Acta path. microhiol. scand. Sect. C , 85: 49-56, 1977

IN P’ITRO EFFECTS OF RUBELLA VIRUS, STRAIN RA 27/3, ON HUMAN LYMPHOCYTES

I . Viral Inhibition o/ A4itogen Stimulation in Relation to Rubella Haenzagglutination Inhibition Antibodies

ROLF MALLER and LARS S O R ~

Department of Clinical Bacteriology and Department of Infectious Diseases, Linkoping University, Linkoping, Sweden

Maller, R. Rr SorCn, I,. I n riitro effects of rubella virus, strain RA 27/3 on human lymphocytes. Viral inhibition of mitogen stimulation in relation to rubella haemagglutination inhibition anti- bodies. Acta path. microbiol. scand. Sect. c, 85: 49-56, 1977.

The inhibiting effect of rubella virus on lymphocyte stimulation in uitro was studied, using purified virus of the attenuated strain RA 27/9. Addition of the virus to human lymphocytes from twenty healthy blood donors before stimulation with leuco-agglutinin (LA) , a component of phytohaemagglutinin, caused a considerable inhibition of the L A response in some experi- ments, whereas in other experiments the inhibition was slight or non-existent. If further analysed, the results showed a correlation between the degree of inhibition and the immunity of the lymphocyte donor against rubella, as measured by haemagglutination inhibition (HI ) . Thus the LA-response was significantly more depressed in a group of lymphocyte donors with HI-titres ranging from 20 to 160 than in another group with HI-titre less than 5. Possible explanations of the virus-induced inhibition of the LA response and possible connection between this phe- nomenon and the immune response against rubella are discussed.

Key words: Rubella virus; lymphocytes in cJitro ; mitogen stimulation; haemagglutination in- hibition antibodies.

R. Maller, Regionsjukhuset, Linkoping, S-581 85 Linkoping, Sweden.

.Received 16.vii.76 Accepted 13.ix.76

Rubella virus infection is known to inhibit the phytohaemagglutinin ( PHA) response of lymphocytes cultured in vitro (10, 19, 20, 23, 24). This was first demonstrated in babies presenting the congenital rubella syndrome (20) and, subsequently, the phenomenon has been studied following vaccination against rubella (9, 16, 31). Some investigators have reported a normal or only slightly depressed response to PHA (9, 11, 15, 24, 34), whereas others have demonstrated a considerable in-

4 Acta path. iniciobiol. xand. Sect. C, 85, 1

hibition (16, 20, 31). This variation could, in part, be explained by differences in the virulence of the rubella strains used (16, 23). Addition of live rubella virus to nor- mal lymphocytes in uitro inhibits the PHA response of these cells (10, 19, 23, 24). The mechanism involved is not known. The object of the present paper is to study the effect on PHA stimulation of the attenuated rubella virus strain RA 2713 added to normal human blood lymphocytes in uitro and, in particular, the differences, if any, between rubella im-

49

mune and non-immune lymphocytes, with re- spect to viral inhibition of mitogen stimula- tion.

M A T E R I A L A N D M E T H O D S

Materia!$: Gelatin K 936 was obtained from Edible Compounds Ltd., H d l , England. Ficoll was ob- tained from Pharmacia AB, Uppsala, Sweden and sodium metrizoate (Isopaque) from Nyegaard Br Co., Oslo, Norway. Tissue culture medium 199 F ( T C M 199) obtained from GIBCO, Grand Island, New York, and buffered with HEPES, was sup- plemented with L-glutamine and benzylpenicillin, 1250 IEJml. Foetal calf serum (Flow Laboratories, Irvine, Scotland) and autologous serum were heat- inactivated at 56” C for 30 min. Leuco-agglutinin (LA) , purified from phytohaemagglutinin (33) from Pharmacia AB, Uppsala, Sweden, was diluted in physiological saline to different concentrations and added in 0.1 ml aliquots to 0.9 ml of cell suspension. Micro ‘Test I1 tissue culture plates 3040 (Falcon Plastics, Oxnard, USA) were used for lymphocyte cultures. 1QC-TdR, spec.act. 58 mCiJ mmole, was obtained from The Radiochemical Centre, Amersham, England. At harvest, cultured cells were collected on glass fibre filters, 934 AH, from Reeve Angel, New Jersey, USA.

