Clin. exp. Immunol. (1976) 25, 40-49.
Serum inhibitory factors (SIF) in patients with acute and chronic hepatitis and their clinical significance N. BRATTIG & P. A. BERG Department of Medicine, University of Tubingen, West Germany
(Received 16 February 1976) SUMMARY
Serum inhibitory factors (SIF) were demonstrated in a follow-up study in eighteen patients with HBSAg-positive viral hepatitis, in nine patients with chronic persistent hepatitis (CPH) and in six patients with progressive viral hepatitis (CAH). In addition these factors were studied in fifteen patients with HB.Ag-positive and HBsAg-negative CAH. SIF appeared during the incubation period up to 4 weeks before onset and disappeared in most instances within 4 weeks after onset of jaundice. Sera from patients with CPH showed no marked inhibitory activity when studied over a period of up to 3 years as compared to patients with a progressive course of hepatitis. The presence of SIF may depend upon persistence of virus, and may help to predict the development of chronic hepatitis.
INTRODUCTION Reduction of phytohaemagglutinin (PHA) induced lymphocyte transformation during viral infection may be related to in vivo depression of lymphocyte function (reviewed by Woodruff & Woodruff, 1975a). Recent findings, however, suggest that humoral factors which are released during viral infections also seem to interfere with the lymphocyte reactivity (Kantor, 1975). Thus, sera from patients with acute viral hepatitis were found to inhibit PHA-induced stimulation of autologous and control lymphocytes (Baroyan, Barinski & Shatkin, 1970; Mella, 1969; Newberry et al., 1973; Newble et al., 1975; Willems, Melnick & Rawls, 1969; Wands et al., 1975). Similar results were also obtained with sera from patients with chronic liver diseases (Fox et al., 1973; McSween & Thomas, 1973; Hsu & Leevy, 1971). The clinical relevance of these factors is still unclear but there is evidence that persistence of inhibitory activity correlated with a progressive course of hepatitis (Chisari & Edgington, 1975). In a follow-up study we tested sera from patients with acute viral hepatitis, chronic persistent hepatitis and chronic active hepatitis and correlated inhibitory activity with clinical, biochemical and histological parameters. The preliminary results indicate that early prediction of the outcome of acute viral hepatitis may be possible by testing for serum inhibition factors (SIF) during the acute phase of the illness. MATERIALS AND METHODS Patients. Serial studies of serum inhibition factors were performed in eighteen patients with acute viral HB.Ag-positive hepatitis (118 sera), nine patients with chronic persistent hepatitis (CPH) (thirty-seven sera), six juvenile patients (age 10-27 years) developing chronic active hepatitis after onset of acute hepatitis (fifty sera), and fifteen patients with histologically proven CAH (sixty-four sera). Sera. In the follow-up studies many sera were stored up to 4 years at - 20'C. The sera tested were decomplemented at 560C for 30 min. Lymphocyte transformation test. The lymphocyte transformation test was used in a modification of the method described by Penhale et al. (1974). Lymphocytes from a panel of six healthy donors were separated from heparinized blood by centri-
Correspondence: N. Brattig, Department of Medicine, University of Tubingen, D-7400 Tubingen, Auf dem Schnarrenberg, West Germany.
40
Serum inhibitory factors in hepatitis
41
fugation on Ficoll-Amido trizoe acid (specific gravity 1P077 g/ml) and washed three times in Hanks's BSS. In the presence of 10 pg phytohaemagglutinin (PHA) P/ml (Difco Laboratories) 0-25 x 106 lymphocytes were cultured for 90 hr in 0 25 ml of TC 199 (Difco Laboratories) containing 20%. test serum, 100 pg streptomycin per ml and 100 u/penicillin per ml. After addition of 10 pCi/ml [3H]thymidine (specific activity 2 Ci/mmole) (Radiochemical Centre, Amersham, Bucks.) incubation was continued for 6 hr. The cultures were transferred to glassfibre filters (Whatman GF/C), washed with 5%O trichloracetic acid and ethanol. The dried filters were counted in a liquid scintillation counter (Tricarb 3375) for incorporated thymidine. Lymphocytes from nine patients with acute HB.Ag-positive hepatitis and eleven patients with CAH were also stimulated with 2, 10 and 50 ug PHA-P per millilitre. The serum inhibitory activity was expressed as inhibition index comparing the PHA induced lymphocyte transformation of normal lymphocytes in the presence of patients serum and the transformation in presence of normal serum: inhibition index = mean ct/min of stimulated control lymphocytes+test serum mean ct/min of stimulated control lymphocytes+control serum Lymphocyte cultures from two or three controls were set up in triplicate and mean ct/min values of these triplicates were calculated. In the presence of normal serum the mean index was 0 95. Inhibitory activity was demonstrated when the index was lower than 0 7. Demonstration of HBAg and HBsAb. HBAg and HBSAb were detected by radioimmunoassay AUSRIA 11-125 and AUSAB (Abbott), respectively. Serum autoantibodies. Antibodies against smooth muscle (SMA), nuclei (ANA), mitochondria (AMA), and vascular endothelium were detected by immunofluorescence using human kidney and thyroid as well as rat stomach, liver and kidney. The sera were tested in a dilution of 1:10, fluorescein-labelled antihuman globulin (Hyland) was applied in a dilution of 1:15. The sections were examined with the Orthoplan microscope (Leitz) and filters UG1, BG 36 and K 460. Sera were also tested by CFT for cytoplasmic complement-fixing antibodies using homogenates of rat kidney.
RESULTS SIF in acute viral hepatitis In a follow-up study inhibitory factors (SIF) were evaluated in sera from eighteen patients with an uncomplicated course of acute HB.Ag-positive viral hepatitis (Fig. 1). All sera markedly depressed the PHA-induced transformation of normal lymphocytes during the first week showing a mean index of 0-25 (s.d. + 0 12; P< 0.001). The serum inhibitory effect disappeared gradually and after 4 weeks 67% of the patients sera had lost these factors. 7 weeks after onset none of the sera showed inhibition activity. In six patients SIF were also demonstrated during the incubation period up to 4 weeks before onset Norma index E
Percentage of patients showing inhibitory
week
0 2100 _
6
7
1~
~I75
50
_
+
I Weeks after clinical onset
FIG. 1. Serum inhibitory factors (SIF) in eighteen patients during the course of acute viral hepatitis. Each point gives the mean index (± s.d.) of all sera tested per week. Solid columns show the percentage of patients with SIF per week. The results indicate that 72%/ of patients had lost the activity in the 5th week and that SIF disappeared at the 8th week.
