EXPERIMENTAL
AND
Diffuse B. N. Departments
of
MOLECULAR
23,
PATHOLOGY
155-163
( 1975)
Liver Injury in Amebic Abscess of the An Electron Microscopic Study TANDON, Gastroenterology
H. D.
TANDON,
V. V.
RAVI,
and Pathology, All-India New Delhi 110016, lndia Received
September
AND
P. C.
Institute
of
Liver:
GANDHI Medical
Sciences,
9, 1974
Liver biopsies of eight patients with amebic abscess of the liver were studied by light and electron microscopy. In four patients, they were repeated on complete clinical recovery, after specific antiamebic treatment. Light-microscopic changes were nonspecific and suggested diffuse parenchymal injury in some biopsy specimens. The ultrastructural changes in the hepatocytes were consistent in all patients. These consisted of dilatation of endoplasmic reticulum, vesicle formation and fragmentation, reduction in microsomes, and mitochondrial abnormalities in addition to other associated changes. Many cells showed features of regenerative activity. These changes are similar to those reported in patients with the nonsuppurative form of disease (Tandon, Tandon, and Puri, 1974) in which group they were less severe and suggestive of more active regeneration. The changes were reversed on treatment. These observations suggest that nonsuppurative hepatic amebiasis is a distinct pathologic entity even though the light microscopic changes by themselves are not specific.
INTRODUCTION Hepatic amebiasis has been classified into two categories by the WHO Expert Committee on Amebiasis (WHO, 1969): (a) amebic abscess of liver, (b) nonsuppurative hepatic amebiasis. The latter category has been highly controversial as a clinical and pathologic entity (Powell, Wilmont, and Elson-Dew, 1957; Kean, 1957). The occurrence of diffuse tender hepatomegaly with no evidence of localized suppuration, responding to specific antiamebic treatment, with or without associated colonic amebiasis is common clinical experience in the tropics. Yet it has not been possible to demonstrate unequivocal evidence of diffuse hepatocellular injury in hepatic amebiasis, and the morphologic basis of hepatomegaly is not understood (Kean 1955). There are several reports of the light-microscopic liver biopsy studies in nonsuppurative hepatic amebiasis (Kean, 1955; Nelson, Anderson, and Thomas, 1955; Kasliwal and Bhatia, 1956; Doxiades, Candreviots, Tiliakos, and Polymeropoulos, 1961)) amebic abscess of the liver (Kamat, Johri, Pathak, and Kothare, 1970; Keeley, Schmann, and Scott, 1962), and colonic amebiasis without clinical hepatomegaly (Madangopalan, 1973). These have shown only nonspecific and reactive changes, e.g., abnormal degree of anisonucleosis and lipofuchsin pigment in the hepatocytes, minimal Kupffer cell hyperplasia, and mild inflammatory cell infiltration in the portal canals. 155 Copyright All rights
0 1975 by Academic Press. Inc. of reproduction in any form reserved.
1.56
TANDON
ET. AL.
The present study was aimed at looking for the ultrastructural morphological evidence, if any, of diffuse parenchymal injury in association with amebic abscess of the liver in which condition, the ctiological role of Entmnoel~a histolytica is noncontroversial. MATERIALS
AND METHODS
Eight adult patients with amebic abscessof the liver were selected using the following clinical criteria: (1) Enlarged tender liver with palpable abscessover its surface. (2) Aspiration of characteristic “anchovy sauce pus” from the abscess cavity, which was sterile on bacterial culture. (3) Indirect hemagglutination test for amebiasis positive in titers higher than 1: 256. (4) Complete clinical recovery on treatment with specific dihydroemetine or metrinidizole therapy. Clinical examination, routine liver-function tests, chest X-ray and fluoroscopy, and indirect hemagglutination test for amebiasis were carried out on all patients. The size and the