rd 1&,f@$

CENTRE FOR NEWFOUNOUNO STUDlES

T87 #l. OF rd 1&,f@$ a N Y

~ R V $E XEUQXEE)

_ I I folmd foe o t h e r gcoupa. (v) many r e l a t i w a of, the p a t i e n t s showed immmoglobulin deficiencies of v a r i o u s grades and

L

t

^{2 ,}

-

.

7

t

1

d

L

I I 7. t h r e e geographically and g e n e t i c a l l y c l o s e

.

^J

$

Nedolmdland co&zties. thare'was an aggregate o f 19 cases

A

bf Hadgkms d i s e a s e , embryonic tunour, lymphosarcoma, ' '

-

leukaeofla. thymaw and immmodef=c~ency fn t h i s study. k

.

939 sera from c o m i t y members and c o n t r o l san@le"s from 1

-.

321 blaod donors and healthy children were t e s t e d f o r t h e i r concentrations O F i m m o g l o b u l i n s G A,

d

and D by imnuno-

\ \ L

d i f f u s i o n The r e s u l t s were submitted t o 9 \ m u l t i f a c t m i a l

a n a l y s i s of v a r i a h e a L ,

ences i n imunoglobulin con

i n c m a i l size and t h e mean e e n c a n t r a t i o ~ of the-4 immune- globulin a l a i e s . ( i i i ) t h e mean concentrations of IgG.

.

I g A and IgM were'elevared i n the' f i r s t and second degree r c h z i v e s or m e p a t t e n r s p a r t i c u l a r l y r e l a t i v e s o t chose

-- --

i/

with =embryonic tumilur, lymphosarcma, leukeemis and thynoma.

and of thoee w i t h i m m o d e f i d e n c y , and to a l e s s e r e x t e n t

1

1:

2.4

.

i n relatives o f p a t i e n t s w i t h Hodgkins d i s e a s e , ( i v ) the

1

r e l a t i v e s of p a t i e n t s d t i ) Aodgkins d i s e a s e had a s i g n i f i e a n t - l y elevated mean I@ l e v e l compared w i t h the mean IgD l e v e l s

" . _1-

-

. , \ _iii

typed i n c l u d i n g bne c a s e eacd of hypo~ammaglobqlinaerma zrnd i s o l a t e d IgA d e f i c i e n c y

Elevated immunoglobulins i n r e l a t i v e s of p a t i e n t s

.

^' witk l y r q h o r e t i c u l a r m l i g n s n e i e a and iomnmodeficienoies , m y r e s u l t from i n c r e a s e d a n t i g e n i c s t i n u l a t i o n of the i-e aydtem, perhaps by an i n f e c t i v e agent A s u b t l e farm o f imrmmodeficieney which p e r m i t s the e n t r y o f anrlgenr i n t o these, i n d i v h a l s more e a s i l y than i n h e a l t h y people, may be a p r e d i s p o s i n g f a c t o r I t i s also p o s s i b l e t h a t t h e c l o s e l y a s s o c i a t e d inrmmodefieieney land malignancy could

'

both r e s u l t from t h e same cause. The p e c u l i a r g e n e i c make u p of t h i s ~ m m m i t y ing r a i s e s t h e p o s s i b i l i t y of with an i n h e r i t e d d i n p o s i t + n a h i g h incidence of

\

i breed- t o

'

d0ch c o n d i t i o n s Contimued e x p o s m e of the e-mity to an

'

i n f a c c i v e a g e n t ( V i r u s ( e s ) ) +y l e a d t o r a l s e d immuno- g l o b u l i n l e v e l s i n m y people, and t o o v e r t d i s e a s e s as malignancy nr s e v e r e i m o d a f i e i e n c y in a few

dz

the f u n c t i o n a l s t a t e of the i m e

_--

system may b e i n h e r i t e d ,

,

I

i t i s l i k e l y t h a t t h b p r e d i s p o s i t i o n t o v i r u s c a r r i a g e i s

g e n e t i c a l l y determined.

I

IC i s smggeatcd t h a t b o r h g e n e t i c a n d environmental i

.

faeeora may b e c o n t r i b u t i n g significantly t o i m o p a t h o l o g y i n t h e s e o o m i t i e l

\i-,

^{~ -}

. . -

^{a w .A.}

^..

^{w'*,;< ." Y ' .',}

^,%'+.:

-- --

#--*

i v I ACaOWLEDGEHENTS

\-

I wiilh t o expresslmy s i n c e r e a t thanks and a p p r e o i a t i o p ' t o bath Dr W H Mershall. Prof6ssor of Imlmology, Memorial Zmiversity of Newfoundland and Professor B b Osunkoys. Head of Departrent of Chemical Pathology. University of Ibsdan,

Nigeria, for making the M Sc prograbrme p o s s i b l e

f

I waold l i k e t o express my indebtedness' t o Professor R K Chandra, or/ supervisor, and P r o f e s s o r W H Marshall whose combined m t i r i n e encouragement, suggestions, s u p p o r t . and c a r e f u l g e d a n c e contributed l a r g e l y t o the completion

I would a l s o l i k e to express my s i n c e r e appreciation t o Canadian I n t e r n a t i o n a l Developm~nt Agency f o r awarding m e a PellawshipLwithout which t h i s t h e s i s would have been an impos?iible undertaking. the University Medical Library, end t h e Audio Visual D e p e r m t for t h e i r unceaeing cooperation.

.

¹ am g r a t e f u l t o Dr David Brgant. Rofeaaor R

n.

