238
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The Protection of Mice Against Experimental Infection by Means of I m m u n i z a t i o n with Enriched Membrane Fraction of Salmonella typhimurium T h e r e is a s u b s t a n t i a l difference of o p i n i o n in ttte literature concerning the protection that can be obtained f r o m v a c c i n e s c o n s t i t u t e d b y n o n - l i v i n g g e r m s or f r o m fractions extracted from them, agreement being achieved o n l y r e g a r d i n g v a c c i n e s c o n s t i t u t e d of l i v i n g g e r m s w i t h r e d u c e d virulence. I n a p r e v i o u s paper, we r e p o r t e d d a t a o n t h e p r o d u c t i o n of a n t i b o d i e s w h i c h i n h i b i t in v i t r o t h e g r o w t h of Micrococcus lysodeikticus in r a b b i t s i m m u n i z e d w i t h p u r i f i e d b a c t e r i a l m e m b r a n e s 1. I n t h e p r e s e n t s t u d y , t h e i m m u n o g e n i c i t y of l i v i n g pathogenic germs and their enriched membranes fractions h a s b e e n c o m p a r a t i v e l y i n v e s t i g a t e d in o r d e r to e s t i m a t e t h e i r r e s p e c t i v e p r o t e c t i n g effects a g a i n s t infection. Salmonella typhimurium 74 N C T C g r o w n in n u t r i e n t b r o t h Difco a t 37 ~ w a s used. T h i s s t r a i n g a v e a n LDa0 of 5 • 10~ w h e n i n j e c t e d s.c. ill mice. M e m b r a n e s were p r e p a r e d b y t h e SCHNAITMAN m e t h o d S f r o m c u l t u r e s in t h e tog g r o w t h p h a s e w a s h e d 3 t i m e s i n saline. Succinic d e h y d r o g e n a s e a n d lactic d e h y d r o g e n a s e d e t e r m i n a t i o n was used as i n d i c a t i o n of m e m b r a n e isolationK T h e m e m b r a n e s f r a c t i o n i s o l a t e d b y t h i s m e t h o d were cont a m i n a t e d w i t h cell-wall l i p o p o l y s a c c h a r i d e s . Salmonella-free CD-1 mice w e i g h i n g a b o u t 15 g were used. T h e t e s t s were r e p e a t e d 10 t i m e s o n 4 g r o u p s of 20 mice. A n i m a l s of t h e first g r o u p received e v e r y w e e k 0.25, 0.50 a n d 1.0 m g of l y o p h i l i z e d m e m b r a n e s . Tile first i n o c u l a t i o n was g i v e n s,c. in c o m p J e t e F r e u n d a d j u v a n t (CFA). T h e o t h e r i n o c u l a t i o n s were m a d e i.m. in saline. T h e second g r o u p was i m m u n i z e d w i t h cell lipopolys a c c h a r i d e s p r e p a r e d as d e s c r i b e d b y WESTPHAL et al. ~, a c c o r d i n g t o t h e s a m e s c h e d u t e as t h e firs4c group. The" t h i r d g r o u p of a n i m a l s was i m m u n i z e d w i t h l i v i n g ceils.
