The Influence of Psychological Intervention upon Psycho-Neuro ...
The Influence of Psychological Intervention upon Psycho-Neuro ... The Influence of Psychological Intervention upon Psycho-Neuro ...
P s y c h ol o gi cal in ter ven ti on an d p s y c h o-n e u r o-e n d o c r i n -i o mmu n e n et wo rk. A k i r A a I T O N E xpr es s ion o f Annexin V- PI o n -l y T m p h o c y t e s m e a s u r e d b y f l o w c y t o m e t r y Th e A n n -F e I x i T C n ( -1 F L ) V a n t i b o d y & p r o p i d i u m i o d i n e : ( P I -3) F L s tai n in g met h od for d etecti on of ap op tos is was u s ed to d eter m in e th e effect of c ortis ol u p o n a p o p t o t i c p r o c e s . s e s Fo u r -c o l o r f l o w c y t o m e t i r s i c o n a c q lo a u n w d J o ® F f l o w c y t o m e t r y a n a l y s w e e s r e p e r f o . r A m e d r e g i o n w a s et on a d ot p lot of SS C vs . FL-2 ( C D -P 3 E ) t o t -l a ymp r g he oc t yt es T , a n d fu rt h er p e r c e n t a g e s o f n e c r o t i c c e l l s , a p o p t o t i c c e l l s a n d l i v e d c e bl y l a s d ow t e pr lot e of d e FL- t e r m i n e 3 (P I) vs . FL-1 ( An n exi n V) [Figu re 1 5 ]. Side Scatter (SSC) W h o l e l y mp h o cytes F L -2 : C D 3 s t a i n i n g F L -3 : P I s t a i n i n g Fi gur e 15b: s e t t -up i n a g re gio n f or a popt ot ic c e lls u s i n g n e ga t i ve i s o t yp e c o nt r o l i n a d ot p l o t Fi gur e 15: Ac quis ion s e tt ings of the f l o w c yt o m e ter i n t Annexin h e V- P I a n a l y s i s A regi on was s et on a d ot p lot o f FL-2 ( 3) C vs D . SSC t o tar get T-cells ( F i g u r e a ) fr 1 om 5 w h o l e l ym p h o c yt e s g a te d b y t -S C h C e p l F o S t C p r e v i , an o u d s fu l y rt h er r e g i o n s f o r a p o p t o t i c cell s u bs e t s e w r e d etermi n ed b y a d ot p lo t of FL-3 (P I ) vs . FL-1 (A n n e x ) i ( Fig n u re V 1 5 c: FL-1: Annexin V staining FL-1: Annexin V staining ( g a t e d b -S y S C t p l o h t ) e F S C T-l y m p h o c y t e s Fi gur e 15a: a r e g i -c o e n l l s i f n a o d r ot p T l ot Ne gati ve c ont r ol ( f o r g a t i n g p u r p o s e ) Apopt ot i c c e l l s L i v e c e l l s Apopt ot i c cel l s L i v e c e l l s N e c r o t i c c e l l s E x p e r i m e n t s a m p l e s N e c r o t i c c e l l s F L -3 : P I s t a i n i n g F i gur e 15c: a r e gi o n f o r a p op t ot i c ce l l s i n a do t p l o t + - + ap op tot ic ( A n n e x & i P n I ) a n V d n e c r o t i c c e l l ( P ) I s u b s et s i n t -l h y e m p h T o c y t e s ). F i g u r b e 1 5 s ho w s t h e m e t h o d -u s e p t for t i th n e g re gi o n of ap op tot ic cells b y u s in g n egati ve is ot yp e con tr ols . P h . D . a t U n i v e r s i t y o f L o n d o n - 7 6 - I m p e r i a l o C n o d l o n l e g e L
P s y c h ol o gi cal in ter ven ti on an d p s y c h o-n e u r o-e n d o c r i n -i o mmu n e n et wo rk. A k i r A a I T O N 2.5 S t a t i s t i n c a l ys e s a All p s ych ol o gi cal an d p h ys iol o gical lab o r ator y s amp les we re a n o n ym i s e an d d p roc es s ed u s i n g a n a ss ign ed s tud y n u m b e r to e n s u r that e t h e a n a l y s e ws ere p e r f o r m e d b l i n terms d l y of i n t h e g r o u p s a s n u d b gr o u ps t w o h i c h t h e s a m s p b l e l o n g . t o