extent of the abscesswas demarcated in each case with the help of the clinical examination, fluoroscopy, and X-ray study of the chest and the upper abdomen. Liver biopsy was obtained using either a Menghini or VimSilverman needle from the lateral part of the right lobe at 15-20 cm distance from the abscess,from where the pus had been aspirated. The biopsy specimen was split into two pieces. One half of the biopsy was preserved in buffered formalin, processed in paraffin, and studied by light microscopy. The other half of the specimen was first fixed in 370 gluteraldehyde and then in 25% osmium tetraoxide ( Karnovsky, 1965)) and after processing was embedded in epoxy resin (Luft, 1961). An LKB 380 I ultramicrotome was used to cut 600 A- to 700 A-thin sections which were sustained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963), and examined with a Phillips EM 300 electron microscope. Repeat biopsy study after specific antiamebic therapy and clinical recovery was done in four patients. RESULTS All the patients were males in the age range of 26-40 yr. Clinically each patient was suspected to have ‘a single abscesslocated in the epigastric region. There was no clinical evidence to suggest the extension of the abscess to the lateral part of the right lobe of the liver which was selected as the site for the biopsy. Skin over the lower part of the right inframammary and right infraaxillary region did not show any edema, nor was the intercostal tenderness demonstrated in any case. Fluoroscopy showed decreased respiratory movements but no significant elevation or the fixity of the right dome of the diaphragm. Results of the important laboratory data have been presented in Table I. All had normal serum bilirubin levels. There was no significant alteration in the serum transaminase levels. Serum alkaline phosphatase values were high in three patients Light-microscopic changes were not specific and often varied quantitatively. Five biopsies showed changes as observed in nonsuppurative hepatomegaly presumably caused by amebiasis, as reported earlier (Tandon, Tandon, and Puri, 1974). In these, there was evidence of diffuse though mild parenchymal injury. Hepatocytes were often swollen and the cytoplasm coarsely
LIVER
IN
AMEBIC TABLE
ESSENTIAL
Serum normal values :
157
ABSCESS I
LABORATORY FINDINQS IN SERUM OF EIGHT AMOEBIC ABSCESS OF THE LIVER
Bilirubin L 1.2 mg/ 1OOml
Total protein 7.5 g/ 100 ml
0.4 0.6 0.3 0.7 0.5 0.6 0.8 0.6
7.8 7.4 6.8 7.8 5.6 6.4 6.8 6.0
Albumin 4.3 g/ 100 ml
3.8 3.6 3.0 3.0 3.0 3.0 3.2 3.8
Globulin 3.2/ 100 ml
4.0 3.8 3.8 4.4 3.2 3.0 3.0 2.4
Alkaline phosphatase L 13 K-A units
33.0 20.0 13.0 10.0 9.0 16.0 26.0 9.0
PATIENTS
SGOT ~40
SGPT
Karmen
30 32 31 26 25 70 18 26
WITH
IHA test L 1: 162 titer
units
20 24 26 24 25 40 20 22
1: 1: 1: 1: 1: 1: 1: 1:
4374 4374 4374 4374 4374 4374 1458 1458
granular and rarefied. They contained variable quantities of lipofuscin pigment which was often conspicuous. There was increased regenerative activity as evident by larger number of bi- or trinucleated cells, prominent anisonucleosis, and hyperchromatism of nuclei which contained large nucleoli (Fig. 1). In others, the diffuse hepatocytic chsanges were only equivocal but all cases showed reactive changes in the sinusoids and portal canals. There were portal infiltrates which were often dense. These included a variety of cells but polymorphonuclear leukocytes and eosinophils often dominated. In one case, polymor-
FIG. 1. Liver biopsy showing dense inflammatory infiltrates cytes show cytoplasmic vacuolation and other degenerative variation in nuclear size and many are hyperchromatic. (H&E
in the sinusoids. changes. There X130.)
Many hepatois an intensive
158
TANDON
FIG. 2. Hepatocyte showing dilatation of glycogen and an increase of lipofuscin
ET.
AL.
of endoplasmic reticulum pigment ( L) . X9900.