Hwbrsy and Hr. Larry Chambers f o r s t a t i s t i c a l advice end help, Hr. lany C d e y f o r h i e h e l p i n the d a t a processing of my r e e u l t s , m Dr .Robert HandevLlle f o r w r i t i n s t h e

' s

progr-e f o r e s l c u l a t i o n s m t h e Kang 600 desk ealeulator.

and t o Professors J Podor, B Lawan, and G R. praser for t h e i r h e l p i n s e v e A l ways as members of my Bupermsary

%

caannfFCee - I

I em p a r t i c u l a r l y g r a t e f u l t o my w i f e for her p a t i e n c e

\

<

-4 i a,, i'

-' -- --- A-- --...*,.-..-*

I

. I

A

IgD and Igx

Methods which i n e l v d e d p o l y p e p t ~ d e chain s e p a r a t i o n by c y a t e i n e r e d u e t i o n followed by_sklylarion, and a p a l y s i s '

1

^{of N-te-nal} and C-terminal amino acids l e d P o r t e r (1963)

t o P ~ O P O S B a 4 c h a i n model f o r r a b b i t IgG. T h i s moBel

\ shoved two p e i r s of poLypeptide c h a i n s , one p a h . of long (heavy> sand rme pair of s h o r t ( l i g h t ) chains arranged spnmecrieally, t h e c h a i n s being l i q k e d c o v a l e n t l y by d i -

.

' s d l p h i d e bonds The =&rent model does n o t d i f f e r v e i y much Erom P o r t e r ' ? o r i g i n a l d e s c r i p t i o n except in t h e p o s i t i o n h p and t h e n m b e r of d i s u l p h i

and Edalman 1965 w d 1966),

It is g e n e r a l l y accepted t n e t e a e h ' i m d m o g l o b u l h i s e p r o t q i n w i t h the sane b a d e s t r u c t u r e as d e s c r i b e d f o r / IgG IgU f o r example, is r e l s t i v e i y e a s i l y broken down t o *

are b u i l t on t h e same g e n e r a h p s t t e r n as 180

o r 5 and =he Lambda o r h c h a i n s Each c l a s s or s u b c l a s s of

c- E

I . ^.

_kr.

' ^I

% -

--

- ,----

-.

,--- -".--<*- I

7

4 w e i l h t of abovt 22.000 and c o n s i s t s of about 21% amino a c i d resihues iPumam et a 1

.

¹⁹⁶⁶⁾

The hesvy chains d i f f e r from one e l s s s or subclass

i

^{* Go} thk o t h e r i n m i n o acid sequence ss w e l l as a few other

4

p r o p e ' r i e s The heavy c h a m of LgG and 'probably t h a t of '

i

IgA c tainli one v a r i a b l e region a t the amino t e r m m a l end and a o n s t w t p o r t i o n conteinmg t h r e e h o p ogy regions or domains a t t h e carbony1 t e r n i n a l end bf fhe polypeptide

P

\ (~delmdn and CunAinghaa 1969) That of -I4 and probably thaae of akD and IgE each c o n t a i n s one v a r i a b l e and four

' c m s z a n t r e g i o n domains (Putnam e t e l

.

^{1 73).}

Mydmlysi8 by t h e m y m e papain

dblrail

t h e I @ . IgD. IgE and subunits of IgA and IgPl i n t d three roughly equal p o r t l a n s (Bernier e t a 1

.

1965, Bennich and Johanoaon 1967) Tuo of t h e s e 3 f r a c t i o n s a r e i d e n t i b a l and each o f the m a c o n t a i n s the a n t i g e n combining s i t e , t h e s e e r e c a l l e d t h e Fab ('fragment antfgen b i n d i n g ' ) .

he

Pab produCed b y t h i s digestion is n m o v a l e n t Thoagh i t ii.

capable of combining with t h e antigen i t cadnot farm p r e c t p i t a t e s Tne t h i r d fragment c o n s i s t s of r v o hesvy chair!. of theaC terminal halves of two h e a q chains joined

'

by a diaulphide bond. It i s termed the Fc ( ' f r a @ = n t ' c r y s r a l l i z a b l e T ) because it r e a d l y c r y a t a l i i r e s i n water

b t h e cold. I t is knam t h t in man the .f matee- nel IgG through the p l a c e n t a t o t h e f o e t u s . t h e c o q l e m m c f i x i n g a c E i v i t y , and c y t o p h n i c a c t i v i t i e s including IgE

, I

-

^{Y E'}

---

^.

^--

_{\ I}

I

1 8 d i s t r i b u t i o n of IgA Van Mlmster e t a1 , (L965) r e p o r t e d i

i

t h a t IgA f o l l y a normal b e l l - s h a p e d ) frequency d i s t r l -

- 1 and 44 c a u c a s i a n s i n Denver (U S A ) reported a log normal

4

d i s t r i b u t i o n . Othez wrkrs have a l a o r e p o r t e d a log normal d i s t r i b u t i o n Tkeae i n c l u d e Johanseon (1967). Goldman e t a1

.

(1967). Liehtman s t a l . . (1967), 'Allansmith e t a l . . (1968) and Yeye (1973) A ~ t u d y o r i g i n a t i n g from Gambia a h w e d t h a t _% I& doea n o t f o l l o w * normal d i s t r i b u t i o n (Row and ~ c ~ r e g o ~ , ~ ~

1968) ' 9

_.

- T n - _

. -

I r appears t h a t (i) the l e v e l of 1 g d a t

,:

b i r t h i s v e r g low. ( i i ) t h e IgA i s manufactured in u t e r o by the foetus. ( i i i ) the level i n c r e a s e d e m r i n u o u s l y w i t h age throughout l i T e .

1

w .

^I

me I@ l e v e l f i e lo(. a t b i r t h I ~ M is s y n t h e s i z e d i n u t e r o b y thb i n i m t : a n d i s p r e s e n t i n a l l normal neonates - .+ ^(Van Purth et a l . . 1965) It i a always p r e s e n t i n a d e t e c t a b l e

J -.mt s t b q & C ~ v m < t h e t a 1

.

1965. F u l g i n i t i cc sl

.