T o e x a m i n e t h a e p r i o r i h yp ot h es is th at p s ych olo gical in ter ven tion ma y c ou n teract th e d e t r i m e n t a l e f f e c t o f s t r e s s o n w e l l-b e i n g , -t o r e n a t m e n t a n a l ys is (a n a l ys i s of res u lts fr om on l y th os e wh o co mp let ed th e tri al) was p erf or med . W h e n statistical d i f f e r e es n c w e r e f o u n d in th e p r i m a r y o u t c o m e m e a s in u r u e n i v a r i a t a e n a l y s e s , I n -t t e o-t n r t e i a t o n a n a l y s w i s a s p e r f o r m in e d o r d e r t o c o n f i r m t h e r e s u l t s . T h e i n te n t i o -t n o-t reat is th e an al ys is of res u lts fro m e v er y s u b ject en rolled i n t h e t ri al re gard l es s o f wh eth er th e y co mp leted th e trial, an d all in d ivid u al s wh o w i t h d r e w w e r e c o u n t e d i n t h e n u m b e r o f s u b jects wh o failed th e h yp oth es is . Un i v a r i a t s t e a t i s t i c a l an a l y s e s w e r e r m p e r d f t o t e s t s p e c i f i c h y p o t s h t a e t s e d s a t each p o i n t i n t h e r e s u l t s s e c t io n [3 .1 a n d 3 . 2 i n v i sv tu o d ies ; an d 3 .3 an d 3 .4 in vir to e x p e r i m e n t s]. S a m p l e s i s z of e each grou p were rel at i vel y s mall in th e i n v i an v o al ys es [3 .1 an d 3 .2 ], an d t h e r e f o re e s s e n t i a l l y m u l t i v s a e r s i a t r e a u n a l i t y ach k o e l ie y ve mean in gfu l res u lt s . If mu ltivari at e an al ys es were p erf or med , s t a n d a rd e r r o r s w o u l d b e r e l h a givi t i nv g e ver l y y h wi i dg e 9 5 % c o n f i d e n ti a l i n t e r v a ls (C . I.) in d icating a l ot of u n certain t y in fin d in gs . Fo r th is reas on , it was felt i n a p p r o p r i a t o e p erf or m mu lt iv a ri at e an al ys es . Th e S t a t i s t i c a l P a c k a g e o f S o c i a l S c i e n c e v e r s i o n 1 1 . 0 ( S P [ S F S i e ® l ) d , 2 0 0 3 ] was u s ed with a p val u e o f les s th an 0 .0 5 con s id ered as a s t a t i s t i c a l l y s i g n i f i , c an a n d t a v p a val l u e u e o f l e s s t h a n 0 . 1 0 a s h a e S tu s d y t Sa ize t i Det s er t mi i nc at a ion l (SSD) t r pe ro n gra d . mm e T [ Leh man n , 2 0 0 1 ] w a s u s e d f o r amp s le s ize cal cu lat io n a t t h e 5 % s i g n i pf ower. i c a n c e Fo r in d ep en d en t n ormal l y d i s trib u ted d ata, a n a l y s e s o f v a ri a n c e ( A N O V A ) w e r e p e r f o r m e d f o l l o w e d b y p o s t-h o c St u d en t t-t e s t e p s e a . t e -meas d R u res AN O V A w a s u s e d f o r l o n g i t u d i n a l d a t a n ormal l y d is tri b u ted [ T a b a c h n i c k & F i d e l l , 1 9 9 . 6 For ] n o n-n o rmal l y d i s t r i b u dt ata, e d r e p e a t -m e d e a s u r e A N O V A f o l l o w e d b y 1t 0 h % e tri mmi n g m et h od [ W i l c o x , 1 9 9 7 ] p e r f o r m e trim d mi . n g met T h od h we as ap 1 p li ed 0 in % ord er to r em ove o u t l iers s o t h at th e ANO V A m et h od can b e ap p lied . T o c o n f i r m t h e f i n d i n g f r A o m N O t V h A e f i o n g l th e l 1 0 o % w trimmi n g met h od , t h e N -p o n a r a m e t r i c A N O V A th ( e K r u s k a l -Wallis r a n k t e s ) t wa s s u s e d [ T a b a c h n i c k & F i d e l l , 1 9 9 . 6 ] T h e W i l c o x o n r a n k t e s t s w e r e u s e d n-n f or o mal r p l y a i r e d a n d n o d is trib u ted d at a [ A j e t u n m o b i , 2 0 0 2 . ] P h . D . a t U n i v e r s i t y o f L o n d o n - 7 7 - I m p e r i a l o C n o d l o n l e g e L a w s
