multifocal. CT, unlike echography, is little influenced by the technical ability of the operator, even if a correct study of the liver cannot prescind from the contrastographic examination after bolus injection of contrast medium. In fact, hepatic parenchyma must be studied during the first phase of the contrastographic exami- nation because only at this time it is possible to detect the differences of attenuation that a focal lesion may present. Otherwise, in a later contrastographic phase, a focal lesion may become isodense with the healthy parenchyma, therefore no longer resulting evident. The hepatocellular carcinomas are generally demonstrated as irregular zones of hypodensity on the CT examina-

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tion, more evident in the contrastographic phase, with central necrotic degeneration. About 15% of hepatocellular carcinomas may present tiny calcifications. Rarely, the tumor is isodense with the healthy parenchyma, thus not apparent at the direct examination, and becomes evident only after contrast administration. Sometimes, it is the neoplastic lesion that demonstrates an earlier and more marked enhancement with regard to the healthy hepatic parenchyma. I f the CT exam- ination is performed with an appropriate technique, not only is a precise demonstration of the carcinoma possible, with exact definition of its spatial relationships with the adjacent structures, but also a correct staging, useful for the planning of a proper therapeutic approach. CT enables the angiographic data to be correlated more precisely by visualizing the irregular hypervascu- larization and possible portal thrombosis due to the neoplasm. Finally, it is possible with CT to assess the possible invasion of the loco-regional lymph nodes, thus defining the tumor stage. Magnetic resonance (MR) is a valuable complementary examination, besides echography and CT, in the study of hepatocellular carcinoma. This technique, which can still be defined as a 'luxury' examination, provides the surgeon with a tridimensional analysis of the lesion, by virtue of its multiplanar imaging capability (it is possible to obtain axial, coronal and sagittal slices). Moreover, MR permits the definition of the intra- and extrahepatic extension of the neoplasm (identification of a possible tumor capsule and precise evaluation of the tumor's spatial relation- ship with biliary ducts, arterial and venous vessels): in one word, it permits a correct staging of the carcinoma. In analogy to echography, MR is non-invasive, it does not use ionizing radiations and identifies correctly the organ's vessels without the need to administer contrast media. Finally, it is possible to visualize any given plane. On the other hand, in analogy with CT, MR permits a panoramic study of the liver with axial scans and, due to the various parameters which influence the image (proton density, T1 and T2 relaxation times), it permits a 'characterization' of the focal lesions as well. In fact, the hepatocellular carcinomas present a longer T1 and T2 in the Spin- Echo pulse sequences: the Tl-weighted images ensure the best anatomical detail, whereas the T2-weighted images allow a clear tissue differentiation. Unfortunately, there are still evident limits that prevent the greater use of MR in the clinical and diagnostic field. First of all, the high cost, both for purchase and for management of the MR imager, limits its widespread diffusion and consequently hinders the availability of this examination. Secondly, not to mention the patients who refuse the examination because of claustrophobia, the long time needed for image acquisition has a two-fold consequence: a. the examination is poorly tolerated by the patients with severe pain; b. the respiratory and cardiac movements may generate such artifacts that the resulting image is no longer useful for diagnosis. Lastly, there are methodological problems in MR study of the liver (differences in the technical parameters of image acquisition) which can modify the relaxation times, the value of which is therefore not solely dependent on the patho- logical features of the focal lesion, compared to those of the healthy parenchyma.

S. Bartoletti: Forbes Regional Health Center, Honroeville, PA, USA.

Role of fine needle biopsy in the diagnosis of hepatic masses.

The new technologies that have become available in the past 12 to 13 years have significantly simplified the diagnosis of nodular lesions within the liver and in the abdomen in general. The initial optimism regarding the ability of these technologies to provide us with a histological diag- nosis has however faded away as the experience has shown that histological diagnosis of ab- dominal masses could not be obtained at the present time with the available equipment. This is why biopsy of intraabdominal and specifically, intrahepafic masses, has remained the most reli- able method to diagnose the lesions that we are not able to visualize by either ultrasonography, CT or MRI. Compared with the conventional liver biopsy performed during laparoscopy, the percutaneous method obviously obviates the discomfort generated by the laparoscopic proce- dure. No premedication or local anesthesia is necessary. It allows to reach deep lesions that are not visible through the laparoscope and it can be used in patients who have undergone previous surgery without having to worry about the presence of adhesions that might make laparoscopy rather difficult to perform. The two technologies that are primarily used in our institution to guide the advancement of the needle within the liver parenchyma are ultrasonography and CT. Fluoroscopy historically has also been used for such purpose, but its use nowadays is very limited and only on one occasion we used fluoroscopy to guide advancement of the needle in a patient who had already a radiopaque catheter in place allowing drainage of the biliary tree through a

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cholangiocarcinoma in the duodenum. The advantages of ul t rasonography are: the rapid local- ization of the lesion; the lack of ionizing radiations; the flexibility of posi t ioning of both the probe and the patient; the ability to visualize lesions of medium or relatively small size; the ability at times to accurately visualize the tip of the needle. CT, on the other hand, allows visual- ization of lesions of very small size and of difficult location to reach by ul t rasonography such as near the dome of the liver. The tip of the needle is easily visualized. It is possible by CT with injection of contrast material and rapid sequence to identify vascular lesions and hemangiomas. The major advantage of CT is the ability to penetrate through gas in bowel or o ther artifacts that might be present and that at times make ul trasonography difficult. Both techniques allow visual- izadon of the anatomical relationships, but we feel that CT has superiority and the ability to establish anatomical relationships as well as to visualize the diaphragm. There has been a long controversy in the literature on the type of needles that are felt to be the most useful in percuta- neous biopsy of hepatic lesions. The large needles are divided into two main categories, a bevel- led tip type such as the Menghini needle and the cut needles with lateral windows. Large needles are defined as needles that have an outer diameter superior to 1 ram. The cur ren t trend, how- ever, is to use fine needles for percutaneous biopsies of the liver. A fine needle is a needle with an outer diameter inferior to 1 mm. Several designs are available of which we primarily use two types in our institution. T he Cheeba type needle with a bevelled tip a n d / o r the regular spinal needles that have a similar design with a slighdy sharper bevelled tip. The o ther type of needle that we occasionally use is ei ther the Green or Franzen needles that have a 90-degree bevel, and in the case of the Franzen, small teeth at the end of the needle. It is the opinion of most authors that the bevelled tip provides bet ter samples than a 90-degree tip. However, results that were recently reported from Freiburg (Federal Republic of Germany) show that a new needle has excellent results using a #20 gauge (0.095 mm) with a cut tip in a 90-degree end. The technique that is suggested using a large needle is somewhat complex because it requires the localization of the lesion by way of a fine needle. The large needle is then introduced with a rapid motion of insertion and removal at a precalculated depth using continuous suction. The technique used with free needles is significantly different. The needle is inserted through the skin unde r ei ther ul trasonographic or CT control following the use of some local anesthetic. The fine needle is advanced to the lesion, preferably to the edge of the lesion. Suction is then applied using an aspiration gun attached to a#20 ml syringe. The needle is then moved with small up and down motions, and with rotatory motion at the same time while suction is applied. After the motion is stopped, the aspiration is discontinued and the needle is removed. At this point, the contents of the needle are sprayed by the pathologist on several slides. The remain ing material that might be left in the needle or in the tubing is then fixed in alcohol as are the slides. The material in the tubing is then put in a paraffin block for what we call a cell block evaluation. The slides are usually stained with hematoxylin and eosin. Let me point out that the immediate t rea tment of the specimen by the pathology depar tment is absolutely essential. Mso, it is of great importance that the pathologist is adequately trained to read cytological specimens. The success of the aspiration biopsy is greatly dependen t on the quality of the pathological evaluation. While the utilization of a large needle is pretty much restricted to one pass per lesion, that is essentially bl ind because of technical reasons that were above mentioned, a fine needle biopsy can be performed with mul- tiple passes and in the literature as many as 12 to 15 passes per lesion have been mentioned. We are somewhat more conservative and ordinarily try to limit our n u m b e r of attempts to three or four if no diagnostic sample has been obtained with the previous pass. Whe the r we choose ul t rasonography or CT as our guiding method, we try to approach the lesion by the most direct route. It is ordinarily much easier to accurately obtain a sample simply going in a vertical direc- tion at 90 degrees to the floor. Occasionally, because of anatomical considerations, we will angle the needle to avoid traversing the pleural space or the gallbladder or the main biliary ducts. The flne needle approach to hepatic lesions at times is jus t the preliminary phase to the insert ion of draining catheters or devices that we utilize particularly in patients who are poor surgical risks to drain hepatic abscesses. T he indications for percutaneous needle biopsy of the liver are several. Apart from the histological diagnosis of solitary intrahepatic masses, the diagnosis of hepatic abscesses represents a significant amount of the number of cases that we are asked to diagnose. With the aggressive approach to neoplasm that characterizes m o d e m medicine, the confirmation of the nature of hepatic metastases in patients with known primaries is also frequently the rea- son for performing the procedure. This is particularly true when patients have histories of two separate pr imary tumors that require different therapeutic approaches ei ther by radiation ther- apy or chemotherapy. It is also very important in patients with known primary lesions elsewhere

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in the body, to exclude the possibility that the lesion identified in the liver might not represent a metastasis, but indeed a benign lesion, as pointed out by the group from Freiburg in West Germany. While the utilization of larger needles probably provides us with a better histological diagnosis as Pagani pointed out in his articles, the experience of most other authors with the use of needles above the 1-mm diameter size, has been marked by a significant increase in the mor- bidity if not the mortality. This is why fine needle biopsy of abdominal lesions is now considered the optimal technique. We prefer to use #20 gauge bevelled tip needle with a connecting tube, a

20 ml syringe and a Franzen syringe holder that allows one person to perform the procedure alone. I have reviewed 36 cases of percutaneous needle biopsies performed by this technique. Twenty-nine patients had a positive diagnosis of malignancy corresponding to a diagnostic sensi- tivity of 81%. In 7 patients we were unable to obtain a significant sample. Of the 29 patients with malignancy, the pathologist was able to give a probable histological diagnosis that was correct in 19 or 51% of the cases. In 10 patients, the diagnosis of malignancy was made; however, the histological type could not be established on the basis of the sample. These results are within the usual range reported in most large series. It appears that the most difficulty in establishing a histological diagnosis is when lymphoma is involved, as its differentiation from undifferentiated carcinomas can be at times rather difficult. We had no false-positive diagnoses, therefore the specificity is 100%. Although a number of complications have been mentioned in the literature including infection, hemorrhage, hemoperitoneum, bilioperitoneum and hemobilia, we have ex- perienced a very low rate of complications at our institution. No complications required signif- icant medical intervention to correct them. We have had no instances of infection or bilioperi- toneum. A few patients have experienced some minor pain that might have indicated a small amount of hemoperitoneum without severe consequences. In one case, the gallbladder was inad- vertently punctured, but the patient experienced no complications. There is one report in the literature by Dr..Ferrucci from Massachusetts General Hospital in Boston of seeding of tumor cells along ithe biopsy tract. We have not experienced this kind of complication and apparently this must be a rare occurrence because it is not mentioned in other large series. We, however, have noticed in a patient a neoplasm that tracked outside the liver to the abdominal wall follow- ing a drainage procedure that was performed on a patient because the diagnosis of abscess was originally made in a patient who had two large infected metastatic lesions to the liver from a primary of the colon. A few months after the draining tube was removed, the tumor was seen growing to and outside the chest wall. Surprisingly enough, two years after this finding, the patient is today still alive and the metastases are controlled by chemotherapy. In conclusion, we feel that percutaneous needle biopsy of hepatic lesions is a valuable and relatively innocuous way to establish diagnosis in patients with focal hepatic lesions.