42
N. Brattig & P. A. Berg
7
5 4 6 3 Weeks before onset
(Incubation period)
2
Onset (Icterus)
2
3 4 5 6 Weeks after onset (Acute hepatitis)
l
l
7
8
FIG. 2a. FIG. 2(a). Sera from six patients with acute viral hepatitis (stored at -20'C) were tested retrospectively for SIF. The ordinate shows the inhibition index (HBAg-positive period, solid bars; HBAb-positive period, open bars; overlap between demonstration of HB.Ag and HBSAb, hatched bars). The figure shows strong SIF 3 weeks before onset (P< 0.001) and disappearance of the activity within 4 weeks after icterus. There is no correlation between appearance and disappearance of HBAg or HBSAb. (b) Comparison of SIF and nonorgan-specific antibodies during incubation period and acute phase in six patients with acute viral hepatitis (SMA, hatched bars; ANA, stippled bar). No correlation exists between the appearance and disappearance of SIF, SMA and ANA.
of jaundice (Fig. 2a) and in one case before the appearance of HBAg. No correlation was found between HBSAg, non-organ-specific antibodies and SIF (Fig. 2b). Furthermore, inhibitory activity was not
related to inflammatory activity (Fig. 3).
persistent hepatitis (CPH) and chronic active hepatitis (CAH) Thirty-seven sera from nine patients with CPH were tested and four were followed up to 3 In most instances SIF were at the lower limit of the normal range (mean 074+ 0-10) (Fig. 4).
SJF in chronic
years.
Strong and lasting inhibitory activity was, however, observed in six young patients (age 10-27 years) a progressive course of acute viral hepatitis who were followed up to 4 years and chronic active
with
43
Serum inhibitory factors in hepatitis Patients
Incubation period
Acute phase
I
Serum Inhibitory Factors
H.S.
N.B.
SIF
SIF
E.G.
A.M.
SIF
M. - K..ST.
SIF
H.-D.B.
SIF ..I
_____
5
4
2
3
2
3
4
Iweeks
Onset
AIcterus) Fia. 2b.
hepatitis was clinically and histologically evident in all patients when re-examined 1-3 years after first (Table 1). A typical follow-up study is shown in Fig. 5. This boy developed symptoms of acute hepatitis at the age of 10. HB5Ag was demonstrated only during the first weeks. Afterwards neither HB.Ag nor HBSAb could be detected, but SMA became strongly positive in the absence of ANA. High immunoglobulin
symptoms
TABLE 1. Persistence ofserum inhibitory factors (SIF) in six patients after acute onset ofhepatitis associated with a progressive course of the disease
SIF* tested months after acute onset Patient
Sex
Born
No. sera tested
R.D. H.F.
M M
1962 1957
6 13
n.d. 0-14 0 05 0 01 0-27 011 0*15 0-36 n.d. 1*0
R.M.D. M.B. V.H. A.A.
F F F M
1960 1955 1953 1956
5 13 8 5
001 06 n.d. 0*05 0-6 0-13 n.d. 0-21 0 25 0-19
1
12
24
36
0-59
42
45
1-0
Present clinical state: (histology) April 1975: CH April 1974: CAH with cirrhosis (in remission since July 1974) December 1974: CAH with cirrhosis May 1974: CAH with cirrhosis March 1975: CAH June 1974: CAH
n.d. = Not determined. * SIF given as index. t All patients have been or are on immunosuppressive therapy.
N. Brattig & P. A. Berg
44
0 18,2
800
700
16
*II
14'-
-
600
12
500 _
10-
' 400
It
k
-e
e24.8.46 H.S.O
I
1'
8
E
300
200
4
-
100 50_
HB Ag-negative
F-
F-
If. =
v
ME
_
21 days before onset he~~~X = ~ HB Ag-postive Id j m * _0 - -W --W A -r.1
HB Ag
-
-=
negative
lk
-
-
IX
1974
iF-1975
Normal index E |3c '
04 _
IV~~~~~~~~~~~~ncbto 0-8~~~~~~~~~~pro
3
Clinical onset of hepatitis
FIG. 3. Relationship between SIF and the inflammatory activity demonstrated in a patient with HBAgpositive viral hepatitis (HB5Ag, solid bar). The figure shows reduction of inhibitory activity (P< 0 01) after clinical onset despite high levels of transaminase (serum glutamate pyruvate transaminase) (SGPT) (0) and bilirubin (x ).
levels at the onset of the disease already indicated the unfavourable course. Although steroids (prednisolone) induced clinical remission, SIF continued to be present showing strong inhibition indices. Serial studies were also performed in fifteen patients having chronic active hepatitis at the time of admission to the hospital (Table 2). Four of them had evidence of previous viral infection. Clinical status and liver biopsies correlated well with the serum inhibitory activity in twelve patients: 3001- (a)
200
-
X
* Liver biopsy
_Liver
* biopsy
L.D.y *11.9.20
0
-~~~~~~~~~~~~
100
XXX- X
X
-X
X
x X
X
E 5
In
in
°
1-2
A,i-
1.0 0-8 0-6
x
04
-
I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
vHN^
I
(b)
°o I~~~~~~C °.0.0
__
-o--Do-
-O
Normal index
0-2 6 2
1972
~
1973
AAAAt---~~~~~~~~~~~
7vvvv
1974
FIG. 4. Absence of inhibition factors in sera from a patient with HBAg-positive chronic persistent hepatitis: a 3 year follow-up study. (+) Liver biopsy. (a) Transaminase activity; (b) serum inhibitory activity.
Serum inhibitory factors in hepatitis
45
W 4)
U)
v
.o -
-
+
tv
0C.U
bfo
C)0
~0X
0 V OC) 4)04
0.
04)
v4)4
e4
CU
CC
0
0
V) .° 0Y 0
U
4)
4-
c
e4
4)
4-.
CACU
w0~
,
*
U)
U) U )
on Y
C)
r. 4.)
4)J
uz
U)
.
U)
.~ .~ *
eq .r
I.
I
CU
opw
.0
-. -. +S. +c + + .
V)
C)
0 +
+ + + -s +
--s
+
CU
cq *-
00en
+ +
z
+ -+-F-
C)
0 0
oo e N U1 N en Ien .
N 00
00
00
1I) %0 00
00
e~l
Nl
4.-4
,0
14)
uz
C)
4Cf)
co
04.A 0
00
+
00
N N 0 Ns 0 N 0000C) eq 00 I-
o0 0 N
0
.0 -0
00
cn
.0
4)
cl0
N
N
00
N
I -
0 N ON 00 )
-
N
-
0N
N- 00 I"
CU
0
4-
0 -
4)1
44
*
_
N
o4
0
I*
-1
en~
4) N CN
0 0
N
+
N
00 N
e
e,
ON4 O~N
1f - 00 0% N 00 0 - 0% + 0 0% +% 0% 00 0% 0%
4)
cl)
cli "i ;4 -,. --.
:. :. .:!
z 04 1. -,. .-. P4 :. .,! --. .4 .4 :.
-. cli e-; 4 tr;
r--: 06 C. 1-4 C; -. (-Ii ." -4
g4 .4
00 -I" N oN 0% .% I.
46
N.
Brattig
&
P. A. Berg
900
CIE) 800 700j
600
R.D.d' *15.5.62
-Onset
-1%2. of jaundice
01CD
(o
x, 500 a
0
a. OD
400
Ig
45Srel. %
300 (mg0 /o) 4'
200
~~~Liver
3
biopsy
2m
+48
+74
0
5.
i1.