Two a l i q u o t s of 5 x 103 cells in 0.5 saline were i n o c u l a t e d a t 15 d a y i n t e r v a l s . M o r t a l i t y was a p p r o x i m a t e l y 2 5 % so t h a t i m m u n i z e d mice were s u r v i v o r s of a m i n i m a l l y v i r u l e n t infection. A n i m a l s i n o c u l a t e d w i t h s a l i n e a l o n e in C F A a n d k e p t u n d e r t h e s a m e c o n d i t i o n s were u s e d as control. A week a f t e r t h e l a s t i n o c u l a t i o n , t h e m i c e were t e s t e d for t h e p r e s e n c e of a n t i m e m b r a n e f r a c t i o n a n d anti-0 antibodies. A n i m a l s of t h e 3 g r o u p s t e s t e d p r e s e n t e d a n t i b o d i e s r e a c t i n g b o t h w i t h cells in 0 p h a s e a n d w i t h m e m b r a n e fraction. T i t e r s were 1 : 320 for b o t h a n t i b o d y types, i n t h e a n i m a l s of t h e first g r o u p ; 1 : 80 for a n t i m e m b r a n e f r a c t i o n a n d 1:1280 for allti-0 in t h e a n i m a l s of t h e second g r o u p ; 1:1280 or h i g h e r for b o t h a n t i - m e m b r a n e a n d allti-0 in t h e a n i m a l s of t h e t h i r d group. T h e i m m u n i t y of mice was c h a l l a n g e d i n v i v o 'by s.c. i n o c u l a t i o n of 100 LDs0 of Salmonella; t h i s dose killed all t h e c o n t r o l mice w i t h i n 10 days. M o r t a l i t y was followed for 30 d a y s a n d p r o t e c t i o n w a s m e a s u r e d as p e r c e n t a g e of survival. S u r v i v a l was 70~ 4- 6 for mice t r e a t e d w i t h m e m b r a n e s u s p e n s i o n 7 4 % 4- 6 for a n i m a l s t r e a t e d w i t h live o r g a n isms a n d 40~ 4- 5 for a n i m a l s t r e a t e d w i t h L P S . Statist i c a l a n a l y s i s of t h e r e s u l t s o b t a i n e d f r o m t h e 10 t e s t s p e r f o r m e d for e a c h oI t h e 4 g r o u p s of a n i m a l s s h o w e d n o s i g n i f i c a n t difference b e t w e e n t h e t r e a t m e n t s ( P v a l u e b a s e d o n Z 2 t e s t < 0.05). R e s u l t s show t h a t p r o t e c t i v e i m m u n i t y of a p p r o x i m a t e ly t h e s a m e o r d e r is s t i m u l a t e d b y l i v i n g m i c r o o r g a n i s m or b y m e m b r a n e f r a c t i o n o b t a i n e d f r o m t h e m .
Riassunto. T o p i i m m u n i z z a t i con m e m b r a n e isolate d a Salmonella typhimurium p r e s e n t a n o u n a r e s i s t e n z a alia infezione s p e r i m e n t a l e n g u a l e a quella d i m o s t r a t a d a t o p i i m m u n i z z a t i con Salmonelle v i v e n t i .
1 F. GALDIERO, Zbl. Bakt. Hyg., A 21g, 449 (1972). 2 C. A.. SCI-tNAITMAN,J. Baet. 104, 890 (1970). a C. A. SCHNAIT~IAN,J. Cell Biol. 38, 158 (1968). O. WESCPHAL, O. LODERITZ and F. BISrER, Z. Naturforsch. 7b, 148 (1952).
F. GALDIERO a n d CATERINA ~:~OMANO
Istituto di Microbiologia dell' Universit&, Sant'Andrea delle Dame 2, 1-80138 Napoli (Italy), 24 September 1974.
Local Monoclonal I m m u n o g l o b u l i n Production in Cancer Patient P r e s e n c e of M - c o m p o n e n t in s e r u m of c a n c e r p a t i e n t s has been described with prevalence in those bearing g a s t r o i n t e s t i n a l (GI), b l a d d e r a n d l u n g cancers1, ~. T h e r e is n o d i s t i n c t l i n k b e t w e e n t h e p r e s e n c e of M - c o m p o n e n t a n d t h e t y p e of cancer. T h e o c c u r a n c e of t h e f o r m e r is a p p a r e n t l y s i m i l a r t o t h a t o b s e r v e d in t h e n o r m a l p o p u l a t i o n 3. "While s e a r c h i n g i m m u n o f l u o r e s c e n t s t a i n e d sections of r e g i