(ER)
apparent
depletion
phonuclear infiltrate was present ‘around and even within bile ducts suggesting cholangitis. There were focal necroses infiltrated with similar inflammatory cells. Kupffer cells were generally hyperplastic and large. Another conspicuous feature was the presence of diffuse and focal inflammatory infiltrates in the sinusoids among which polymorphonuclear leukocytes were generally conspicuous. None histolytica. These changes of the cases showed the presence of Entamoeba reversed on clinical recovery. A consistent pattern of ultrastructural abnormalities was seen in all the cases though the severity was variable. There was unequivocal evidence of degenerative changes in the hepatocytes. The most conspicuous change was that of dilatation of the endoplasmic reticulum (ER) with formation of vesicles (Figs. 2 and 3). In some casesthe ER was also fragmented (Fig. 4). There was an apparent per unit area decrease of microsomes attached to the ER membrane. In such cells the stack-like arrangement of the ER in the pcrinuclear and pericanalicular regions was not seen. Mitochondria of these hepatocytes were swollen, bizarre shaped, and of variable sizes (Fig. 5). Often the mitochondrial matrix was dense and vacuolated and the cristae were destroyed. Such degenerative changes were observed in approximately one third to two thirds of the total number of hepatocytes screened. Many cells showed autophagic vacuoles, microbodies, and lipofuscin pigment. Glycogen appeared to be decreased in most of the cells. Intercellular space appeared widened at several places (Fig. 6). A number of lamellated bodies appeared to be unremarkable. were seen in such spaces. Rile canaliculi In many hepatocytes there were giant nuclei associated with intense diffuse proliferation of the ER. In such cells, the mitochondria appeared normal and did
LIVER
FIG.
3. Hepatocyte
shows
dilated
IN
AMEBIC
endoplasmic
159
ABSCESS
reticulum
forming
vesicles
(V).
(X13,200.)
not contain any inclusion bodies. There was no condensation of proliferating ER around them. Significant anisonucleosis was often observed. No structures resembling Entamoeba histolytica were observed.
FIG. 4. Hepatocyte ( x8250.)
showing
fragmentation
of endoplasmic
reticulum
as pointed
by
arrows.
160
FIG. chondria
TANDON
5. Hepatocyte are swollen
showing and bizarre
a very shaped
ET.
AL.
marked dilatation of endoplasmic and cristate are not apparent (M).
reticulum. ( X15,400.)
Mito-
Repeat biopsy after successful therapy in all four patients showed apparently normal ultrastructure. Degenerative changes had completely reversed, and the hepatocytes appeared within normal limits (Fig. 7).
FIG.
6. Widened
intercellular
space
containing
number
of lamellated
bodies.
( X15,400.)
LIVER
FIG. 7. Normal ultrastructure
IN AMEBIC
of the hepatocyte
ABSCESS
after antiamebic
161
therapy.
( x6600.)
DISCUSSION From the above description it is clear that there was ultrastructural evidence of diffuse parenchymal damage in the cells distant from the abscess, associated with regenerative change, consistently observed in all the cases, and which reversed on clinical recovery. There is no report of electron microscopic study of liver biopsies in amebic abscess of the liver. Several investigators have reported hyperplastic and regenerative changes on light-microscopic studies (Kamat, Johri, Pathak, and Kothare, 1970; Keeley, Schmann, and Scott, 1962). On light-microscopic study, the evidence of unequivocal diffuse parenchymal injury was seen in only some cases. These resemble those described in nonsuppurative hepatomegaly associated with this disease in our earlier report (Tandon, Tandon, and Puri, 1974). Diffuse sinusoidal and portal infiltration was, however, seen consistently. None of these changes by themselves are specific, as they can be expected to occur in any inflammatory disease of the liver associated with suppuration, but the over-all picture can be highIy suggestive as is also evident by the fact that they reversed on clinical recovery. Tanikawa (1968) has described ballooning of the hepatocytes with less electron-dense cytoplasm, swollen mitochondria, distended cisternae of the ER, and glycogen depIetion as the characteristics of degenerative reaction. In contrast, he interpreted shrivelled hepatocytes with marked electron-dense cytoplasm containing compressed nucleus, ER, and mitochondria, and depletion of glycogeu particles as indicative of hepatocytic necrosis. In the present series, degenerative changes of the hepatocyt,es were consistently observed but changes suggestive of the necrosis as stated above were not seen in any of the specimens.
162
TANDON
ET.
AL.
