Journal of Infection (I992) z4, 7-I I
P l a s m a c o n c e n t r a t i o n s o f f i b r o n e c t i n a n d C3d in p a t i e n t s w i t h amoebic liver abscess M. Irshad, M. P. S h a r m a and S. K. Acharya
Department of Gastroenterology and Human Nutrition, All India Institute of Medical Sciences, Ansari Nagar, New Delhi ILOO29, India Accepted for publication 14 November I99O Summary By means of simple and specific E L I S A techniques, the plasma concentrations of soluble fibronectin and Cad, a breakdown product of C~ complement, were determined in patients with amoebic liver abscesses (ALA) and in healthy controls. The mean plasma fibronectin concentrations in 23 patients with ALA and in 2o controls were found to be 44I _+89 rag/1 and 442+66 ms/l, respectively. The difference between these two values was not statistically significant. The mean C3d value in 2i patients with ALA, however, was found to be 84+ I4 AU/1 which was significantly different from the value of I2 + 4"7 AU/1 noted in 2o healthy persons. Plasma concentrations of these two proteins are discussed in relation to their possible implications in the immunopathogenesis of amoebic liver abscess.
Introduction Immunological reactions play a major part in both the causation and healing of an amoebic liver abscess (ALA). 1 Soluble fibronectin and the C~ c o m p o n e n t of c o m p l e m e n t are proteins mainly synthesised by the liver. T h e y have important r61es in the reticuloendothelial system (RES), being involved in the phagocytosis of circulating particles, such as cell debris, fibrin and i m m u n e complexes as well as bacterial and parasitic debris. ~-4 Since an amoebic abscess is a disease of the liver, it might be s u p p o s e d that the plasma concentrations of both these proteins m a y change during the formation of an abscess; firstly, due to a possible change in their synthesis and, secondly, their regular use as opsonic proteins b y the R E S in the healing process. Little is known, however, a b o u t their concentrations in the blood during the development of an amoebic liver abscess. W e have therefore measured the plasma concentrations of fibronectin and C~d, a b r e a k d o w n p r o d u c t of C 3 c o m p l e m e n t and a marker of C 3 activation, in patients with A L A so as to investigate their significance in this disease.
Patients and methods Patients A total of 23 patients with A L A and 20 healthy persons were selected for study. T h e patients with A L A were those admitted to the wards of the India Institute of Medical Sciences, N e w Delhi. T h e diagnosis of A L A made according to the current W H O criteria 5 based on clinical s y m p t o m s
this All was and
* Author to w h o m correspondence and reprint requests should be addressed.
oi63-4453/92/oiooo7 +o5 $03.00/0
© I992 T h e British Society for the Study of Infection
8
M. I R S H A D
ET
AL.
1200
120
O 1000
OO O00
100
80O
ooo
8o O
(3o3 o
%E
oo o
600
< 60
v m
m
-o
7" U_
o
400
40
200
20 oo 0(3o (3o I
[
I
I
NC ALA A/A NC Fig. I. Plasma concentrations of fibronectin (FN) and C3d in patients with amoebic liver abscess (ALA) and normal controls (NC). F N level ( Q ) was expressed in/zg/ml whereas Cad level ( O ) was expressed in arbitrary units per litre (AU/1).