2.4.
Prednisolone
60-10mg
20-4mg
25-7-5mg
Inhibitory activity
E 0*
a .s 046
-
Normal index
-
0
1973
--4i
im
1974
1 .-- 1975
FIG. 5. Strong inhibitory activity over a period of 30 months in a 10-year-old boy with progressive HB.Agpositive hepatitis, who developed cirrhosis 2 years after first symptoms. Upper graph: SGPT (0); SGOT (0); bilirubin (x). Lower graph: SIF (o).
six patients with almost normal indices had no signs of any active liver disease when examined 12-24 months later whereas six patients with indices below 0 5 still had abnormalities of liver function and histological evidence of CAH and cirrhosis 12-30 months after the first SIF test. No association was, however, found in three patients in whom initially CAH was diagnosed. TABLE 3. Persistent inhibitory activity in sera from a patient with HB5Ag positive liver cirrhosis of low inflammatory activity indicating the unfavourable course of the disease
Date
2.2.71 14.5.71 21.5.71 7.8.73 3.9.73
HBAg
GOT/GPT
Bilirubin
y-Globulin
Sed. rate (SR)
Inhibition factor indices
d-
18/6 32/19 29/19 3/10 51/98
0-6 0-8 09 1.1 7-7
21-1 25-1 20-0 27-6 39 0
5/13 8/24 5/15 23/50 -
0-02
+ + + +
Died 7 September 1973.
0-01 0.01 0-26 0-01
Serum inhibitory factors in hepatitis
47
Thus sera from a female patient with typical lupoid hepatitis did not suppress PHA-induced lymphocyte transformation and another female patient who had lupoid hepatitis still expressed strong inhibitory activity although clinically there is at present no evidence of inflammatory activity. The same phenomenon was observed in a 52-year-old man who still shows strong SIF but histologically has only discrete signs of persistent hepatitis. In one case SIF were studied in the final stage of HB.Ag-positive liver cirrhosis (Table 3). 21 years before death of the patient (1971) SIF were found (index between 0-01 and 0.26) although clinically very few symptoms indicated the lethal event. There was no episode of acute hepatitis and signs of cirrhosis were first detected in 1968 by laprascopy and liver biopsy.
DISCUSSION Serum factors which interfere with the in vitro transformation of lymphocytes in response to non-specific mitogens or specific antigens have been demonstrated in various infectious diseases including tuberculosis (Heilman & McFarland, 1966), leprosy (Bullock & Fasal, 1971), syphilis (Levene et al., 1969) and histoplasmosis (Newberry et al., 1968). These factors may be related to the transient anergy found in patients during the early stage of infections and to the depression of lymphocyte function in vitro (Chisari & Edgington, 1975; Dudley, Giustino & Sherlock, 1972; Kantor, 1975). The demonstration of SIF as early as 4 weeks before onset of acute HBAg-positive viral hepatitis may be consistent with the view that these factors are produced and released in response to a viral or other antigenic stimulus. This hypothesis is in accordance with our results which indicate a correlation between disappearance of SIF and recovery from viral hepatitis and with the observation that high transaminase-and bilirubin levels had no effect on PHA-induced DNA synthesis. Newberry et al. (1973) showed that bilirubin concentration up to 041 mg/ml and bile acids up to 0-1 mg/ml did not influence the PHA-induced stimulatory effect. Furthermore, we also found that immunosuppressive treatment by itself did not abolish or increase
inhibitory activity. In one of the cases of progressive CAH SIF disappeared 1 year after clinical remission induced by immunotherapy and the observation raises the question of whether loss of SIF may support in this instance the discontinuance of immunosuppressive treatment. There is additional evidence from the serial studies in patients with HBAg-positive and HB.Agnegative chronic hepatitis that normal indices favour a good prognosis whereas low indices may be indicative of a progressive course. The determination of SIF seems especially useful in HBAg-positive patients in whom generally no autoantibodies can be detected and in whom the progressive course is more difficult to recognize than in patients with autoimmune liver diseases. The hypothesis is consistent with our findings of almost normal SIF indices in patients with CPH and the marked inhibitory activity in six young patients developing severe chronic active hepatitis after acute onset of viral hepatitis. It seems that the early diagnosis of CPH and the differential diagnosis between CPH and CAH may be facilitated by the application of this test. However, the prognostic validity of SIF in the various forms of chronic active hepatitis awaits further experience in view of the fact that some patients with biopsy-proven CAH did not express these factors in their sera. We also do not know yet if the degree of inhibitory activity can be associated with the severity of the chronic lever process although the very strong and persisting SIF in a female patient with lethal outcome of HBAg-positive liver cirrhosis may favour this view. It also would be of interest to know whether these factors are similar or identical to those described in other diseases like alcoholic cirrhosis (Hsu & Leevy, 1971), drug-induced hepatitis (Paronetto & Popper, 1970), primary biliary cirrhosis (McSween & Thomas, 1973; Fox et al., 1973) or in malignant diseases (Humphrey et al., 1975). The fact that SIF can appear during the preicteric phase of acute viral hepatitis and can disappear at the onset of the disease makes it unlikely that they have antibody activity or consist of immune complexes which may interact with lymphocyte surface receptors (Woodruff & Woodruff, 1975a).
48
N. Brattig & P. A. Berg
Intrinsic and extrinsic factors may be responsible for the observed transient depressed T-cell function (Woodruff & Woodruff, 1975b) and according to Kantor (1975) this phenomenon may be a normal regulatory mechanism indicating feedback suppression of T cells by lymphocytic products. We also measured patients' lymphocyte reactivity to PHA during the course of acute viral hepatitis but could not observe significant T-cell alteration. Careful washing of patients lymphocytes increased their PHA sensitivity and the addition of autologous serum had a marked suppressive effect as compared to homologous control serum, thus also implying that extrinsic factors may be mainly responsible for suppressed T-cell function. Newble et al. (1975) postulated an intrinsic defect of patients' lymphocytes in acute viral hepatitis since in the absence of autologous serum an increase in PHA response was not observed. However, foetal calf serum and no homologous serum was used as control serum and the PHA-induced thymidine uptake of the control lymphocytes was also lower when cultured in foetal calf serum instead of autologous serum. This suggests that their foetal calf serum caused an inhibitory effect and was unsuitable as control serum. Since a virus-induced suppression of T-cell function may affect only a small population of T cells it seems unlikely that a method which stimulates the majority of T cells would detect this defect by decreased PHA sensitivity. The association between depressed T cell reactivity and SIF in hepatitis is also supported by the findings of Wands et al. (1975) who reported that serum factors in patients with acute and chronic hepatitis inhibited PHA-induced blast transformation and reduced lymphocyte cytotoxicity. Similar findings are reported by Paronetto & Vernace (1975). Recently a factor responsible for induction of defective formation of T-rosettes was described by Chisari & Edgington (1975) in patients with hepatitis B virus infection. This rosette inhibitory factor (RIF) appeared to selectively suppress a subpopulation of T-lymphocytes and was associated with persistent viral infection. However, this factor did not inhibit thymidine incorporation of lymphocytes after stimulation by PHA. It seems unlikely, therefore, that RIF and SIF are identical and these observations imply that probably different immunoregulatory factors may be produced during viral infection or hepatocellular damage. Direct viral interference with lymphocyte function was also observed and taken as an explanation for altered T-cell reactivity (Woodruff & Woodruff, 1975b). Thus, Willems et al. (1969) observed that the transmission of the inhibitory effect occurred with viruses which replicated in the cytoplasm of the cell and Mella (1969) reported the presence of cytoplasmatic inclusion bodies during viraemia of infectious hepatitis in the affected lymphocytes and assumed that the virus can multiply in the leucocytes thus causing inhibition of mitosis. Osunkoya et al. (1974) examined lymphocyte cultures from children with acute measles infections up to 7 days after onset of the disease and found that the virus can persist within leucocytes for at least a week after the appearance of neutralizing antibodies. McFarland (1974) studied the effect of measles virus on lymphocytes and the immune response in mice. He found a transient suppression of T cells which corresponded to the time during which measles antigen was demonstrated in the spleen. In these experiments the anti-hapten response to DNP was measured and the reduction of anti-hapten antibody formation interpreted as a consequence of T helper cell suppression. Thus SIF could indicate suppressed helper cells, i.e. an impaired immune response which may be normally of limited duration and may be overcome by T cells. In conclusion these observations favour the idea that SIF are produced in response to viral infections and released from lymphocytes as long as viral antigenic stimulus persists. We thank Dr H. Lindner, DRK Hospital Hamburg, for supplies of sera from patients with chronic active hepatitis and Professor D. Doniach, Department of Immunology, Middlesex Hospital, London, for reviewing the manuscript. N.B. was supported by Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.
