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UBC Theses and Dissertations

In vitro lymphocyte responses with particular reference to tuberculous infections Clements, Donna Valerie Margaret 1970

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__ VITRO LYMPHOCYTE RESPONSES WITH PARTICULAR REFERENCE TO TUBERCULOUS INFECTIONS by DONNA V. M. CLEMENTS B.Sc.j U n i v e r s i t y o f B r i t i s h Columbia, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department o f Pathology We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1970 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n . f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t , o f Pathology T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a D a t e September 30, 1970, ABSTRACT Active tuberculosis i s a frequent complication of Hodgkin's disease and i t has been demonstrated recently that the c i r c u l a t -ing lymphocytes are abnormal i n t h i s disease £48). Furthermore, re a c t i v a t i o n of tuberculosis following steroid therapy i s a common complication (56) and i t i s now w e l l established that steroids are t o x i c to lymphocytes (57) . With t h i s background evidence to suggest the importance of normal lymphocyte function i n host resistance and the accidental finding of a poor lympho-cyte response i n a tuberculous patient, a study of the _Ln v i t r o reaction of the lymphocytes i n tuberculous patients was under-taken. In v i t r o cultures of peripheral white blood c e l l s w i l l undergo transformation and mitosis when i n the presence of phyto-hemagglutinin (PHA), an extract of the red kidney bean Phaseolus  v u l g a r i s . The number of c e l l s which transform or enter mitosis i n the culture i s representative of the i n d i v i d u a l ' s immune res-ponsiveness. In the tuberculous patients studied, irrespective of the state of t h e i r disease, the mean mitotic index (the number of c e l l s i n mitosis per thousand c e l l s ) was found to be s i g n i f i c a n -t l y lower than normal controls suggesting that t h e i r mean immuno-l o g i c a l capacity i s below normal. During the course of the tuberculous study one of the indiv i d u a l s used as a normal control came down with the " f l u " on the evening of the day her blood was taken. The PHA mitotic index was found to be severely depressed. To study the effect of such acute upper respiratory i n f e c t i o n s , presumably of v i r a l o r i g i n , mitotic indices were determined on blood samples taken from laboratory volunteers who f e l t they were coming down with a cold. The indices were found to be depressed i n a l l cases as compared to healthy normals. The reaction of delayed hypersensitivity and the r e l a t i o n -ship of t h i s reaction to immunity and tuberculosis has been a matter of study for many years. I t seems clear that the indi v i d u a l s i n the population that l a t e r develop tuberculosis are drawn largely from those members who have a positive skin t e s t while those with a negative skin t e s t have been found u n l i k e l y to develop tuberculosis to the point of not being required to have X-rays i n many instances. Tuberculin PPD ( p u r i f i e d protein derivative) was one of the f i r s t antigens shown to be capable of transforming lympho-cytes i n v i t r o (60,61) providing the i n d i v i d u a l had had p r i o r s e n s i t i z a t i o n . As a l l tuberculin negative student nurses entering t r a i n i n g at the Vancouver General Hospital receive a b a c i l l u s Calmette Guerin vaccination (BCG), they were chosen for study i n order to elucidate the relationship of i n vivo and i n v i t r o responses to the tubercle antigen, PPD. As the number of c e l l s responding to PPD i s much less than responding to PHA, the mitotic index i s a very insensitive method to use i n determination of i n vitro^ responsiveness to antigen. Therefore, the response i n v i t r o was determined by the incorporation of ^H-thymidine into DNA of stimulated lympho-cytes. I t was found that i n 12 to 18 months after vaccination with BCG nurses who had become skin t e s t negative to PPD did not d i f f e r s i g n i f i c a n t l y i n t h e i r i n v i t r o response from nurses who had remained