Viral hepatitis is chsaracterized by the presence of both degenerative and necrotic reaction with preponderance of the latter (Tanikawa, 1968). Degenerative changes of the hepatocytes have been reported in a number of liver diseases, viz., alcoholic hepatitis, fatty liver, and protein-calorie malnutrition ( Tanikawa, 1968; Tandon, Ramanujan, Tandon, Puri, and Gandhi, 1974). These diseases were excluded among the present group of patients. Evidence of significant regenerative reaction was observed both in the lightand the electron-microscopic studies. This was characterized by an unusual degree of anisonucleosis and the presence of giant nuclei in the hepatocytes. The ER showed a diffuse and intense proliferative activity in these cells which had normal mitochondria. The above light-microscopic observations are in conformity with those reported in earlier studies (Nelson, Anderson, and Thomas, 1955; Kasliwal and Bhatia, 1956; Doxiades et al., 1961; Kamat, Johri, Pathak, and Kothare, 1970; Keeley, Schmann, and Scott, 1962) both in suppurative and nonsuppurative forms of this disease. In our earlier studies we have reported the ultrastructural degenerative and regenerative changes in the parenchymal cells in nonsuppurative hepatic amebiasis. There was no qualitative difference in the degenerative changes in the two forms of disease. However, they were significantly more severe in association with the liver abscess. In contrast, the regenerative changes were more conspicuous in the nonsuppurative disease. Further, intramitochondrial inclusion bodies which were absent in cases with abscess were #aprominent feature of the nonsuppurative group (Tandon, Tandon, and Puri, 1974). In this disease, abscess is undoubtedly caused by the Entamoeba histolytica. However, it cannot be concluded from the results of the present study that the diffuse liver injury is also caused directly by this organism. The parasite was not seen in any of the liver biopsy specimens, which confirms the earlier reports in the literature (Kamat, Johri, Pathak, and Kothare, 1970; Keeley, Schmann, and Scott, 1962). Some unknown toxins liberated by the ameba or associated bacteria in the gut may be the possible factor in the pathogenesis of diffuse liver injury which needs further investigation. Since the ultrastructural changes in the liver were completely reversible after specific antiamebic therapy for 7-10 days, it is quite likely that Enfamoeba histolytica has some role, albeit indirect, in the pathogenesis of diffuse hepatocytic damage. The observation of identical and diffuse changes in liver parenchymal cells as observed in suppurative and nonsuppurative forms of disease, as recorded by us (Tandon, Tandon, and Puri, 1971) supports the belief that the latter is indeed a distinct pathogenic entity. Hepatocytic degeneration, the mechanism of which is not understood, triggers off the regenerative changes in the cells as a reactive phenomenon. Th e circunlstanccs or pathogenetic mechanisms which result in abscess formation in some cases and a nonsuppurative form in others, are not llnderstood. ACKNOM’LEDGhlENTS The authors are grateful to Dr. T. C. Anand Kumar, Officer-in-Charge, Laboratory, Department of Anatomy, All-India Institute of Medical Dr. K. Bahadur, Officer-in-Charge, Electron Microscope Lalxxatory, oratory, Delhi for their help and permission to use their laboratory sistance of Mr. S. Sharma is gratefully acknowledged.
Electron hlicroscope Sciences, New Delhi and Defence Science Lahfacilities. Technical as-
LIVER The Medical
study was Research,
partly India
IN
AMEBIC
163
ABSCESS
supported by PL-480 Research Grant from and the National Institutes of Health, U.S.A.
the
Indian
Council
of
REFERENCES DOXIADES, T., CANDREVIOTS, N., TILIAKOS, M., and POLYMEROPOULOS, I. (1961). Chronic diffuse non-suppurative amoebic hepatitis. Brit. Med. J. 1, 460-462. KAMAT, G. R., Jonm, B. S., PATHAK, V. P., and KOTHARE, S. N. (1970). Histopathological changes in serial liver biopsy amoebic liver abscess. J. Assoc. Phys. Ind. 18, 749-754. KAFLNOVSKY, M. J. ( 1965). A formaldehyde-gluteraldehyde fixative of high osmolality for use in electron microscopy. J. Cell. Biol. 27, 137 ( Abstr.). KASLIWAL, R. M., and BHATJA, M. L. (1956). Liver changes in chronic intestinal amoebiasis. J. Ind. Med. Ass. 27, 127-129. KEAN, B. H. (1955). Amoebic hepatitis. Absence of diffuse lesions at autopsy and in biopsies. Arch. Intern. Med. 19, 667-673. KEAN, B. H. ( 