Clin. exp. Immunol. (1975) 22, 78-83.
IMMUNOLOGICAL STUDIES IN CHILDREN WITH ACUTE VIRAL HEPATITIS FLORENCE KANAKOUDI,* A. NIKOLAIDIS,* B. DANIILIDIS,t S. MANIOS,t S. S. ZURUKZOGLU* AND C. CASSIMOS* * Paediatric Clinic, The Aristotle University andt Infectious Diseases Hospital, Paediatric Department, Thessaloniki, Greece (Received 10 December 1974)
SUMMARY
Sera from 116 consecutive unselected cases of sporadic acute viral hepatitis in children were examined for hepatitis B antigen (HBAg), smooth-muscle autoantibodies (SMA), other autoantibodies and immunoglobulins, and skin tests were performed with dinitrochlorobenzene (DNCB). HBAg was detected in twenty-one and SMA in ninety-eight out of 116 sera that had been obtained during the 1st or 2nd week from the onset of jaundice. Hepatitis B antigen was present in seventeen out of the eighteen SMA negative patients (94.40%) and in only four out of the ninety-eight SMA-positive patients (4 1 %)4 The presence of SMA was not related to the sex and age of the patients or to the serum bilirubin and transaminase levels. SMA did not persist for more than 6 weeks from the onset of jaundice in most of the cases. In twenty-eight out of forty-one sera which were tested the IgM level was found to be elevated during the acute phase of illness and within normal limits during the recovery stage. A negative correlation between the presence of SMA and the elevated serum IgM level and the presence of HB Ag in the same patients was observed. The DNCB skin test was found to be positive in all fifty-two patients who did not have HBAg in their serum and in twenty out of the twenty-one patients who had circulating HbAg. From these findings there appears to be no gross impairment of cell-mediated immunity in acute viral hepatitis, and hepatitis A is associated with SMA production and an increase in serum IgM levels, when compared to hepatitis associated with HBAg.
INTRODUCTION Smooth muscle antibodies (SMA) have been demonstrated in sera of patients with acute viral hepatitis (Ajdukiewicz et al., 1972; Farrow et al., 1970; Smith, Frances & David-West, 1973). Farrow et al. (1970) did not find any correlation between SMA and hepatitis B Correspondence: Dr Florence S. Kanakoudi, First Paediatric Clinic, The Aristotle University, Thessaloniki, Greece.
78
Immunology of acute viral hepatitis
79
antigen (HBAg), whereas Holborow (1972) observed a higher incidence of SMA among blood donors who were carriers of HBAg compared to those negative for HBAg. IgM levels have been found to be elevated during the early weeks of illness (Ajdukiewicz et al., 1972; Giles & Krugman, 1969). Ajdukiewicz et al. (1972) found no correlation between IgM levels and the presence of SMA. Giles & Krugman (1969) found that there was an increase in IgM levels during the course of hepatitis type A and Wolheim (1968) found that there was no increase in IgM levels during hepatitis type B. However, Peters & Ashcavai (1970) have not confirmed these findings in a larger number of patients. It has been postulated that the HBAg carrier state is associated with impairment of delayed type hypersensitivity (Dudley, Fox & Sherlock, 1972a; Halikowski, Korczowski, & Zajaczkowski, 1972), but this has not been confirmed by others (Sodomann & Havemann, 1973) either in the acute stage of hepatitis B or in the chronic carrier state. For these reasons, SMA, HBAg, serum immunoglobulin levels and delayed cutaneous reactivity to DNCB have been investigated in a group of children with acute viral hepatitis. MATERIALS AND METHODS Children studied. (a) During a period of 1 year (March 1972 to March 1973) 116 unselected consecutive cases of sporadic acute viral hepatitis, admitted to the Infectious Diseases Hospital of Thessaloniki, Greece, were studied. The age and sex of the patients were as follows: sixty-five of the 116 children were males (fourteen