positive amoebic serological tests. In addition, the diagnosis was confirmed in all the patients by ultrasound examination, a p r o m p t response to treatment with metronidazole and the presence of amoebic antigen in the pus aspirated from the liver abscess. N o n e of the patients had s y m p t o m s which suggested intestinal amoebiasis either before or during the diagnosis of A L A . All the healthy controls were free from s y m p t o m s of amoebic infection with negative serological tests and were without a history of invasive amoebiasis. In each case, a 5 ml sample of venous blood was collected and added to o'5 ml 3"8 % (w/v) sodium citrate solution as an anti-coagulant. Plasma was obtained by centrifugation immediately after collection of the b l o o d and was then stored at - - 70 °C within IO min of its separation. Methods
Fibronectin assay
Plasma concentrations of fibronectin were determined by a sensitive E L I SA technique established in our laboratory. 6 In brief, 5o #1 rabbit antihuman fibronectin diluted I in IOOO in o.i M carbonate buffer, p H 9"6, was added to each well of a 96-well microtitre plate (Nunc, D e n m a r k ) (already activated b y exposure to U V light for I h) and then incubated for 18 h at 2o °C. Postcoating was done by adding 2oo #1 of 1 % bovine serum albumin (BSA) in
Fibronectin and Cad in amoebic liver abscess
9
phosphate-buffered saline (PBS) to each well and incubating the plate for 4 h at 37 °C. Each step was followed by washing of the plate with PBS containing 0"05 % Tween-2o (PBS-T). Then, 50 #1 plasma diluted in PBS was added to each well. In a separate row of wells, 5o #1 aliquots of purified fibronectin solution in varying concentrations were also included and tested under identical conditions. Plates were incubated at 37 °C for 2 h and then washed. Finally, 50 #1 horse-radish peroxidase (HRPO)-labelled rabbit antihuman fibronectin conjugate (prepared by the two-step glutaraldehyde method 7) was added to each well and incubation continued for 2 h at 37 °C. T h e wash cycle was then repeated. Enzyme activity was measured by adding 50 #1 freshly prepared substrate solution (o'4 mg O-phenylene diamine per ml of o.I M phosphate citrate buffer, pH 5"0, and containing 0.06 % H~O2) to each well. Plates were then incubated in the dark at 20 °C for IO min. T h e reaction was stopped by adding IOO #1 of 4N H2SO4 and absorbance values read at 492 nm. T h e fibronectin concentration in each sample was calculated by reference to the standard curve as previously described. 6 Cad assay
Cad, the breakdown product of C a complement, was assayed by E L I S A as described by Peakman et al. 8 T h e Csd standard was derived from normal human serum by incubating it with 3 m g / m l inulin at 37 °C for I h followed by centrifugation at 1500 g for 30 rain. 8 T h e supernatant obtained was divided into aliquots and used as the Cad standard. Before measuring Cad concentrations by E L I S A , IOO #1 each of the Cad standard and of test plasma was added to equal volumes of 22 % polyethylene glycol (PEG) 6000 in PBS to achieve a final P E G concentration of I 1%. T h e mixture was incubated for I h at 4 ° C followed by centrifugation at I 5 o o g for 3 o m i n at 4 ° C to precipitate native C a and related molecules, leaving Cad in the supernatant. 9 T h e supernatant was promptly diluted in PBS containing o-oI % BSA and then tested by ELISA. T h e E L I S A may be briefly described here as follows: A micro-titre plate, after activation with UV light, was coated by adding Ioo #1 rabbit antihuman Cad (Dakopatt's) diluted I in IOOO in carbonate buffer, pH 9"6, to each well. Incubation and post coating with BSA was done exactly as described for the fibronectin assay. Aliquots of 50 #1 each of plasma and standard supernatant, after dilution with PBS, were added to the wells and the plate incubated at 37 °C for 2 h. After washing, 50 #1 of HRPO-conjugated rabbit antihuman Cad (conjugate prepared by the two-step gluteraldehyde method 7) diluted I in 500 in PBS, was added to each well and incubation was continued for I h at 37 °C. After three washings, colour was produced and measured as described above. Cad in plasma was determined in the test samples by reference to the standard curve prepared by running a standard assay concurrently with the test samples and by following the publication of Peakman et al. 8 Results
Plasma concentrations of soluble fibronectin and Cad were determined by means of simple and specific ELISAs Fibronectin concentrations were expressed as mg/1 whereas Cad measurements were expressed in arbitrary units
IO
M. I R S H A D
ET /tL.