1957). The nature of diffuse amoebic hepatitis. Amer. J. Dig. Dis. 2, 342-347. KEELEY, K. J., SCHMANN, A., and SCOTT, A. ( 1962). Definitive diagnosis of amoebic liver abscess: Volume of liver biopsy. Brit. Med. 1, 375-376. LUFT, J. H. ( 1961). Improvement in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409414. MADANAGOPALAN, N. ( 1973). Liver biopsy study in acute amoebic dysentery. Paper read at Fourteenth Annual Conference of Indian Society of Gastroenterology. NELSON, T. L., ANDERSON, H. H., and THOMAS, D. (1955). Amoebic hepatitis; laboratory findings and treatment with erythromycin. Amer. J. Trap. Med. Hyg. 4, 812-921. POWELL, S. J., WILMONT, A. J., and ELSON-DEW, R. (1957). Hepatic amoebiasis. Trans. Roy. Sot. Trap. Med. Hyg. 54, 190-195. REYNOLDS, E. S. ( 1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell. Biol. 17, 208-212. TANDON, B. N., RAMANUJAN, R. A., TANDON, H. D., Porn, B. K., and GANDHI, P. C. (1974). An electron microscopic study. Amer. Liver injury in human protein-calorie malnutrition: J. Clin. Nutr. 27, 550-558. TANDON, B. N., TANDON, H. D., and PURI, B. K. ( 1974). An electron microscopic study of liver in hepatomegaly presumably caused by amebiasis. Exp. Mol. Pathol. 22, 118-132. TANIKAWA, K. ( 1968). In “Ultrastructural aspects of the liver and its disorders.” Published by Igaku Shoin Ltd., Tokyo. WATSON, M. L. ( 1958). Staining of tissue sections for electron microscopy with heavy metal. J. Biophys. Biochem. Cytol. 4, 475478. WORLD HEALTH ORGANIZATION. (1969). Tech. Rep. Ser. No. 421: Amoebiasis.
AND
Diffuse B. N. Departments
of
MOLECULAR
23,
PATHOLOGY
155-163
( 1975)
Liver Injury in Amebic Abscess of the An Electron Microscopic Study TANDON, Gastroenterology
H. D.
TANDON,
V. V.
RAVI,
and Pathology, All-India New Delhi 110016, lndia Received
September
AND
P. C.
Institute
of
Liver:
GANDHI Medical
Sciences,
9, 1974
Liver biopsies of eight patients with amebic abscess of the liver were studied by light and electron microscopy. In four patients, they were repeated on complete clinical recovery, after specific antiamebic treatment. Light-microscopic changes were nonspecific and suggested diffuse parenchymal injury in some biopsy specimens. The ultrastructural changes in the hepatocytes were consistent in all patients. These consisted of dilatation of endoplasmic reticulum, vesicle formation and fragmentation, reduction in microsomes, and mitochondrial abnormalities in addition to other associated changes. Many cells showed features of regenerative activity. These changes are similar to those reported in patients with the nonsuppurative form of disease (Tandon, Tandon, and Puri, 1974) in which group they were less severe and suggestive of more active regeneration. The changes were reversed on treatment. These observations suggest that nonsuppurative hepatic amebiasis is a distinct pathologic entity even though the light microscopic changes by themselves are not specific.
INTRODUCTION Hepatic amebiasis has been classified into two categories by the WHO Expert Committee on Amebiasis (WHO, 1969): (a) amebic abscess of liver, (b) nonsuppurative hepatic amebiasis. The latter category has been highly controversial as a clinical and pathologic entity (Powell, Wilmont, and Elson-Dew, 1957; Kean, 1957). The occurrence of diffuse tender hepatomegaly with no evidence of localized suppuration, responding to specific antiamebic treatment, with or without associated colonic amebiasis is common clinical experience in the tropics. Yet it has not been possible to demonstrate unequivocal evidence of diffuse hepatocellular injury in hepatic amebiasis, and the morphologic basis of hepatomegaly is not understood (Kean 1955). There are several reports of the light-microscopic liver biopsy studies in nonsuppurative hepatic amebiasis (Kean, 1955; Nelson, Anderson, and Thomas, 1955; Kasliwal and Bhatia, 1956; Doxiades, Candreviots, Tiliakos, and Polymeropoulos, 1961)) amebic abscess of the liver (Kamat, Johri, Pathak, and Kothare, 1970; Keeley, Schmann, and Scott, 1962), and colonic amebiasis without clinical hepatomegaly (Madangopalan, 1973). These have shown only nonspecific and reactive changes, e.g., abnormal degree of anisonucleosis and lipofuchsin pigment in the hepatocytes, minimal Kupffer cell hyperplasia, and mild inflammatory cell infiltration in the portal canals. 155 Copyright All rights
0 1975 by Academic Press. Inc. of reproduction in any form reserved.