aged between 3 and 6 years and fifty-one between 7 and 13 years); fifty-one were females (seven aged between 3 and 6 years and forty-four between 7 and 13 years). Of the 116 patients, 106 were clinically jaundiced when first examined and the remaining ten patients had been jaundiced prior to admission. The time of onset of jaundice was determined in all cases as accurately as possible. The diagnosis of acute viral hepatitis was based on clinical, laboratory and epidemiological data. (b) Eighty-two normal unselected children, aged between 4 and 14 years, were used as controls. Collection of blood samples. The first specimen of blood was obtained from all patients at the time of admission or 1-2 days later and was always within 7 days from the onset of jaundice. Subsequent specimens were obtained 1 week, 2 weeks and 6 weeks after the first sample. Sera were coded and stored at - 20'C. Biochemical estimations. Serum bilirubin and aspartate transaminase (SGOT) levels were estimated by standard methods. Immunological studies. Cutaneous delayed hypersensitivity to DNCB. During the first week of hospitalization the DNCB skin test was performed in seventy-three patients according to the method of Brown et al. (1967) modified as follows: the sensitizing dose was 1000 pg of DNCB in 0-1 ml of acetone (instead of 2000 pug) and the sensitized area was covered for 24 hr. The result was read after 15 days while the subjects were still hospitalized. Detection of autoantibodies. Autoantibodies were detected by immunofluorescence on composite tissue blocks (Roitt & Doniach, 1969). Tissue sections were cut at 4 ,um on a Bright's cryostat at - 20'C. Fluorescein isothiocyanate-conjugated sheep anti-human immunoglobulin, anti-IgM and anti-IgG (Burroughs Wellcome Ltd) were used at suitable dilutions. Each serum sample was tested in duplicate with all conjugates. Sections were examined with darkfield illumination through a Zeiss fluorescence microscope with an Osram HBO 200 lamp and BG12 and 470 pm filters. Undiluted serum samples were examined for the presence of thyroid, gastric, mitochondrial and antinuclear autoantibodies. The sera were diluted 1/10 when examined for the presence of SMA on fresh rat stomach (Johnson, Holborow & Glynn, 1965). The results for SMA were graded as strongly positive (4ff + +) when SMA was detected at serum dilutions of 1:20 or greater, positive (+ +) at dilutions 1 :10, weakly positive (+) at dilutions of less than 1: 10 and negative (0) when SMA was not detected at any of these dilutions. The results were read by the same person, who was unaware of the code number of each serum. Detection of HBAg. An immunoelectrophoretic method was used for the detection of HBAg in all serum samples (Memoranda, 1970). Determination of serum immunoglobulin levels. Radial immunodiffusion technique on 'Tripartigen' agar plates (Behringwerke) was used for the determination of serum immunoglobulin levels. F
Florence Kanakoudi et al.
80
RESULTS Autoantibodies Smooth muscle autoantibodies were found in ninety-eight out of 116 sera (8455%) obtained during the 1st or 2nd week from the onset of jaundice. These autoantibodies were of the IgM type in all but two serum samples in which SMA were found to be both of the IgG and IgM type. Results are shown in Table 1. All sera initially negative for SMA remained negative throughout the study. The majority of the positive sera became negative after the 4th week from the onset of.jaundice. TABLE 1. Relationship between presence of SMA and time from the onset of jaundice
Weeks from the onset of jaundice
Number of patients tested
1 2 3 4 5 6-11
107 104 52 13 8 36
Strength of fluorescence* +++
++
64 5 45 1 25 0 7-7
28-9 44-2 34 6 46-1 12-5 2-7
2-7
+ -
9-6 23-0 12-5 5-5
0 6-5 10-6 30 7 23-0 75 0 88 8
* Results are expressed as a percentage of patients in each class and show the presence of SMA.