Lymphocy te donors: Healthy volunteers, thirteen men and seven women aged 20 to 57 years (aver- age 32 years), were the donors of lymphocytes.

Lymphocy te separation: Newly drawn venous blood was immediately defibrinated by vigorous shaking with glass pearls and lymphocytes were separated by gelatin sedimentation (5 ) followed by centrifugation on a layer of Ficoll-Isopaque ( 2 ) . The lymphocytes were washed three times with T C M 199 and were suspended in T C M 199 with 15 per cent foetal calf serum in a conc. of 1 x 106 mononuclear cellsJml. The final cell suspensions contained about 90 per cent mononuclear cells.

Rubella virus: Attenuated rubella virus, strain RA 27/3, was kindly provided by Dr. Pertti Vuu- nunen, Orion Diagnostica, Helsinki, Finland. The virus was propagated in cultures of BHK 21/13S cells, in total 55 passages, purified by low speed centrifugation followed by ultracentrifugation in a sugar gradient and finally dialysed (29) . The cul- tures were checked for absence of contaminating microorganisms including mycoplasma. The infec- tivity of the virus preparation was tested by plaque assay on BHK 21JW-3 cells and the titre was ad- justed to 107 pfu/ml. The final product was sus- pended in 0.025 M phosphate buffer, p H 7.3 with EDTA 0.003 M ; it was rapidly frozen and stored at -70” C. Infectivity was also checked after thaw- ing. T h e virus was transported to Linkoping in liquid nitrogen, -196” C, and stored after arrival at -90” C. The virus was diluted in 0.025 M phos-

phate buffer supplemented with EDTA 0.003 M, NaCl 0.15 M and 2 per cent sacharose (“virus buffer”). When applicable, the virus was UV- inactivated by irradiating the virus suspension con- tained in a quartz-vessel for 20 min at 254 nm. The radiation energy obtained in the quartz-vessel was 480 pWJcm2. Under these conditions rubella virus is inactivated without losing its antigenic properties

HI-test: T h e rubella haemagglutination-inhibit- ing ( H I ) antibodies in autologous sera and foetal calf sera were determined by the method of Cooper et al. ( 4 ) . Every HI-titre figure was set accord- ing to the results of three separate determinations. The HI-titres of the foetal calf sera used were <5.

Lymphocy te cultures: In order to minimize pos- sible reactions between virus and rubella antibodies in the serum of the lymphocyte donor ( l o ) , the incubation of lymphocytes together with rubella virus was performed in TCM-15 per cent foetal calf serum in siliconized conical glass tubes. Rubella virus or “virus buffer” in 0.1 ml portions were added to 0.9 ml lymphocyte cultures which were incubated overnight. The cells were spun down, washed twice in T C M 199 and finally resuspended in T C M 199 with 15 per cent autologous serum which proved to be superior in supporting the growth of LA stimulated cells. The mononuclear cell concentration was adjusted to 1 x 10‘;/ml. LA in different concentrations in 0.1 ml aliquots were added to 0.9 ml cultures of virus-infected and non- infected cultures, respectively. Four aliquots of 0.2 ml from each specimen were dispensed on microplates which were then closed with a film and incubated a t 37” C in humidified air for an indi- cated number of days. The survival of the cells was checked intermittently using trypan blue exclusion. Five hours before the end of the incubation period, 0.03 pCi of IIC-TdR in 20 pl of saline was added to each culture. Cultures were washed and pre- cipitated onto glass fibre filters by a semiautomatic multiple sample processor (28). The filters were placed in 10 ml Permablend I11 (Packard Instru- ment Co., Ill., USA) , and the radioactivity was measured in a Packard Tri-Carb 3375 scintillation counter. T h e incorporated radioactivity was ex- pressed as cpm/ml of cell suspension.

Statistics: The methodological error of I‘C-TdR incorporation and radioactivity determination, standard deviation (SD) , was calculated from the values ( X ) of cultures without virus, stimulated by LA in optimal concentration. An estimated devia- tion from 8 in a future series of cultures performed under the same conditions (Y) could be prognov ticated, using Student’s t-distribution. Accordingly, the prognosticated interval for P would be:

( 7 ) .

x * t . SD, . l/n, + 1/n,,

Values below and above these limits would be con-

50

TABLE 1. Results of LA-stimulation for Six Days of Lymphocytes f rom Donors with HI-titres Ranging from 20 t o 160

Incubation with

HI-titre “virus buffer” loF pfu/ml virus cpm/ml x 103a

No. cpm/ml x 103a

Incubation with “Inhibition” 105 pfu/ml virus

cpm/ml x lO3a 2 70.