¹⁹⁶⁶

and Stishm e r a l . . 1966). S e v e r a l s t u d i e s have s h a m t h a t the I @ p r e s e n t a t b i r t h i s manufactured by the f o e t u s The , dord blood c a n t s i n a l e s s than 1 0 p e r c e n t of maternal l e v e l of IgM

ROB^^:

1971). Ig* doea n o t cross t h e p l a c e n t a Cord r"

blood IgU. l e v e l s of =re than 20 mg. per 1 0 0 n l are i n d i c a t i v e

$

\ I

r

-

-3 +

w

^{--7-- -w4"}

>>?".

^{,..% .i,s..}

-.

J b

I 5'

.

²⁰

1,

t h a t the IgN l e v e l increases from b i r t h t o a c u t the heginning

.

of 2nd decade of l i f t Thay alao fomd t h a t IgM reaches maxirrmm l e v e l and msximra. v a r i a n c e also a t a b u t the 5th ' decade of l i f e In t h q i r r e s u l t s the l e v e l d e c l i n e s somhhat l s f t e r the 5 t h decade of lit=. The m e e t of the increase m

i ^'

IgM l e v e l i n childhood l a u a u a l l y e a r l i e r than that of 1gG but t h e y d h maximum l e v e l about the 2nd decade of l i f e West e t e l , 1962. Stiehm and Fudmberg, 1966),

However. not a l l workers agree. for example Allansmith e t e l

.

(1968) q u a n t i t a t e d i m m o g l o b u l i n s G , A and A i n sera '

+

ranging from c o r d blood t o a d u l t s and r e p o r t e d that'the a d u l t G

l e v e l of I @ i r reached a f t e r the 1 s t o r the 2nd year of l i f e mi^ i s much e a r l i e r than t h a t reported by 8uckley and oarsey (1970) Other workers have found qo a g e difeerences i n IgM

.4'

a f t e r the 5th y e a r o f Vife Cassidy er a 1

,

(1974) reported

$

^{no age} differcnoea i n IgG. IgA and IgM They reached these

B

conclusions a f t e r analysing 3213 samples aged 5-94 yeera.

Nozberg (1967) a l s o r e p o r t e d no ate d i f eerences i n t h e IgG.

IgA and IgM a f t e r analysing 370 samples from apparenrly h e a l t h y nubjeets aged 18-92 years

,

As with t h e o t h e t ilmnunoglobulins, t h e r e are s e v e r a l

a

repozts on the shape of the frequency d i s t r i b u t i o n of IgN some workers r e p o r t a normal frequency distribution (Van

b Z

N m s t e r a n d Stoelinga. 1965 end Crrynaraki. 1968) Kalff (1970) analysed imnmoglobulin cqnrents of 252 s u b j e c t s over 5 years of age from 3 v i l l a g e s i n the southern

-

"~*'%+.c-

-

^{- '}

^q-

%A-"VY @"&;& ,

..

ⁱ

i ' 4

-

- - - _- _--_ _

- - -

30

-1

a s s o c i a t i o n between h m o g l o b u l i n and the X chromosoms.

Thvs it appears t h a t I.@ i s usually higher i n female0 than i n melee This might b e because

-

of d i f - ferencea i n hsrmnral l e v e l s beoreen the two rexea a n d / o r

t

r

b

1 t h e X chromosome might probably carry q-tifative genes ror I ~ E I ea suggested by G-aachar. (1972a)

'

IKD and IRE

No sex differences have beGn a s s o c i a t e d w i t h IgD and IgE l e v e l s (Rare. Crabbe and mrner. 1968. Johananan.

1968b. Berg e t n l

.

^{1969) a}

A U T D S O U GENETIC FACmRS \

There i s abundant e'ridenco char aut6somal g e n e t i c factors are involved i n t h e e o n r r o l of l e v e l s of t h b d i f - ' I ierenf i m o g l o b u l i n a i n h e a r t h an4 disease Such evidence could h e found i n t h e work of R a r e . Boyle and Buchanan.

(1968) who s t u d i a d i m m o g l o b u l i n l=evels i n m o z y g o t i c and

I

d i z g g o t i c twins They o b s e m d more conaordanca i n IgC,

IgA and I@ l e v e l s i n mmozygous than i n dizygoua edo- 1 -

6

l e s e e n t w i n s (both males bnd3females) .\mongTd"lts t h e male mmozygotie twins had s i g n i 6 i c a n r l y smeller i n t a r t w i n d i f f e r e n c e s i n IgG l e v e l s than d i z y g o r i c o r i n s No diEfer-

t

^eoces ware observed i n 1gA o r ISM i n a d u l t N i n p d i r s The

5 ,

arudy provides suggestive evidence of g e n e t i c j e g u l a t i m of

~

I g b . IgA and IgH. but the e f f e c t g e t s masked during sdulc-

a * .

hood, e s p e c i a l l y i n the case ol - IgA and Ign Similar a f v d i e a

i

'

!

.*

⁺ e-y - n ---3

.-" .

4

s, I

--

^A

^-- --- -

I

/ 34

/

I

found t o v a r y from l e s s rhan 1t300 t o 1150 of normal ( S e l l

42

and Fahey. 1964. Paheg and S e l l . 1965) On t h e other hand

mice which are h y p a r i m q i n e d with haemacyanin o r r a i s e d i n

i

an environment w i t h high b a c t e r i a l c m t e n t aynthe8ize ioramo-

/

globulins of a l l c l a s s e s a t r a t e s which are<bout: 5-10 rimes

1

higher than are seen i n normal animals.

._ - _{1 '}

It would nee: l o g i c a l t o expect t h a t antibody for-

\.

mation a k inm+noglobulin l e v e l s would be g r e a t l y irbpaited

\

by tamt ti on. s t u d i e s m8de on prisoners of war and c a c h e t i c

i

^\ h ~ ~ ~ i t a l harever s h w e d ~ o l u i d e r a b l e d e p l e t i o n of 'I s e m albumin whereas t h e gemnaglobulins were n o y l (Humphrey

and White. 19711). Similarly m i l d r e n w i t h p r o t e i n - c a l o r i e

d

m s l n u t r i t i o n (P.C PI.) were f ~ m d t o have decreased albumin levhl<, b u r no s i g n i b c a n t l g la. l e v e l s of i m m o g l o b u l l n s r

.