The incidence of SMA was not related to the sex or age of the patients or to the serum bilirubin concentration and transaminase levels. Of the eighty-two control serum samples tested for SMA, three were found to be positive (3.6%) and none of those children had ever been jaundiced. Gastric parietal cell antibodies were found in two of the ninety-eight SMA positive patients. Antinuclear, mitochondrial and thyroid antibodies were not found in the patients or control groups. Presence of HBAg HBAg was detected in twenty-one out of 116 patients (18
1%). The relationship between the presence of HBAg and SMA autoantibodies is shown in Table 2. Thus, HBAg was detected in seventeen out of eighteen SMA-negative patients (94.40), in contrast to only four out of ninety-eight SMA-positive patients (4-1%). This result is statistically highly significant. Serum immunoglobulin levels The IgM level was found to be elevated in twenty-eight out of forty-one sera (68.3%) tested during the acute phase of illness. This returned to normal limits after 7-12 weeks from the onset of jaundice. The IgM level during illness and during the convalescent stage, as well as its relation to the presence of SMA and HBAg are shown in Table 3. Those patients
Immunology of acute viral hepatitis
81
TABLE 2. Correlation between presence of SMA and HBAg
Number of patients with:
HBAg(+)
HBAg(-)
Total
4 17 21
94 1 95
98 18
SMA-positive patients SMA-negative patients Total
Y2=
116
82-59; P
IMMUNOLOGICAL STUDIES IN CHILDREN WITH ACUTE VIRAL HEPATITIS FLORENCE KANAKOUDI,* A. NIKOLAIDIS,* B. DANIILIDIS,t S. MANIOS,t S. S. ZURUKZOGLU* AND C. CASSIMOS* * Paediatric Clinic, The Aristotle University andt Infectious Diseases Hospital, Paediatric Department, Thessaloniki, Greece (Received 10 December 1974)
SUMMARY
Sera from 116 consecutive unselected cases of sporadic acute viral hepatitis in children were examined for hepatitis B antigen (HBAg), smooth-muscle autoantibodies (SMA), other autoantibodies and immunoglobulins, and skin tests were performed with dinitrochlorobenzene (DNCB). HBAg was detected in twenty-one and SMA in ninety-eight out of 116 sera that had been obtained during the 1st or 2nd week from the onset of jaundice. Hepatitis B antigen was present in seventeen out of the eighteen SMA negative patients (94.40%) and in only four out of the ninety-eight SMA-positive patients (4 1 %)4 The presence of SMA was not related to the sex and age of the patients or to the serum bilirubin and transaminase levels. SMA did not persist for more than 6 weeks from the onset of jaundice in most of the cases. In twenty-eight out of forty-one sera which were tested the IgM level was found to be elevated during the acute phase of illness and within normal limits during the recovery stage. A negative correlation between the presence of SMA and the elevated serum IgM level and the presence of HB Ag in the same patients was observed. The DNCB skin test was found to be positive in all fifty-two patients who did not have HBAg in their serum and in twenty out of the twenty-one patients who had circulating HbAg. From these findings there appears to be no gross impairment of cell-mediated immunity in acute viral hepatitis, and hepatitis A is associated with SMA production and an increase in serum IgM levels, when compared to hepatitis associated with HBAg.
INTRODUCTION Smooth muscle antibodies (SMA) have been demonstrated in sera of patients with acute viral hepatitis (Ajdukiewicz et al., 1972; Farrow et al., 1970; Smith, Frances & David-West, 1973). Farrow et al. (1970) did not find any correlation between SMA and hepatitis B Correspondence: Dr Florence S. Kanakoudi, First Paediatric Clinic, The Aristotle University, Thessaloniki, Greece.
78
Immunology of acute viral hepatitis
79
antigen (HBAg), whereas Holborow (1972) observed a higher incidence of SMA among blood donors who were carriers of HBAg compared to those negative for HBAg. IgM levels have been found to be elevated during the early weeks of illness (Ajdukiewicz et al., 1972; Giles & Krugman, 1969). Ajdukiewicz et al. (1972) found no correlation between IgM levels and the presence of SMA. Giles & Krugman (1969) found that there was an increase in IgM levels during the course of hepatitis type A and Wolheim (1968) found that there was no increase in IgM levels during hepatitis type B. However, Peters & Ashcavai (1970) have not confirmed these findings in a larger number of patients. It has been postulated that the HBAg carrier state is associated with impairment of delayed type hypersensitivity (Dudley, Fox & Sherlock, 1972a; Halikowski, Korczowski, & Zajaczkowski, 1972), but this has not been confirmed by others (Sodomann & Havemann, 1973) either in the acute stage of hepatitis B or in the chronic carrier state. For these reasons, SMA, HBAg, serum immunoglobulin levels and delayed cutaneous reactivity to DNCB have been investigated in a group of children with acute viral hepatitis. MATERIALS AND METHODS Children studied. (a) During a period of 1 year (March 1972 to March 1973) 116 unselected consecutive cases of sporadic acute viral hepatitis, admitted to the Infectious Diseases Hospital of Thessaloniki, Greece, were studied. The age and sex of the patients were as follows: sixty-five of the 116 children were males (fourteen aged between 3 and 