M. Strazzabosco, M. Muraca, M. Venuti, A. Varotto, R. M. Iemmolo, A. Fragasso, D. Passera, L. Okolicsanyi: Istituto di Medicina Interna, Universit/~ di Padova; Istimto di Clinica Medica e Nefrologia, Universit~ di Parma, Italy.

Evaluation of simplified procedures for determination of antipyrine clearance.

Antipyrine exhibits unique kinetic properties: given orally it is rapidly and completely absorbed, its binding to plasma proteins is negligible and its distribution volume equals total body water. The drug is extensively metabolized by hepatic microsomal enzymes, its disposal can be describ- ed by a one-compartment model, and it is not influenced by hepatic blood flow. Therefore, the determination of antipyrine clearance is being increasingly used to assess hepatic drug metabo- lism and as a quantitative test of liver function. The procedure usually performed is cumbersome and requires repeated blood sampling for at least 27h. The aim of our work was to evaluate the applicability of antipyrine clearance calculated from one or two serum samples by using a newly available kit that includes a spectrophotometric assay suitable for clinical laboratories. Patients and methods: 68 hospitalized patients (54 males, 14 females, mean age 49 + 8 years) were studied. Thirty of them presented no signs or symptoms of liver disease and had not received drugs for at least 2 weeks before the study. The other 38 were chronic liver patients of different clinical severity, some of them with ascites receiving spironolactone. They were classified as decompen- sated (n ~ ----- 24) if they presented at least two of the following features: bilirubin >1.2 mg/dl, albumin <3.2 g/dl, ascites, and portal hypertension. In 15 patients with liver disease the proce- dure was repeated at 6-month intervals, so that a total of 90 tests was performed. Antipyrine, 18

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mg/kg body weight as an aqueous solution, was given orally after overnight fasting. After the ad- ministration of the drug, blood samples were taken at 3-h intervals up to the 27th h. Samples were kept at -20 ~ until the time of the assay. Antipyrine concentrations in plasma were deter- mined with a spectrophotometric assay derived from Brodie et al. Calculations: in all patients anti- pyrine clearance (ApCI) and apparent volume of distribution (Vd) were calculated by the three following procedures. 1. The standard procedure: it takes into account the whole series of con- centrations determined and clearance (C1) is calculated as: CI = K • Vd = (dC/dT) x (D/Co), where C is the concentration, T is time, D is the dose given, Co is the extrapolated concentration at time 0, and K is the elimination constant estimated from the linear regression of the logarithm of C on T. 2. The one-sample procedure: this is based on the same principle, but Co is estimated from a postulated Vd and K is assessed from Co and Ct:

C1 = In (D/Vd)-- In Ct x Vd t

In each patient, for each of the 7 samples determined, ApC1 was calculated both assuming a Vd of 40 1 and by approximating it from age, sex, body weight and height, using the formula of Bruce et al. The latter estimation was possible since the volume of distribution of antipyrine can be considered equal to total body water. 3. The two-sample procedure: in this case antipyrine clearance was calculated from Eq. 1, utilizing only two time-concentration points. Vd was estimat- ed from the administered dose and the extrapolated Co. At any calculated point, the values of one-sample and two-sample clearance were tested for correlation with the values obtained from the whole set of data by least-squares analysis. Results and discussion: one-point method: the results of regression analysis (see the table) indicate that the longer the time interval between adminis- tration and sampling, the better the correlation with the clearance calculated on the whole series of plasma samples. The 27th h time point gave the best correlation coefficient, using both a fixed Vd of 401 and the one calculated from Bruce's formula (r = 0.93 and 0.92, respectively). When separating our patients according to the presence or absence of liver disease, the one-sample method was also found to be reliable in patients with liver disease. Assuming that the 27th h is the best sampling point, the mean antipyrine clearance values were 34.5 + 12.8 vs. 37.4 + 13.7 ml /min (standard calculation vs. one-point, r-----0.91) in the control group and 22.8 + 9.7 vs. 24.7 + 9.8 ml /min (r = 0.97) in patients with liver disease. Data obtained employing Bruce's for- mula were comparable.

sampling n-* of r slope of intercept of residual ApCI time (h) samples regression regression variance (ml/min, mean • SD)

one-sample method

3 52 0.24 0.62 30.06 887.5 47.69 • 30.44 6 60 0.44 0.68 16.70 262.4 35.42 • 17.88 9 57 0.69 0.88 8.31 110.0 33.81 • 14.37

12 54 0.82 0.88 6.93 50.8 31.65 • 12.56 24 61 0.92 0.98 0.45 23.6 27.90 • 12.40 27 53 0.93 1.06 -0.10 21.4 27.76 • 13.03

two-sample method

3- 6 58 0.34 0.41 20.63 188.2 31.64 • 14.48 24-27 39 0.26 0.31 11.72 176.0 20.18 • 13.59 6-12 52 0.88 1.00 1.12 41.6 28.75 + 13.60 3-12 54 0.87 0.85 6.26 33.9 29.16 • 11.94

12-24 48 0.96 0.98 -1.15 11.0 27.31 • 12.29 6-24 53 0.97 0.98 -0.76 8.1 27.35 • 11.90 3-24 57 0.96 0.98 -0.31 11.8 26.77 • 12.41 6-27 50 0.97 1.05 -1.04 6.3 27.24 • 12.41 3-27 49 0.97 1.03 -0.05 7.5 26.49 • 12.62

Whole-series clearance = 27.79 + 11.56 ml/min.

Two-point method: the regression analysis of the two-sample method is also shown in the table. The longer the interval between the two samples, the better the correlation was with the clear- ance calculated from the whole series of plasma samples. The 3rd-27th h pair of time points

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appeared to be the best combination (r = 0.97, see the table). The simplified method also gave reliable results when dividing our patients according to the presence or absence of liver disease (34.5 + 12.8 vs. 33.5 + 14.9 ml/min, r -- 0.98 in controls, and 22.8 • 9.7 vs. 24.2 + 11 ml /min in patients with liver disease). Moreover, the two-sample method allowed an accurate estimation of the apparent volume of distribution of antipyrine (r ---- 0.95, p<0.001). In our series, the mean apparent volume of distribution of the group of patients with liver disease was significantly dif- ferent from that of the control group (43.6 + 9.3 vs. 36.9 • 9.01, p<0.025); the total body water was significantly higher in decompensated patients than in compensated or normal subjects (60.5 • 4.2 vs. 54.75 + 8.9 and 54.5 + 8.4%, respectively, p<0.025). In conclusion, the results of the one-sample method confirmed the data obtained by Dossing et al. in patients with chronic liver disease of varying clinical severity. However, the one-point method can only be applied to pa- tients in a stable condition, and frequently this is not the case in chronic liver patients. Moreover, since water retention commonly occurs in liver disease, the determination of total body water (i.e., antipyrine Vd) could be important in such conditions. With the two-sample method, we ob- tained a highly significant correlation with ApCl calculated on the whole series of samples, the 3rd and 27th h being the best pair of time-concentration points. Moreover, we obtained an accurate estimation of distribution volume. This is of particular interest since clearance and Vd are two independent pharmacokinefic variables. In our series, the mean Vd of antipyrine was higher in the group of patients with decompensated liver disease than in compensated patients or normal subjects. Nevertheless, the potential value of estimating antipyrine Vd in the follow-up of patients with liver disease remains to be established.

L Capocaccia, F. Ariosto, M. Merli, O. Riggio, A. Romiti: II Cattedra di Gastroenterologia, Uni- versit~t di Roma 'La Sapienza'; Istituto di Medicina Interna, Universit~ de L'Aquila, Italy.

Pathogenesis of hepatic encephalopathy. Certainties and uncertainties.