o n a l l y m p h n o d e s of 14 colon c a n c e r p a t i e n t s , we h a v e o b s e r v e d t h e p r e s e n c e of n e a r l y 3 t i m e s as m a n y i m m u n o g l o b u l i n b e a r i n g p l a s m a cells in n o d e s f r o m c a n c e r p a t i e n t s as c o m p a r e d to c o n t r o l n o d e s o b t a i n e d f r o m t h e v i c i n i t y of g a s t r i c or d u o d e n a l ulcers. A m o n g t h e immuoglobulins produced, IgM predominateda. I n one of t h e cases e x a m i n e d , large c l u m p s of p l a s m a cells s h o w e d a p a t t e r n of m o n o c l o n a I i m m u n o g l o b u l i n G (IgG) p r o d u c t i o n . T h e afore m e n t i o n e d case was a 68y e a r - o l d male, w i t h r e c t a l a d e n o c a r c i n o m a w h o s e tissues
b e c a m e a v a i l a b l e a i t e r surgery. T u m o r a d j a c e n t m u c o s a h a d n o signs of i n f l a m m a t i o n . P l a s m a cells were i d e n t i f i e d in t h e t m n o r itself, s u r r o u n d i n g m u c o s a a n d r e g i o n a l l y m p h n o d e s b y m e a n s of a b a t t e r y of m o n o s p e c i f i c i m m u n o f l u o r e s c e n t r e a g e n t s . T h e r e l a t i v e q u a n t i t y of p l a s m a cells was d e t e r m i n e d b y m e a n s of cellular d e n s i t y i n d e x (CDI), i.e., t h e m e a n n u m b e r of cells in tile field of v i s i o n u n d e r h i g h p o w e r of m i c r o s c o p e - • 480. T h e C D I for t h e r e g i o n a l l y m p h n o d e s w a s v e r y h i g h (8.92) for I g G p r o d u c i n g cells w h i c h c o n s t i t u t e d 9 0 % of t h e t o t a l p l a s m a cell n u m b e r . T h e r e m a i n i n g 10~o c o n s i s t e d of lgA, IglVI a n d I g E p r o d u c i n g cells (Table). I n t h e case
t E. F. OSS~R~aANand K. TAKATSUKI, Medicine ,12, 357 (1963). 2 A. TALERMANand W. G. HAIffE, Br. J. Cancer 27, 276 (1973). a M. K. G6RNY and J. ZEROMSKI, to be published. 4 E, A. JoNEs, Gut 13, 825 (1972).
15.2. 1975
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239
Examination of the number of plasma ceils in lymph node and immunoglobulin levels in patient's serum Ig
CDI ~in lymph-node
Serum immunoglobulin Ievels in InternationaI Units
Examined case
13 cases of other colon cancer (mean values)
Examined case
Blood donors values
IgG IgA IgM IgE
8.92 0.47 0.10 0.33
0.43 0.45 0.90 0.06
88 68 206 n.t.
149 • 43 139 4- 51 152 4- 48 n.t.
'K: L ratio
31:1
2.5:1
n.t.
n.t.
CDI, cell density index; n.t., not tested.
d e s c r i b e d t h e k a p p a : l a m b d a q i g h t c h a i n r a t i o was 31:1, while in o t h e r c a n c e r l y m p h n o d e s it did n o t e x c e e d 2.5 : 1. L o c a l i z a t i o n of k a p p a specific cells (Figure) c o r r e s p o n d e d closely t o t h o s e of IgG a n d s t r o n g l y f a v o r e d t h e v i e w t h a t t h e s e IgG p r o d u c i n g cells arose f r o m single clone. T h e l y m p h n o d e c o n t a i n e d a s m a l l m e t a s t a t i c loci, w h i c h did n o t c h a n g e its s t r u c t u r e . G e r m i n a l c e n t r e s of l y m p h a t i c n o d u l e s w e r e v e r y few a n d inconspicious. T h e p a t i e n t d i s p l a y e d h y p o p r o t e i n e m i a (total p r o t e i n 3,4 g/100 ml). Q u a n t i t a t i v e s e r u m i m m u n o g l o b u l i n d e t e r m i n a t i o n s r e v e a l e d a decrease of I g G a n d I g A a n d a n increase of IgM (Table). B o t h , p a p e r e l e c t r o p b o r e s i s a n d i m m u n o e l e c t r o p h o r e s i s did n o t s h o w m o n o c l o n a l p r o t e i n in s e r u m .