skin t e s t p o s i t i v e . A correl a t i o n was found between the PHA and PPD i n v i t r o responses which suggests that the response to the general stimulus, PHA, i s an indicator of the reaction that can be mobilized to s p e c i f i c antigen. Varying the dose of BCG that was administered affected only i n vivo responses. F i n a l l y , contrary to many reports, no quantitative c o r r e l a t i o n was found between i n vivo and i n v i t r o responses to PPD following BCG vaccination. TABLE OF CONTENTS Page TITLE PAGE i ABSTRACT ... i i TABLE OF CONTENTS v LIST OF FIGURES ... .... v i i LIST OF TABLES i x 1 INTRODUCTION 1 1 SUBJECTS STUDIED 11 1. Normal Controls.... 11 2. Tuberculous Patients 11 3. Chronically 111, Bedridden Non-tuberculous Patients 12 4. Individuals Suffering from Acute Upper Respiratory Infections 12 5. Student Nurses Receiving BCG Vaccination 12 METHODS AND MATERIALS ' 12 1. Procedure for Skin Testing 12 2. Lymphocyte Culture Technique for Measuring PHA Induced Mitosis - 12 a) Preparation of Cultures 12 b) Determination of Mitosis 13 3. Leukocyte Culture Technique for Measuring % -Thymidine Incorporation i n Stimulated C e l l s 14 a) Preparation of Cultures 14 b) Measurement of 3H-Thymidine Incorporation 16 4. Technique to Measure PPD Antibody T i t r e s 17 a) Preparation of Erythrocytes with Tannic Acid.. 17 b) Adsorption of Antigens by Erythrocytes 17 c) Exposure of the Tanned, Sensitized C e l l s to the Test Sera 17 5. Serum Immunoglobulin Measurements 18 6. Routine Hematology...... 18 7. Electron Microscopy 18 8. Antibody T i t r e s 18 v i Page EXPERIMENTS AND RESULTS 18 1. Lymphocyte Transformation by PHA i n the Tuberculous Patient 18 2. Lymphocyte Transformation by PHA i n Acute Upper Respiratory Infections 29 3. The In V i t r o Response to PHA and PPD Following BCG Vaccination 31 a) Group I Student Nurses 32 4. The In V i t r o Response to PHA and PPD Before and Aft e r BCG Vaccination ^ 35 a) Group I I Student Nurses . 35 b) Group I I I Student Nurses 45 c) Group IV Student Nurses 47 5. Case Report of an Abnormal In V i t r o Response 63 DISCUSSION 70 CONCLUSIONS 93 ACKNOWLEDGEMENTS 94 BIBLIOGRAPHY 95 LIST OF FIGURES FIGURE 1 Phytohemagglutinin m i t o t i c i n d i c e s o f lympho-cytes from normal subjects and tuberculous pa t i e n t s 2 The e f f e c t of a c e t y l s a l i c y l i c acid on the phytohemagglutinin m i t o t i c i n d i c e s o f lympho-cytes from normal subjects . . j 3 Repeat phytohemagglutinin m i t o t i c i n d i c e s o f lymphocytes from patients on and o f f drugs f o r one week. 4 Phytohemagglutinin m i t o t i c i n d i c e s of lympho-cytes from tuberculous ( a t y p i c a l mycobacteria Type 3) and non-tuberculous c h r o n i c a l l y i l l p a t i e n t s . 5 Repeat phytohemagglutinin m i t o t i c i n d i c e s o f lymphocytes from normal subjects. 6 Repeat phytohemagglutinin m i t o t i c i n d i c e s o f lymphocytes from tuberculous patients 7 E f f e c t of BCG vacc i n a t i o n as a function o f time on the i n v i t r o PPD-S response when measured by ^H-thymidine incorporation 8 E f f e c t of BCG va c c i n a t i o n as a function o f time on the i n v i t r o PPD-B response when measured by ^H-thymidine incorporation 9 C o r r e l a t i o n o f the i n v i t r o PPD-S and PHA responses following BCG va c c i n a t i o n i n group I (.) and group II (o) nurses measured by %-thymIdine incorporation v i i Page 20 21 23 25 26 27 41 42 43 v i i i Page FIGURE. 10 Correlation of the i n v i t r o PPD-B and PPD-S response as measured by ^H-thymidine incor-poration i n student nurses (group IV) 6 weeks after vaccination with either a small, standard, or large dose of BCG (Connaught) 60 11 Correlation of the i n vivo skin t e s t re-sponse to PPD-S and PPD-B following BCG vaccination i n groups I I and IV student nurses. 