Hepatic encephalopathy (HE) can be described as an alteration in mental or neuromuscular behavior which occurs in some patients during severe hepatic dysfunction, no other neurological causes being evident It is generally considered a metabolic encephalopathy, the structural changes occurring in the central nervous system not being sufficient in themselves to explain the complexity and seriousness of the clinical symptoms which arise. Clinical features: HE may be divided into two clinical settings: HE complicating fulminant hepatic failure and HE compli- cating chronic liver disease. HE complicating chronic liver disease can be further distinguished either as a latent 'subclinical' HE; a sporadic HE occurring after major precipitating events, usually spontaneously subsiding; a chronic HE, frequently relapsing or continuously symptomat- ic; or finally a permanent HE, rare and usually irreversible with conventional therapy. Diagnosis of HE is clinical, as no diagnostic, biochemical, neurological or radiological tests are simulta- neously specific, sensitive, easy and inexpensive. Clinical manifestations of HE may be graded into 5 stages, from grade 0 where no abnormality is detectable through to grade 4 where the patient is in coma and the mental state is not testable. These stages are useful for follow-up assessment The pathogenesis of HE has still not been fully clarified and thus the mechanism(s) involved may differ. The possible pathogenetic mechanisms of HE proposed include: alterations of the blood-brain-barrier (BBB); toxins: ammonia, mercaptans; altered neurotransmitters: FNTs-AAAs, GABAVBz receptor complex, neuromodulatory peptides. The BBB alteration hypothesis: no proven structural changes in chronic HE have been described other than those linked to artifacts relat- ed to the experimental models employed. However, functional changes in the BBB transport systems possibly involved in the pathogenesis of HE have been well documented. The ammonia toxicity hypothesis: the existence of a statistical correlation between HE and ammonia favours this hypothesis, as does the fact that the administration of ammonia on experimental animals pro- vokes coma. Moreover, treatments thought to reduce ammonia blood levels are usually effective in the treatment of HE. The increased quantity of ammonia in the brain, causing substrate and energy depletion in the central nervous system (CNS), has been proposed as a mechanism of ammonia toxicity. More recendy, ammonia-induced changes in the neurotransmission (neuro- excitation or neuroinhibition) have been described. However, the relevance of such alterations is still widely disputed, and to date no single toxicity mechanism has enjoyed full consent Besides, experimental hyperammonemia with coma is different from experimental HE and it has been shown that patients often have high blood ammonia without HE and vice versa. Thus, while

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ammonia is involved somehow in the pathogenesis of HE, it cannot be said to play the role of key toxin. The mercaptan toxicity hypothesis: a synergistic effect has been demonstra ted in experi- mental animals between mercaptans, ammonia and short-chain fatty acids in inducing coma. Visually evoked potentials (VEPs), similar to those observed in experimental HE in some studies, accompany this effect. Human data in some studies show a correlation between blood mercaptan levels and HE. Recent methodology used in determining blood mercaptan concentrat ions has cast some doubt on this finding. Moreover, a combinat ion of synergistic toxins has not always reproduced coma a n d / o r VEP modifications. Finally, mercaptan concentrat ions used experimen- tally have little relevance for human HE. To sum up, no confirmed evidence exists on the role of mercaptans. Moreover, early studies implicating mercaptans in the pathogenesis of HE need to be repeated using contemporary technology. The false neurotransmitter-aromatic amino acid (FNT- AAA) hypothesis: according to this hypothesis, the elevation of plasma aromatic amino acid and the contemporary reduction of branched-chain amino acid (BCAA) facilitate the flux of AAA into the brain. Simultaneously, and particularly when precipitating events occur, high ammonia plas- ma levels and the subsequent rise in brain glutamine content activate the neural amino acid transport system across the BBB. This leads to a further increase in the entry of AAA into the brain. Such an accumulation would cause a decrease in aminergic neurotransmit ters and an increase in serotonin and FNTs. Certain data favour the FNT-AAA hypothesis. Experimental data show an accumulation of AAA, serotonin and FNT in the brain, and the activation of the BBB transport system. Moreover, the administration of an AAA infusion in normal dogs induces co- ma. Lastly, changes "occur in dopamine receptors in experimental coma. With regard to human data, the presence of AA imbalance, which is always observed in patients with HE, the correla- tion between FNT plasma levels and FNT- and AAA-CSF levels and stages of HE, the effective- ness of BCAA infusion and of increasing brain dopamine content in some studies, all favour the FNT-AAA hypothesis. On the other hand, the lack of experimental coma induction with the administration of FNT and depletion of catecholamines, the lack of decreased bra in catechol- amine content in deceased patients with HE, the ineffectiveness of BCAA infusion and of treat- ments aimed at increasing brain dopamine in o ther studies, as well as the fact that AAA doses to induce experimental coma are usually h igher than the AAA levels reported in humans , are all aspects which put this f inding into dispute. In conclusion, al though a considerable amount of experimental and human data lends force to the FNT-AAA hypothesis, the original f inding should be reinvestigated in the light of contemporary neurophysiological technology before it can be accepted. GABAergic neurotransmission hypothesis: this hypothesis proposes that increased GABAergic tone contributes to the neural inhibit ion of HE. To explain this alteration two sug- gestions have been proposed: 1. increased gut-derived GABA crosses an abnormal ly permeable BBB and results in increased brain GABA content; 2. decreased brain content of GABA-synthe- sizing enzyme and denervation supersensitivity of GABA-benzodiazepine receptor system due to toxins (ammonia, mercaptans causing zinc depletion). With regard to animal data, the increased n u m b e r o f GABA/Bz binding sites in some acute and chronic models of HE, as well as the amelioration in HE induced by GABA antagonist administration, support this hypothesis. More- over in man, patients show increased sensitivity to benzodiazepines, PET scan data are consistent with increased density of Bz receptors in some patients with recent HE, and finally, Bz antagonist induced transient amelioration of HE in experimental models and in some patients. The pro- posed mechanism leading to coma would be a CNS depression due to GABAergic activation or to endogenous benzodiazepine agonist ligand. Contrary to the GABAergic neurotransmiss ion hypothesis is the fact that GABA/Bz binding sites are normal in other models of HE. Moreover, no changes in plasma and CSF GABA levels in humans with HE have been demonstrated. In conclusion, the availability of new experimental data have modified fairly extensively this hypoth- esis, thus necessitating reappraisal. The neuromodulator peptide hypothesis: certain peptides, known to trigger neurotransmit ter release, have been involved in the pathogenesis of HE. This hypothe- sis is based on the improved effect of high permeability membranes on HE (induced by fulmi- nan t hepatic failure) in eliminating the middle molecules. Serotonin may be involved in this process, as yet undef ined substances appear to provoke its metabolism. The neuromodulatory peptides have not yet been defined, and few controlled studies on humans regarding the effect of high permeabili ty membranes exist. Thus, the role of this hypothesis has yet to be clarified. Conclusions: to summarize, al though HE seems to be the consequence of altered neurotransmis- sion in which the exci ta t ion/ inhibi t ion balance shifts towards inhibition, no current hypothesis can be considered as fully accounting for all the characteristic features of hepatic encephalo- pathy.

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GASTRODUODENAL PEPTIC DISEASE

L. Pagtiaro, G. D'Amico, M. Traina, L. Montalbano, G. Gatto, R. Pisa, S. Maisano, F. Polifi, P. Colletti, F. Tin6: Clinica Medica, Universit~t di Palermo; Ospedale 'V. Cervello', USL 60, Palermo, Italy.

Congestive gastropathy in cirrhosis.

Several studies on gastric mucosal changes in cirrhosis have been published in the last few years. McCormack et al. described the endoscopic features of 'mild' and 'severe' gastric changes having the histological hallmark of dilated and congested submucosal veins. Accordingly, they called 'congestive gastropathy' (CG) the gastric mucosal changes of cirrhosis. The concept that the gastric changes common in cirrhosis (and in portal hypertensive rats) are related to the portal hypertension itself has been supported by other clinical and experimental studies. Additional evidence can be drawn from reports showing that the reduction of portal hypertension by sur- gery or propranolol seems to be of benefit in cirrhotics bleeding from gastric erosions. Here we report preliminary data from a prospective study performed in patients (pts) with cirrhosis, ex- ploring the endoscopic features and prevalence of CG as well as its value in predicting acute or chronic bleeding from gastric mucosal lesions. Materials and methods: two groups of pts entered the study. Group 1 included 132 consecutive pts with newly diagnosed, most often compensated cirrhosis. None of th6m had bled before entering the study. All were endoscoped at admission into the study and regularly followed-up. Endoscopy was repeated at 1-year intervals and as an emergency procedure if pts bled. Group 2 included 62 consecutive pts admitted to the hospital for variceal bleeding and then treated with elective endoscopic sclerotherapy (EVS) for the pre- vention of rebleeding. None of the pts in either group was taking drugs inducing gastric damage. Mean follow~up was 46 + 15 months for group 1 and 21 + 12 months for group 2. The presence, location and severity of CG were assessed by two independent observers. In the last 18 endosco- pies from 13 pts with CG, biopsies were taken from fundus, body and antrum for histological examination. Results: 1. two patterns of CG were observed: a 'mild' form, with multiple hyperemic areas surrounded by a thin, yellowish network ('mosaic-like'), also described by McCormack et al. and Papazian et al.; and a 'severe' form, characterized by intense hyperemia, granular or papular elevations of mucosa with erosions, and by focal or diffuse bleeding, spontaneous or on touch. Both patterns were easily recognized by the endoscopists, with a very high inter-observer agree- ment, as shown by preliminary data from a formal assessment. The mild CG was most often diffuse, or localized in the antrum; the severe CG was usually localized in the antrum or in the fundic area, and infrequendy diffuse. Histology showed prominent congestion of small vessels in all specimens, with remarkable mononuclear infiltration. The preferential localization of severe CG was in the antral area:

antrum fundus/body

small vessel congestion +++ +-- mononuclear infiltration usually full thickness usually superficial hyperplasia of m. mucosae +++ ++

chorion fibrosis ++ +--

2. The total prevalence of CG in both groups was 43.8%; the changes of CG were detected at the first observation in 26.3% and appeared during the follow-up in the remaining 17.5%. CG (in mild form) disappeared in 4 pts only after first detection. The prevalence and the severity of CG were significantly greater in group 2 than in group 1, although the follow-up of group 2 was shorter. This difference might be due to the higher number of pts with large varices and in Child-Pugh class B in group 2. In fact, the prevalence and the severity of CG were significantly related to the presence and size of esophageal varices, and were also greater in pts in Child-Pugh class B than in those in class A (only 6 pts were in class C, and this small number precluded any comparative assessment). CG was not more frequent in pts with a history of alcohol abuse. How- ever, all pts had stopped alcohol abuse and only a few of them were continuing moderate drink- ing. Therefore, we assume that alcohol did not play any role in inducing CG in our patients.