per litre (AU/1). T h e mean plasma fibronectin concentration in 2o healthy controls and in 23 patients with ALA were found to be 442_+ 66 mg/1 and 441___89 mg/1, respectively. There was no statistically significant difference between these two values. By contrast, the mean Cad value in 21 patients with ALA was 84+_I4 AU/1 which was significantly higher (P < o-oi) than the mean C3d value of I2 +_4"7 AU/1 observed in 2o healthy controls (Fig. i). This difference may be helpful in further understanding the immunopathogenesis of ALA. Discussion
T h e host immunity plays a vital r61e in both the causation as well as the healing of ALA. 1 Although Entamoebia histolytica is itself cytopathic, necrosis of the liver as a result of amoebic infection is regarded as the sequel of immunological reactions directed against amoebic antigen and the surface proteins of live cells. 1 Both soluble fibronectin and complement C a are synthesised mainly by hepatocytes. 1° In that case, it is important to know how these two major component proteins of the reticuloendothelial system act during amoebic infection. T h e information about their r61e in the causation of liver abscesses may be obtained by designing a separate experimental study where an abscess may be produced artificially in experimental animals such as chimpanzee or rhesus monkey, and then their activities are monitored. T h e present study merely deals with plasma concentrations in patients with liver abscess and thus the data obtained are helpful in understanding their behaviour during this infection. No significant difference could be detected between the plasma concentrations of fibronectin in healthy persons and in the patients with liver abscess. This indicates that the plasma pool of fibronectin does not undergo any significant change despite its possible utilization in RES during healing of the abscess. This does not however exclude a role for fibronectin in the genesis of amoebic liver abscess. Fibronectin is an important component of RES and thus fully involved in the opsonisation of cell debris and other breakdown products for their removal from circulation. 2-3 One possible explanation for the finding of normal plasma fibronectin concentrations in patients with amoebic liver abscess may be that the overall synthesis of fibronectin by the liver is not affected much in localised injuries caused by amoebic infection. Normal fibronectin concentration in ALA patients may thus be a good sign indicating fast recovery of the abscess region, and thus differs from viral hepatitis cases, where a significantly low fibronectin level impairs the RES and renders the patients susceptible to further complications such as superimposed endotoxaemia and other bacterial infection, n-~2 This may be the reason why amoebic liver abscess is bacteriologically sterile despite severe necrosis of the liver. T h e plasma C3d concentration in ALA patients is significantly higher than that in healthy persons. Activation and involvement of complements in the pathogenesis of liver in ALA cases have been described. ~a T h e presence of circulating C3d in significantly higher concentrations indicates a possible r61e for C3d during liver abscess formation. Mere demonstration of raised Cad concentrations does not indicate whether complement activates via
Fibronectin a n d C3d in amoebic liver abscess
II
classical or a l t e r n a t i v e p a t h w a y s b u t it w o u l d s e e m t h a t t h e a c t i v a t i o n o f c o m p l e m e n t is p o s s i b l y i n v o l v e d in t h e tissue d a m a g e t h r o u g h a n t i b o d y m e d i a t e d c y t o t o x i c r e a c t i o n s o r in t h e r e m o v a l o f n e c r o t i c m a t e r i a l f r o m c i r c u l a t i o n b y R E S , t h u s h e l p i n g in t h e h e a l i n g process. T h e s e data s u p p o r t t h e p r e v i o u s s t u d y 1~ t h a t c o m p l e m e n t is a c t i v a t e d d u r i n g t h e f o r m a t i o n o f t h e liver abscess. H o w e v e r , at t h e s a m e t i m e it does n o t s u g g e s t t h a t c o m p l e m e n t a c t i v a t i o n is s e c o n d a r y to E. histolytica i n v a s i o n as r e p o r t e d b y M u n o z a n d Salazar la in t h e i r s t u d y . T h e significantly h i g h C3d c o n c e n t r a t i o n s in all cases o f A L A clearly p o i n t o u t t h a t c o m p l e m e n t a c t i v a t i o n is a sequel o f a m o e b i c i n v a s i o n a n d n o t a s e c o n d a r y effect. References