_$

(Gomee e t a1

.

¹⁹⁵⁵⁾

In c h i l d r e n wirh Washiokm, t h e mean abeolute eon- c e n t r a t i o n s of t o t e l p r o t e i n s , albumins, oZ-globulins and

I) ,.

;

globulins were s i & i f i r s n t l y diminished, but =here were >

no a i @ ~ i E i c a n t changes i n t h e mean ga-globulim coneeq- rraeims Edozisn (1960).

Hovever t h e r e are a o m d h u d i e a whith have s h a m t h a t I

_ -

m a h u t r i t i o n - a f f e c t 3 the imnmoglabulin l e v e l s In s t u q e s i n EgyptiBn Ehlldren w i t h h a r h i o k o r . d i d n i a h e d l e v e l s Of

$

^{IgG: IgA and Ign} have been r e p o r t e d i n c h i l d r e n who had

t '

i

-

P

.

.z

,.

~ a ? ,z -L

- ".

- -

^*

:

-

--- \

^-

-

-* *m.---

".- -

-"?aawm m**>

- 3i

1 ^{, . , \ -} . + . $ ' ^{. _}

^{4 1}

i ~ o g l a b u l i n s ) However, m mast cases $n which d e h y d r s t ~ o n

$

IS a diominent feat-, such complicating f a c t o r s as mal-

l

^a n u t r i t i o n , \ diarrhoea add vomiting exert opposing influences

f 1

Elevation of anly.th7 IgM e l a s s of i m m o g l o b u l i n s was re:'

$

1 , p o r t e d i n cHildren wirh d i a r r h a e ~ (Raider. 1971) Also

;i

Waldmann. Wencie e t a l . . (1971) repoiaed e l e v a t e d IgA i h

4

~ n o l a r s i c diarrhoea p a t i e n t s , during the acute phase of the i l l n e s p . which increased f u r t h e r during convalescence. an

,

elevated IgM i n "on-choleraic and a Dore e l e v a t e d IgM than normal in e h o l e r a i b p a t i e n t s w i t h diarrhoea was a l s o noted

, .

educed blood volume can be caused by shock, burns,

I.

d i a b e d c a c i d o s i s . Addlsar'r disease, intes$inal o b a t r u e t i o n , i n t e s t i n a l f i s t u l 8 , p y l o r i s obstruction, r i g i d r e s t r i c t i o n of f l q i d ink&, h e a t exhaustion Each oE t h e s e can in-

!

Elpence ae- imrmnoglobblin. c o n e e n r ? a t i p . For example

4

4 " Ritrmann et a 1

.

(1913) repbrted t h e following i m m o g l o b n l i n p a t t e r p s i n thermal burns ' T h e I g c l e v e l dropped s i g n i f i -

j .

c a n t l y in.the f i r s t few dsya a f t e r t h a burna

,

- g r a d u a l l y rose reaching a 1-1 h f ~ h e r than

n$lt:ythe

" ' ^.

a d p l t s The IgA and IgM f e l l odly s l i g h t l y a few days a f t e r the burns and then rose u n t i 1 . t h e y reached v a l u e s h i g h e r than the normal b t h e r workers reported a marked decrease in'IgG l e v e l immediately a f t e r burns which very s l o v l y

1

^{returned m} n o m l . The deersasu, i n IgA &ld IgM f o l l d r t g

^{. ,}

_~ 1 t h e b y m s were n o t e i g n i f i c r m t , and returned to,~nqrmal l e v e l

mre quickly (Arturson et a 1 , 1969. Muater e t a l . . 1970)

8

5

<

I _

T

^{*if*,, :}

^, . - .,

^4'.

r .

.. --- . l - - I - - . - l ~ . ~

^{I --r_.w} t l - 4 -

44 ^$4

I

'

^*V

l i f e of IgD IS 2.8 d a y s and t h a t o f IgE i s 2 5 days (Solomon.

' rWaldmsnn and &ey. 1963. S o l m n end Tomasi. 1964).

m e r a t e s of synthesis however d i f f e r

fry

o n e

t

i n d i v i d u a l t o a n o t h e r f o r orher r e a s o n s such as d i f f e r e n t eErperieneeb w i t h i n f e c t i o n s In m a l a r i a , f d r example, t h e r e c o u l d b e up t o a 7-fold i n c r e a s e i n t h e s y n t h e t i c r a t e o f I g G (Cohen and HCGregor. 1963)

As regarda c a t a b o l ~ a m , p a r t of t h e catabolism 05 i m o g l o b u l i n tavee place i n t h e l i v e r (Cohen. Gordon e t e l . . 1962) Denamred imnnmoglobulins and. t h q e which have fo-r complexes v i r h a n t i g e n s a r e m s c probably taken up

!

a n d degraded b y the c e l l s of the r e d c u l o e n d o t h e l i a l syst%v (Benacerraf. S e b e s t y e n and Cooper. 1959). In ma. e n d mouse, i t h e f r a c t i o n a l c a t a b o l i c r a t e of IgG i s dizsmtly r e l a t e d co ifa aerum l e v e l (Fahey and Robinson, 1963, This c o u l d b e d u e to e feed-back mechanism Henee t h o s e w i r h hi'gh serum

I

c o n c a n t r a t i o n s of I g G due to i n f e c t i o n s or h y p e r i-nil- a t i a n u s u a l l y have i n c r e a s e d c a t q b o l i c rates o f 1gG (Humphrey e t a1 , 1961, Waldmsnn ec a l . , 1969), whereas some p a t i e n t s with hypogamma g l o b u l i n a e d a may have decieeeed c a t a b o l i c r a t e s (Waldmnn e t a1

,

The B e r m l e v e l . o f I& d 1gN however do n o t in-.