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3. Follow-up data showed that pts with CG had a significantly higher incidence of episodes of acute bleeding from erosions, and also of admission to the hospital for overt hemorrhage. Both were more frequent in patients with the severe form of CG. Some patients in this subset had a characteristic clinical course, with episodes of slow-grade decrease in Hb, recurring for years and requiring hospitalization with hemotransfusions or iron treatment:

no CG mild CG severe CG

patient n z 109 68 17 Hb decrease (%) 10 32 88 acute UGIH (%) 11 19 48 deaths(%) 17 25 40

Discussion: in the present study we found that the endoscopic changes of CG are common in patients with cirrhosis, have characteristic features and are easily and reproducibly recognizable. These findings confirm and extend previous reports by McCormack et al. and by Papazian et al. On the other hand, we did not confirm the preferential localization of CG in the fundic area, as reported by these authors. We found a statistically significant relationship of CG to the presence and size of esophageal varices; this observation, together with the histological congestion of small vessels, supports the dependence of CG from the portal hypertensive status. The most re- markable finding of this study is the prospectively assessed increase in the incidence of both acute episodes of bleeding from erosions, and of recurrent episodes of slow-grade, insidious develop- ment of anemia without overt manifestation of bleeding. The high risk of these events can justify clinical trials of treatments decreasing portal hypertension in patients with severe CG.

L. Barbara, R. Corinaldesi, R. De Giorgio, V. Stanghellini: Istimto di Medicina Interna, Divisione di Gastroenterologia, UniversitY. di Bologna, Italy.

The medical treatment of peptic ulcer: present status.

Peptic ulcer may be considered a consequence of the prevalence of intraluminal noxious agents - i.e., chlorhydropeptic secretion - upon mucosal resistance of the gastroduodenal tract. Despite extensive research, the pathogenesis of peptic ulcer is still only partially understood. The pres- ent views suggest different possible levels of therapeutic approaches: 1. inhibition of gastric acid secretion by drugs acting on parietal cells (H2-blockers, antimuscarinic compounds, prostaglan- dins and substituted benzimidazoles); 2. decrease of the damaging effects of intraluminal con- tents (antacids, binding agents); 3. protection of the gastric and duodenal mucosa, directly or by promoting cytoprotective events (sucralfate, colloidal bismuth, carbenoxolone and prostaglan- dins); 4. modulation of gastrointestinal motility by agents acting on muscle layers (prokinetic drugs, fibres). The purposes of the medical management are twofold: a short-term treatment ('tactical') is necessary to induce the healing of the lesions, while a long-term treatment ('strate- gic') is required to prevent the relapses of the disease. Short-term treatment: the use of increasing- ly powerful drugs, at present, brings about a rapid healing in almost all the patients treated, not only in duodenal ulcer but also in gastric ulcer. Antisecretory compounds are the most common- ly used drugs for short-term treatment. They decrease hydrochloric secretion either by blocking the receptors (H~, muscarinic) present on the serosal surface of the parietal cells, or by inhibiting intracellular adenylcyclase, or by blocking the proton pump H+/K+-ATPase which represents the final step of the secretory mechanism. The introduction of cimetidine marked the beginning of a new era in the treatment of peptic ulcer. This drug has not only been successfully used in several million patients, but has also stimulated a profitable research towards new molecules. Among these, ranitidine has proved to be more effective than cimetidine in inhibiting gastric secretion. Moreover, it induces the same healing rates, both in gastric and duodenal ulcer, at a dosage that is 3 to 4 times lower than cimetidine, with fewer side effects. Recently, a newer H~-antagonist, famotidine, has been shown to exert an antisecretory effect 8 times greater than ranitidine: at a dose of 40 mg 'nocte' it induces healing rates similar to those obtained with ranitidine 300 mg daily. Famotidine, like ranitidine, also seems to be characterized by high tolerance levels, even though the clinical experience is not large enough to allow a definitive opinion. Research aimed

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to identify more and more effective and well tolerated H2-antagonists is still very active (niza- tidine, etintidine, mifentidine, roxatidine), even if the results obtained so far do not suggest remark- able advantages as compared to those of the previous molecules. In clinical practice, the intro- duction of drugs, such as the benzimidazolic substitutes, represents a further step in the treat- ment of peptic ulcer, since these compounds are able to block the proton pump which controls gastric acid output. Omeprazole, at a single dose of 20 mg daily, can almost completely (90%) suppress 24-h gastric secretion. Omeprazole (20 mg i.m.) has been described as being very effec- tive in the short-term treatment of both duodenal and gastric ulcer, with healing rates of 100% (after 6 weeks) and 96% (after 8 weeks), respectively. Pirenzepine, a selective antagonist of musca- rinic receptors (M1), is as effective as cimetidine in the treatment of duodenal ulcer. However, it is less rapid than H~-antagonists in promoting the remission of dyspeptic symptoms and, at high doses, it may provoke anticholinergic side effects (e.g., mouth dryness, accommodation dis- turbances). Besides antisecretory compounds, other drugs capable of improving the defensive mechanisms of the mucosa have been successfully used in the therapy of peptic ulcer. Sucralfate exerts its protective effects by creating a film over the injured mucosa, thanks to its property of linking to cell proteins ('coating effect'). Its therapeutic efficacy is comparable to that of cimeti- dine, but its use is limited by the necessity for multiple daily administration. Colloidal bismuth has been reported to exert its anti-ulcer effects through different mechanisms: it creates a protective film on the ulcer lesion; it stimulates the synthesis of endogenous prostaglandins; it exerts an intense antimicrobial effect on Campylobacter pylori (even if the actual role of this germ in the pathogenesis of peptic ulcer remains controversial). The therapeutic results are very encourag- ing, since the healing rates are similar to those obtained with ranitidine. However, the moderate increase of bismuth serum levels suggests that the administration of full doses of this drug should be limited to 8 weeks or less. The term 'cytoprotection' indicates the capacity of some prostaglandins (when administered at doses unable to inhibit gastric secretion) to prevent in- flammation and necrosis of gastroduodenal mucosa induced by lesive agents. Carbenoxolone, suc- cessfully employed in the treatment of peptic ulcer, can slow down the catabolism of endogenous prostaglandins and it probably also exerts an antipeptic effect. The possible induction of side effects (water and salt retention, hypokalemia and hypertension), however, limits the use of this drug. Natural prostaglandins are not suitable for ulcer treatment, because of their very rapid catabolism after injection. At present, attention has been focused on the synthetic derivatives of cytoprotective prostaglandins. Misoprostol and enprostil have been successfully used in short-term treatment of peptic ulcer, with healing rates similar to those of H~-antagonists. However, these favorable results are somehow limited by both the occurrence of frequent side effects (especially diarrhea) and the need for repeated daily administrations. Antacids exert their therapeutic effects by buffering hydrogen ions secreted in the gastric lumen, decreasing peptic activity and, proba- bly, by binding bile acids. Compared with H~-blockers (cimetidine), antacids show a similar ulcer healing incidence, significantly higher than placebo. However, their use is limited by the high frequency of side effects, mainly diarrhea. In conclusion, many drugs can effectively heal peptic ulcers: only a small number of patients do not respond to appropriate short-term treatments. The reasons for the lack of response in this limited group of patients remain obscure. However, the use of increasingly effective antisecretory drugs tends to eliminate this problem. Five to 10% of ulcer patients do not heal after a short-term treatment with H~-antagonists, while almost no negative results have been observed after omeprazole. Furthermore, this latter drug appears efficacious in healing gastric or duodenal ulcer 'resistant' to ->3 month therapy with full doses of H~-blockers. Long-term treatment: healing of the ulcer crater cannot be considered the only aim of a correct therapeutic approach. Peptic ulcer is a chronic disease characterized by complications and by a reduction of life-expectancy. The mean duration of the disease is about 15 years, with periods of exacerbation followed by spontaneous remission. It has been documented that at least one relapse occurs in about 80% of patients, within 25 years from the onset of the disease; in almost half of these cases, the relapse may be found during the first year. The 'strategic' goal of medical treatment is represented by the prophylaxis of relapses. An adequate therapy may pre- vent the relapse in more than 4/5 of the patients, induces a marked reduction of complications and, consequently, of surgical operations. However, recurrences are possible also during mainte- nance therapy. An ideal therapeutic strategy should be chosen after having answered the follow- ing questions: 1. what is the best drug to use?; 2. what is the best therapeutic scheme?; 3. in which patients have we to apply a particular therapeutic scheme?; 4. for how long must we treat ulcer patients? Once again, the antisecretory drugs are the most commonly used in the prophylaxis of both gastric and duodenal ulcer relapses. Many studies have been performed, in order to corn-

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pare the clinical activity of the different H2-blockers: ranitidine has been demonstrated to be significantly more active than cimetidine in preventing relapses in duodenal ulcer disease; on the other hand, famotidine and ranifidine seem to have similar long-term therapeutic activities. As to the modalifies of the long-term treatment, we can choose between the following schemes: 'intermittent' or 'on demand treatment', 'seasonal treatment', 'week-end treatment', 'long-term maintenance treatment'. The term 'intermittent' or 'on demancr therapy indicates a cycle of full dosage treatment at a moment of clinical relapse; the limit of such a treatment is represented by the possibility of having asymptomafic relapses. 'Seasona~ prophylaxis is based on the assumption that peptic ulcers, and particularly duodenal ulcers, present more frequent relapses in spring and autumn. 'Week-end' treatment represents a variation of continuous maintenance therapy; a possi- ble usefulness of such treatment has been suggested by the advent of drugs, such as omeprazole, with a long-lasting anfisecretory action. 'Long-term maintenance' therapy consists of continuous low dosage administration of the drugs employed, at full doses, in the short-term treatment. With H2-antagonists, half dosage (at bed time) of attack therapy (400 mg cimetidine, 150 mg ranitidine and 20 mg famotidine) appears to be effective and safe. Long-term maintenance therapy has been shown to be more useful than seasonal treatment in preventing relapses. It also reduces the annual incidence of relapses, in comparison with on demand therapy, and it provides a valid prevention of hemorrhagic complications. Such a treatment ensures a good quality of life in the vast majority of patients, and leads to a decrease of absenteeism from work, as well as, more in general, to an acceptable cost/benefit ratio. A number of clinical conditions may be identified, in which long-term maintenance therapy is recommended: 1. long-lasting peptic disease; 2. peptic ulcer familiarity; 3. slow healing during short-term treatment; 4. frequent and early relapses after suspension of the treatment; 5. previous complications (hemorrhage, perforation); 6. associated diseases (renal failure, cirrhosis); 7. chronic intake of gastrolesive drugs (NSAIDs, steroids); 8. 'at risk' life conditions (working activity, etc.) and inappropriate life habits (smoking, alcohol, diet, etc.). It is not yet known for how long maintenance therapy should last. Previously, it has been documented that the relapse rates of untreated patients are the same as the treated ones, within the 12 months after suspension of the long-term therapy. However, we can state so far that the prolonged treatment with H~-blockers, especially with the newest molecules of this family, pre- sents few side effects. In particular, a possible bacterial contamination has been excluded, in spite of the protracted inhibition of gastric acid secretion. In fact, the nocturnal administration of antisecretory agents, at low dosages, guarantees a diurnal low pH, capable of sterilizing the foodstuff. Several factors may modify the expected results of an adequate therapy. Among these, cigarette smoking seems to play an important role, as it causes a delay in the ulcer healing process and an increased relapse frequency. Even if no definite correlations have been dem- onstrated between food and peptic ulcer disease, a proper diet might be of some help during maintenance treatment. For example, fibre-enriched diets have been reported to prevent re- lapses in duodenal ulcer patients, by both decreasing gastric emptying and augmenting the neu- tralizing meal capacity. On the other hand, such diets are not indicated in the case of gastric ulcer, because they tend to delay gastric emptying and, consequently, might increase the contact time between the gastric mucosa and potentially damaging gastric contents (chlorhydropeptic secretion, enterogastric reflux).