i. Trissi D. Immunology of Entamoeba histolytica in human and animal hosts. Rev Infect Dis I982; 4: I I 5 4 - I I 8 4 . 2. Mosesson MW, Amrani DL. The structure and biological activities of plasma fibronectin. Blood 198o; 56: I45-I58. 3. Saba TM. Plasma fibronectin and hepatic Kupffer cell function. In : Popper H, Schaffner F, Eds. Progress in liver diseases Vol. 7- Orlando, FL: Grune and Stratton, I982 ; IO9-I3I. 4. Proctor RA. Fibronectin: an enhancer of phagocyte function. Rev Infect Dis I987; 9 (Suppl. 4): 5412-419. 5. WHO. Amoebiasis. Geneva: World Health Organization, Technical Report Series No. 421, 1969; 1-52. 6. Anand AC, Irshad M, Acharya SK, Gandhi BM, Joshi YK, Tandon BN. Fibronectin in acute and subacute hepatic failure. J Clin Gastroenterol 1989; xI : 314-319 . 7. Engvall E. Enzyme immunoassay : ELISA and EMIT. Meth Enzymol 1980 ; 70:419-439 . 8. Peakman M, Lobo-Yeo A, Senaldi G, Nilsson M, Tee DEH, Vergani D. Quantification of C3d in biological fluids by an enzyme-linked immunosorbent assay. J Immunol Meth 1987 ; I04:51-56. 9. Perrin LH, Lambert PH, Miescher PA. Complement breakdown products in plasma from patients with systemic lupus erythematosus and patients with membranoproliferative or other glomerulonephritis. J Clin Invest I975; 56: 165-176. IO. Tamkun JW, Hynes RO. Plasma fibronectin is synthesised and secreted by hepatocytes. J Biol Chem 1983 ; z58 : 4641-4647. I I. Canalese J, Gove CD, Dimson AES, Wilkinson SP, Wardle EN, William R. Reticuloendothelium system and hepatocyte function in fulminant hepatic failure. Gut I982 ; 23: 265-269. 12. Wyke RJ, Canalese JC, Gimson AES, William R. Bacteraemia in patients with fulminant hepatic failure. Liver I982; 2:45-52. 13. Munoz LE, Salazar OG. Complement activation in patients with amoebic liver abscess. J Hepatol 1987; 5: 30-36.
P l a s m a c o n c e n t r a t i o n s o f f i b r o n e c t i n a n d C3d in p a t i e n t s w i t h amoebic liver abscess M. Irshad, M. P. S h a r m a and S. K. Acharya
Department of Gastroenterology and Human Nutrition, All India Institute of Medical Sciences, Ansari Nagar, New Delhi ILOO29, India Accepted for publication 14 November I99O Summary By means of simple and specific E L I S A techniques, the plasma concentrations of soluble fibronectin and Cad, a breakdown product of C~ complement, were determined in patients with amoebic liver abscesses (ALA) and in healthy controls. The mean plasma fibronectin concentrations in 23 patients with ALA and in 2o controls were found to be 44I _+89 rag/1 and 442+66 ms/l, respectively. The difference between these two values was not statistically significant. The mean C3d value in 2i patients with ALA, however, was found to be 84+ I4 AU/1 which was significantly different from the value of I2 + 4"7 AU/1 noted in 2o healthy persons. Plasma concentrations of these two proteins are discussed in relation to their possible implications in the immunopathogenesis of amoebic liver abscess.
Introduction Immunological reactions play a major part in both the causation and healing of an amoebic liver abscess (ALA). 1 Soluble fibronectin and the C~ c o m p o n e n t of c o m p l e m e n t are proteins mainly synthesised by the liver. T h e y have important r61es in the reticuloendothelial system (RES), being involved in the phagocytosis of circulating particles, such as cell debris, fibrin and i m m u n e complexes as well as bacterial and parasitic debris. ~-4 Since an amoebic abscess is a disease of the liver, it might be s u p p o s e d that the plasma concentrations of both these proteins m a y change during the formation of an abscess; firstly, due to a possible change in their synthesis and, secondly, their regular use as opsonic proteins b y the R E S in the healing process. Little is known, however, a b o u t their concentrations in the blood during the development of an amoebic liver abscess. W e have therefore measured the plasma concentrations of fibronectin and C~d, a b r e a k d o w n p r o d u c t of C 3 c o m p l e m e n t and a marker of C 3 activation, in patients with A L A so as to investigate their significance in this disease.