- C"'

f l u e n e e t h e i r c a t a b o l i c r a t e s For example t h e c a t a b o l i c r a t e of IgM i s the same i n nomal a u b j e c t s as i n hypo- g a m a g l o b u l i n a d c p a t i a n t s with reduced I@!. q d i n p a t i e n t s w i t h mscroglobulinaemia w i t h i d o r e s a e d IgU c o n c e n t r a t i o n s

than th& of IgC end IgM, i t i s r e p o r t e d co be s i m l l a r t o o r e l i g h r l y h i g h e r tHan t h a r of I g A (Earth e t a l . . 14641

i

Solomon and T o y s i . 1964).

- .

2 ^{!ASS PROM ll(B BODY}

Z

I m n o g l o b u l i n d e s t r u c t i o n occurs i n s i d e phagocytea

i

Which have engulfed b a c t e r i a and f o l a i g n p a r t i c l e s coated with t h e antibody) present i n t h e c e l l s of =he r e r i c u l o 9

-

y n d o t h e l i a l system. I m m o g l o b u l i n can a l s o be l a e t i n t o

t h e u r i n a v o r g a a t r o i n t e s r i n a l t r a c t . Loss through damaged glomeruli of che kidney i s s e l e c t i v e and s i g n i f i c a n t i n , ' p a t i e n t s with n e p h m i c sgndrom In t h e s e ceaes t h e rs-

l e t i v e l y small rmlecular Weight immunoglobulins l i k e IpC ate removed i n the u r i n e m u c l p o r e r a p i d l y than thoae wlch high m o l e c u l ~ r weight I l k & IgM (Jahachim ec a 1

.

¹⁹⁶⁴⁾

9 . .

^{Several atdies} u s i n g l a b e l l e d p r o t e i n s

-

have sh-

5

^{t h a t exceasfve} p m r e i n loan drough the s a s r r o i n t e a c i n a l

,

P 3 ' " a

- __ -.

-*

-

^{---A 7 -}

^-

7-4""

'q

1 .

('4

46

\

c r a f t i s one of t h e major f a c t o r s leading t o hypogam-

/ j

globulinnaada (including low i m m o g l o b u l i n s ) i n protein- 1 {$

k

l o s i n g enteropathy For example. Strober et e l . . (1967)

8,

reparred l o w insnunoglobulin l e v e l s o f a l l the major elasnea 6

i

^- i n 19 patience w i t h i n t e s t i n a l ly~nphangiecrssia EFFECTS OF GX A L L O W E S

/

^{h e} coocenrrltion; of c e r t a i n imunoglohulins notably I @ appear t o be influeneed by the conatant region genes

i

The evidence for t h i s i s an a s s o c i a t i o n of concentration

I

diffegencea wlrh d i f f e r e n t a l l o r y p i c markers (Gm a l l o t y p e s ) .

/'

These markers are present i n t h e constant region of t h e 7 (IgC heavy) chains I n IgC3 svbclaas f o r example homazygauo Cm(b) i n d i v i d u a l s have a i e n i f i c e n t l y higher l e v e l s of Ig03 6 than do peraone who are homzygoue m ( g ) (Yount ef 81 , 1967)

I n t h e case of IgCZ, homozygovl Gm(e) i n d i v i d u a l s

.

>,

have lower l e v e l s of IgC2 than do people who are hombrygous Dm(*) The type of Gm f l l o t y p e i n IgGl is e l s o found to i n h u e n e e t h e IgCl subclass concentration ( L i M n and Balaban. 1972)

Preliminary findings by Van Loghem. (1971) *on ^the

*

IgG4 subclass a l s o i n d i c a t e s a m k e d e f f e c t of homrygous a l l o t y p e of Igc2 on the leva1 of IgG4

Whether t h e a c t u a l Gmmarker i s responeible for the e f f a c r s o r wherher it simply a c t s as r marker For a p a r t i c u l a r eamplex of 0 region s t r u c t u r a l gwes i s n o t c l e a r However

>

.

^.*

,--

_,

. -

^\

-

.

64

I

a n t i IgM (Behci-rke Batch No.

2716A)

(j) W H.0. Reference Preparation No.97167 (Lauaanna).

@) B r i t i s h Research Standard Solution No., 67/37 (W.A.O.

Reference Centre Lauseme). ,

(1) IgD p l a t e s Behringverke Batch N m b e r s 3014 end 3066.

(m> Behringverke IgD Standard Batch No. 674A.

(p) Magneoie S t i q r e r ( o m g n ~ ~ t i r , Labline m s t m e n t s Inc.. Chicago, I l l T

i n a drying r a c K

(9) Coated Photo raohif l a a s l a r e s

-

^{8 . 2 X 11) 2 m}

pharogrsphicDglass DEaefman Kodak Co,,

Rochester ~ e v York) were bCoiled In rarer m t i l ell the phocoiraphic emulaion was removed. The p l a t e s were allowed ra cool. They.were waslred m t a p water

and rlnaed s e v e r a l times i n d x s r i l l e d water. men

.

W; ; ~ e ; ~ ; e w ~ ~ n ; ; a ~ d b ~ ~ e ~ ; ~ u ~ ~ ~ ; ~ ;

;;;:-

scope s l i d e s .

.

,

(r) Determination of ~ o n o a p e c i i i c i t v of Anciaera

-

^{~mnulo- '}

e eccrophoreees were ruo with each test anti-serum A a i n s r whole human serum. The s i n g l e r e c i i t = n arcs produced (Figs. 6 ) aha* that

eat!

^aptfee-

i e monoepeoiiic.