L.A. Scuro, I. Vantini: Istituto di Clinica Medica, UniversitY. di Verona, Policlinico di Borgo Roma, Verona, Italy.

Periodic t reatment of peptic ulcer.

A simple definition of periodic treatment for peptic ulcer is a treatment course with drug-free periods, based on a predetermined strategy with the aim of healing the ulcer as well as prevent- ing recurrence. There are two different approaches to this strategy which can be defined as follows: 1. treatment on demand; 2. intermittent and seasonal treatment. Treatment on demand is linked to the presence of symptoms, particularly pain, and is probably the most widely used. Unfortunately, about 50% of patients with ulcer-type pain, even if typical, do not in reality pre- sent with an ulcer crater. Ten to 15% of ulcers are asymptomatic. Although widely adopted, this therapeutic approach has little scientific backing and is often the consequence of autoregulation closely linked to the presence of pain. The duration of this type of treatment is thus extremely variable and exposes the patient to the real risk of an incompletely healed ulcer and thus to

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early, more frequent recurrences. In one report, however, the frequency of healed ulcers without relapse was similar, regardless of the duration of short-term treatment with cimetidine, even if obviously the frequency of unhealed ulcers was h igher in patients treated for very shor t periods. Because of the characteristics of this type of therapeutic approach, it might be bet ter carried out with very powerful drugs such as omeprazole which can strongly inhibi t gastric acid secre- tion, so as to br ing about the heal ing of a percentage of ulcers in a very short per iod of time, for example in a couple of weeks. There have been no specific studies performed, but the data of some clinical trials with omeprazole vs. H~-antagonists show that the heal ing of a duodenal ulcer is more rapid with omeprazole, but the differences are only of clinical value with doses of ome- prazole of 40 mg/die and over. With lower doses, the difference is no more than 10-15%. More- over, a more rapid heal ing of the ulcer could expose the patient to the risk of an earl ier recur- rence. Thus, on demand treatment does not seem to be the best or most rat ional even with an intermittent therapeutic strategy with repeated short-term cycles. On the o ther hand, there is no information on the possible effect of this sort of approach on the natural history of the ulcer. Intermit tent t reatment with active drugs in the presence of an endoscopically-proven peptic ul- cer, in tended as a therapeutic approach to encourage heal ing of the ulcer, is however a rational strategy based on scientific data, widely used and commonly defined as short-term treatment of peptic ulcer. The vast literature and clinical experience built up on the studies performed from the middle of the 1970's on H~-antagonists and also other drugs with antisecretive or 'protective' action, document how with many drugs available today it is possible to obtain the heal ing of gastric and duodenal ulcers in a few weeks. With all the treatments studied, the prolonging of treatment increases the probability of healing. However, this is earlier with more powerful anti- secretive drugs. A series of studies has also been carried out to try and define the factors which interfere with the heal ing process, information which would be useful when deciding whether to use this type of intermittent t reatment or that which prevents recurrences once heal ing has occurred. Unfortunately, there are no precise criteria to define the limits for a choice between intermittent and maintenance therapy for peptic ulcer. There is no doubt that there are situa- tions in which maintenance treatment cannot be advised; for example, in a young subject with previous ulcer history, suffering from a single episode of peptic ulcer, probably due to stress, a short-term treatment without maintenance treatment is probably sufficient. The re is a wide in- between area, in which the choice is subjective and based on the contribution of multiple factors for and against main tenance treatment, the quantification of which can only be approximate. In fact, this choice should be based on the exact aim of the treatment - the heal ing of the ulcer or prevent ion of recurrence, or both. Whereas there is a vast literature which documents how it is possible with active drugs, especially H2-antagonists, to prevent a statistically greater n u m b e r of recurrences compared to untreated patients, even with long periods of time of more than 4-5 years, there is very little information on treatment for the prevention of recurrence based on different therapeutic strategies, such as is the case of the so-called seasonal treatment. This type of approach is based on common observations in the natural history of the ulcer, of its tendency, more striking in the duodenal ulcer, to recur at particular times of the year, especially in spring and autumn. Seasonal t reatment plans the administration of full doses of H~-antagonists (e.g., ranitidine) for a period of 4 weeks, as in a recent Sicilian study. From this study, compared with cont inuous t reatment with ranit idine (150 mg/nocte), at 24 months of treatment, it emerges that the frequency of ulcer recurrence in patients receiving seasonal treatment is about twice that of patients on maintenance therapy. The difference is even greater during the first 6 months in which the frequency of recurrence in patients treated with a 'seasonal' approach is about 3 times h igher than that observed in the group on maintenance therapy receiving reduced doses of ranitidine/nocte. Similar data have also been reported by others and it can be said that seasonal t reatment can be placed in terms of ability to prevent ulcer recurrence, halfway between mainte- nance therapy with reduced doses/nocte and placebo or no treatment at all. This is justified by the fact that ulcer recurrence can also recur outside the period predetermined for the seasonal t reatment and that dur ing the natural history of the ulcer, the seasonal rhythm may not always be found, or can also disappear with time. Another type of therapeutic approach for the preven- tion of ulcer recurrence, and which has the objective of limiting drug assumption, is intermit tent t reatment with predetermined cycles. This treatment, conducted with full doses of rani t idine for one month, followed by 3 months without treatment and so on, has been compared with mainte- nance therapy with ranit idine 150 mg/nocte. This study has been conducted for about 2 years and shows that the frequency of recurrence observed with intermittent treatment is about twice that with traditional main tenance therapy and is even higher (5-8 times) during the first 10-11 months

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of treatment. Therefore, if seasonal t reatment with its limitations could be a reasonable proposal for those patients in whom recurrence occurs with a precise seasonal rhythm, the therapeutic approach with intermittent t reatment (at predetermined periods) on the basis of data obtained in clinical controlled trials does not seem to be advantageous. Because it has been demonstrated that the frequency of recurrence after short-term treatment is not affected by drugs used to heal the ulcer, with the exception of tripotassium dicitrate bismuthate which is followed by a signifi- cantly lower frequency of recurrence after t reatment withdrawal compared to patients not treated with this drug short-term, the use of TDB can also be suggested as a periodic t reatment of peptic ulcer, for example, intermittent approach. Its use would need to be limited to patients in whom the ulcerative lesion has been documented and subsequently healed, because at present we do not know the effect of TDB in preventing recurrence in the absence of ulcerative lesions. There- fore, it does not seem possible at present to propose a real main tenance therapy with this drug, which has these characteristic and interesting properties. In conclusion, despite the various at- tempts at periodic or intermittent therapy for the prevention of peptic ulcer recurrence, the maintenance therapy with continuous evening administration of an H2-antagonist in reduced doses, lasting for several years (if necessary), seems to be the best treatment, proving itself the most rational and best documented in the prevention of ulcer recurrence. It should however be stressed that prescription of a maintenance therapy should not be automatic, but should be based on an evaluation of the cost /benef i t ratio in the individual patient. Alternative treatments, as is the case with seasonal treatment, may be taken into consideration in a limited n u m b e r of cases, in which recuri 'ence is not particularly frequent, the seasonal rhythm is clearly respected and the risk of recurrence outside these periods does not expose the patient to serious conse- quences.

G. Dobrilla, S. Amplatz: Divisione di Gastroenterologia; Servizio di Fisiopatologia ed Endoscopia Digestiva, Ospedale Generale Regionale, Bolzano, Italy.

Prevent ion of ulcer relapse: rationale.

There are four basic components to the rationale underlying the long-term t reatment of peptic ulcer, that is to say the long-term prophylaxis of ulcer relapse: i. the natural history of ulcer disease; ii. the ulcer course after drug-induced healing; iii. the specific nature of ulcer relapse symptoms and complications in long-term treated ulcer patients; and iv. surgical therapy as the only alternative to medical prophylaxis of ulcer relapse. Natural history: 'once an ulcer, always an ulcer' is a t ime-honoured dictum. In fact, a series of epidemiological investigations seems to suggest that on average ulcer disease tends to persist for many years or even for the rest of the patient's life. However, individual variability proves to be very considerable, probably on account of its pathogenetic heterogeneity and the very large number of life variables involved. Therefore, we can predict only the average outcome of the disease but not that of individual patients. Ulcer course after drug-induced healing:, a whole series of clinical trials have shown that, without mainte- nance treatment, some 80 to 90% of ulcer patients will relapse within 1-2 years as against only 15 to 50% of patients on maintenance treatment with Ho:antagonists. Italy figures somewhere around midway in this range with an average relapse rate of about 23.9%. The tendency of an ulcer to relapse bears no relation to the duration of the previous acute t reatment or to the n u m b e r of acute courses of therapy. On average, ulcers still tend to relapse even after discon- t inuation of main tenance treatment lasting many years. Once again, however, we should bear in mind the possibility of substantial individual variability. Ulcer relapse symptoms and complications during long-term prophylaxis with H2-antagonists: studies or routine endoscopic follow-up examina- tions carr ied out after heal ing in patients on long-term placebo treatment show that the asymp- tomatic relapse rate ranges on average from 15 to 35%. During maintenance treatment with active drugs, this percentage is significantly h igher and may reach as much as 80%. As far as complicat- ed relapses in patients on active-drug maintenance treatment are concerned, a n u m b e r of studies suggest that there is significantly less bleeding than in patients receiving no active treatment. Only 2% of relapsing ulcers bleed in ranitidine-treated patients dur ing the first year of mainte- nance t reatment as against roughly 30% of relapsing ulcers in patients on placebo. Reports of ulcer perforat ions during maintenance treatment are very rare. Surgery as the only alternative to medical prophylaxis: surgery, which invariably implies failure of the medical means available, does