Patients and methods Patients A total of 23 patients with A L A and 20 healthy persons were selected for study. T h e patients with A L A were those admitted to the wards of the India Institute of Medical Sciences, N e w Delhi. T h e diagnosis of A L A made according to the current W H O criteria 5 based on clinical s y m p t o m s
this All was and
* Author to w h o m correspondence and reprint requests should be addressed.
oi63-4453/92/oiooo7 +o5 $03.00/0
© I992 T h e British Society for the Study of Infection
8
M. I R S H A D
ET
AL.
1200
120
O 1000
OO O00
100
80O
ooo
8o O
(3o3 o
%E
oo o
600
< 60
v m
m
-o
7" U_
o
400
40
200
20 oo 0(3o (3o I
[
I
I
NC ALA A/A NC Fig. I. Plasma concentrations of fibronectin (FN) and C3d in patients with amoebic liver abscess (ALA) and normal controls (NC). F N level ( Q ) was expressed in/zg/ml whereas Cad level ( O ) was expressed in arbitrary units per litre (AU/1).
positive amoebic serological tests. In addition, the diagnosis was confirmed in all the patients by ultrasound examination, a p r o m p t response to treatment with metronidazole and the presence of amoebic antigen in the pus aspirated from the liver abscess. N o n e of the patients had s y m p t o m s which suggested intestinal amoebiasis either before or during the diagnosis of A L A . All the healthy controls were free from s y m p t o m s of amoebic infection with negative serological tests and were without a history of invasive amoebiasis. In each case, a 5 ml sample of venous blood was collected and added to o'5 ml 3"8 % (w/v) sodium citrate solution as an anti-coagulant. Plasma was obtained by centrifugation immediately after collection of the b l o o d and was then stored at - - 70 °C within IO min of its separation. Methods
Fibronectin assay
Plasma concentrations of fibronectin were determined by a sensitive E L I SA technique established in our laboratory. 6 In brief, 5o #1 rabbit antihuman fibronectin diluted I in IOOO in o.i M carbonate buffer, p H 9"6, was added to each well of a 96-well microtitre plate (Nunc, D e n m a r k ) (already activated b y exposure to U V light for I h) and then incubated for 18 h at 2o °C. Postcoating was done by adding 2oo #1 of 1 % bovine serum albumin (BSA) in
Fibronectin and Cad in amoebic liver abscess
9
phosphate-buffered saline (PBS) to each well and incubating the plate for 4 h at 37 °C. Each step was followed by washing of the plate with PBS containing 0"05 % Tween-2o (PBS-T). Then, 50 #1 plasma diluted in PBS was added to each well. In a separate row of wells, 5o #1 aliquots of purified fibronectin solution in varying concentrations were also included and tested under identical conditions. Plates were incubated at 37 °C for 2 h and then washed. Finally, 50 #1 horse-radish peroxidase (HRPO)-labelled rabbit antihuman fibronectin conjugate (prepared by the two-step glutaraldehyde method 7) was added to each well and incubation continued for 2 h at 37 °C. T h e wash cycle was then repeated. Enzyme activity was measured by adding 50 #1 freshly prepared substrate solution (o'4 mg O-phenylene diamine per ml of o.I M phosphate citrate buffer, pH 5"0, and containing 0.06 % H~O2) to each well. Plates were then incubated in the dark at 20 °C for IO min. T h e reaction was stopped by adding IOO #1 of 4N H2SO4 and absorbance values read at 492 nm. T h e fibronectin concentration in each sample was calculated by reference to the standard curve as previously described. 6 Cad assay