REFERENCES lymphocytes and integrity of their chromosomes in the BAROYAN, O.V., BARINSKY, I.F. & SHATKIN, A.A. (1970) course of serial passaging in vitro. Acta Virol. 14, 324. Inhibitory effect of sera from patients with Botskin's viral hepatitis on the differentiation and mitotic activity of BULLOcK, W.E. & FASAL, P. (1971) Studies of immune
Serum inhibitory factors in hepatitis
49
mechanisms in leprosy. III. The role of cellular and NEWBERRY, W.M., CHANDLER, J.W., CHIN, T.D.Y. & KIRKPATRICK, C.H. (1968) Immunology of the mycoses. humoral factors in impairment of the in vitro immune response. J. Immunol. 106, 888. I. Depressed lymphocyte transformation in chronic CHISARI, F.V. & EDGINGTON, T.S. (1975) Lymphocyte E histoplasmosis. J. Immunol. 100, 436. rosette inhibitory factor: a regulatory serum lipoprotein. NEWBERRY, W.M., SHOREY, J.W., SANFORD, J.P. & COMBES, J. exp. Med. 142, 1092. B. (1973) Depression of lymphocyte reactivity to phytoDUDLEY, F.J., GIUSTINO, V. & SHERLOCK, S. (1972) Cellhaemagglutinin by serum from patients with liver disease. mediated immunity in patients positive for hepatitis Cell. Immunol. 6, 87. associated antigen. Brit. med. I. iv, 754. NEWBLE, D.I., HOLMES, K.T., WANGEL, A.G. & FORBES, Fox, R.A., DUDLEY, F.J., SAMUELS, M., MILLIGAN, J. & I.J. (1975) Immune reactions in acute viral hepatitis. SHERLOCK, S. (1973) Lymphocyte transformation in Clin. exp. Immunol. 20, 17. response to phytohaemagglutinin in primary biliary OSUNKOYA, B.O., ADELEYE, G.I., ADEJUMO, T.A. & SALIcirrhosis: The search for a plasma inhibitory factor. Gut, MONU, L. (1974) Studies on leukocyte culture in measles. 14, 89. II. Detection of measles antigen in human leukocytes by HEILMAN, D.H. & McFARLAND, W. (1966) Inhibition of immunofluorescence. Arch. Ges. Virusjorsch. 44, 323. tuberculin-induced mitogenesis in cultures of lymphocytes PARONETTO, F. & POPPER, H. (1970) Lymphocyte stimufrom tuberculous donors. Int. Arch. Allergy, 30, 58. lation induced by halothane in patients with hepatitis Hsu, C.C.S. & LEEVY, C.M. (1971) Inhibition of PHAfollowing exposure to halothane. New Engl. J. Med. 283, stimulated lymphocyte transformation by plasma from 277. patients with advanced alcoholic cirrhosis. Clin. exp. PARONETTO, F. & VERNACE, S. (1975) Immunological studies Immunol. 8, 749. in patients with chronic active hepatitis. Cytotoxic HUMPHREY, G.B., PETERSON, L., WHALEN, M., PARKER, D., activity of lymphocytes to autochthonous liver cells grown LANKFORD, J., KRIVIT, W. & NESBIT, M. (1975) Lymin tissue culture. Clin. exp. Immunol. 19, 99. phocyte transformation in leukemic serum. Cancer, 35, PENHALE, W.J., FARMER, A., MACCUISH, A.C. & IRVINE, 1341. W.J. (1974) A rapid micro-method for the phytohaemagKANTOR, F.S. (1975) Infection, anergy and cell-mediated glutinin induced human lymphocyte transformation test. immunity. New Engl. J. Med. 292, 629. Clin. exp. Immunol. 18, 155. LEVENE, G.M., TURK, J.L., WRIGHT, D.J.M. & GRIMBLE, WANDS, J.R., PERROTTO, J.L., ALPERT, E. & ISSELBACHER, A.G.S. (1969) Reduced lymphocyte transformation due to K.J. (1975) Cell-mediated immunity in acute and chronic a plasma factor in patients with active syphilis. Lancet, ii, hepatitis. J. clin. Invest. 55, 921. 246. WILLEMS, F.TH.C., MELNICK, J.L. & RAWLS, W.E. (1969) McFARLAND, H.F. (1974) The effect of measles virus Viral inhibition of the phytohaemagglutinin response of infection on T and B lymphocytes in the mouse. I. human lymphocytes and application to viral hepatitis. Suppression of helper cell activity. J. Immunol. 113, 1978. Proc. Soc. exp. Biol. (N.Y.), 130, 652. MCSWEEN, R.N.M. & THOMAS, M.A. (1973) Lymphocyte WOODRUFF, J.F. & WOODRUFF, J.J. (1975a) T lymphocyte transformation by Phytohaemagglutinin (PHA) and interaction with viruses and virus-infected tissues. purified protein derivate (PPD) in primary biliary cirrProgr. med. Virol. 19, 120. hosis. Clin. exp. Immunol. 15, 523. WOODRUFF, J.F. & WOODRUFF, J.J. (1975b) The effect of MELLA, B. (1969) Use of PHA stimulated lymphocyte viral infections on the function of the immune system. cultures for detecting the agent of hepatitis. Vox Sang. Viral Immunology and Immunopathology (ed. by A. L. (Basel), 17, 45. Notkins), p. 393. Academic Press, New York.