1.56
TANDON
ET. AL.
The present study was aimed at looking for the ultrastructural morphological evidence, if any, of diffuse parenchymal injury in association with amebic abscess of the liver in which condition, the ctiological role of Entmnoel~a histolytica is noncontroversial. MATERIALS
AND METHODS
Eight adult patients with amebic abscessof the liver were selected using the following clinical criteria: (1) Enlarged tender liver with palpable abscessover its surface. (2) Aspiration of characteristic “anchovy sauce pus” from the abscess cavity, which was sterile on bacterial culture. (3) Indirect hemagglutination test for amebiasis positive in titers higher than 1: 256. (4) Complete clinical recovery on treatment with specific dihydroemetine or metrinidizole therapy. Clinical examination, routine liver-function tests, chest X-ray and fluoroscopy, and indirect hemagglutination test for amebiasis were carried out on all patients. The size and the extent of the abscesswas demarcated in each case with the help of the clinical examination, fluoroscopy, and X-ray study of the chest and the upper abdomen. Liver biopsy was obtained using either a Menghini or VimSilverman needle from the lateral part of the right lobe at 15-20 cm distance from the abscess,from where the pus had been aspirated. The biopsy specimen was split into two pieces. One half of the biopsy was preserved in buffered formalin, processed in paraffin, and studied by light microscopy. The other half of the specimen was first fixed in 370 gluteraldehyde and then in 25% osmium tetraoxide ( Karnovsky, 1965)) and after processing was embedded in epoxy resin (Luft, 1961). An LKB 380 I ultramicrotome was used to cut 600 A- to 700 A-thin sections which were sustained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963), and examined with a Phillips EM 300 electron microscope. Repeat biopsy study after specific antiamebic therapy and clinical recovery was done in four patients. RESULTS All the patients were males in the age range of 26-40 yr. Clinically each patient was suspected to have ‘a single abscesslocated in the epigastric region. There was no clinical evidence to suggest the extension of the abscess to the lateral part of the right lobe of the liver which was selected as the site for the biopsy. Skin over the lower part of the right inframammary and right infraaxillary region did not show any edema, nor was the intercostal tenderness demonstrated in any case. Fluoroscopy showed decreased respiratory movements but no significant elevation or the fixity of the right dome of the diaphragm. Results of the important laboratory data have been presented in Table I. All had normal serum bilirubin levels. There was no significant alteration in the serum transaminase levels. Serum alkaline phosphatase values were high in three patients Light-microscopic changes were not specific and often varied quantitatively. Five biopsies showed changes as observed in nonsuppurative hepatomegaly presumably caused by amebiasis, as reported earlier (Tandon, Tandon, and Puri, 1974). In these, there was evidence of diffuse though mild parenchymal injury. Hepatocytes were often swollen and the cytoplasm coarsely
LIVER
IN
AMEBIC TABLE
ESSENTIAL
Serum normal values :
157
ABSCESS I
LABORATORY FINDINQS IN SERUM OF EIGHT AMOEBIC ABSCESS OF THE LIVER
Bilirubin L 1.2 mg/ 1OOml
Total protein 7.5 g/ 100 ml
0.4 0.6 0.3 0.7 0.5 0.6 0.8 0.6
7.8 7.4 6.8 7.8 5.6 6.4 6.8 6.0
Albumin 4.3 g/ 100 ml
3.8 3.6 3.0 3.0 3.0 3.0 3.2 3.8
Globulin 3.2/ 100 ml
4.0 3.8 3.8 4.4 3.2 3.0 3.0 2.4
Alkaline phosphatase L 13 K-A units
33.0 20.0 13.0 10.0 9.0 16.0 26.0 9.0
PATIENTS
SGOT ~40
SGPT
Karmen
30 32 31 26 25 70 18 26
WITH
IHA test L 1: 162 titer
units
20 24 26 24 25 40 20 22
1: 1: 1: 1: 1: 1: 1: 1:
4374 4374 4374 4374 4374 4374 1458 1458
granular and rarefied. They contained variable quantities of lipofuscin pigment which was often conspicuous. There was increased regenerative activity as evident by larger number of bi- or trinucleated cells, prominent anisonucleosis, and hyperchromatism of nuclei which contained large nucleoli (Fig. 1). In others, the diffuse hepatocytic chsanges were only equivocal but all cases showed reactive changes in the sinusoids and portal canals. There were portal infiltrates which were often dense. These included a variety of cells but polymorphonuclear leukocytes and eosinophils often dominated. In one case, polymor-
FIG. 1. Liver biopsy showing dense inflammatory infiltrates cytes show cytoplasmic vacuolation and other degenerative variation in nuclear size and many are hyperchromatic. (H&E
in the sinusoids. changes. There X130.)