Cad, the breakdown product of C a complement, was assayed by E L I S A as described by Peakman et al. 8 T h e Csd standard was derived from normal human serum by incubating it with 3 m g / m l inulin at 37 °C for I h followed by centrifugation at 1500 g for 30 rain. 8 T h e supernatant obtained was divided into aliquots and used as the Cad standard. Before measuring Cad concentrations by E L I S A , IOO #1 each of the Cad standard and of test plasma was added to equal volumes of 22 % polyethylene glycol (PEG) 6000 in PBS to achieve a final P E G concentration of I 1%. T h e mixture was incubated for I h at 4 ° C followed by centrifugation at I 5 o o g for 3 o m i n at 4 ° C to precipitate native C a and related molecules, leaving Cad in the supernatant. 9 T h e supernatant was promptly diluted in PBS containing o-oI % BSA and then tested by ELISA. T h e E L I S A may be briefly described here as follows: A micro-titre plate, after activation with UV light, was coated by adding Ioo #1 rabbit antihuman Cad (Dakopatt's) diluted I in IOOO in carbonate buffer, pH 9"6, to each well. Incubation and post coating with BSA was done exactly as described for the fibronectin assay. Aliquots of 50 #1 each of plasma and standard supernatant, after dilution with PBS, were added to the wells and the plate incubated at 37 °C for 2 h. After washing, 50 #1 of HRPO-conjugated rabbit antihuman Cad (conjugate prepared by the two-step gluteraldehyde method 7) diluted I in 500 in PBS, was added to each well and incubation was continued for I h at 37 °C. After three washings, colour was produced and measured as described above. Cad in plasma was determined in the test samples by reference to the standard curve prepared by running a standard assay concurrently with the test samples and by following the publication of Peakman et al. 8 Results
Plasma concentrations of soluble fibronectin and Cad were determined by means of simple and specific ELISAs Fibronectin concentrations were expressed as mg/1 whereas Cad measurements were expressed in arbitrary units
IO
M. I R S H A D
ET /tL.
per litre (AU/1). T h e mean plasma fibronectin concentration in 2o healthy controls and in 23 patients with ALA were found to be 442_+ 66 mg/1 and 441___89 mg/1, respectively. There was no statistically significant difference between these two values. By contrast, the mean Cad value in 21 patients with ALA was 84+_I4 AU/1 which was significantly higher (P < o-oi) than the mean C3d value of I2 +_4"7 AU/1 observed in 2o healthy controls (Fig. i). This difference may be helpful in further understanding the immunopathogenesis of ALA. Discussion
T h e host immunity plays a vital r61e in both the causation as well as the healing of ALA. 1 Although Entamoebia histolytica is itself cytopathic, necrosis of the liver as a result of amoebic infection is regarded as the sequel of immunological reactions directed against amoebic antigen and the surface proteins of live cells. 1 Both soluble fibronectin and complement C a are synthesised mainly by hepatocytes. 1° In that case, it is important to know how these two major component proteins of the reticuloendothelial system act during amoebic infection. T h e information about their r61e in the causation of liver abscesses may be obtained by designing a separate experimental study where an abscess may be produced artificially in experimental animals such as chimpanzee or rhesus monkey, and then their activities are monitored. T h e present study merely deals with plasma concentrations in patients with liver abscess and thus the data obtained are helpful in understanding their behaviour during this infection. No significant difference could be detected between the plasma concentrations of fibronectin in healthy persons and in the patients with liver abscess. This indicates that the plasma pool of fibronectin does not undergo any significant change despite its possible utilization in RES during healing of the abscess. This does not however exclude a role for fibronectin in the genesis of amoebic liver abscess. Fibronectin is an important component of RES and thus fully involved in the opsonisation of cell debris and other breakdown products for their removal from circulation. 