These d a t a s h o w t h a t regional l y m p h n o d e s in cancer m a y b e a site of m o n o c l o n a l I g s y n t h e s i s a n d t h a t t h i s p h e n o m e n o n m a y n o t be d e t e c t a b l e in t h e p a t i e n t ' s serum. The o b s e r v e d h y p o p r o t e i n e m i a could be due t o t h e loss of p r o t e i n via t h e GI t r a c t , a p o s s i b i l i t y s u g g e s t e d for cancer of t h e GI t r a c t b y Jo~;Es *. T h e failure t o o b s e r v e a m o n o c l o n a l spike in t h e s e r u m c a n n o t be e x p l a i n e d a t t h i s t i m e b u t m a y b e due t o t h e a m o u n t of I g G p r o d u c e d a n d t h e f a c t t h a t it is p r o d u c e d focally. T h e cause of local m o n o c l o n a l i m m u n o g l o b u l i n p r o d u c t i o n a n d w h e t h e r t h i s Ig possesses a n t i - t u m o r a n t i b o d y specificity remains unknown. Some observations hint h o w e v e r , t h a t t h e r e exists a link b e t w e e n m o n o c l o n a l i m m u n o g l o b u l i n s y n t h e s i s in t h e l y m p h n o d e a n d a n e x i s t i n g t u m o r . T h e a b s e n c e of d i s t i n c t i n f l a m m a t i o n in t h e i m m e d i a t e v i c i n i t y of t h e p r i m a r y c a n c e r site a n d t h e e x i s t e n c e of m e t a s t a t i c loci as a source of a n t i g e n i c m a t e r i a l in l y m p h node, s u g g e s t t h i s possibility. The d e s c r i b e d case i n d i c a t e s t h a t m o n o c l o n a l i m m u n o g l o b u l i n p r o d u c t i o n n o t r e l a t e d to general p l a s m a cell d y s c r a s i a m a y be m a n i f e s t e d o n l y on t h e t o t a l level.
Rdsumd. L a t e c h n i q u e d i r e c t e de l ' i m m u n o f l u o r e s c e n c e a p e r m i s de m e t t r e en 6vidence d a n s u n ganglion l y m p h a t i que a d j a c e n t ~ la t u m e u r canc6reuse du r e c t u m la pr6sence de n o m b r e u s e s cellules s6cr6tant de I g G m o n o c l o n a l . M. K. G6RNu ~ a n d
J.
ZEROMSKI 6
Institute o/Biostructure, Department o/ Pathological Anatomy, Medical A cademy, Przybyszewskiego dg, P-60-355 Pozndn (Poland), 30 September 797d.
Regional lymph node of cancer of the rectum. Specific staining of plasma cells with anti-human kappa antiserum labelled FITC. • 960.
5 Present address: The Wistar Institute, 36th Street at Spruce, Philadelphia, Pa. 19104, USA. 6 This work was supported by a grant No. 09.3.2. of the Polish Academy of Sciences.
Localizing Properties of Anti-Nervous Tissue Antibodies in Rat Cervical Ganglion Since t h e d e m o n s t r a t i o n t h a t a r a b b i t a n t i - c a t c a u d a t e nucleus s e r u m a f f e c t s t h e bioelectrical a c t i v i t y of c a u d a t e nucleus of t h e cat b r a i n 1, t h e a n t i - n e r v o u s tissue a n t i b o d y has b e c o m e a v a l u a b l e tool in s t r u c t u r a l a n d f u n c t i o n a l s t u d i e s of t h e b r a i n a n d t h e n e u r o n S. T h e i m m u n o l o g i c a l i n v e s t i g a t i o n s of t h e n e u r o n concern, i n t e r alia, t h e a n t i g e n i c d e f i n i t i o n of n e u r o n a l c o m p o n e n t s , a n d t h e
m a p p i n g of a n t i g e n s in t h e n e u r o n a n d its m i c r o e n v i r o n m e n t . T h e p r e s e n t r e p o r t deals w i t h t h e l a t t e r s u b j e c t 1 Lj. 3dlHAILOVld and B. D. JANKovld, Nature, Lond. 192, 665 (1961). 2 t3. D. JANKOVId, in Macromolecules and Behavior (Ed. J. GAITO; Appleton-Century-Crofts, New York 1972), p. 99.