Many hepatois an intensive
158
TANDON
FIG. 2. Hepatocyte showing dilatation of glycogen and an increase of lipofuscin
ET.
AL.
of endoplasmic reticulum pigment ( L) . X9900.
(ER)
apparent
depletion
phonuclear infiltrate was present ‘around and even within bile ducts suggesting cholangitis. There were focal necroses infiltrated with similar inflammatory cells. Kupffer cells were generally hyperplastic and large. Another conspicuous feature was the presence of diffuse and focal inflammatory infiltrates in the sinusoids among which polymorphonuclear leukocytes were generally conspicuous. None histolytica. These changes of the cases showed the presence of Entamoeba reversed on clinical recovery. A consistent pattern of ultrastructural abnormalities was seen in all the cases though the severity was variable. There was unequivocal evidence of degenerative changes in the hepatocytes. The most conspicuous change was that of dilatation of the endoplasmic reticulum (ER) with formation of vesicles (Figs. 2 and 3). In some casesthe ER was also fragmented (Fig. 4). There was an apparent per unit area decrease of microsomes attached to the ER membrane. In such cells the stack-like arrangement of the ER in the pcrinuclear and pericanalicular regions was not seen. Mitochondria of these hepatocytes were swollen, bizarre shaped, and of variable sizes (Fig. 5). Often the mitochondrial matrix was dense and vacuolated and the cristae were destroyed. Such degenerative changes were observed in approximately one third to two thirds of the total number of hepatocytes screened. Many cells showed autophagic vacuoles, microbodies, and lipofuscin pigment. Glycogen appeared to be decreased in most of the cells. Intercellular space appeared widened at several places (Fig. 6). A number of lamellated bodies appeared to be unremarkable. were seen in such spaces. Rile canaliculi In many hepatocytes there were giant nuclei associated with intense diffuse proliferation of the ER. In such cells, the mitochondria appeared normal and did
LIVER
FIG.
3. Hepatocyte
shows
dilated
IN
AMEBIC
endoplasmic
159
ABSCESS
reticulum
forming
vesicles
(V).
(X13,200.)
not contain any inclusion bodies. There was no condensation of proliferating ER around them. Significant anisonucleosis was often observed. No structures resembling Entamoeba histolytica were observed.
FIG. 4. Hepatocyte ( x8250.)
showing
fragmentation
of endoplasmic
reticulum
as pointed
by
arrows.
160
FIG. chondria
TANDON
5. Hepatocyte are swollen
showing and bizarre
a very shaped
ET.
AL.
marked dilatation of endoplasmic and cristate are not apparent (M).
reticulum. ( X15,400.)
Mito-
Repeat biopsy after successful therapy in all four patients showed apparently normal ultrastructure. Degenerative changes had completely reversed, and the hepatocytes appeared within normal limits (Fig. 7).
FIG.
6. Widened
intercellular
space
containing
number
of lamellated
bodies.
( X15,400.)
LIVER
FIG. 7. Normal ultrastructure
IN AMEBIC
of the hepatocyte
ABSCESS
after antiamebic
161
therapy.
( x6600.)