61 12 Correlation of the i n vivo skin t e s t r e -sponse to PPD-S and PPD-G following BCG vaccination i n groups I I and IV student nurses 62 13 Correlation of i n vivo skin t e s t re-sponse to PPD-B and PPD-G following BCG vaccination i n groups I I and IV student nurses 64 ix LIST OF TABLES Page TABLE I Change of mitotic index with drug therapy 22 I I E f f e c t of plasma on the mitotic index of PHA stimulated lymphocytes . . . i 28 I I I Relationship of the mitotic index to the absolute lymphocyte count 29 IV M i t o t i c indices during and after acute upper respiratory infections 30 3 V H-Thymidine incorporation following PHA and PPD stimulation i n group I nurses 34 VI Immunoglobulin l e v e l s of group I student nurses reported i n mg. % 35 VII PHA response i n student nurses (group I I ) before and after vaccination with a standard dose of BCG (Danish) 37 VIII PPD-S response i n student nurses (group I I ) before and after vaccination with the standard dose of BCG (Danish) ; 38 IX PPD-B response i n student nurses (group I I ) before and af t e r vaccination with the standard dose of BCG (Danish) 39 3 X Mean values of H-thymidine Incorporation i n group I I nurses following i n v i t r o stimulation 12 weeks after receiving a standard dose of Danish BCG vaccine. 40 XI I n i t i a l skin test response of group I I nurses to PPD-S, PPD-B, PPD-G, and 250 TU measured i n mm 44 Page TABLE XII Skin t e s t response of group I I nurses s i x weeks af t e r vaccination with a standard dose of BCG measured i n mm 44 XIII Skin t e s t response of group I I nurses one year following vaccination measured i n mm. ....... 45 XIV Antibody t i t r e s to PPD i n a group ( I I I ) of student nurses before and after vaccination with BCG. 46 XV PHA response i n a group (IV) of student nurses before and after vaccination with either a small, standard, or large dose of BCG (Connaught) 48 XVI PPD-S response i n a group (IV) of student nurses before and after vaccination with either a small, standard, or large dose of BCG (Connaught) 50 XVII PPD-B response i n a group (IV) of student nurses before and after vaccination with either a small, standard, or large dose of BCG (Connaught) 52 3 XVIII Mean values of H-thymidine uptake i n group IV nurses following i n v i t r o stimulation 6 weeks af t e r vaccination with either a small, standard, or large dose of BCG (Connaught) 55 XIX I n i t i a l skin t e s t response of group IV nurses to PPD-S, PPD-B, PPD-G and 250 TU measured i n mm 56 Page TABLE XX Skin t e s t response of group IV nurses 6 weeks af t e r vaccination with either a small, standard, or large dose of BCG (Connaught) measured i n mm. 57 XXI Mean values of skin t e s t responses i n group IV nurses 6 weeks following vaccination with the small, standard, or large dose of BCG (Connaught) measured i n mm 58 XXII Case report of an abnormal i n v i t r o PHA response 65 INTRODUCTION Immun i t y i s a s t a t e o f a l t e r e d r e s p o n s e t o a s p e c i f i c s u b s t a n c e w h i c h i s f o r e i g n t o t h e h o s t . Any m a t e r i a l c a p a b l e o f p r o v o k i n g an immune r e s p o n s e may be t e r m e d an a n t i g e n . T h i s s y s t em o f d e f e n s e d e v e l o p s e a r l y i n t h e p h y l o g e n e t i c s c a l e o f v e r t e b r a t e s and i s a s s o c -i a t e d w i t h t h e p r e s e n c e o f l y m p h o i d t i s s u e . B u r n e t ( l ) b e l i e v e s t h e deve l opmen t o f t h e immune r e s p o n s e a r o s e a s t h e c o m p l e x i t y o f t h e o r g a n i s m i n c r e a s e d . A s t h e c o m p l e x i t y i n c r e a s e d , t h e o p p o r t u n i t y f o r h a r m f u l s o m a t i c m u t a t i o n s t o d e v e l o p a l s o a r o s e ; f o r e x a m p l e , c a n c e r c e l l s . I f t h i s i s a c c e p t e d t h e n t h e deve l opment o f t h e s e unwanted c e l l s r e p r e s e n t s a b reakdown i n t h e h o s t ' s immune s y s t e m . The immune r e s p o n s e may be e i t h e r h u m o r a l o r c e l l u l a r , t h e f o r m e r c h a r a c t e r i z e d by c l a s s i c a l c i r c u l a t i n g a n t i b o d y p r o d u c e d by p l a s m a c e l l s and t h e l a t t e r m e d i a t e d b y s m a l l l y m p h o c y t e s . W h i l e most a n t i -gens s t i m u l a t e b o t h f a c e t s o f t h e immune r e s p o n s e u s u a l l y one f a c e t p r e d o m i n a t e s i n t h e i n i t i a l r e s p o n s e . The t y p e o f r e s p o n s e w h i c h o c c u r s f o l l o w i n g t h e e n t r a n c e o f a f o r e i g n s u b s t a n c e a l s o depends on t h e f o l l o w i n g : a ) The t y p e o f a n t i g e n - c e r t a i n b a c t e r i a s uch a s M y c o b a c t e r i u m  t u b e r c u l o s i s t e n d t o r e s u l t i n a c e l l u l a r r e s p o n s e whe rea s p o l y s a c c h a r -i d e s cau se h u m o r a l a n t i b o d y p r o d u c t i o n . b ) The dose o f a n t i g e n - s m a l l amounts o f c e r t a i n a n t i g e n s may r e s u l t o n l y i n c e l l u l a r i m m u n i t y w h i l e l a r g e r amounts a r e c o m b a t t e d w i t h h u m o r a l a n t i b o d y . c ) The r o u t e o f e n t r a n c e o f a n t i g e n may a l s o d e t e r m i n e t h e r e s -ponse w h i c h d e v e l o p s . I n t r a v e n o u s i n j e c t i o n o f a n t i g e n f a v o u r s a n t i -body p r o d u c t i o n whe rea s i n t r a d e r m a l i n j e c t i o n f a v o u r s t h e deve l opment o f c e l l u l a r i m m u n i t y . d) H e r e d i t y may a l s o p l a y a r o l e i n immune d e v e l o p m e n t , e s p e c -i a l l y i n t h o s e i n d i v i d u a l s p r one t o a t o p y . 2. The control of the developing response Is also separate. This i s exemplified most c l e a r l y i n birds (2,3). Antibody production i s controlled by the bursa of Fabricius while c e l l u l a r responses come under control of the thymus. Both develop from ectoendodermal junctions i n association with gut epithelium. I f either are removed there i s a loss of the p a r t i c u l a r functions which the organ i n question controls. As of yet there i s no known immunological equivalent of the bursa i n mammals although many organs have been implicated. Evidence that these two responses are under separate control has developed i n man from the study of immunologic deficiency diseases. Before looking into these states i t would be i n order to consider the development of the hemopoietic system. The o r i g i n of the lymphocyte and the development of the lympho-myeloid complex has been a matter of controversy for a number of years. U n t i l 10-15 years ago i t was generally believed that stem c e l l s ^ c e l l s capable of p r o l i f e r a t i n g , d i f f e r e n t i a t i n g , and renewing t h e i r own kind) arose l o c a l l y i n the embryonic myeloid organs (bone marrow and spleen) and primary lymphoid organs (thymus and bursa of Fabricius) by trans-formation of fixed e p i t h e l i a l or mesenchymal c e l l s so that each devel-oping organ produced i t s own complement of stem c e l l s . The idea that lymphocytes arose within various tissues as a re s u l t of stem c e l l migration v i a the c i r c u l a t i o n was unacceptable. With the development of marker systems, p a r t i c u l a r l y chromosome marker systems, old concepts had to be revised. Using adult animals and marker chromosomes i t has been shown that bone marrow c e l l s are capable of repopulating the complete lymphomye-l o i d complex i n l e t h a l l y i r r a d i a t e d r e c i p i e n t s .(4). On the other hand, thymus c e l l s , while able to repopulate spleen and lymph nodes, were incapable of repopulating bone marrow or thymus. I t was concluded from these experiments that the thymus c e l l s had originated from a myeloid c e l l resident i n the bone marrow. Once having entered the thymus maturation occurred r e s u l t i n g i n a population of c e l l s capable of repopulating only thymus or peripheral lymphoid t i s s u e . Further evidence for the c i r c u l a t i o n of stem c e l l s i n adult animals came from parabiosis experiments. The development of chimer-ism i n paraboised animals supported the concept of stem c e l l migration. The thymus though, and the bone marrow to an even lesser extent, never developed complete chimerism suggesting that i n the thymus (and bone marrow) there was a considerable degree of autonomy i n lymphopoiesis (5). These experiments however do not suggest the importance of stem c e l l migration i n the i n i t i a t i o n of hemopoiesis. Conclusive evidence that stem c e l l migration into embryonic organs does occur r e s u l t i n g i n the development of the lymphomyeloid complex has been presented recent-l y by Moore and h i s associates (6,7). In mice, chickens, and man the yolk sac i s the f i r s t organ of embryonic hemopoiesis. This function i s taken over by