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EXPERIENTIA 31/2
The Protection of Mice Against Experimental Infection by Means of I m m u n i z a t i o n with Enriched Membrane Fraction of Salmonella typhimurium T h e r e is a s u b s t a n t i a l difference of o p i n i o n in ttte literature concerning the protection that can be obtained f r o m v a c c i n e s c o n s t i t u t e d b y n o n - l i v i n g g e r m s or f r o m fractions extracted from them, agreement being achieved o n l y r e g a r d i n g v a c c i n e s c o n s t i t u t e d of l i v i n g g e r m s w i t h r e d u c e d virulence. I n a p r e v i o u s paper, we r e p o r t e d d a t a o n t h e p r o d u c t i o n of a n t i b o d i e s w h i c h i n h i b i t in v i t r o t h e g r o w t h of Micrococcus lysodeikticus in r a b b i t s i m m u n i z e d w i t h p u r i f i e d b a c t e r i a l m e m b r a n e s 1. I n t h e p r e s e n t s t u d y , t h e i m m u n o g e n i c i t y of l i v i n g pathogenic germs and their enriched membranes fractions h a s b e e n c o m p a r a t i v e l y i n v e s t i g a t e d in o r d e r to e s t i m a t e t h e i r r e s p e c t i v e p r o t e c t i n g effects a g a i n s t infection. Salmonella typhimurium 74 N C T C g r o w n in n u t r i e n t b r o t h Difco a t 37 ~ w a s used. T h i s s t r a i n g a v e a n LDa0 of 5 • 10~ w h e n i n j e c t e d s.c. ill mice. M e m b r a n e s were p r e p a r e d b y t h e SCHNAITMAN m e t h o d S f r o m c u l t u r e s in t h e tog g r o w t h p h a s e w a s h e d 3 t i m e s i n saline. Succinic d e h y d r o g e n a s e a n d lactic d e h y d r o g e n a s e d e t e r m i n a t i o n was used as i n d i c a t i o n of m e m b r a n e isolationK T h e m e m b r a n e s f r a c t i o n i s o l a t e d b y t h i s m e t h o d were cont a m i n a t e d w i t h cell-wall l i p o p o l y s a c c h a r i d e s . Salmonella-free CD-1 mice w e i g h i n g a b o u t 15 g were used. T h e t e s t s were r e p e a t e d 10 t i m e s o n 4 g r o u p s of 20 mice. A n i m a l s of t h e first g r o u p received e v e r y w e e k 0.25, 0.50 a n d 1.0 m g of l y o p h i l i z e d m e m b r a n e s . Tile first i n o c u l a t i o n was g i v e n s,c. in c o m p J e t e F r e u n d a d j u v a n t (CFA). T h e o t h e r i n o c u l a t i o n s were m a d e i.m. in saline. T h e second g r o u p was i m m u n i z e d w i t h cell lipopolys a c c h a r i d e s p r e p a r e d as d e s c r i b e d b y WESTPHAL et al. ~, a c c o r d i n g t o t h e s a m e s c h e d u t e as t h e firs4c group. The" t h i r d g r o u p of a n i m a l s was i m m u n i z e d w i t h l i v i n g ceils.