160 160 80 80 80 80 40 40 20

16.7 16.9 19.7 8.9 5.7

12.0 10.3 12.6 4.9

4.6***b 6.3** 2.4*** 1.9*** 2.6** 8.6** 7.3**

2.8*** 12.4

-73 -63 -88 -78 -55 -28 -29

-2 -43

12.4 9.5**b

21.9 7.8** 6.0 9.2** 6.0

11.6 -

Incubation with “Inhibition” UV-inact. virusd

cpm/ml x 102a -+ %c

-2 7 - -44 15.6 + 11

-13 8.3 + 5 6.1

-23 8.1**b + 5 6.1 -8 14.2 - 4.5

-

“Inhibition” * %c

+ 6 -3 3

+ 6 + 13 -10

Mean I SDe 12.0 i 5.1 5.4 f 3.5 11.2 -C 4.8 9.5 -+ 4.1

Thymidine incorporation expressed as cpm/ml of cell suspension. Values approximated to the nearest hundred. Denotes level of significance of the deviation from mean thymidine incorporation in cultures without rubella virus: ** significant at the 1 per cent level arid *** significant a t the 0.1 per cent level.

cpm virus Deviation in per cent, i.e. expressed as (100- . 100); (-) before figures denotes inhibition.

cpm control Rubella virus loG pfu/ml inactivated by UV-irradiation. SD = standard deviation.

TABLE 2. Results of LA-stimulation for Six Days of Lymphocytes from Donors with HI-titre Less than 5

Incubation with Incubation with Incubation with I “Inhibition” UV-inact, virusd “Inhibition”

~~ ~~

10 <5 4.9 4.2 -14 - - 6.8 + 39 11 11.6 11.7 + 1 13.6 + 18 12.8 + 11 12 1, 9.6 9.0 -7 9.4 -3 10.2 + 6 13 16.8 12.5**h -2 6 16.5 -2 16.1 -4 14 16.0 15.4 -4 16.6 + 4 18.7 + 17 15 14.8 15.1 + 2 18.5**b + 25 13.6 -8

,> ~~

Mean +_ SDe 12.3 * 4.5 11.4 f 4.2 14.9 * 3.5 13.0 2 4.2

Symbols and terms are the same as those in Table 1. z

TA

BL

E 1

. R

esul

ts o

f LA

-sti

mul

atio

n fo

r Si

x D

ays

of

Lym

phoc

ytes

fro

m D

onor

s w

ith

HI-

titr

es R

angi

ng f

rom

20

to 1

60

Incu

bati

on w

ith

HI-

titr

e “v

irus

buf

fer”

lo

F pf

u/m

l vi

rus

cpm

/ml

x 10

3a

No.

cp

m/m

l x

103a

Incu

bati

on w

ith

“Inh

ibit

ion”

10

5 pf

u/m

l vi

rus

cpm

/ml

x lO

3a

2 7

0.

160

160 80

80

80

80

40

40

20

16.7

16

.9

19.7

8.

9 5.

7 12

.0

10.3

12

.6

4.9

4.6*

**b

6.3*

* 2.

4***

1.

9***

2.

6**

8.6*

* 7.

3**

2.8*

**

12.4

-73

-63

-88

-78

-55

-28

-29 -2 -43

12.4

9.

5**b

21

.9

7.8*

* 6.

0 9.

2**

6.0

11.6

-

Incu

bati

on w

ith

“Inh

ibit

ion”

U

V-i

nact

. vi

rusd

cpm

/ml

x 10

2a

-+

%c

-2 7

- -4

4 15

.6

+ 11

-13

8.3

+5

6.

1 -2

3 8.

1**b

+

5

6.1

-8 14

.2

-

4.5

-

“Inh

ibit

ion”

*

%c

+6

-3

3 +

6

+ 13

-1

0

Mea

n I S

De

12.0

i 5

.1

5.4 f 3

.5

11.2

-C 4

.8

9.5

-+ 4

.1

Thy

mid

ine

inco

rpor

atio

n ex

pres

sed

as c

pm/m

l of

ce

ll su

spen

sion

. V

alue

s ap

prox

imat

ed t

o t

he n

eare

st h

undr

ed.