DISCUSSION From the above description it is clear that there was ultrastructural evidence of diffuse parenchymal damage in the cells distant from the abscess, associated with regenerative change, consistently observed in all the cases, and which reversed on clinical recovery. There is no report of electron microscopic study of liver biopsies in amebic abscess of the liver. Several investigators have reported hyperplastic and regenerative changes on light-microscopic studies (Kamat, Johri, Pathak, and Kothare, 1970; Keeley, Schmann, and Scott, 1962). On light-microscopic study, the evidence of unequivocal diffuse parenchymal injury was seen in only some cases. These resemble those described in nonsuppurative hepatomegaly associated with this disease in our earlier report (Tandon, Tandon, and Puri, 1974). Diffuse sinusoidal and portal infiltration was, however, seen consistently. None of these changes by themselves are specific, as they can be expected to occur in any inflammatory disease of the liver associated with suppuration, but the over-all picture can be highIy suggestive as is also evident by the fact that they reversed on clinical recovery. Tanikawa (1968) has described ballooning of the hepatocytes with less electron-dense cytoplasm, swollen mitochondria, distended cisternae of the ER, and glycogen depIetion as the characteristics of degenerative reaction. In contrast, he interpreted shrivelled hepatocytes with marked electron-dense cytoplasm containing compressed nucleus, ER, and mitochondria, and depletion of glycogeu particles as indicative of hepatocytic necrosis. In the present series, degenerative changes of the hepatocyt,es were consistently observed but changes suggestive of the necrosis as stated above were not seen in any of the specimens.
162
TANDON
ET.
AL.
Viral hepatitis is chsaracterized by the presence of both degenerative and necrotic reaction with preponderance of the latter (Tanikawa, 1968). Degenerative changes of the hepatocytes have been reported in a number of liver diseases, viz., alcoholic hepatitis, fatty liver, and protein-calorie malnutrition ( Tanikawa, 1968; Tandon, Ramanujan, Tandon, Puri, and Gandhi, 1974). These diseases were excluded among the present group of patients. Evidence of significant regenerative reaction was observed both in the lightand the electron-microscopic studies. This was characterized by an unusual degree of anisonucleosis and the presence of giant nuclei in the hepatocytes. The ER showed a diffuse and intense proliferative activity in these cells which had normal mitochondria. The above light-microscopic observations are in conformity with those reported in earlier studies (Nelson, Anderson, and Thomas, 1955; Kasliwal and Bhatia, 1956; Doxiades et al., 1961; Kamat, Johri, Pathak, and Kothare, 1970; Keeley, Schmann, and Scott, 1962) both in suppurative and nonsuppurative forms of this disease. In our earlier studies we have reported the ultrastructural degenerative and regenerative changes in the parenchymal cells in nonsuppurative hepatic amebiasis. There was no qualitative difference in the degenerative changes in the two forms of disease. However, they were significantly more severe in association with the liver abscess. In contrast, the regenerative changes were more conspicuous in the nonsuppurative disease. Further, intramitochondrial inclusion bodies which were absent in cases with abscess were #aprominent feature of the nonsuppurative group (Tandon, Tandon, and Puri, 1974). In this disease, abscess is undoubtedly caused by the Entamoeba histolytica. However, it cannot be concluded from the results of the present study that the diffuse liver injury is also caused directly by this organism. The parasite was not seen in any of the liver biopsy specimens, which confirms the earlier reports in the literature (Kamat, Johri, Pathak, and Kothare, 1970; Keeley, Schmann, and Scott, 1962). Some unknown toxins liberated by the ameba or associated bacteria in the gut may be the possible factor in the pathogenesis of diffuse liver injury which needs further investigation. Since the ultrastructural changes in the liver were completely reversible after specific antiamebic therapy for 7-10 days, it is quite likely that Enfamoeba histolytica has some role, albeit indirect, in the pathogenesis of diffuse hepatocytic damage. The observation of identical and diffuse changes in liver parenchymal cells as observed in suppurative and nonsuppurative forms of disease, as recorded by us (Tandon, Tandon, and Puri, 1971) supports the belief that the latter is indeed a distinct pathogenic entity. Hepatocytic degeneration, the mechanism of which is not understood, triggers off the regenerative changes in the cells as a reactive phenomenon. Th e circunlstanccs or pathogenetic mechanisms which result in abscess formation in some cases and a nonsuppurative form in others, are not llnderstood. ACKNOM’LEDGhlENTS The authors are grateful to Dr. T. C. Anand Kumar, Officer-in-Charge, Laboratory, Department of Anatomy, All-India Institute of Medical Dr. K. Bahadur, Officer-in-Charge, Electron Microscope Lalxxatory, oratory, Delhi for their help and permission to use their laboratory sistance of Mr. S. Sharma is gratefully acknowledged.
Electron hlicroscope Sciences, New Delhi and Defence Science Lahfacilities. Technical as-
LIVER The Medical
study was Research,
partly India
IN
AMEBIC
163
ABSCESS
supported by PL-480 Research Grant from and the National Institutes of Health, U.S.A.
the
Indian
Council
of
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