Two a l i q u o t s of 5 x 103 cells in 0.5 saline were i n o c u l a t e d a t 15 d a y i n t e r v a l s . M o r t a l i t y was a p p r o x i m a t e l y 2 5 % so t h a t i m m u n i z e d mice were s u r v i v o r s of a m i n i m a l l y v i r u l e n t infection. A n i m a l s i n o c u l a t e d w i t h s a l i n e a l o n e in C F A a n d k e p t u n d e r t h e s a m e c o n d i t i o n s were u s e d as control. A week a f t e r t h e l a s t i n o c u l a t i o n , t h e m i c e were t e s t e d for t h e p r e s e n c e of a n t i m e m b r a n e f r a c t i o n a n d anti-0 antibodies. A n i m a l s of t h e 3 g r o u p s t e s t e d p r e s e n t e d a n t i b o d i e s r e a c t i n g b o t h w i t h cells in 0 p h a s e a n d w i t h m e m b r a n e fraction. T i t e r s were 1 : 320 for b o t h a n t i b o d y types, i n t h e a n i m a l s of t h e first g r o u p ; 1 : 80 for a n t i m e m b r a n e f r a c t i o n a n d 1:1280 for allti-0 in t h e a n i m a l s of t h e second g r o u p ; 1:1280 or h i g h e r for b o t h a n t i - m e m b r a n e a n d allti-0 in t h e a n i m a l s of t h e t h i r d group. T h e i m m u n i t y of mice was c h a l l a n g e d i n v i v o 'by s.c. i n o c u l a t i o n of 100 LDs0 of Salmonella; t h i s dose killed all t h e c o n t r o l mice w i t h i n 10 days. M o r t a l i t y was followed for 30 d a y s a n d p r o t e c t i o n w a s m e a s u r e d as p e r c e n t a g e of survival. S u r v i v a l was 70~ 4- 6 for mice t r e a t e d w i t h m e m b r a n e s u s p e n s i o n 7 4 % 4- 6 for a n i m a l s t r e a t e d w i t h live o r g a n isms a n d 40~ 4- 5 for a n i m a l s t r e a t e d w i t h L P S . Statist i c a l a n a l y s i s of t h e r e s u l t s o b t a i n e d f r o m t h e 10 t e s t s p e r f o r m e d for e a c h oI t h e 4 g r o u p s of a n i m a l s s h o w e d n o s i g n i f i c a n t difference b e t w e e n t h e t r e a t m e n t s ( P v a l u e b a s e d o n Z 2 t e s t < 0.05). R e s u l t s show t h a t p r o t e c t i v e i m m u n i t y of a p p r o x i m a t e ly t h e s a m e o r d e r is s t i m u l a t e d b y l i v i n g m i c r o o r g a n i s m or b y m e m b r a n e f r a c t i o n o b t a i n e d f r o m t h e m .
Riassunto. T o p i i m m u n i z z a t i con m e m b r a n e isolate d a Salmonella typhimurium p r e s e n t a n o u n a r e s i s t e n z a alia infezione s p e r i m e n t a l e n g u a l e a quella d i m o s t r a t a d a t o p i i m m u n i z z a t i con Salmonelle v i v e n t i .
1 F. GALDIERO, Zbl. Bakt. Hyg., A 21g, 449 (1972). 2 C. A.. SCI-tNAITMAN,J. Baet. 104, 890 (1970). a C. A. SCHNAIT~IAN,J. Cell Biol. 38, 158 (1968). O. WESCPHAL, O. LODERITZ and F. BISrER, Z. Naturforsch. 7b, 148 (1952).
F. GALDIERO a n d CATERINA ~:~OMANO
Istituto di Microbiologia dell' Universit&, Sant'Andrea delle Dame 2, 1-80138 Napoli (Italy), 24 September 1974.
Local Monoclonal I m m u n o g l o b u l i n Production in Cancer Patient P r e s e n c e of M - c o m p o n e n t in s e r u m of c a n c e r p a t i e n t s has been described with prevalence in those bearing g a s t r o i n t e s t i n a l (GI), b l a d d e r a n d l u n g cancers1, ~. T h e r e is n o d i s t i n c t l i n k b e t w e e n t h e p r e s e n c e of M - c o m p o n e n t a n d t h e t y p e of cancer. T h e o c c u r a n c e of t h e f o r m e r is a p p a r e n t l y s i m i l a r t o t h a t o b s e r v e d in t h e n o r m a l p o p u l a t i o n 3. "While s e a r c h i n g i m m u n o f l u o r e s c e n t s t a i n e