the f e t a l l i v e r i n mammals. The f i r s t lymphoid organ to appear i s the thymus followed by the bone marrow and bursa of Fabricius. These workers (6,7) showed the development of chimerism i n embryos providing vascular union, accomplished by yolk sac parabiosis, was present before the development of lymphopoiesis i n the thymus. Follow-ing introduction of l a b e l l e d c e l l s into the embryonic c i r c u l a t i o n thymic l o c a l i z a t i o n occurred. One of the o r i g i n a l objections to the develop-ment of lymphopoiesis In the thymus after the entrance of c i r c u l a t i n g stem c e l l s was the avascularity of the thymus during early embryonic l i f e . But as l a b e l l e d c e l l s were shown to enter the avascularized thymus Moore believes that the organ has a capacity to abstract these stem c e l l s from the c i r c u l a t i o n so that they migrate out of blood vessels and across the thymus basement membrane to develop i n the thymic epithelium. Migration of stem c e l l s into the thymus epithelium by i t s e l f does not lead to lymphopoiesis. Something else i s needed. There must be an inductive interaction with the surrounding mesenchyme. I f mesen-chyme i s removed even though stem c e l l s are present i n the epithelium the thymic rudiment w i l l f a i l to become lymphoid i n v i t r o . But by re-aggregation of the thymic rudiment with mesenchyme or by allowing 4. mesenchyme to act across a m i l l i p o r e f i l t e r membrane, the induction of stem c e l l p r o l i f e r a t i o n into an active cycle of lymphopoiesis occurs. Further evidence that the yolk sac i s the s i t e of o r i g i n of the stem c e l l s comes from the fact that the yolk sac i s the f i r s t organ to contain detectable colony forming c e l l s (CFCs). Using the T6 marker system, yolk sac c e l l s are found to repopulate the complete lymphomyeloid complex of l e t h a l l y i r r a d i a t e d mice. The pattern of d i f f e r e n t i a t i o n taken by the multipotential CFC i s determined by the host microenvironment. In v i t r o culture systems of whole embryos also support t h e i r concept. Embryos separated from their! yolk sac w i l l show no hemopoiesis while the yolk sac by i t s e l f continues to show hemopoiesis. Recent evidence (8) suggests that the adult and embryonic stem c e l l s are different as ref l e c t e d i n volume and density changes and once having d i f f e r e n t i a t e d into the more mature adult stem c e l l there i s no d e d i f f e r e n t i a t i o n . Once the thymus has become lymphoid, about the twelfth week of gestation i n man, lymphocytes appear i n the blood, the spleen, and the lymph nodes no l a t e r than the twentieth week of gestation sup-porting the concept that one function of the thymus i s to populate the secondary or peripheral lymphoid t i s s u e . Further evidence for thymus lymphocytes leaving the thymus to take up residence elsewhere comes from the presence of la b e l l e d c e l l s i n the periphery following thymus i n s i t u l a b e l l i n g . Because of the loss of the thymus c e l l s rapid p r o l i f e r a t i o n of c e l l s within the thymus would be expected to maintain the lymphocyte supply. Compared to other organs the mitotic rate i s high. What are the functions of the thymus derived lymphocytes? Following neonatal thymectomy cell-mediated immune reactions are depressed. These include homograft re j e c t i o n (9), delayed, hyper-s e n s i t i v i t y (10), and immunological memory ( l l ) . There i s a gener-a l i z e d lymphopenia and depletion of. lymphocytes i n the deep cortex of lymph nodes and p e r i a r t e r i o l a r sheaths of the spleen (12). The ef f e c t s of thymectomy may be overcome by grafts or implants of thymus. These grafts w i l l eventually be repopulated by host c e l l s as long as the e p i t h e l i a l r e t i c u l a r framework of the graft remains v i a b l e . Immunological deficiencies are even overcome when the thymus implant i s maintained i n a m i l l i p o r e f i l t e r (13) which suggests the importance of a humoral regulatory factor. Thymus c e l l s per se are not immunologically competent. I t requires 20-30 times t h e i r number to produce a graft-versus-host