Serum inhibitory factors (SIF) in patients with acute and chronic hepatitis and their clinical significance N. BRATTIG & P. A. BERG Department of Medicine, University of Tubingen, West Germany
(Received 16 February 1976) SUMMARY
Serum inhibitory factors (SIF) were demonstrated in a follow-up study in eighteen patients with HBSAg-positive viral hepatitis, in nine patients with chronic persistent hepatitis (CPH) and in six patients with progressive viral hepatitis (CAH). In addition these factors were studied in fifteen patients with HB.Ag-positive and HBsAg-negative CAH. SIF appeared during the incubation period up to 4 weeks before onset and disappeared in most instances within 4 weeks after onset of jaundice. Sera from patients with CPH showed no marked inhibitory activity when studied over a period of up to 3 years as compared to patients with a progressive course of hepatitis. The presence of SIF may depend upon persistence of virus, and may help to predict the development of chronic hepatitis.
INTRODUCTION Reduction of phytohaemagglutinin (PHA) induced lymphocyte transformation during viral infection may be related to in vivo depression of lymphocyte function (reviewed by Woodruff & Woodruff, 1975a). Recent findings, however, suggest that humoral factors which are released during viral infections also seem to interfere with the lymphocyte reactivity (Kantor, 1975). Thus, sera from patients with acute viral hepatitis were found to inhibit PHA-induced stimulation of autologous and control lymphocytes (Baroyan, Barinski & Shatkin, 1970; Mella, 1969; Newberry et al., 1973; Newble et al., 1975; Willems, Melnick & Rawls, 1969; Wands et al., 1975). Similar results were also obtained with sera from patients with chronic liver diseases (Fox et al., 1973; McSween & Thomas, 1973; Hsu & Leevy, 1971). The clinical relevance of these factors is still unclear but there is evidence that persistence of inhibitory activity correlated with a progressive course of hepatitis (Chisari & Edgington, 1975). In a follow-up study we tested sera from patients with acute viral hepatitis, chronic persistent hepatitis and chronic active hepatitis and correlated inhibitory activity with clinical, biochemical and histological parameters. The preliminary results indicate that early prediction of the outcome of acute viral hepatitis may be possible by testing for serum inhibition factors (SIF) during the acute phase of the illness. MATERIALS AND METHODS Patients. Serial studies of serum inhibition factors were performed in eighteen patients with acute viral HB.Ag-positive hepatitis (118 sera), nine patients with chronic persistent hepatitis (CPH) (thirty-seven sera), six juvenile patients (age 10-27 years) developing chronic active hepatitis after onset of acute hepatitis (fifty sera), and fifteen patients with histologically proven CAH (sixty-four sera). Sera. In the follow-up studies many sera were stored up to 4 years at - 20'C. The sera tested were decomplemented at 560C for 30 min. Lymphocyte transformation test. The lymphocyte transformation test was used in a modification of the method described by Penhale et al. (1974). Lymphocytes from a panel of six healthy donors were separated from heparinized blood by centri-
Correspondence: N. Brattig, Department of Medicine, University of Tubingen, D-7400 Tubingen, Auf dem Schnarrenberg, West Germany.
40
Serum inhibitory factors in hepatitis
41
fugation on Ficoll-Amido trizoe acid (specific gravity 1P077 g/ml) and washed three times in Hanks's BSS. In the presence of 10 pg phytohaemagglutinin (PHA) P/ml (Difco Laboratories) 0-25 x 106 lymphocytes were cultured for 90 hr in 0 25 ml of TC 199 (Difco Laboratories) containing 20%. test serum, 100 pg streptomycin per ml and 100 u/penicillin per ml. After addition of 10 pCi/ml [3H]thymidine (specific activity 2 Ci/mmole) (Radiochemical Centre, Amersham, Bucks.) incubation was continued for 6 hr. The cultures were transferred to glassfibre filters (Whatman GF/C), washed with 5%O trichloracetic acid and ethanol. The dried filters were counted in a liquid scintillation counter (Tricarb 3375) for incorporated thymidine. Lymphocytes from nine patients with acute HB.Ag-positive hepatitis and eleven patients with CAH were also stimulated with 2, 10 and 50 ug PHA-P per millilitre. The serum inhibitory activity was expressed as inhibition index comparing the PHA induced lymphocyte transformation of normal lymphocytes in the presence of patients serum and the transformation in presence of normal serum: inhibition index = mean ct/min of stimulated control lymphocytes+test serum mean ct/min of stimulated control lymphocytes+control serum Lymphocyte cultures from two or three controls were set up in triplicate and mean ct/min values of these triplicates were calculated. In the presence of normal serum the mean index was 0 95. Inhibitory activity was demonstrated when the index was lower than 0 7. Demonstration of HBAg and HBsAb. HBAg and HBSAb were detected by radioimmunoassay AUSRIA 11-125 and AUSAB (Abbott), respectively. Serum autoantibodies. Antibodies against smooth muscle (SMA), nuclei (ANA), mitochondria (AMA), and vascular endothelium were detected by immunofluorescence using human kidney and thyroid as well as rat stomach, liver and kidney. The sera were tested in a dilution of 1:10, fluorescein-labelled antihuman globulin (Hyland) was applied in a dilution of 1:15. The sections were examined with the Orthoplan microscope (Leitz) and filters UG1, BG 36 and K 460. Sera were also tested by CFT for cytoplasmic complement-fixing antibodies using homogenates of rat kidney.
RESULTS SIF in acute viral hepatitis In a follow-up study inhibitory factors (SIF) were evaluated in sera from eighteen patients with an uncomplicated course of acute HB.Ag-positive viral hepatitis (Fig. 1). All sera markedly depressed the PHA-induced transformation of normal lymphocytes during the first week showing a mean index of 0-25 (s.d. + 0 12; P< 0.001). The serum inhibitory effect disappeared gradually and after 4 weeks 67% of the patients sera had lost these factors. 7 weeks after onset none of the sera showed inhibition activity. In six patients SIF were also demonstrated during the incubation period up to 4 weeks before onset Norma index E
Percentage of patients showing inhibitory
week
0 2100 _
6
7
1~
~I75
50
_
+
I Weeks after clinical onset
FIG. 1. Serum inhibitory factors (SIF) in eighteen patients during the course of acute viral hepatitis. Each point gives the mean index (± s.d.) of all sera tested per week. Solid columns show the percentage of patients with SIF per week. The results indicate that 72%/ of patients had lost the activity in the 5th week and that SIF disappeared at the 8th week.