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P s y c h ol o gi cal in ter ven ti on an d p s y c h o-n e u r o-e n d o c r i n -i o mmu n e n et wo rk. A k i r A a I T O N<br />
E xpr es s ion o f Annexin V- PI o n -l y T m p h o c y t e s m e a s u r e d b y f l o w c y t o m e t r y<br />
Th e A n n -F e I x i T C n ( -1 F L ) V a n t i b o d y & p r o p i d i u m i o d i n e : ( P I -3) F L s tai n in g met h od for<br />
d etecti on <strong>of</strong> ap op tos is was u s ed to d eter m in e th e effect <strong>of</strong> c ortis ol u p o n a p o p t o t i c p r o c e s . s e s<br />
Fo u r -c o l o r f l o w c y t o m e t i r s i c o n a c q lo a u n w d J o ® F f l o w c y t o m e t r y a n a l y s w e e s r e p e r f o . r A m e d<br />
r e g i o n w a s et on a d ot p lot <strong>of</strong> SS C vs . FL-2 ( C D -P 3 E ) t o t -l a ymp r g he oc t yt es T , a n d fu rt h er<br />
p e r c e n t a g e s o f n e c r o t i c c e l l s , a p o p t o t i c c e l l s a n d l i v e d c e bl y l a s d ow t e pr lot e <strong>of</strong> d e FL- t e r m i n e<br />
3 (P I) vs . FL-1 ( An n exi n V) [Figu re 1 5 ].<br />
Side Scatter (SSC)<br />
W h o l e l y mp h o cytes<br />
F L -2 : C D 3 s t a i n i n g<br />
F L -3 : P I s t a i n i n g<br />
Fi gur e 15b: s e t t -up i n a g re gio n f or a popt ot ic c e lls<br />
u s i n g n e ga t i ve i s o t yp e c o nt r o l i n a d ot p l o t<br />
Fi gur e 15: Ac quis ion s e tt ings <strong>of</strong> the f l o w c yt o m e ter i n t Annexin h e V- P I a n a l y s i s<br />
A regi on was s et on a d ot p lot o f FL-2 ( 3) C vs D . SSC t o tar get T-cells ( F i g u r e a ) fr 1 om 5<br />
w h o l e l ym p h o c yt e s g a te d b y t -S C h C e p l F o S t C p r e v i , an o u d s fu l y rt h er r e g i o n s f o r a p o p t o t i c<br />
cell s u bs e t s e w r e d etermi n ed b y a d ot p lo t <strong>of</strong> FL-3 (P I ) vs . FL-1 (A n n e x ) i ( Fig n u re V 1 5 c:<br />
FL-1: Annexin V staining FL-1: Annexin V staining<br />
( g a t e d b -S y S C t p l o h t ) e F S C<br />
T-l y m p h o c y t e s<br />
Fi gur e 15a: a r e g i -c o e n l l s i f n a o d r ot p T l ot<br />
Ne gati ve c ont r ol<br />
( f o r g a t i n g p u r p o s e )<br />
Apopt ot i c<br />
c e l l s<br />
L i v e c e l l s<br />
Apopt ot i c<br />
cel l s<br />
L i v e c e l l s<br />
N e c r o t i c c e l l s<br />
E x p e r i m e n t s a m p l e s<br />
N e c r o t i c c e l l s<br />
F L -3 : P I s t a i n i n g<br />
F i gur e 15c: a r e gi o n f o r a p op t ot i c ce l l s i n a do t p l o t<br />
+ - +<br />
ap op tot ic ( A n n e x & i P n I ) a n V d n e c r o t i c c e l l ( P ) I s u b s et s i n t -l h y e m p h T o c y t e s ). F i g u r b e 1 5<br />
s ho w s t h e m e t h o d -u s e p t for t i th n e g re gi o n <strong>of</strong> ap op tot ic cells b y u s in g n egati ve is ot yp e con tr ols .<br />
P h . D . a t U n i v e r s i t y o f L o n d o n - 7 6 - I m p e r i a l o C n o d l o n l e g e L
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