response. The function of the thymus then i s to s t a r t the c e l l s on t h e i r d i f f e r e n t i a t e d course, and to seed them to the periphery where under the influence of the microenvironment immunological competence i s acquired. As previously mentioned, immunologic deficiency diseases have supported the r o l e of the thymus i n cell-mediated immune responses. In patients with congenital aplasia of the thymus gland (DiGeorge's syndrome) humoral immunity i s normal but c e l l u l a r immunity i s def i c i e n t (14). Post-mortem examinations of such patients usually show a deficiency of lymphocytes i n the deep c o r t i c a l regions of the lymph nodes and p e r i a r t e r i o l a r sheaths of the spleen with normal c o r t i c a l germinal center a c t i v i t y and the presence of plasma c e l l s . The opposite disease to DiGeorge's syndrome would be Bruton agammaglobulinemia (15). Here c e l l u l a r immune functions are normal but plasma c e l l antibody production i s abnormal. Biopsies reveal the absence of plasma c e l l s and lack of germinal centers. Swiss type agammaglobulinemia on the other hand i s characterized by lack of delayed hypersensitivity and low glo b u l i n l e v e l s . This disease appears to be a defect of the central lymphoid tissue i n that these patients appear to lack the multipotential stem c e l l . As already discussed, for normal thymic development there must be an interaction between the stem c e l l and organ anlage. Owen and R i t t e r (16) suggest therefore that normal immunological functions i n Swiss agammaglobulinemia could possibly be restored by i n j e c t i o n of normal f e t a l hemopoietic c e l l s , that i s , by the i n j e c t i o n of mu l t i -p o t e n t i a l stem c e l l s . S i m i l a r l y , i n DiGeorge's syndrome, function has been restored by implantation of a thymus graft (17,18). With the development of i n v i t r o culture systems, the immuno-l o g i c a l competence of small lymphocytes and t h e i r r o l e i n c e l l -mediated immune reactions has been further c l a r i f i e d . In v i t r o studies developed ra p i d l y after 1960. At t h i s time Nowell (19) observed a higher proportion of bla s t o i d and mitotic c e l l s i n h i s cultures of peripheral blood than had been reported by previous investigators. This effect was found to be due to the substance phytohemagglutinin (PHA) an extract of the red kidney bean Phaseolus v u l g a r i s . The p r i n c i p l e use of PHA up u n t i l t h i s time had been to agglutinate red blood c e l l s (20) thereby f a c i l i t a t i n g t h e i r separation from the.leukocyte-rich plasma. Studies by MacKinney (21) demonstrated that i t was the same small lymphocyte of such importance i n cell-mediated immunity that was undergoing mitosis i n v i t r o . , PHA i s a proteinaceous, non-dialyzable substance containing hemagglutinating as w e l l as leukoagglutinating a c t i v i t y . Associated with the l a t t e r i s the blastogenic a c t i v i t y . Following the discov-ery of t h i s a c t i v i t y i n PHA the search began for other agents which would have a si m i l a r e f f e c t . The review by Robbins (22) divided the agents into 4 groups: 1) Nonspecific stimulants such as phytohemagglutinin, poke-weed mitogen, staphlococcal f i l t r a t e , streptolysin S and concanav-a l i n A. 2) Antiserum - wherein the change i s presumably a resu l t of the antibody complexing with the c e l l surface antigens which i n turn t r i g g e r s the lymphocytes. 3) Tissue antigens, present on such c e l l s as p l a t e l e t s , 3) Continued... macrophages and lymphocytes, due to foreign histocompatability a n t i -gens t r i g g e r c e l l s to enter mitosis. This i s made use of extensively i n mixed leukocyte cultures where the degree of blastogenesis i s an i n d i c a t i o n of the histoincompatability of the two donor c e l l s . This type of response represents a primary immune response i n v i t r o . 