42
N. Brattig & P. A. Berg
7
5 4 6 3 Weeks before onset
(Incubation period)
2
Onset (Icterus)
2
3 4 5 6 Weeks after onset (Acute hepatitis)
l
l
7
8
FIG. 2a. FIG. 2(a). Sera from six patients with acute viral hepatitis (stored at -20'C) were tested retrospectively for SIF. The ordinate shows the inhibition index (HBAg-positive period, solid bars; HBAb-positive period, open bars; overlap between demonstration of HB.Ag and HBSAb, hatched bars). The figure shows strong SIF 3 weeks before onset (P< 0.001) and disappearance of the activity within 4 weeks after icterus. There is no correlation between appearance and disappearance of HBAg or HBSAb. (b) Comparison of SIF and nonorgan-specific antibodies during incubation period and acute phase in six patients with acute viral hepatitis (SMA, hatched bars; ANA, stippled bar). No correlation exists between the appearance and disappearance of SIF, SMA and ANA.
of jaundice (Fig. 2a) and in one case before the appearance of HBAg. No correlation was found between HBSAg, non-organ-specific antibodies and SIF (Fig. 2b). Furthermore, inhibitory activity was not
related to inflammatory activity (Fig. 3).
persistent hepatitis (CPH) and chronic active hepatitis (CAH) Thirty-seven sera from nine patients with CPH were tested and four were followed up to 3 In most instances SIF were at the lower limit of the normal range (mean 074+ 0-10) (Fig. 4).
SJF in chronic
years.
Strong and lasting inhibitory activity was, however, observed in six young patients (age 10-27 years) a progressive course of acute viral hepatitis who were followed up to 4 years and chronic active
with
43
Serum inhibitory factors in hepatitis Patients
Incubation period
Acute phase
I
Serum Inhibitory Factors
H.S.
N.B.
SIF
SIF
E.G.
A.M.
SIF
M. - K..ST.
SIF
H.-D.B.
SIF ..I
_____
5
4
2
3
2
3
4
Iweeks
Onset
AIcterus) Fia. 2b.
hepatitis was clinically and histologically evident in all patients when re-examined 1-3 years after first (Table 1). A typical follow-up study is shown in Fig. 5. This boy developed symptoms of acute hepatitis at the age of 10. HB5Ag was demonstrated only during the first weeks. Afterwards neither HB.Ag nor HBSAb could be detected, but SMA became strongly positive in the absence of ANA. High immunoglobulin
symptoms
TABLE 1. Persistence ofserum inhibitory factors (SIF) in six patients after acute onset ofhepatitis associated with a progressive course of the disease
SIF* tested months after acute onset Patient
Sex
Born
No. sera tested
R.D. H.F.
M M
1962 1957
6 13
n.d. 0-14 0 05 0 01 0-27 011 0*15 0-36 n.d. 1*0
R.M.D. M.B. V.H. A.A.
F F F M
1960 1955 1953 1956
5 13 8 5
001 06 n.d. 0*05 0-6 0-13 n.d. 0-21 0 25 0-19
1
12
24
36
0-59
42
45
1-0
Present clinical state: (histology) April 1975: CH April 1974: CAH with cirrhosis (in remission since July 1974) December 1974: CAH with cirrhosis May 1974: CAH with cirrhosis March 1975: CAH June 1974: CAH
n.d. = Not determined. * SIF given as index. t All patients have been or are on immunosuppressive therapy.
N. Brattig & P. A. Berg
44
0 18,2
800
700
16
*II
14'-
-
600
12
500 _
10-
' 400
It
k
-e
e24.8.46 H.S.O
I
1'
8
E
300
200
4
-
100 50_
HB Ag-negative
F-
F-
If. =
v
ME
_
21 days before onset he~~~X = ~ HB Ag-postive Id j m * _0 - -W --W A -r.1
HB Ag
-
-=
negative
lk
-
-
IX
1974
iF-1975
Normal index E |3c '
04 _
IV~~~~~~~~~~~~ncbto 0-8~~~~~~~~~~pro
3
Clinical onset of hepatitis
FIG. 3. Relationship between SIF and the inflammatory activity demonstrated in a patient with HBAgpositive viral hepatitis (HB5Ag, solid bar). The figure shows reduction of inhibitory activity (P< 0 01) after clinical onset despite high levels of transaminase (serum glutamate pyruvate transaminase) (SGPT) (0) and bilirubin (x ).
levels at the onset of the disease already indicated the unfavourable course. Although steroids (prednisolone) induced clinical remission, SIF continued to be present showing strong inhibition indices. Serial studies were also performed in fifteen patients having chronic active hepatitis at the time of admission to the hospital (Table 2). Four of them had evidence of previous viral infection. Clinical status and liver biopsies correlated well with the serum inhibitory activity in twelve patients: 3001- (a)
200
-
X
* Liver biopsy
_Liver
* biopsy
L.D.y *11.9.20
0
-~~~~~~~~~~~~
100
XXX- X
X
-X
X
x X
X
E 5
In
in
°
1-2
A,i-
1.0 0-8 0-6
x
04
-
I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
vHN^
I
(b)
°o I~~~~~~C °.0.0
__
-o--Do-
-O
Normal index
0-2 6 2
1972
~
1973
AAAAt---~~~~~~~~~~~
7vvvv
1974
FIG. 4. Absence of inhibition factors in sera from a patient with HBAg-positive chronic persistent hepatitis: a 3 year follow-up study. (+) Liver biopsy. (a) Transaminase activity; (b) serum inhibitory activity.
Serum inhibitory factors in hepatitis
45
W 4)
U)
v
.o -
-
+
tv
0C.U
bfo
C)0
~0X
0 V OC) 4)04
0.
04)
v4)4
e4
CU
CC
0
0
V) .° 0Y 0
U
4)
4-
c
e4
4)
4-.
CACU
w0~
,
*
U)
U) U )
on Y
C)
r. 4.)
4)J
uz
U)
.
U)
.~ .~ *
eq .r
I.
I
CU
opw
.0
-. -. +S. +c + + .
V)
C)
0 +
+ + + -s +
--s
+
CU
cq *-
00en
+ +
z
+ -+-F-
C)
0 0
oo e N U1 N en Ien .
N 00
00
00
1I) %0 00
00
e~l
Nl
4.-4
,0
14)
uz
C)
4Cf)
co
04.A 0
00
+
00
N N 0 Ns 0 N 0000C) eq 00 I-
o0 0 N
0
.0 -0
00
cn
.0
4)
cl0
N
N
00
N
I -
0 N ON 00 )
-
N
-
0N
N- 00 I"
CU
0
4-
0 -
4)1
44
*
_
N
o4
0
I*
-1
en~
4) N CN
0 0
N
+
N
00 N
e
e,
ON4 O~N
1f - 00 0% N 00 0 - 0% + 0 0% +% 0% 00 0% 0%
4)
cl)
cli "i ;4 -,. --.
:. :. .:!
z 04 1. -,. .-. P4 :. .,! --. .4 .4 :.
-. cli e-; 4 tr;
r--: 06 C. 1-4 C; -. (-Ii ." -4
g4 .4
00 -I" N oN 0% .% I.
46
N.
Brattig
&
P. A. Berg
900
CIE) 800 700j
600
R.D.d' *15.5.62
-Onset
-1%2. of jaundice
01CD
(o
x, 500 a
0
a. OD
400
Ig
45Srel. %
300 (mg0 /o) 4'
200
~~~Liver
3
biopsy
2m
+48
+74
0
5.
i1.
2.4.