d sections of r e g i o n a l l y m p h n o d e s of 14 colon c a n c e r p a t i e n t s , we h a v e o b s e r v e d t h e p r e s e n c e of n e a r l y 3 t i m e s as m a n y i m m u n o g l o b u l i n b e a r i n g p l a s m a cells in n o d e s f r o m c a n c e r p a t i e n t s as c o m p a r e d to c o n t r o l n o d e s o b t a i n e d f r o m t h e v i c i n i t y of g a s t r i c or d u o d e n a l ulcers. A m o n g t h e immuoglobulins produced, IgM predominateda. I n one of t h e cases e x a m i n e d , large c l u m p s of p l a s m a cells s h o w e d a p a t t e r n of m o n o c l o n a I i m m u n o g l o b u l i n G (IgG) p r o d u c t i o n . T h e afore m e n t i o n e d case was a 68y e a r - o l d male, w i t h r e c t a l a d e n o c a r c i n o m a w h o s e tissues
b e c a m e a v a i l a b l e a i t e r surgery. T u m o r a d j a c e n t m u c o s a h a d n o signs of i n f l a m m a t i o n . P l a s m a cells were i d e n t i f i e d in t h e t m n o r itself, s u r r o u n d i n g m u c o s a a n d r e g i o n a l l y m p h n o d e s b y m e a n s of a b a t t e r y of m o n o s p e c i f i c i m m u n o f l u o r e s c e n t r e a g e n t s . T h e r e l a t i v e q u a n t i t y of p l a s m a cells was d e t e r m i n e d b y m e a n s of cellular d e n s i t y i n d e x (CDI), i.e., t h e m e a n n u m b e r of cells in tile field of v i s i o n u n d e r h i g h p o w e r of m i c r o s c o p e - • 480. T h e C D I for t h e r e g i o n a l l y m p h n o d e s w a s v e r y h i g h (8.92) for I g G p r o d u c i n g cells w h i c h c o n s t i t u t e d 9 0 % of t h e t o t a l p l a s m a cell n u m b e r . T h e r e m a i n i n g 10~o c o n s i s t e d of lgA, IglVI a n d I g E p r o d u c i n g cells (Table). I n t h e case
t E. F. OSS~R~aANand K. TAKATSUKI, Medicine ,12, 357 (1963). 2 A. TALERMANand W. G. HAIffE, Br. J. Cancer 27, 276 (1973). a M. K. G6RNY and J. ZEROMSKI, to be published. 4 E, A. JoNEs, Gut 13, 825 (1972).
15.2. 1975
Specialia
239
Examination of the number of plasma ceils in lymph node and immunoglobulin levels in patient's serum Ig
CDI ~in lymph-node
Serum immunoglobulin Ievels in InternationaI Units
Examined case
13 cases of other colon cancer (mean values)
Examined case
Blood donors values
IgG IgA IgM IgE
8.92 0.47 0.10 0.33
0.43 0.45 0.90 0.06
88 68 206 n.t.
149 • 43 139 4- 51 152 4- 48 n.t.
'K: L ratio
31:1
2.5:1
n.t.
n.t.
CDI, cell density index; n.t., not tested.
d e s c r i b e d t h e k a p p a : l a m b d a q i g h t c h a i n r a t i o was 31:1, while in o t h e r c a n c e r l y m p h n o d e s it did n o t e x c e e d 2.5 : 1. L o c a l i z a t i o n of k a p p a specific cells (Figure) c o r r e s p o n d e d closely t o t h o s e of IgG a n d s t r o n g l y f a v o r e d t h e v i e w t h a t t h e s e IgG p r o d u c i n g cells arose f r o m single clone. T h e l y m p h n o d e c o n t a i n e d a s m a l l m e t a s t a t i c loci, w h i c h did n o t c h a n g e its s t r u c t u r e . G e r m i n a l c e n t r e s of l y m p h a t i c n o d u l e s w e r e v e r y few a n d inconspicious. T h e p a t i e n t d i s p l a y e d h y p o p r o t e i n e m i a (total p r o t e i n 3,4 g/100 ml). Q u a n t i t a t i v e s e r u m i m m u n o g l o b u l i n d e t e r m i n a t i o n s r e v e a l e d a decrease of I g G a n d I g A a n d a n increase of IgM (Table). B o t h , p a p e r e l e c t r o p b o r e s i s a n d i m m u n o e l e c t r o p h o r e s i s did n o t s h o w m o n o c l o n a l p r o t e i n in s e r u m .