4) Soluble and particulate antigens such as PPD, v a c c i n i a , and tetanus toxoid. Response to these antigens represents a secondary immune response i n v i t r o as p r i o r s e n s i t i z a t i o n of the lymphocyte donor must have occurred. \ • The antigenic mitotic agents d i f f e r from PHA and other non-s p e c i f i c or general stimulants i n requiring a longer latent period before stimulation occurs (5-7 days versus 3 days), i n stimulating a much smaller percentage of the population, as w e l l as requiring the presence of monocytes i n the culture system. I t i s not yet known whether t h i s represents a different mechanism of activation between antigenic stimulation and the general mitogenic agents. I t may s i g n i f y only a difference i n the i n t e n s i t y of the blastogenic stimu-l u s . Perhaps the threshold of a c t i v a t i o n for PHA stimulated c e l l s i s much lower than for antigen stimulated c e l l s . Besides the suggestion that PHA acts as an antigen, i t has been postulated that PHA acts non-specifically to decrease the s t a b i l i t y of the c e l l membrane or as a stimulator of intermediate metabolism within the c e l l leading to increased protein synthesis and mitotic a c t i v i t y (23). Recent evidence that PHA acts by affecting the surface mem-brane has been shown by the increased rate of uptake of alpha-amino-isobutyric acid (AIB) (24) within 30 minutes a f t e r PHA addition. S i m i l a r l y the rate of potassium transport has been shown to increase (25). Both these phenomena occur even when protein and RNA synth-esis i s blocked. Further evidence for PHA-acting non-specifically i s shown by i t s action on non-lymphoid c e l l s . PHA has been shown to have a strong mitogenic effect on cultures of free l i v i n g amoebae (26). I t has been shown also to have an agglutinating and mitogenic action on connective tissue c e l l s , e p i t h e l i a l c e l l s , and r e t i c u l a r c e l l s ( 2 7 ) . Recent evidence by Lucas (28) suggests that i n i t i a l membrane reactions may vary .with different mitogens. 32 PHA was found to r e s u l t i n the increased incorporation of P0„ 4 into phosphatidyl i n o s i t o l whereas tetanus toxoid stimulated the incorporation of phosphate into phosphatidyl choline and phosphat-i d y l ethanolamine, but not into phosphatidyl i n o s i t o l . As mentioned, antigenic stimulation requires the presence of monocytes. Thus pure lymphocytes w i l l respond to PHA but not to s p e c i f i c antigens. I f , rather than using pure lymphocytes, cultures are set up with a l l peripheral blood leukocytes there w i l l be a response (providing the i n d i v i d u a l i s sensitive to the p a r t i c -u l a r antigen used). Studies by McFarland and co-workers (29) suggest that the lymphocytes which undergo transformation are those that have been in t e r a c t i n g with either lymphoblasts or macrophages. A clustering of c e l l s may be seen usually with a central macrophage. Attach-ment occurs by means of the uropod. Hersh and Harris (30) have shown that continued contact between dif f e r e n t c e l l types was not necessary. They found that i f leukocytes were preincubated with antigen and then separated on glass bead columns the r e s u l t i n g p u r i f i e d lymphocytes would s t i l l undergo transformation. MacLaurin (31) found that PHA resulted i n a rapid induction of i n t e r c e l l u l a r cytoplasmic bridging. Antigen caused a si m i l a r interlinkage. The suggestion i s that the cytoplasmic bridging could enable rapid dissemination of antigen throughout the lymph node macrophage population and thereby enhance the speed and inten-s i t y of the secondary immune response. Cytoplasmic connections i n vivo (32) between macrophages and lymphocytes have also been demonstrated and there i s an increase i n these connections follow-ing antigenic stimulation. The necessity for the presence of monocytes i s not l i m i t e d to secondary i n v i t r o responses as they are also necessary to obtain a response i n mixed leukocyte cultures (33) . This reaction i s presumed to be a primary response. In mixed cultures separation of the two c e l l populations also abolishes the response (34). Recently supernatants from macrophage cultures i n 8/10 experiments have reconstituted the response of p u r i f i e d lymphocytes (35). C e l l interactions are needed for primary immune responses between antibody producing c e l l s i n v i t r o as w e l l (36). Both adherent