I h s method i s a a l i g h t modlflcqtion of t h e s i l l g l e

$$ r a d i a l d i f h a l o n technique

9f

Fahey a n d McRelrey (1965).

$

h

.

.

b.' Y

1

,

\ l a t t e r 6-10 knvinversione of t h e tube were routineky made in,

. r

^'

I ^.~

. , o r d e r to m f x t h e r e a g e n d . Farnation of a i r bubbles was ! , t o t a l l y a v o ~ d e d as €hey i n r e r f e r e with t h e p r & i p i t i n r i n g s .

- -

A series of Wells was cut i n the ag& p l a t e with

.

¹

~.

-\ ¹ fie metal at 12%- bemeen centres for t h e l a c ,

. i

IgA and IgM. Agar was c a r e f u l l y removed from w 4 l s with a -smooch edged

. .

penteur p i p e t t e a t t a c h e d to a vecuum plimp.

' c 4 i n p care nor: t o dameee t h e 'sides of the well. The p l a t e s

* e r e now ready f o r use..

. . .

. ,

Use 6f the P l a t e s r

-

e me welie were each f ~ i i s d with a measured w-e'

.

,

:

(see' l a t e r ) of t e s t o r sianderd serum using e Hemiltan Acre- ,

\ .

l i c r e ' s y i n g e which wad r i n e e d rhreeLtimes m s a l i n e ' i n

bemeen s~amples. TheTheThe'platea were placed i n humid boxes The

+ i

IgC p l a t e s we& pleeed i n e 37'C incubator v h i l e t t h e IgA and I @ p l a t e s were placed i n Gw r a f r i g e r a t o r a t 4-6. The ti%=

. o f incubation was decernined by p t e l L i k a r y t a s t e which a r e

.

^' d e r a i l e d I s e e r

The diameters of rhe p r e c i p i r i n r i n g s were measured i n ,two dirLctiqna et rikht.angles to the nearest b.1 m using

f

. .

_, a ~ y l & d viewer wiah I micromerdr eye piece (Figs 7-10>

a For permanent keepiny, rhe p?ates were s r a i n e d ea follows The p&tes were placed i n normal s a l i n e . n ~ e e a z i n e was chan&J a t l e a s h 4 f i d a 6/24 hadrs me p l a t e s

'

wow then placed i n t a p i t e r . me ^water was changed M c e i n c3 hrs. Platel, werearemoved ad: wet P i l t e r paper ?as.

. ^,.

^{a *}

d

Y

-

.-,car .is*.<

.,.

^--z=

77 applied m the a g a r surface. l h e y w e r e ' l e f t on the bench with t h e f i i t e r pap& on t M for a day o r @do u n t i l dm.

me f i l t e ; papers were removed.' P l a t e s were then stained i n Pmcesu S f o r -20 m h a . Excess s e a & was washed o f f in s e v e r a l changes of 5 percent a c e t i c acid. Wet f i l t e r pap+ra were applied on t h e surface of the p l a t e s The p l a t e a ' v e r e l e f t on the bench Overnight t o dry.

IID Estimations

Serum IgD l e v e l s were measured using connnercially

,

, p z e p a r e d m m 0 5 p e ~ i f i c IgD p l a t e ( a n d standards which were

.

a t o r e d a t 4'C. Before use, each p l a c e w a s opened and l e f t a t rmrn tempereture f 4 T 5-10 mine. Ihe IgD atandard conr t s i n i n g 2 0 8 I n t e r n a t i o n a l Units of IgD p e r 111 wall d i l u t e d

. ^-

t ^-

^{1 i n 2. 1 i n 6} a n d ' l in 8 t o give 1 0 4 u n i t s , 52 u n i t s , and y. 20 m i e r o l i t r e s (0.02 ml) of

P

e ~ l u t i o n was plneed in e a c h of

F-, the.wl'ls using e B m i l t o n mirso-syringe. Wells 1, 4. 8 and '11 o f /he f i r s t p l a t e of every b a t c h of e s t i m a t i o n s were

.

I

f i l l e d w i t h the standards. Each of the o t h e r p l e t e a con- I

mina$ a t l e a s t one B e h r i a p e r k e s t a n d a r d

solution.

I n i

closed t i g h t l y and allorjed t o d i f f u s e f m 3 days s t room temperaturo (Pig.11). ^{l h e} d i m t e r s of t h e p r e c i p i t i n r i y s

*",

h

; were meaaured i n hso d i r e c t i o n s s t r i g h t angles to t h e

f

-

-. , . -

^k

'.

I

' .

.

^,

people) : (5)' Nun-descendant# -of thi fm'der couple uho lid

:

arien.age g r o u p s .Omup 1 (1-5

~ r m p 3 '(10-14 yea..). Group 4 ((15-19 y a a t s ) . ~ r & p .5 ,

I I (20-36 g a p i s ) . Group 6 (37-52 y e a r s ) ; and Group 2 (53-7qyear8) '

j \ he msil p u p s rere as follws : (1)

I"

,

*

%

,

"

r

⁴

' . .

i

.*

"

a

15

6Xut'',*ii, , ,,

* ,

,

^{: P}

(2) Vestig*al. (3) Nornal, (4) Enlarged (5) T o n s i l l e e t o w . People with d a t a on t d i l s i z e nmbered 1049 and a l l of them come from t h e t h r e e study commnities None of the controls had been examined i n t h i s way

It was considered thar-e Multiple E a c t o t i s l Analysis of variance would be approprxate f o r the Lnvestigacion of >

the data AB a r a t a d i n t h e Vethodso Seotioa. rho data was 2

*

_prepared f o r a n a l y s i s by computer using'the S P 6 S s r a e i e t i c a l package (Kim w d Kohout. 1975) It was decided

('

t o undertAe t h r e e s e p a r a t e anelyses (19 h a l y s i a of variance

'

o f four major c l a s s e s of imonmoglobulins (IgG, IgA. I@ end

'

IgD) by s e h age and +e c l i n i c a l geauplngs: ( 2 ) Analyaia of , variance of t h e s e immmoglolbubina b) eex, age and t o n s i l

s i z a e . me second a n a l y s i s was e a r r i a d o u t only f o r the

.

a ~ i t y ' p o p u l a t i m as t o n s i l rizea were n o t measured i n

?