These d a t a s h o w t h a t regional l y m p h n o d e s in cancer m a y b e a site of m o n o c l o n a l I g s y n t h e s i s a n d t h a t t h i s p h e n o m e n o n m a y n o t be d e t e c t a b l e in t h e p a t i e n t ' s serum. The o b s e r v e d h y p o p r o t e i n e m i a could be due t o t h e loss of p r o t e i n via t h e GI t r a c t , a p o s s i b i l i t y s u g g e s t e d for cancer of t h e GI t r a c t b y Jo~;Es *. T h e failure t o o b s e r v e a m o n o c l o n a l spike in t h e s e r u m c a n n o t be e x p l a i n e d a t t h i s t i m e b u t m a y b e due t o t h e a m o u n t of I g G p r o d u c e d a n d t h e f a c t t h a t it is p r o d u c e d focally. T h e cause of local m o n o c l o n a l i m m u n o g l o b u l i n p r o d u c t i o n a n d w h e t h e r t h i s Ig possesses a n t i - t u m o r a n t i b o d y specificity remains unknown. Some observations hint h o w e v e r , t h a t t h e r e exists a link b e t w e e n m o n o c l o n a l i m m u n o g l o b u l i n s y n t h e s i s in t h e l y m p h n o d e a n d a n e x i s t i n g t u m o r . T h e a b s e n c e of d i s t i n c t i n f l a m m a t i o n in t h e i m m e d i a t e v i c i n i t y of t h e p r i m a r y c a n c e r site a n d t h e e x i s t e n c e of m e t a s t a t i c loci as a source of a n t i g e n i c m a t e r i a l in l y m p h node, s u g g e s t t h i s possibility. The d e s c r i b e d case i n d i c a t e s t h a t m o n o c l o n a l i m m u n o g l o b u l i n p r o d u c t i o n n o t r e l a t e d to general p l a s m a cell d y s c r a s i a m a y be m a n i f e s t e d o n l y on t h e t o t a l level.
Rdsumd. L a t e c h n i q u e d i r e c t e de l ' i m m u n o f l u o r e s c e n c e a p e r m i s de m e t t r e en 6vidence d a n s u n ganglion l y m p h a t i que a d j a c e n t ~ la t u m e u r canc6reuse du r e c t u m la pr6sence de n o m b r e u s e s cellules s6cr6tant de I g G m o n o c l o n a l . M. K. G6RNu ~ a n d
J.
ZEROMSKI 6
Institute o/Biostructure, Department o/ Pathological Anatomy, Medical A cademy, Przybyszewskiego dg, P-60-355 Pozndn (Poland), 30 September 797d.
Regional lymph node of cancer of the rectum. Specific staining of plasma cells with anti-human kappa antiserum labelled FITC. • 960.
5 Present address: The Wistar Institute, 36th Street at Spruce, Philadelphia, Pa. 19104, USA. 6 This work was supported by a grant No. 09.3.2. of the Polish Academy of Sciences.
Localizing Properties of Anti-Nervous Tissue Antibodies in Rat Cervical Ganglion Since t h e d e m o n s t r a t i o n t h a t a r a b b i t a n t i - c a t c a u d a t e nucleus s e r u m a f f e c t s t h e bioelectrical a c t i v i t y of c a u d a t e nucleus of t h e cat b r a i n 1, t h e a n t i - n e r v o u s tissue a n t i b o d y has b e c o m e a v a l u a b l e tool in s t r u c t u r a l a n d f u n c t i o n a l s t u d i e s of t h e b r a i n a n d t h e n e u r o n S. T h e i m m u n o l o g i c a l i n v e s t i g a t i o n s of t h e n e u r o n concern, i n t e r alia, t h e a n t i g e n i c d e f i n i t i o n of n e u r o n a l c o m p o n e n t s , a n d t h e
m a p p i n g of a n t i g e n s in t h e n e u r o n a n d its m i c r o e n v i r o n m e n t . T h e p r e s e n t r e p o r t deals w i t h t h e l a t t e r s u b j e c t 1 Lj. 3dlHAILOVld and B. D. JANKovld, Nature, Lond. 192, 665 (1961). 2 t3. D. JANKOVId, in Macromolecules and Behavior (Ed. J. GAITO; Appleton-Century-Crofts, New York 1972), p. 99.