Prednisolone
60-10mg
20-4mg
25-7-5mg
Inhibitory activity
E 0*
a .s 046
-
Normal index
-
0
1973
--4i
im
1974
1 .-- 1975
FIG. 5. Strong inhibitory activity over a period of 30 months in a 10-year-old boy with progressive HB.Agpositive hepatitis, who developed cirrhosis 2 years after first symptoms. Upper graph: SGPT (0); SGOT (0); bilirubin (x). Lower graph: SIF (o).
six patients with almost normal indices had no signs of any active liver disease when examined 12-24 months later whereas six patients with indices below 0 5 still had abnormalities of liver function and histological evidence of CAH and cirrhosis 12-30 months after the first SIF test. No association was, however, found in three patients in whom initially CAH was diagnosed. TABLE 3. Persistent inhibitory activity in sera from a patient with HB5Ag positive liver cirrhosis of low inflammatory activity indicating the unfavourable course of the disease
Date
2.2.71 14.5.71 21.5.71 7.8.73 3.9.73
HBAg
GOT/GPT
Bilirubin
y-Globulin
Sed. rate (SR)
Inhibition factor indices
d-
18/6 32/19 29/19 3/10 51/98
0-6 0-8 09 1.1 7-7
21-1 25-1 20-0 27-6 39 0
5/13 8/24 5/15 23/50 -
0-02
+ + + +
Died 7 September 1973.
0-01 0.01 0-26 0-01
Serum inhibitory factors in hepatitis
47
Thus sera from a female patient with typical lupoid hepatitis did not suppress PHA-induced lymphocyte transformation and another female patient who had lupoid hepatitis still expressed strong inhibitory activity although clinically there is at present no evidence of inflammatory activity. The same phenomenon was observed in a 52-year-old man who still shows strong SIF but histologically has only discrete signs of persistent hepatitis. In one case SIF were studied in the final stage of HB.Ag-positive liver cirrhosis (Table 3). 21 years before death of the patient (1971) SIF were found (index between 0-01 and 0.26) although clinically very few symptoms indicated the lethal event. There was no episode of acute hepatitis and signs of cirrhosis were first detected in 1968 by laprascopy and liver biopsy.
DISCUSSION Serum factors which interfere with the in vitro transformation of lymphocytes in response to non-specific mitogens or specific antigens have been demonstrated in various infectious diseases including tuberculosis (Heilman & McFarland, 1966), leprosy (Bullock & Fasal, 1971), syphilis (Levene et al., 1969) and histoplasmosis (Newberry et al., 1968). These factors may be related to the transient anergy found in patients during the early stage of infections and to the depression of lymphocyte function in vitro (Chisari & Edgington, 1975; Dudley, Giustino & Sherlock, 1972; Kantor, 1975). The demonstration of SIF as early as 4 weeks before onset of acute HBAg-positive viral hepatitis may be consistent with the view that these factors are produced and released in response to a viral or other antigenic stimulus. This hypothesis is in accordance with our results which indicate a correlation between disappearance of SIF and recovery from viral hepatitis and with the observation that high transaminase-and bilirubin levels had no effect on PHA-induced DNA synthesis. Newberry et al. (1973) showed that bilirubin concentration up to 041 mg/ml and bile acids up to 0-1 mg/ml did not influence the PHA-induced stimulatory effect. Furthermore, we also found that immunosuppressive treatment by itself did not abolish or increase
inhibitory activity. In one of the cases of progressive CAH SIF disappeared 1 year after clinical remission induced by immunotherapy and the observation raises the question of whether loss of SIF may support in this instance the discontinuance of immunosuppressive treatment. There is additional evidence from the serial studies in patients with HBAg-positive and HB.Agnegative chronic hepatitis that normal indices favour a good prognosis whereas low indices may be indicative of a progressive course. The determination of SIF seems especially useful in HBAg-positive patients in whom generally no autoantibodies can be detected and in whom the progressive course is more difficult to recognize than in patients with autoimmune liver diseases. The hypothesis is consistent with our findings of almost normal SIF indices in patients with CPH and the marked inhibitory activity in six young patients developing severe chronic active hepatitis after acute onset of viral hepatitis. It seems that the early diagnosis of CPH and the differential diagnosis between CPH and CAH may be facilitated by the application of this test. However, the prognostic validity of SIF in the various forms of chronic active hepatitis awaits further experience in view of the fact that some patients with biopsy-proven CAH did not express these factors in their sera. We also do not know yet if the degree of inhibitory activity can be associated with the severity of the chronic lever process although the very strong and persisting SIF in a female patient with lethal outcome of HBAg-positive liver cirrhosis may favour this view. It also would be of interest to know whether these factors are similar or identical to those described in other diseases like alcoholic cirrhosis (Hsu & Leevy, 1971), drug-induced hepatitis (Paronetto & Popper, 1970), primary biliary cirrhosis (McSween & Thomas, 1973; Fox et al., 1973) or in malignant diseases (Humphrey et al., 1975). The fact that SIF can appear during the preicteric phase of acute viral hepatitis and can disappear at the onset of the disease makes it unlikely that they have antibody activity or consist of immune complexes which may interact with lymphocyte surface receptors (Woodruff & Woodruff, 1975a).
48
N. Brattig & P. A. Berg
Intrinsic and extrinsic factors may be responsible for the observed transient depressed T-cell function (Woodruff & Woodruff, 1975b) and according to Kantor (1975) this phenomenon may be a normal regulatory mechanism indicating feedback suppression of T cells by lymphocytic products. We also measured patients' lymphocyte reactivity to PHA during the course of acute viral hepatitis but could not observe significant T-cell alteration. Careful washing of patients lymphocytes increased their PHA sensitivity and the addition of autologous serum had a marked suppressive effect as compared to homologous control serum, thus also implying that extrinsic factors may be mainly responsible for suppressed T-cell function. Newble et al. (1975) postulated an intrinsic defect of patients' lymphocytes in acute viral hepatitis since in the absence of autologous serum an increase in PHA response was not observed. However, foetal calf serum and no homologous serum was used as control serum and the PHA-induced thymidine uptake of the control lymphocytes was also lower when cultured in foetal calf serum instead of autologous serum. This suggests that their foetal calf serum caused an inhibitory effect and was unsuitable as control serum. Since a virus-induced suppression of T-cell function may affect only a small population of T cells it seems unlikely that a method which stimulates the majority of T cells would detect this defect by decreased PHA sensitivity. The association between depressed T cell reactivity and SIF in hepatitis is also supported by the findings of Wands et al. (1975) who reported that serum factors in patients with acute and chronic hepatitis inhibited PHA-induced blast transformation and reduced lymphocyte cytotoxicity. Similar findings are reported by Paronetto & Vernace (1975). Recently a factor responsible for induction of defective formation of T-rosettes was described by Chisari & Edgington (1975) in patients with hepatitis B virus infection. This rosette inhibitory factor (RIF) appeared to selectively suppress a subpopulation of T-lymphocytes and was associated with persistent viral infection. However, this factor did not inhibit thymidine incorporation of lymphocytes after stimulation by PHA. It seems unlikely, therefore, that RIF and SIF are identical and these observations imply that probably different immunoregulatory factors may be produced during viral infection or hepatocellular damage. Direct viral interference with lymphocyte function was also observed and taken as an explanation for altered T-cell reactivity (Woodruff & Woodruff, 1975b). Thus, Willems et al. (1969) observed that the transmission of the inhibitory effect occurred with viruses which replicated in the cytoplasm of the cell and Mella (1969) reported the presence of cytoplasmatic inclusion bodies during viraemia of infectious hepatitis in the affected lymphocytes and assumed that the virus can multiply in the leucocytes thus causing inhibition of mitosis. Osunkoya et al. (1974) examined lymphocyte cultures from children with acute measles infections up to 7 days after onset of the disease and found that the virus can persist within leucocytes for at least a week after the appearance of neutralizing antibodies. McFarland (1974) studied the effect of measles virus on lymphocytes and the immune response in mice. He found a transient suppression of T cells which corresponded to the time during which measles antigen was demonstrated in the spleen. In these experiments the anti-hapten response to DNP was measured and the reduction of anti-hapten antibody formation interpreted as a consequence of T helper cell suppression. Thus SIF could indicate suppressed helper cells, i.e. an impaired immune response which may be normally of limited duration and may be overcome by T cells. In conclusion these observations favour the idea that SIF are produced in response to viral infections and released from lymphocytes as long as viral antigenic stimulus persists. We thank Dr H. Lindner, DRK Hospital Hamburg, for supplies of sera from patients with chronic active hepatitis and Professor D. Doniach, Department of Immunology, Middlesex Hospital, London, for reviewing the manuscript. N.B. was supported by Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.