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not, as the layman is often inclined to believe, automatically solve the problem of ulcer relapse, and the results, that is to say the post-surgical relapse rates, vary considerably from one type of operation to another and, even within the same type of operation, according to the experience and expertise of the surgeon. Relapse rates also vary according to the postoperative time interval. The true post-surgical relapse rate, however, is difficult to establish, because, as a rule, only op- erated patients with symptoms undergo endoscopic reassessment and, thus, asymptomatic post- surgical relapses will not be counted in the evaluation. Few studies are available directly compar- ing relapse rates observed in patients receiving long-term treatment with H2-antagonists and those recorded in surgically treated subjects, and such results as we have are by no means easy to interpret. Even in the study yielding the most favorable results for surgical therapy, there is still a 10% recurrence rate within the space of only 12 months. Quite apart from actual percentage figures, various other factors should be carefully weighed when deciding whether or not to opt for surgical treatment; these include: i. the worry and anxiety any operation involves; ii. the surgical risk (low, but by no means negligible); iii. the appreciably higher surgical risk if reop- eration proves necessary; iv. the possible impact of postoperative sequelae other than relapse; v. the irreversibility of the new functional situation created (particularly important with regard to the relationship between hypoacidity and gastric carcinogenesis); vi. the higher operative risk in elderly patients and, especially, the high operative mortality rates due to hemorrhagic complica- tions in patients aged above 65. Conclusions: in conclusion, on the basis of what has been said, we can safely claim that the rationale for long-term prophylaxis with H2-antagonists is pretty con- vincing. The results .actually obtained in recent years in ulcer patients receiving long-term treat- ment with such drugs provide further confirmation a posteriori of the validity of this rationale. In the first place, the quality of life of the patients is significantly improved from every point of view. Secondly, maintenance treatment with H~-antagonists has been shown to reduce not only symp- tomatic, but also asymptomatic, ulcer recurrence in terms both of frequency and potential asso- ciated complications. Ulcers recur only in 1 out of 5 patients on H,-antagonists, and not in 4 out of 5, as is the case in placebo-treated patients. The number of surgical operations for peptic ulcer, particularly duodenal ulcer, has fallen steadily since the advent of H2-antagonists in short- term treatment and long-term prophylaxis. A number of important issues, however, remain unre- solved: should long-term prophylaxis be the rule for all peptic ulcer patients? With which drugs? In what kind of treatment regimen (continuous or intermittent)? For how long? For an analysis of these aspects, which go beyond the scope of this appraisal of the basic rationale, the reader is referred to a number of recent, exhaustive studies on the subject.

R. Naccarato, F. Di Mario: Clinica Medica I, Policlinico Universitario, Padova, Italy.

Strategies for 'non-responders' in ulcer disease.

Between 10 and 25% of duodenal ulcer (DU) patients do not heal after a 4-8-week full dose therapy with one of the active drugs currently used. Although the non-responder rate may be significantly reduced by prolonging or changing treatment it remains to be explained why, in standardized conditions, some patients heal whereas others do not. The situation is similar dur- ing so-called 'maintenance treatment' with at least 20% of patients relapsing during the first year, regardless of therapy. Before talking about strategies for non-responders, we think it is better to briefly define these patients as subjects who, under maintenance therapy with 'active' drugs, presented one or more relapses (symptomatic or asymptomatic) within a period of 6-12 months from the evidence of scarring ulcer. In our opinion, the 'slow-healing patients' (subjects who, under full doses of active drug during the acute phase of the disease, fail to heal the scarring of the lesion within 4-8 weeks of therapy) differ with frequent relapses as regards the pathophysio- logical background of these two phenomena. In fact, as regards for example the gastric ulcer, two different patterns of epidemiological factors are related to the 'slow-healing' of the lesion and to the relapses during the follow-up (male sex, smoking habit and early onset o f the disease for the 'slow-healing patients', and familiarity, aging and smoking habit for the 'non-responders', respectively). In order to simplify, however, the problem will be treated as the same. The ratio- nale to explain the 'non-responder' problem is possibly due to a standardized therapeutic ap- proach to the individual peptic ulcer patient (and in particular duodenal ulcer subject), regardless of the various and different factors involved in the imbalance between acid and mucosal barrier mechanisms, as shown in the following table:

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I parietal and oxyntic cells

basal secretory 'drive'

I postprandial secretory 'drive'

I gastric emptying

bicarbonate secretion

the same therapeutic approach

In addition to these well-known aspects, in fact, we recently described an antibody-mediated hypersecretion mechanism in a group of 'non-responder ' DU patients, as well as an IgE-mediat- ed pathway in other subjects with a poorer response to the therapy. These data may confirm that a different therapeutic approach could be postulated. In o ther words, therefore, briefly a mono- therapy for multifactorial disease determines the existence of 'non-responders'. Of the various parameters studied to establish a possible influence on the treatment in DU patients, the alcohol intake showed no difference between the groups, both in short- and long-term therapy. On the con- trary, smoking has been clearly connected with a poorer clinical outcome, as shown by our experience on non-treated DU patients, in agreement with the data present in the literature. In order to consider, in this view, the possible influence of some pathophysiological parameters, we represent the different relapse rate registered dur ing maintenance therapy subdividing the DU patients according to maximal acid output (MAO) values. In our experience, in fact, subjects with elevated MAO levels present a poorer outcome of this disease. The same has been found for serum pepsinogen levels in a standardized study on the same population. Al though we are still some way off from identifying the etiology of ulcer disease, recognition of particular subgroups of patients with different pathogenesis is an important step forward and, in our opinion, repre- sents a useful means of focusing on the non-responder subjects. Another point, as yet generally accepted, is that any drug able to reduce peptic acid secretion is effective in promot ing ulcer heal ing or in reducing recurrences, but it cannot modify the natural history of the disease itself or lead to a complete recovery. Possible exceptions to this view are represented by the recent experiences after acute t reatment with tripotassium bismuth and dur ing a very long-term main- tenance therapy with H2-blockers. Coming now to the therapeutic possibilities, we would like to draw attention to the problems related to continuous maintenance treatment, in particular costs and pat ient compliance. Length of long-term main tenance t reatment should be established according bo th to a clinical history (with particular attention to 'at risk condit ions ' such as the continuous use of anti-inflammatory drugs, previous bleeding episodes, etc.) and outcome of the disease. At present, on the basis of the data of the literature and from our experience, we can only make suggestions in order to identify 'non-responder ' patients by assessing aggressive fac- tors (peptic acid secretion, smoking, etc.). The following table reports a provocative summary of the problem of identifying an effective strategy for non-responders, in an attempt to have, in addition to the indispensable clinical experience, the support of objective and reproducible data:

- presence of Campylobacter pylori

- smoking habit

- 'stress' factors

- peptic acid hypersecretion

- hypergastrinemia

- immuno-allergic mechanisms

- gastric emptying

- mucosal barrier imbalance

This aim could be obtained by evaluating in individual patients functional, epidemiological, di- etary and psychological factors to assess the 'pathophysiological profile' of the disease.

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A. Blasi, A. Mangiameli: Istituto di Medicina Interna, Divisione di Gastroenterologia, Universitfi di Catania, Italy.

Prevent ion of ulcer relapses: comparison of cont inuous and seasonal rani t id ine t rea tment .

Long-term management of duodenal ulcer (DU) is still a problem. Unfortunately, 80-90% of ulcers recur within a year of heal ing if therapy with antisecretory agents is stopped. Generally, the high rates of recurrence after the end of therapy are not changed by different H: recep tor antagonist agents or by the duration of previous treatment. Is continuous main tenance treatment with H:antagonis ts more effective than a discontinuous treatment in prevent ing DU relapses? Up to the present time, this question is debatable and unresolved. In order to make a small contribu- tion to this problem, a multicentre clinical trial was performed in 5 centres in Sicily. The aim of the study was: 1. to compare the efficacy of continuous versus seasonal rani t idine t reatment over a period of 2 years; 2. to investigate the role Of some risk factors in inf luencing ulcer relapses; 3. to assess the safety of ranit idine in long-term treatment. One hundred and ten patients were admitted to the study. The essential entry criterion was an endoscopically-proven duodenal ulcer, healed during the preceding 3 months. The patients did not receive any main tenance treatment before admission to the trial. The patients were randomly assigned to e i ther cont inuous or sea- sonal treatment; patients on continuous treatment received ranit idine 150 mg at night for 2 years; patients on seasonal treatment received ranitidine 150 mg b.i.d, for 45 days in March-April and in September-October for 2 years. The periods of seasonal t reatment were chosen according to epidemiological :data on duodenal ulcer relapses in Sicily. Scheduled endoscopic, clinical and laboratory assessments were performed before and 6, 12, 18 and 24 months after the trial was started. Unscheduled endoscopy was performed if symptomatic ulcer relapse was suspected. Out of the 110 patients recruited, 104 were suitable for statistical analysis. Six patients dropped out because they did not report for endoscopic controls. The distribution of the patients in the two groups was similar for sex, ulcer history, smoking habits, alcohol and coffee consumption. The mean age was significandy higher in the continuous treatment group, but when the percentage of patients below and over 50 years was taken into account, no significant difference was found. Statistical analysis, performed for each 6-month period, showed significant differences in ulcer relapse rates between the seasonal and continuous treatment group in each per iod examined. After 6 months, ulcer relapse rates were 26.5% in patients with seasonal t reatment and 9.1% in patients with continuous treatment; after 12 months, 46.3% and 25.1%, respectively, and after 18 and 24 months, 58.2% and 30.6%, respectively. The other endoscopic findings, recorded before the start of the trial and then during the scheduled endoscopic controls, concerned the presence of erosive duodenitis. Statistical analysis performed in patients of both groups with endoscopic negativity at the beginning of the study showed that, after 18 and 24 months, the appearance of erosive duodenitis was more frequent in the seasonal treatment group. Among considered risk factors, age and smoking appeared, in our study, to be related to ulcer recurrences. In both t reatment groups, the 24-month relapse rates were significantly h igher under 50 years. A signif- icant difference between smokers and non-smokers was found only in patients with cont inuous treatment. No significant changes in the blood chemistry were found in ei ther t reatment groups. Slight adverse effects were observed in 5 patients only: one had diarrhea and one headache in the seasonal group; 2 had constipation and one a rash in the continuous t reatment group. However, none of these side effects necessitated interruption of the pharmacological treatment. The results of our study show that continuous treatment with ranitidine is bet ter than seasonal t reatment in preventing duodenal ulcer relapses over a period of 2 years. The evidence regard- ing the efficacy of continuous and discontinuous treatment is still scarce and conflicting. We believe that it is difficult at the moment to put forward a plan of discontinuous prophylaxis which is as effective as continuous treatment. Probably, a better knowledge and definition of risk fac- tors might, in the future, permit o ther rational therapeutic approaches for the prevent ion of duodenal ulcer relapses. Based on observations made over more than a decade, it appears that up to now continuous treatment is the best prophylaxis against ulcer recurrences and its symp- toms. If fur ther data confirm the efficacy and safety of ranitidine over a period of many years, the possibility of life-long maintenance therapy could be considered.