L k k l o o d donors and S r John's School children. This ap- _,

i

pmach of c a w i n g out Wo sepAt: analyses i n s t e a d of e combined one was basawe t h e l a t t e r process &mended such a large amormt of computer s t o r a g e space and C.P.U. (Central P ~ o c e s s i n g Unit) time t h a t t h e cost would have been prohibi- t i v e . (3) Calculate Hew t ZSD of IgG. I & andlIgM of Eon- t r o l s from othei- p a r t s of Nwrfoundlend, and cam par^ t h e

number of abnormal i n d i v i d u a l s I

HJL.TIEACTDRIAL lWLUYSIS

M u l t i f e c t o r i a l analysin of variance &#E@ lag transformation) between f i d i aub-groups of the popvlation

,

;

(groups 1. 2 . 3, 5 and 6) end the e o n g o l datb from else-

'

where (gmup 4) were p e r f ~ r m e d This w a l y s i s of variant\

' ^I

I

,

I

~. .

^,

.

----"--.-3m--

----."---.. "?A ^.--*-.

1

i i .

9

:?I 1 1

,I

-

I14

'k

and 2nd degree r e l a t i v e s of Hodgkio's diseaa /petiencs) end

4

group 5 (comnnmitp members n o t i n John and Mary pydigree).

7

IhC 3 c o n t r o l groups a r e similar t o each 0 t h r me group

1

3 r e l a t i v e s of p a t i e n t s (immunodeficisncy. leukaemia' and

I.

).

t h m m ) had the h i g h e s t Heatf I @ levala whereas gioup 1 , r e l a t i v e s (Rodgkim's d i s e t s e p a t i e n t s % d i r e c t l i n e

daseendsnts of John and Nary) hsh* s l i g h t l y l o v e r values than e i t h e r group 3 o r gmup 2 I n the c o n t r o l groups, che

.

higheet man IgA onlcentrsciona are found i n group 5 (cm- i m i q members n o t i n John and Hary pedigree). whilat group 4 (contmla from elsewhere) h a w t h e lowest

With r i g a r d s to the two way i n t e r a c t i o n s , t h e r e axe

'

*

s i g n i f i c a n t two way i n t e r a c t i o n s b e m e n age and c l i n i c a l gtouplr (Table 13). This uhauld be analysed i n d e t a i l i n 4 f u t u r e work

The amount of the t o t a l v a r i a t i o n s in IgA which Can

'

I

be adcounted f o r by sex, age q d c l i n i c e l groups i n t h i s malyeia i s 19.6 percent

Fox IgA t h e r e are age r e l e e d dtffereneee me r e l a t i v e s of p a t i e n t s showed s i g n i f i c a n t l y higher I& than t h e pebple i n t h e t h r e e c o n t r o l groups.

2 IGA AN0 TONSIL SIZE I

1

The a n a l y s i s ( T a b l ~ 15) t h a t there ere s t a t i s - , f i c a l differences in the Mean IgA l e v e l s i n r e l e c i - m - t ~

-

r

-

^{1 ,} * .y.*

.

^{1 :,r*>.}

^, _t

" '

7;?*iff\-*-,,cnxnrxr,ir.a*~-~%

!

J 125

p a t i e n t s ' r e l a t i v e s ) . w h i l s t group 12 i s only d i f f e r e n t from controls from elsewhere (gfoup 4) Gmup 1 had the higheat Nean IgD concentrations w h i l s t group 4 had the lowest (Tabla 22)

. ^me

^amount of the t o t a l v a r i a t i o n i n IgD which could

be accormted f o r by sex. age end clmnicel groupings La 10 6

:

percent

,

I n Svnmurry I

m e r e were no differences due t o sex and vary l i t t l e 5 difrerences due to age i n the IgD concentrations me r e l a - t i y e s of p a t i e n t s i n gmups 1 and 2 had s i g n i f i c a n t l y elevated IgD concentrationk

IGD AND TQNSIL SIZB

\

.

The mean

t

ZSD of IgG. IgA and IgM of blood donors and _, 7 y

-

of Newfoundland) were calculated a f t e r log transformation on

s Wulg 600 desk c a l c u l a t o r (Table 222

Individvala whose im-@obulin eoncentrati&s were I

outside the mean

+

250 f o r t h e i r age group and men i n the study ~ p u l a u o n -a w e l l as i n the cbotzals £ram e l s w h s r e were manually s o r t e d put and are dh- i n Table 26 Tables _,

*; ^> 27 and 28a, b and e oHov t h a t result; from variova @sups are ^'

s i g n i f i c a n t l y d i f f e r e n t B a n a t i o n of t h e Tables s h a r e '

y -73 >-.

- ,-*, --

*",%\7w.w

~11,1(*~~~w+mw

T

3

4

' 1

DISCUSSION

The comnuni%les s t u d i e d i n t h i s work are g e n e t i c a l l y a n d geographically i s o l a t e d me increased ineideilce of

$ m e m a r r i a g e b e m e e n ~ L o a e r e l a t i v e s (Pigs. 2

-

^{4) coupled}

w i t n t h e findings o f l g h occurrence o f lymphoreticulsr malignancies a n d ilmounodeficiency make thia an i n t e r e s t i n g c o m i t y t o study i m m o l o g i c a l l y

This stud). h a s shown t h a t i n t h e extended f a d l y of about 1000 people e r m i n e d , the r e l a t i v e s of p a t i e n t s w i t h embryonic tumour, lymphoaarc-, immmodeficiensy, leukaemia and thymom, had s i e n i f i c a n t l y e l e v a t e d mean eancentrationa o f IgG. IgA a n d I@<

'

The r e l a t i v e s o f p a t i e n t s w i t h

.;

^v

.