REFERENCES lymphocytes and integrity of their chromosomes in the BAROYAN, O.V., BARINSKY, I.F. & SHATKIN, A.A. (1970) course of serial passaging in vitro. Acta Virol. 14, 324. Inhibitory effect of sera from patients with Botskin's viral hepatitis on the differentiation and mitotic activity of BULLOcK, W.E. & FASAL, P. (1971) Studies of immune
Serum inhibitory factors in hepatitis
49
mechanisms in leprosy. III. The role of cellular and NEWBERRY, W.M., CHANDLER, J.W., CHIN, T.D.Y. & KIRKPATRICK, C.H. (1968) Immunology of the mycoses. humoral factors in impairment of the in vitro immune response. J. Immunol. 106, 888. I. Depressed lymphocyte transformation in chronic CHISARI, F.V. & EDGINGTON, T.S. (1975) Lymphocyte E histoplasmosis. J. Immunol. 100, 436. rosette inhibitory factor: a regulatory serum lipoprotein. NEWBERRY, W.M., SHOREY, J.W., SANFORD, J.P. & COMBES, J. exp. Med. 142, 1092. B. (1973) Depression of lymphocyte reactivity to phytoDUDLEY, F.J., GIUSTINO, V. & SHERLOCK, S. (1972) Cellhaemagglutinin by serum from patients with liver disease. mediated immunity in patients positive for hepatitis Cell. Immunol. 6, 87. associated antigen. Brit. med. I. iv, 754. NEWBLE, D.I., HOLMES, K.T., WANGEL, A.G. & FORBES, Fox, R.A., DUDLEY, F.J., SAMUELS, M., MILLIGAN, J. & I.J. (1975) Immune reactions in acute viral hepatitis. SHERLOCK, S. (1973) Lymphocyte transformation in Clin. exp. Immunol. 20, 17. response to phytohaemagglutinin in primary biliary OSUNKOYA, B.O., ADELEYE, G.I., ADEJUMO, T.A. & SALIcirrhosis: The search for a plasma inhibitory factor. Gut, MONU, L. (1974) Studies on leukocyte culture in measles. 14, 89. II. Detection of measles antigen in human leukocytes by HEILMAN, D.H. & McFARLAND, W. (1966) Inhibition of immunofluorescence. Arch. Ges. Virusjorsch. 44, 323. tuberculin-induced mitogenesis in cultures of lymphocytes PARONETTO, F. & POPPER, H. (1970) Lymphocyte stimufrom tuberculous donors. Int. Arch. Allergy, 30, 58. lation induced by halothane in patients with hepatitis Hsu, C.C.S. & LEEVY, C.M. (1971) Inhibition of PHAfollowing exposure to halothane. New Engl. J. Med. 283, stimulated lymphocyte transformation by plasma from 277. patients with advanced alcoholic cirrhosis. Clin. exp. PARONETTO, F. & VERNACE, S. (1975) Immunological studies Immunol. 8, 749. in patients with chronic active hepatitis. Cytotoxic HUMPHREY, G.B., PETERSON, L., WHALEN, M., PARKER, D., activity of lymphocytes to autochthonous liver cells grown LANKFORD, J., KRIVIT, W. & NESBIT, M. (1975) Lymin tissue culture. Clin. exp. Immunol. 19, 99. phocyte transformation in leukemic serum. Cancer, 35, PENHALE, W.J., FARMER, A., MACCUISH, A.C. & IRVINE, 1341. W.J. (1974) A rapid micro-method for the phytohaemagKANTOR, F.S. (1975) Infection, anergy and cell-mediated glutinin induced human lymphocyte transformation test. immunity. New Engl. J. Med. 292, 629. Clin. exp. Immunol. 18, 155. LEVENE, G.M., TURK, J.L., WRIGHT, D.J.M. & GRIMBLE, WANDS, J.R., PERROTTO, J.L., ALPERT, E. & ISSELBACHER, A.G.S. (1969) Reduced lymphocyte transformation due to K.J. (1975) Cell-mediated immunity in acute and chronic a plasma factor in patients with active syphilis. Lancet, ii, hepatitis. J. clin. Invest. 55, 921. 246. WILLEMS, F.TH.C., MELNICK, J.L. & RAWLS, W.E. (1969) McFARLAND, H.F. (1974) The effect of measles virus Viral inhibition of the phytohaemagglutinin response of infection on T and B lymphocytes in the mouse. I. human lymphocytes and application to viral hepatitis. Suppression of helper cell activity. J. Immunol. 113, 1978. Proc. Soc. exp. Biol. (N.Y.), 130, 652. MCSWEEN, R.N.M. & THOMAS, M.A. (1973) Lymphocyte WOODRUFF, J.F. & WOODRUFF, J.J. (1975a) T lymphocyte transformation by Phytohaemagglutinin (PHA) and interaction with viruses and virus-infected tissues. purified protein derivate (PPD) in primary biliary cirrProgr. med. Virol. 19, 120. hosis. Clin. exp. Immunol. 15, 523. WOODRUFF, J.F. & WOODRUFF, J.J. (1975b) The effect of MELLA, B. (1969) Use of PHA stimulated lymphocyte viral infections on the function of the immune system. cultures for detecting the agent of hepatitis. Vox Sang. Viral Immunology and Immunopathology (ed. by A. L. (Basel), 17, 45. Notkins), p. 393. Academic Press, New York.