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G. Bianchi Porto, M. Petrillo: Divisione di Gastroenterologia, Ospedale L. Sacco, Milano, Italy.

The clinical relevance of non-steroidal anti-inflammatory drugs in peptic ulcer disease.

There are three possible roles for non-steroidal anti-inflammatory drugs (NSAIDs) in peptic ul- cer. They may damage the gastroduodenal mucosa. They may cause chronic ulcers, which may be symptomatic, asymptomatic and /o r complicated. They may retard or prevent healing of acute or chronic gastric mucosal lesions. Evidence for these effects has been sought in three ways: from pathogenetic studies in animals and human volunteers, from epidemiological studies and from clinical data. Pathogenetic data which provide the strongest evidence are largely based on animal experiments. Human data, in fact, are scarce and based on healthy volunteers. Epidemio- logical data are based on the incidence or prevalence of pepdc ulcer in patients receiving long- term NSAID therapy or, conversely, on increasing NSAID use in padents with peptic ulcer. The results are inconclusive not only because such studies are retrospective, but also because of faulty study designs. Clinical data rest on endoscopic evaluations in individual patients receiving long- term NSAID treatment and on the prophylactic and therapeutic roles of several anti-ulcer drugs when NSAID therapy is continued or withdrawn. Pathogenetic data: within lh after an oral dose of acetylsalicylic acid (ASA) in the stomach containing hydrochloric acid, patchy mucosal hemor- rhages occur usually in the gastric body, followed by erosions within a few hours. Other NSAIDs induce similar changes; the damage is both direct and related to diminished defence mecha- nisms. ASA is trapped inside the gastric epithelial cells, where it interferes with ARPase-depen- dent processes. Incx;eased membrane permeability, intracellular accumulation of sodium ions and water, osmotic swelling and cell death follow. Both ASA and other NSAIDs reduce mucus and bicarbonate secretion; most of them also inhibit mucosal prostaglandin synthesis. The effect of NSAIDs, however, diminishes with time. In fact, the gastric mucosa is able to adapt itself to the injury, t h e process of adaptation being time- and dose-dependent. In normal volunteers, the maximum damage is reached after one week of continuous ASA treatment. A daily dose of 1.3 g of ASA is associated with less severe damage than a daily dose of 2.6 g. There is no direct evi- dence that erosions become chronic ulcers. The adaptation phenomenon suggests, however, that they do not; furthermore, endoscopy shows that usually the mucosa around the ulcer in rheumat- ic patients is normal. Epidemiological data: there is an increased risk of gastric ulcer in patients taking ASA regularly. The increase in hospitalization for gastric ulcer in patients taking high doses of ASA, however, has been calculated as 10 new cases out of 100,000. The clinical impor- tance may not be as great as generally believed. Other studies have confirmed the association between ASA and ulcer, but have not quantified it. An exception showed that the risk of devel- oping a chronic ulcer increases when the consumption of ASA rises above 20-22 tablets/week. Projecting this figure to the general adult population, one can esdmate that between 1.5 and 3% of the population has a statistically increased risk of developing gastric ulcer because of ASA intake alone. A recent study conducted on padents of 60 years of age or over, admitted to hos- pital with bleeding peptic ulcer, revealed a higher risk of bleeding than those on hospital and community controls. In another study, the same authors showed that the trend towards a de- crease in gastric ulcer mortality observed in England and Wales was not observed in patients aged 65 years and over. The perforation rate, in fact, had increased in the elderly, particularly in those over 75 years of age. These changes are parallel to a three- or four-fold increase in NSAID prescriptions in subjects of this class of age. The epidemiological data can be summarized as follows: 1. gastric ulcer is more common in padents taking ASA regularly; 2. heavy regular ASA intake is associated with a higher prevalence of gastric ulcer in hospitalized patients; 3. padents with arthritis taking higher doses of ASA have a higher gastric ulcer prevalence; 4. there seems to be a threshold phenomenon (dose-dependent) of ASA damaging effects; 5. NSAID users in the United Kingdom have an increased risk of bleeding from pepdc ulcer; 6. a parallel rise in the NSAID prescription rate and the ulcer perforation rate in the elderly has been documented in the United Kingdom. Clinical data: four clinical studies performed in our unit in patients with rheumatic diseases shed some light on the effects of NSAIDs on ulcer. One is an endoscopic evaluation of the gastric damaging effect of various NSAIDs, the others are trials comparing the effects of H~-blockers or colloidal bismuth on the prophylaxis and treatment of gastric lesions in patients taking NSAIDs. Endoscopic study showed that the overall prevalence of gastric lesions associated with NSAIDs was 34%; with placebo and steroids, the corresponding figures were 0% and 14%, respectively. Of all drugs tested, ketoprofen, diclofenac, sulindac, oxyphenbutazone and diflunisal were associated with a significandy smaller prevalence of gastric lesions than ASA. The prevalence of lesions rose from 34% to 51% when a combined NSAID therapy was adopted.

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In 46% of patients, the lesions were asymptomatic. This may partly explain why some ulcers as- sociated with rheumatic disease are already bleeding or perforated on patients' admission. Inter- estingly, 20% of patients showed symptoms without having lesions. Hfblockers as prophylaxis: of 246 patients with rheumatoid arthritis or osteoarthritis shown by endoscopy to be free of le- sions or to have minimal gastric or duodenal mucosal lesions, 119 were randomized to ranitidine 150 mg b.i.d, and 127 to placebo. After 4 weeks of ranitidine treatment associated with regular NSAID treatment, 42% of the evaluable patients on ranitidine and 39% of the evaluable patients on placebo had mucosal lesions. The difference between the two groups was not statistically significant and, therefore, in this study ranitidine did not prevent lesions induced by NSAIDs. Scoring for severity of the lesions also failed to show any difference between the two treatment groups. Hr as treatment: in this study, we investigated the therapeutic effects of ranitidine (R) 150 mg dally or placebo associated or not with regular NSAID administration in rheumatic patients with gastric erosions. Forty-eight patients were admitted and divided into 4 groups, as follows: 12 patients on R 150 mg b.i.d., NSAIDs continued, 8/12 healed; 12 patients on R 150 mg b.i.d., NSAIDs discontinued, 10/12 healed; 12 patients on placebo, NSAIDs continued, 3/12 heal- ed; 12 patients on placebo, NSAIDs discontinued, 6/12 healed. Ranitidine, therefore, appears to be effective in the treatment of NSAID-associated lesions only when NSAIDs are discontinued. However, due to the small sample size, the potential positive role of ranitidine, if such a role exists, also in the group continuing NSAIDs, could not be ascertained. Cimetidine or colloidal bis- muth in the treatment of GU or DU in rheumatic patients: cimetidine and colloidal bismuth (De-NoD were compared in a randomized, controlled 4-week trial in 72 patients with rheumatic disease who were chronically' treated with NSAIDs. 38 patients had gastric and 34 duodenal ulcers. After 4 weeks, 59% of patients with duodenal ulcers had healed in both treatment groups. In the gastric ulcer group, 41% of patients given cimetidine (400 mg t.i.d.), as compared to 43% of patients given colloidal bismuth (4 tablets daily), were healed. Conclusions: a large amount of information suggests that the use of NSAIDs is associated with an increased prevalence of acute mucosal gastroduodenal damage. This association is in fact confirmed either by experimental observations restricted, however, to normal volunteers, and by epidemiological investigations (mainly retrospective analyses). It has for example been demonstrated that ASA, the first and most important NSAID, is capable of inducing acute as well as chronic mucosal gastroduodenal lesions in normal individuals and in patients (mainly rheumatic patients), that the risk of such lesions is both dose- and time-dependent, and that the significant increase in gastric ulcer com- plications (hemorrhage, perforation) observed in various European countries could be linked to the increase of NSAID prescription in those countries. From a clinical point of view, various problems have arisen concerning the use of NSAIDs in rheumatic patients, as for example: 1. to determine the gastrolesivity of individual NSAIDs; 2. to evaluate the prophylactic effect of the H~-receptor antagonists in the prevention of NSAID-associated gastric lesions; 3. to ascertain whether NSAID-associated gastric erosions are best treated by continuing NSAID therapy or not, with or without H2-blocker 'protection'; 4. to assess the efficacy of He-blockers vs. mucosal protec- ting anti-ulcer drugs in the therapy of NSAID-associated gastric or duodenal ulcers. Our experi- ence with these problems suggests: 1. that, on average, the prevalence of acute gastric lesions in rheumatic patients receiving NSAIDs increases significantly from 34%, when these agents are used as a single-drug therapy, to 51%, when they are administered as a combination treatment; 2. that ranitidine administration does not significantly prevent NSAID-associated gastric mucosal lesions; 3. but that this drug is useful in the treatment of these lesions, provided that NSAIDs are discontinued; 4. that cimetidine or De-Nol have equal (low) efficacy in the treatment of NSAID- associated gastric or duodenal ulcers in patients continuing chronic anti-inflammatory therapy.