Hodgkins diseaae showed a s i m i l a r thovgh l e s s pronomeed trend

The r e l n r i v e s of p a t i e n t s with Hodgkills d i s e a s e shpwed rnarbedly elevated mean e e m IgD l e v e l s , w h i l s c r e l a t i v e s of chose with o t h e r t-urs and i m m o d e f i c i e n c g shoved a d l d elevation %ere are n o p r e d o u s published r e p o r t s on IgD measurement8 i n such f a m i l i e s

Other wo~kera have examined s e r m immunoglobulin levels i n f a m i l i e s o f p a t i e o t e s u f f e r i n g f r w l ' m h o r e t i c u l a r malignancies. For exsmp1a-i T i l l e t a l . , (1975) in t h e i r ' e t u d i e s of close r e l 8 t l v e s of 6 c h i l d r e n w i t h a c u t e l e a a e m i a found afgnificanc elqvacion of I g A i n a l l the f a t h e r s . In addition. 2 of t h e f a t h e r s had higher anU one

.-"

^{r l l}

:

^-.r

*- -"-.-? .\.--..,---

~ . ? - . . - - ~ w - - W - ' . ~

:r 4

143

4

I '

.

m u l t i p l e l y m p h o r e t i c v l a r malignancies i n c l u d i n g Xodgkina d i s e a s e a n d f o m d t h a t 3 o f t h e 9 r e l a t i v e s o f the p a t i e n t w i t h Hodgkins d i s e s n e had e l e v a t e d IgM One h a d a mono-

4

c l o n a l IgM s p i k e . 1

1

4

q . Z o r h a l l a - H s l l i o s and S u t t o n (1974) f w d e l e v a t e d E-8 v i m antibody o f the Igll c l a s e in t h e mothers and

/

s i b l i n g s of c h i l d r e n with acute l e u k a e m ~ a ,

P F ~ Y ~ O U S s t u d i e s have been mainly confined t o

"i'

r e l a t i v e s bf p a t i e n t s w i t h a c u t e lenkeemia I n mst rreporta.

a small nunber of i n d i v i d u a l e have been examined Except f o r Fraumeni's (1975) r e p o r t on r e l a t i v e s of one p a t i e n t .

I

t h e r e i s no documentation oi i m l m o g l o b u l i n l e v e l s i n

,

, r e l a t i v e s of Hodgkina'diseaae p a t i e n t a Thus the p r e s e n t s t u d y which i n c l u d e s data on 264 f i r s t a n d second degree r e l a t i v e s of I? pat en s w i t h i m m o p a t h o l o g i c a l d i s e a s e s

f

(183 are r e l a t i v e s

+-\t:

of 7 Aodgkins d i s e a s e p a t i e n t s ) w i t h i t s

i r

b u i l t m i n t e r p a l c o n t r o l p o p u l a t i o n o f 675 people ftam t h e same commmlt$ i s unique i n many r e s p e c t s This i s t h e l a r g e s t n d e d of p a t i e n c s w i t h HodgHns d i s e a s e r e l a r i v e s

Y '

and matched d n t r o l s r e p o r t e d i n gny mingle s t u d y t o date.

The d a t a share e f a m i l i a l p a t t e r n i n the occurrence of

< Sodgkino d i s e a s e , o t h e r malignancies end h m o d e f i c i e n c y . A most s t r i k i n g f i n d i n g i n t h e Rodgkina l s e a n e r e l a t i v e s i s t h e e l e v a t i o n of che IgD However. even though i t i s a l e r h l t a d i n d i p h t h e r i s and t e t a n u s i n f e c t i o n s (see l f t e r s t u r e r e d & ) , t h e pnehaphysiolog%c r o l e o f t h i e

r

,..".

^{?m*w-c,-=mm--m-q-}

*v*,*+l~*,w=&+.

t 1 4 4

/ i m m o g l o b u l i n i s n o t established.

1 Apart from these findings, sex r e l a t e d differences were found i n the l e v e l s of IgG and Igtl, t h e females having higher mean concentrations than +he males Such se* r e l a t e d dfffereneee. possibly dug to hormonal v a r i a t i o n s , are ex-

!

^I

petted and httve been reported by o t h e r s (see l i t e r a t u r e

review) ',

Age r e l a t e d differences i n t h e Ig6, I&. Igll and IgD concentrations found 111 t h i s study alp6 e o r r e l a t e ' w t h ths f i n d i n g s of o t h e r workers (see l i t e r a t u r e review).

Analysis o f the data f o r a p o s s i b l e relationehip of immunoglobulin concentration8 and t o n s i l l a r s i r e f e i l a

\ :

t o r e v e a l s i g n i f i c a n t c o r r e l a t i o n when examined by a

.

stringent s t a t i s t i c a l rest--Scheffe's S - t e a t .

In t h e s t u d i e s by Omoven end s o o t h i l l (1973) lare:

1gA concentrations were fomd i n chlldren vndergoine tansillettomy f o r recurrent t h r o a t i n f e c t i o n s than i n

.

control children They reported an a s s o c i a t i o n between the i m m o l o g i c a l findings and incidence of i n f e c t i w s a f t e r r operations but n o t before Por example, they Eound t h a t the p a t i e n t s ' IgA eoncentrations were not r e l a t e d to t h e incidence of sore t h r o a t s i n t h e previous 6 months before tonsillectomy I n the present study alao, t h e lowest mean

I I$ c a c s n t r a t i m was fmhd i n the group who had mdergona

tonsilleetomy. Hwever, studiea by V e l t c i e t a 1

.

^(1972)~

shared etevaced IgG and IgM but no-1 IgA im p a t i e n t s w t t h

Pl z "

,.*,