6 years and fifty-one between 7 and 13 years); fifty-one were females (seven aged between 3 and 6 years and forty-four between 7 and 13 years). Of the 116 patients, 106 were clinically jaundiced when first examined and the remaining ten patients had been jaundiced prior to admission. The time of onset of jaundice was determined in all cases as accurately as possible. The diagnosis of acute viral hepatitis was based on clinical, laboratory and epidemiological data. (b) Eighty-two normal unselected children, aged between 4 and 14 years, were used as controls. Collection of blood samples. The first specimen of blood was obtained from all patients at the time of admission or 1-2 days later and was always within 7 days from the onset of jaundice. Subsequent specimens were obtained 1 week, 2 weeks and 6 weeks after the first sample. Sera were coded and stored at - 20'C. Biochemical estimations. Serum bilirubin and aspartate transaminase (SGOT) levels were estimated by standard methods. Immunological studies. Cutaneous delayed hypersensitivity to DNCB. During the first week of hospitalization the DNCB skin test was performed in seventy-three patients according to the method of Brown et al. (1967) modified as follows: the sensitizing dose was 1000 pg of DNCB in 0-1 ml of acetone (instead of 2000 pug) and the sensitized area was covered for 24 hr. The result was read after 15 days while the subjects were still hospitalized. Detection of autoantibodies. Autoantibodies were detected by immunofluorescence on composite tissue blocks (Roitt & Doniach, 1969). Tissue sections were cut at 4 ,um on a Bright's cryostat at - 20'C. Fluorescein isothiocyanate-conjugated sheep anti-human immunoglobulin, anti-IgM and anti-IgG (Burroughs Wellcome Ltd) were used at suitable dilutions. Each serum sample was tested in duplicate with all conjugates. Sections were examined with darkfield illumination through a Zeiss fluorescence microscope with an Osram HBO 200 lamp and BG12 and 470 pm filters. Undiluted serum samples were examined for the presence of thyroid, gastric, mitochondrial and antinuclear autoantibodies. The sera were diluted 1/10 when examined for the presence of SMA on fresh rat stomach (Johnson, Holborow & Glynn, 1965). The results for SMA were graded as strongly positive (4ff + +) when SMA was detected at serum dilutions of 1:20 or greater, positive (+ +) at dilutions 1 :10, weakly positive (+) at dilutions of less than 1: 10 and negative (0) when SMA was not detected at any of these dilutions. The results were read by the same person, who was unaware of the code number of each serum. Detection of HBAg. An immunoelectrophoretic method was used for the detection of HBAg in all serum samples (Memoranda, 1970). Determination of serum immunoglobulin levels. Radial immunodiffusion technique on 'Tripartigen' agar plates (Behringwerke) was used for the determination of serum immunoglobulin levels. F
Florence Kanakoudi et al.
80
RESULTS Autoantibodies Smooth muscle autoantibodies were found in ninety-eight out of 116 sera (8455%) obtained during the 1st or 2nd week from the onset of jaundice. These autoantibodies were of the IgM type in all but two serum samples in which SMA were found to be both of the IgG and IgM type. Results are shown in Table 1. All sera initially negative for SMA remained negative throughout the study. The majority of the positive sera became negative after the 4th week from the onset of.jaundice. TABLE 1. Relationship between presence of SMA and time from the onset of jaundice
Weeks from the onset of jaundice
Number of patients tested
1 2 3 4 5 6-11
107 104 52 13 8 36
Strength of fluorescence* +++
++
64 5 45 1 25 0 7-7
28-9 44-2 34 6 46-1 12-5 2-7
2-7
+ -
9-6 23-0 12-5 5-5
0 6-5 10-6 30 7 23-0 75 0 88 8
* Results are expressed as a percentage of patients in each class and show the presence of SMA.
The incidence of SMA was not related to the sex or age of the patients or to the serum bilirubin concentration and transaminase levels. Of the eighty-two control serum samples tested for SMA, three were found to be positive (3.6%) and none of those children had ever been jaundiced. Gastric parietal cell antibodies were found in two of the ninety-eight SMA positive patients. Antinuclear, mitochondrial and thyroid antibodies were not found in the patients or control groups. Presence of HBAg HBAg was detected in twenty-one out of 116 patients (18
1%). The relationship between the presence of HBAg and SMA autoantibodies is shown in Table 2. Thus, HBAg was detected in seventeen out of eighteen SMA-negative patients (94.40), in contrast to only four out of ninety-eight SMA-positive patients (4-1%). This result is statistically highly significant. Serum immunoglobulin levels The IgM level was found to be elevated in twenty-eight out of forty-one sera (68.3%) tested during the acute phase of illness. This returned to normal limits after 7-12 weeks from the onset of jaundice. The IgM level during illness and during the convalescent stage, as well as its relation to the presence of SMA and HBAg are shown in Table 3. Those patients
Immunology of acute viral hepatitis
81
TABLE 2. Correlation between presence of SMA and HBAg
Number of patients with:
HBAg(+)
HBAg(-)
Total
4 17 21
94 1 95
98 18
SMA-positive patients SMA-negative patients Total
Y2=
116
82-59; P