2-3 One possible explanation for the finding of normal plasma fibronectin concentrations in patients with amoebic liver abscess may be that the overall synthesis of fibronectin by the liver is not affected much in localised injuries caused by amoebic infection. Normal fibronectin concentration in ALA patients may thus be a good sign indicating fast recovery of the abscess region, and thus differs from viral hepatitis cases, where a significantly low fibronectin level impairs the RES and renders the patients susceptible to further complications such as superimposed endotoxaemia and other bacterial infection, n-~2 This may be the reason why amoebic liver abscess is bacteriologically sterile despite severe necrosis of the liver. T h e plasma C3d concentration in ALA patients is significantly higher than that in healthy persons. Activation and involvement of complements in the pathogenesis of liver in ALA cases have been described. ~a T h e presence of circulating C3d in significantly higher concentrations indicates a possible r61e for C3d during liver abscess formation. Mere demonstration of raised Cad concentrations does not indicate whether complement activates via
Fibronectin a n d C3d in amoebic liver abscess
II
classical or a l t e r n a t i v e p a t h w a y s b u t it w o u l d s e e m t h a t t h e a c t i v a t i o n o f c o m p l e m e n t is p o s s i b l y i n v o l v e d in t h e tissue d a m a g e t h r o u g h a n t i b o d y m e d i a t e d c y t o t o x i c r e a c t i o n s o r in t h e r e m o v a l o f n e c r o t i c m a t e r i a l f r o m c i r c u l a t i o n b y R E S , t h u s h e l p i n g in t h e h e a l i n g process. T h e s e data s u p p o r t t h e p r e v i o u s s t u d y 1~ t h a t c o m p l e m e n t is a c t i v a t e d d u r i n g t h e f o r m a t i o n o f t h e liver abscess. H o w e v e r , at t h e s a m e t i m e it does n o t s u g g e s t t h a t c o m p l e m e n t a c t i v a t i o n is s e c o n d a r y to E. histolytica i n v a s i o n as r e p o r t e d b y M u n o z a n d Salazar la in t h e i r s t u d y . T h e significantly h i g h C3d c o n c e n t r a t i o n s in all cases o f A L A clearly p o i n t o u t t h a t c o m p l e m e n t a c t i v a t i o n is a sequel o f a m o e b i c i n v a s i o n a n d n o t a s e c o n d a r y effect. References
i. Trissi D. Immunology of Entamoeba histolytica in human and animal hosts. Rev Infect Dis I982; 4: I I 5 4 - I I 8 4 . 2. Mosesson MW, Amrani DL. The structure and biological activities of plasma fibronectin. Blood 198o; 56: I45-I58. 3. Saba TM. Plasma fibronectin and hepatic Kupffer cell function. In : Popper H, Schaffner F, Eds. Progress in liver diseases Vol. 7- Orlando, FL: Grune and Stratton, I982 ; IO9-I3I. 4. Proctor RA. Fibronectin: an enhancer of phagocyte function. Rev Infect Dis I987; 9 (Suppl. 4): 5412-419. 5. WHO. Amoebiasis. Geneva: World Health Organization, Technical Report Series No. 421, 1969; 1-52. 6. Anand AC, Irshad M, Acharya SK, Gandhi BM, Joshi YK, Tandon BN. Fibronectin in acute and subacute hepatic failure. J Clin Gastroenterol 1989; xI : 314-319 . 7. Engvall E. Enzyme immunoassay : ELISA and EMIT. Meth Enzymol 1980 ; 70:419-439 . 8. Peakman M, Lobo-Yeo A, Senaldi G, Nilsson M, Tee DEH, Vergani D. Quantification of C3d in biological fluids by an enzyme-linked immunosorbent assay. J Immunol Meth 1987 ; I04:51-56. 9. Perrin LH, Lambert PH, Miescher PA. Complement breakdown products in plasma from patients with systemic lupus erythematosus and patients with membranoproliferative or other glomerulonephritis. J Clin Invest I975; 56: 165-176. IO. Tamkun JW, Hynes RO. Plasma fibronectin is synthesised and secreted by hepatocytes. J Biol Chem 1983 ; z58 : 4641-4647. I I. Canalese J, Gove CD, Dimson AES, Wilkinson SP, Wardle EN, William R. Reticuloendothelium system and hepatocyte function in fulminant hepatic failure. Gut I982 ; 23: 265-269. 12. Wyke RJ, Canalese JC, Gimson AES, William R. Bacteraemia in patients with fulminant hepatic failure. Liver I982; 2:45-52. 13. Munoz LE, Salazar OG. Complement activation in patients with amoebic liver abscess. J Hepatol 1987; 5: 30-36.