Den

otes

lev

el o

f si

gnif

ican

ce o

f th

e de

viat

ion

from

mea

n th

ymid

ine

inco

rpor

atio

n in

cul

ture

s w

itho

ut r

ubel

la v

irus

: **

sig

nifi

cant

at

the

1 pe

r ce

nt

leve

l ar

id *

** si

gnif

ican

t at

the

0.1

per

cent

leve

l. cp

m v

irus

D

evia

tion

in

per

cen

t, i

.e.

expr

esse

d as

(1

00-

. 10

0);

(-)

befo

re f

igur

es d

enot

es i

nhib

itio

n.

cpm

con

trol

R

ubel

la v

irus

lo

G pf

u/m

l in

acti

vate

d by

UV

-irr

adia

tion

. S

D =

st

anda

rd d

evia

tion

.

TA

BL

E 2

. R

esul

ts o

f LA

-sti

mul

atio

n fo

r Si

x D

ays

of Ly

mph

ocyt

es f

rom

Don

ors

wit

h H

I-ti

tre

Less

tha

n 5

Incu

bati

on w

ith

Incu

bati

on w

ith

Incu

bati

on w

ith

I

“Inh

ibit

ion”

U

V-i

nact

, vir

usd

“Inh

ibit

ion”

~~

~~

10

<5

4.9

4.2

-14

- -

6.8

+ 39

11

11.6

11

.7

+1

13

.6

+ 18

12.8

+ 1

1 12

1,

9.6

9.0

-7 9.

4 -3

10.2

+

6

13

16.8

12

.5**

h -2

6 16

.5

-2 16

.1

-4

14

16.0

15

.4

-4

16.6

+

4

18.7

+

17

15

14.8

15

.1

+2

18.5

**b

+ 25

13.6

-8

,>

~~

Mea

n +

_ SD

e 12

.3 *

4.5

11.4

f 4

.2

14.9

* 3.

5 13

.0 2 4

.2

Sym

bols

and

ter

ms

are

the

sam

e as t

hose

in

Tab

le 1

. z

CPdml lo3

15

10

5

1 0

LA

Fig. 1 . ‘I’hymidine incorporation of lymphocytes incubated with live or inactivated rubella virus or with “virus buffer”, following 6 days incubation after addition of LA in different concentrations. HI-titre of lymphocyte donor = 160. Symbols: 0-0 “virus buffer”, 0- - -0 UV-inactivated rubella virus 10” pfu/ml, A-. , -A live rubella virus 106 pfu/ml and 0- . -0 live rubella virus 105 pfu/ml.

sidered significantly different from 8. These prog- nosticated intervals were calculated using t-values for p = 0.001 and p = 0.01, respectively. Accord- ingly, the differences between control cultures (8) and virus-treated cultures (9) were classified as non-significant a t the 0.1 and 1 per cent level, re- spectively. Student’s t-test was used in other statis- tical calculations.

R E S U L T S

I ) Pattern of Virus Inhibition of thu I.A-response

The inhibiting effect of rubella virus strain RA 27/3 on the LA response of lymphocytes varied considerably in different experiments, ranging from no inhibition at all up to almost complete impairment of the LA response

52

(Table 1 and 2 ) . Fig. 1 shows one experiment where inhibition of the LA-induced thymidine incorporation was pronounced. Evidently, the thymidine incorporation into cultures with UV-inactivated rubella virus was grossly simi- lar to that of cultures without virus. O n the other hand, the incorporation into cultures with rubella virus lo6 pfu/ml was consider- ably lower than that in control cultures. In cultures with rubella virus lo5 pfu/ml, a less pronounced inhibition of thymidine incorpo- ration was observed. In this experiment as in most other experiments, the LA dose-response curves of virus-infected and non-infected cells were quite similar. O n two occasions, how- ever, a depression of the LA-response curve applying to virus treated cells was observed at the optimal LA concentration of non-treated cells (Fig. 2 ) .