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PATHOPHYSIOLOGICAL ASPECTS OF INTESTINAL EPITHELIUM

M. D. Forgac: Tufts University School of Medicine, Boston, MA, USA.

Ion transport and membrane traffic.

Acidification of intracellular compartments has been shown to play a crucial role in intracellu- lar membrane traffic. Thus, following receptor-mediated endocytosis, exposure of internalized ligand-receptor complexes to a mildly acidic pH (5.0-6.0) is necessary to activate ligand-receptor dissociation and receptor recycling to the cell surface. A low pH within vesicles derived from the trans Golgi is also required for recycling of receptors involved in transport of newly synthesized lysosomal enzymes. A number of pathogens, including envelope viruses like influenza and endo- toxins like diphtheria toxin, have also been shown to gain access to the cytoplasm of the host cell via a low pH endosomal compartment. We have focused our attention on the ATP-dependent proton pumps responsible for acidification of intracellular compartments. These proton pumps, including those present in clathrin-coated vesicles, endosomes, lysosomes and Golgi-derived vesi- cles, share a number of important properties which suggest that they are all members of a novel class of ATP-driven proton pumps. Thus, unlike those cation ATPases which form a phosphoryl- ated intermediate [such as the (Na+,K+)-ATPase], the intracellular (H+)-ATPases do not form a phosphorylated intermediate and are resistant to vanadate. On the other hand, the intracellular (H+)-ATPases also differ from the coupling factor (H§ in their resistance to inhibitors such as oligomycin and aurovertin. We have purified the (H+)-ATPase from clathrin-coated vesi- cles and demonstrated that this enzyme contains 9 subunits of molecular weights 100,000, 73,000, 58,000, 40,000, 38,000, 34,000, 33,000, 19,000 and 17,000. We have also isolated a series of mono- clonal antibodies which recognize the native, detergent-solubilized (H+)-ATPase and immuno- precipitate this enzyme in an active form. All three positive monoclonals immunoprecipitate the same set of polypeptides as are observed in the purified enzyme. To investigate the functions of these subunits in the (H+)-ATPase complex, we have carried out site-directed labeling studies of this enzyme. We have demonstrated that the 73,000-dalton subunit shows ATP-protectable label- ing by both NBD-CI and NEM, two ATP-protectable inhibitors of the (H+)-ATPase. These results suggest that the 73,000-dalton subunit possesses an ATP-binding site required for activity, possibly the catalytic site. We have also observed that the 17,000-dalton subunit is specifically labeled by [~4C]DCCD, an inhibitor of proton transport. The extensive similarities which exist between this polypeptide and the c subunit of the coupling factor (H§ including the stoichiometry of labeling and their highly hydrophobic character, suggest that the 17,000-dalton subunit, like the c subunit, is involved in proton translocation. The relationship between the extent of labeling and the degree of inhibition suggests that formation of a functional proton channel requires the presence of 6 copies of the 17,000-dalton subunit per complex.

M. Donowitz, R. P. Rood, J. H. Wesolek, E. Emmer, M. Cohen, J. McCullen, R. S. Braithwaite, G. W. G. Sharp, H. Murer: New England Medical Centre and Tufts University School of Medi- cine, Boston, MA; New York State College of Veterinary Medicine, Cornell University, Ithaca, NY, USA; University of Zurich, Switzerland.

Second messenger regulation of the rabbit ileal brush border membrane Na/H antiporter by second messengers - inhibition by cAMP and Ca++/calmodulin-dependent phosphoryladon.

In rabbit ileum, Ca++/calmodulin (CAM) and cAMP regulate neutral linked NaC1 absorption by inhibiting this process. The role of Ca++/CaM and cAMP-dependent phosphorylation in regula- tion of the ileal brush border N a / H antiporter, a part of neutral linked NaCI absorption, was studied using purified brush border membrane vesicles prepared by Mg precipitation. These vesicles contain the N a / H antiporter and Ca++/CaM-dependent protein kinase(s), cAMP-depen- dent kinase and phosphoprotein substmtes. N a / H exchange was defined as either t h e differ- ence between the rate of acid inside pH gradient (pH 8.0/6.5) stimulated Na t influx and diffu- sive uptake (pH 6.5/6.5) or dimethylamiloride inhibitable Na § uptake; these gave identical results. Using a freeze thaw technique, ATP-depleted vesicles (ATP<I ~.M) were loaded with Ca ~ (0.85 ~.M free Car§ CaM (5 ~.M), and ATP plus an ATP-regenerating system (creatine kinase and creatine phosphate) such that the intravesicular ATP was 47 ~M. N a / H exchange was not altered

371

by either Ca+§ or ATP alone, but the combination of Ca+§ and ATP inhibited N a / H exchange by 57 + 8%. Substitution of the nonhydrolyzable ATP analogue AMP-PMP for ATP abolished this inhibitory effect. Ca++/CaM/ATP freeze thawed into the vesicles did not alter diffusive Na + uptake or Na+-dependent glucose entry. The Ca§ inhibition of N a / H exchange was reversed by W~3, a Ca+§ antagonist, but not by H-7, a protein kinase C antagonist. The inhibition by Ca§ represented an effect on the V,,~, and not the K,, for Na § The Ca++/CaM/ATP experiments were performed at varying Ca ++ concentrations with a maximum effect at 300 nM and a 50% effect at 122 nM. Parallel phosphorylation experiments showed that Ca++/CaM caused a concentration-dependent increase in phosphorylation of spe- cific membrane proteins with a 50% effect at 100 nM free Ca D. Similar experiments were per- formed using cAMP (100 ~zM) freeze thawed into the brush border vesicles in the presence and absence of ATP plus ATP-regenerating system, cAMP in the presence of the ATP-regenerating system inhibited N a / H exchange by approximately 50%, while cAMP had no effect on N a / H exchange in the absence of ATP and did not affect diffusive Na uptake. In addition, cAMP did not affect N a / H exchange when a nonhydrolyzable ATP analogue (AMP-PMP) was studied. The effect of a maximal concentration of AMP, and a maximal concentration of Ca++/CaM exerted approximately equal but not additive effects in inhibiting N a / H exchange. In summary, using freeze thaw to permeabilize vesicles to macromolecules, we have shown that: a. ileal brush border N a / H exchange is inhibited by a Ca++/CaM-dependent process which requires a hydro- lyzable form of ATP; b. the effect is reversed by Ca++/CaM inhibitors; and c. occurs at the same Ca ++ concentrations as the Ca++/CaM regulation of the brush border protein phosphorylation; d. cAMP inhibits ileal brush border N a / H exchange also requiring a hydrolyzable form of ATP; e. the effects of Ca++/CaM and cAMP are quantitatively equivalent and nonadditive on N a / H exchange. These studies represent demonstration of involvement of phosphorylation in regula- tion of an epithelial exchange protein.

H. D. Ward, M. E. A. Pereira: Division of Geographic Medicine and Infectious Diseases, Tufts University School of Medicine, New England Medical Centre, Boston, MA, USA.

Host-parasite interaction in giardiasis.

The protozoan parasite Giardia lamblia is a significant cause of diarrheal disease worldwide. In the USA, Giardia lamblia is the most frequently identified intestinal parasite. Epidemics of the disease in this country have been associated with contamination of municipal water supplies with the cyst form of the parasite. Giardia is also the cause of 'Hiker's' diarrhea due to ingestion of contaminated water from mountain streams. In addition, person to person transmission is re- sponsible for the high incidence of infection in children in daycare centers, institutionalized pa- tients and male homosexuals. Giardia is an extracellular parasite which exists in two developmen- tal forms, trophozoite and cyst. Infection is initiated by ingestion of the cyst which undergoes excystation upon exposure to low gastric pH, to release the modle flagellated trophozoite form which colonizes the proximal small intestine and causes the disease. Encystation occurs by an unknown mechanism and the cysts are excreted into the external environment via the feces. Transmission of the disease occurs by ingestion of the infective cyst form in contaminated food or water or by direct person to person spread via the orofecal route. Clinical manifestations of the disease are variable, ranging from an asymptomatic state or mild self-limited diarrhea, nau- sea, colic and abdominal distension to severe and persistent diarrhea, malabsorption and wast- ing. The pathogenic mechanisms involved in diarrhea and malabsorption are poorly under- stood. Giardia trophozoites selectively colonize the proximal small intestine where they attach to the mucosal surface and exert pathological effects. The molecular basis of the specific adherence o f Giardia to host cells is unknown. Attachment of the parasite to the substratum is thought to be mediated by the ventral suction disc. However, this mechanism does not account for the selective colonization of the proximal small intestine. Therefore, recognition and adherence must be me- diated by specific host and parasite surface membrane determinants. Lectins are sugar-binding proteins which are believed to mediate several specific cell-cell interactions including those be- tween parasite and host cells. Since the heavily glycosylated microvillous brush border mem- brane is the preferred attachment site of Giardia trophozoites, a parasite surface lectin may play a role in the recognition process involved in the host-parasite interaction. We recently described the presence of such a surface lectin, which we have named taglin, in this parasite. This lectin is novel in that it is specifically activated by exposure to trypsin, an enzyme that is present in

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abundance at the site of infection. When activated, the lectin specifically agglutinates enterocytes to which the parasite adheres in vivo. In addition, the activated lectin binds to isolated brush border membranes of these cells. The lectin is most specific for phosphomannosy l residues, re- quires divalent cations for hemagglutinat ing activity and is most active at a pH of 6.5, which is the pH of the intestine at the site of colonization by the trophozoite. Taglin has b e e n found to be present in four different strains of Giardia lamblia as well as in two o ther species, including the murine parasite Giardia muris. In order to fur ther study taglin, we raised monoclonal antibodies to it. The surface membrane location of taglin was confirmed using this monoclonal antibody in an immunofluorescence assay. The subunit molecular weight of taglin was found to be 28/30 kD by Western blott ing using the monoclonal antibody as a probe as well as by direct erythrocyte b inding to nitrocellulose blots. These studies provide a circumstantial evidence to suggest that taglin may be involved in the host-parasite interaction. Further studies are required to determine definitively whether taglin is involved in a t tachment of the parasite to host cells in vitro as well as in vivo, and also to identify and characterize the receptor for taglin on host cells.

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