2 ) Inhibition in Relation to HI-titres of the

The above pattern of virus induced inhibi- tion of thymidine incorporation was not seen in all experiments. The results of 15 experi- ments are shown in Tables 1 and 2. In all these experiments the cells were stimulated

Lymphocyte Donors

5

1

5 0 0 1 2.5

Fig. 2. Depression of the LA dose-response curve of lymphocytes incubated with live rubella virus 106 pfu/ml. Incubation time 6 days after addition of LA. HI-titre of lymphocyte donor = 80. Symbols as those shown in Fig. 1.

mh! x10 t

Fig. 3. Time course of thymidine incorporation of lymphocytes incubated with live rubella virus 10" pfu/ml ( A-A) or with "virus buffer (0-0). The days following addition of LA to cultures are indicated. Lymphocyte donors with HI-titres from 20 to 160. The results of individual cultures are integrated and shown as mean -C standard error.

with a range of different LA-concentrations. Since the dose-response curves generally are not seen to shift after virus infection, nothing but the thymidine incorporations at optimal LA concentrations are shown in the Tables. The experiments are grouped according to the HI-titres of the lymphocyte donors, since these titres are considered to be a good meas- ure of rubella immunity ( 2 7 ) . Following the addition of rubeIla virus in a concentration of' 10" pfu/ml, a considerable inhibition of thy- midine incorporation was seen in five out of six cases where HI = 80-160 (expt 1-5) while inhibition was moderate in one case where HI = 80 (expt 6 ) . In one out of two cases where H I = 40, a moderate inhibition was observed (expt 7 ) , whereas no inhibition (expt 8) was observed in the other case. In the only case where H I = 20, a moderate inhibition was seen (expt 9 ) . Finally, in five out of six cases where serological immunity

against rubella was absent, HI<5, the in- hibition was nil or slight whereas a moderate inhibition of thymidine incorporation in the sixth case was recorded (expt 13). Altogether, these results show that rubella virus-induced inhibition of the LA response is more pro- nounced in cases where the lymphocyte donor displays immunity against rubella virus. The difference in thymidine incorporation in lym- phocyte cultures from donors with definite serological immunity (Table l ) , and in lym- phocyte cultures from donors without sero- logical immunity (Table 2 ) , was significant (p<0.025). This significance was more pro- nounced (p<O.Ol) when the group with the highest HI-titres, i.e. 80-160, was compared with that where HI<5. In general, the inhi- bition of thymidine incorporation following addition of rubella virus in a concentration of lo5 pfu/ml was much less pronounced than that following addition to lo6 pfu/ml. Addi- tion of UV-inactivated virus did not lead to any significant change in thymidine incorpo- ration as compared with that in control cul- tures.

3) Temporal Development of Inhibition of the LA-response

No inhibition of thymidine incorporation was observed on day 3 after addition of LA. However, as soon as the LA-stimulation had reached its optimal effect, i.e. from day four on, the inhibition was considerable in the group of lymphocyte donors with HI-titres 20-160 (Fig. 3 ) .

4) Numbers of Surviving Cells in Virus-infected and Non-infected Cultures

The number of surviving cells in infected and non-infected cultures ( n = 10) were 0.23 t 0.08 x 10"/ml and 0.33 & 0.13 x lO"/ml, respectively (mean -t SD). These cell counts are subject to great uncertainties because of the cell-clumping effect of LA, but the differ- ence was not significant (p>0.05). The per- centages of dead cells were also about the same, 29 2 8 per cent and 26 +- 5 per cent, respectively. Thus, there were no obvious dif-

53

ferences between infected and non-infected cells with regard to number of cells.

D I S C U S S I O N

Both humoral (30) and cellular immunity develop after infection with rubella virus. Cell mediated immunity as demonstrated by lym- phocyte cytatoxicity against rubella infected cells (26), development of cytotoxic lympho- kines (32), MIF production (8), and antigen specific lymphocyte stimulation (24, 31, 32) have been shown to coincide with or precede the development of humoral antibodies. Par- allel to the development of specific immunity against rubella virus, a non-specific anergy arises, as revealed by an impairment of cellu- lar immune reactions against various antigens and mitogens. This is demonstrated by a decreased dermal hypersensitivity against streptokinase-streptodornase (9) and possibly against tuberculin (1) as well as by an inhibi- tion of PHA-induced lymphocyte stimulation (16, 17, 31, 32). This anergy is seen in many viral infections (18) and its mechanism and significance are still obscure.

Studies of the PHA response of lympho- cytes from vaccines given different atten- uated strains of rubella virus have yielded variable results. Some investigators have re- ported a normal or only slightly reduced re- sponse to PHA (9, 11, 17), whereas others have demonstrated a considerable depres- sion (16, 31, 32). Inhibition induced by at- tenuated virus strains is generally less in- tense than that induced by virulent strains (16, 23). Addition of rubella virus to in vitro cultures of normal lymphocytes inhibits the PHA stimulation of these cells, provided that the virus is added before or simultaneous with the PHA (10, 19, 23, 24). UV-inactivation (10, 19, 24) and treatment of the virus with anti-rubella antibodies abolish the inhibition (10). However, the effect of antibodies is complex, since recent results show that IgM anti-rubella antibodies cooperate with rubella virus in the inhibition of PHA response (12).

In the present study, a variable degree of

54

inhibition of the LA response was in different experiments seen to follow incubation of hu- man lymphocytes with the attenuated puri- fied rubella virus strain RA 27/3. In the experiments, where inhibition occurred, the thymidine incorporation into infected and non-infected cultures was similar up to the third day of culture with LA, i.e. there was an initial time interval before the inhibition was fully developed. The most pronounced inhibition was observed in experiments using lymphocyte from individuals with high titres of anti-rubella HI-antibody, whereas the in- hibition in experiments using lymphocytes from sero-negative donors was slight or non- existent. Although no significant correlation between the level of HI titre and the degree of inhibition could be obtained, the mean thymidine incorporation of rubella virus treated lymphocytes from donors with defi- nite serological immunity was significantly lower than that of lymphocytes from non- immune donors. The mechanism involved in the inhibition of the LA response of lympho- cytes induced by rubella virus is still uncertain. At least four explanations can be put forward:

1) T h e Rubella Virus Interferes Directly with the LA Stimulation Process

This alternative seems less likely since, as a rule, no inhibition was observed during the first three days of culture. Furthermore, there was no displacement of the dose response curve for LA, which might be expected if the virus for example interfered with the binding of LA to the lymphocytes.

2 ) Monocytes Are Znfected by Rubella Virus Monocytes augment the proliferative re-

sponse of PHA stimulated lymphocytes ( 13). Polio virus has been shown to infect mono- cytes in leukocyte cultures and thereby abolish the augmenting effect of these cells in PHA stimulation (25) . In the rubella system, how- ever, the viral inhibition of the PHA response of cultures with and without glass adherent cells has not been found to differ ( 3 1 ) ,

3) Rubella Vzrus Exerts a Cytotoxzr EffeLt

According to the present and other studieb ( 10, 19) , this alternative seems unlikely since any definite increase in cell death in the virus infected cultures could not be observed. On the other hand, rubella virus ha5 been shown to replicate i n PHA stimulated lymphocytes (3, 19), and it is possible that this viral repli- cation might affect the proliferation of the stimulated cells without rausing a direct cyto- toxic effect.

on the Lymphocyte5

4) A Produrt zs Synthesized zn the Culturcr which Inhzbzts t h e Proltfuratton of LA Stimulated Lyrnphoc ytey

Rubella virus infected cells synthesize poor- ly characterbed products which inhibit the growth of other cells (21 ) . One known inhibi- tor is interferon which inhibits the prolifera- tion of a number of cells, including PHA stimulated lymphocytes ( 14). I n lymphocyte rultures, interferon is produced by T-lympho- cytes following immune specific stimulation by viral antigens (6, 2 2 ) . A proportion of the cells might be expected to be stimulated by rubella virus to produce interferon, and thus, the amount of interferon would increase with time of culture. After some delay this would lead to an inhibition of the LA-induced pro- liferation. Another possible mechanism of the virus induced inhibition is that antibodies, which form inhibiting complexes with rubella virus (121, are produred in uztro after im- mune activation of the lymphocytes by rubella virus. Hoth these hypotheses would possibly explain the apparent correlation between the degree of LA response and serological immu- nity against rubella. However, other factors must also be operative sinre UV-inactivated virus, which does not inhibit the LA response, would also be able to induce both interferon and antibody production in uitro.

The authors want to thank Mrs. Ulla Strand and Miss Kristina Groth for skilful technical assistance, Stig Danielsson and Thomas Larsson, Department of Mathematics, Linkoping University, for assist-

ance with the statistics. This work was kindly sup- ported by grants from the Medical Faculty, Lin- koping University and the Medical Research Com- mittee of the County Council, Ostergotlands lans landsting.

I .

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

R E F E R E N C E S

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