and non-adherent spleen c e l l populations are necessary. There are only a few investigators who f e e l that t h i s lympho-cyte -macrophage in t e r a c t i o n i s not necessary, and that macrophages only encourage lymphocyte-lymphocyte interaction (37). Coulson and Chalmers (38) have used p u r i f i e d lymphocyte suspensions at concentrations of 2 x 10^ small lymphocytes per ml. As they get a response to antigen, they f e e l that i t i s only a question of providing adequate c e l l contact. I t i s suggested that c e l l - c e l l contact, p a r t i c u l a r l y lympho-cyte-macrophage contact, i s necessary for transfer of information. Extracts obtained from sensitized donor lymph nodes containing RNA can transfer delayed hypersensitivity to non-sensitized c e l l s i n v i t r o (39). Cline and Swett (40) f e e l that the time of expos-ure to PPD required to sensitize monocytes so that they are capable of inducing lymphocyte transformation (2 hours) i s consistent with the demonstration that immunogenic RNA can be extracted from the macrophage a f t e r 30 minutes exposure to antigen. I t has also been suggested the macrophage merely processes the antigen so that the antigen becomes more Immunogenic. Rajewsky (41) suggested recently that these c e l l s merely function as a con-centrating device rather than i n the actual transfer of information. Moller (42) also feels that the antibody response to sheep red blood c e l l s which requires both thymus (T) derived c e l l s and bone marrow (-B) derived c e l l s i s not the r e s u l t of information transfer but only a co-operative e f f e c t . Along a s i m i l a r l i n e , Dutton (43) men-tioned that the combination of T and B c e l l s i n the presence of antigen would r e s u l t i n the release of a factor into the super-natant which would i n the presence of non-adherent c e l l s only restore t h e i r competence. He suggested that t h i s material was a c a r r i e r antibody. What changes occur i n i n v i t r o cultures following the addition of a mi t o t i c agent? The small lymphocytes are f i r s t seen to clump together, enlarge to resemble blast c e l l s (blastogenesis or trans-formation) and f i n a l l y undergo mitosis. In PHA stimulated cultures (44) following a 24 hour lag period the c e l l enters the phase of active RNA synthesis. Six hours a f t e r the i n i t i a t i o n of the c e l l cycle, the S phase i s entered. DNA synthesis begins and continues for about 11 hours. The c e l l then enters a resting phase, G^. During t h i s phase a l l processes necessary for c e l l d i v i s i o n are readied. Three hours l a t e r the c e l l a c t ually begins to. divide, completing the process- i n 2 hours with the formation of two daughter c e l l s . Based on t h i s c e l l u l a r a c t i v i t y there are at present three ways of quantitating the i n v i t r o response following stimulation: 1) Transformation of the c e l l as judged by morphological standards. These r e s u l t s depend to a great extent upon the sub-je c t i v e assessment by the examiner of morphological changes i n the small lymphocyte. 2) The mi t o t i c index. After a predetermined i n t e r v a l of culture, a stathmokinetic agent such as colchicine i s added and the number of mitotic figures accumulated a f t e r a certain time are counted and expressed as an index per thousand c e l l s . 3 3) The incorporation of H-thymidine; . This i s done most accurately by measuring the incorporated r a d i o a c t i v i t y i n the 3) Continued. whole specimen (rather than an aliquot) by means of a l i q u i d s c i n -t i l l a t i o n counter. I t may also be done more laboriously and less accurately by using autoradiography. . Where i t i s necessary to measure antigenic responses l i q u i d s c i n t i l l a t i o n counting i s the method of choice because of i t s greater s e n s i t i v i t y . With the help of these techniques and with the separation of immune responses into two classes, i n v i t r o correlates of c e l l -mediated immunity have been shown. Thus i n DiGeorge's syndrome the i n v i t r o PHA response i s impaired (45) while i t i s normal i n patients with Bruton-type.of agammaglobulinemia (15). Impairment i s also seen In diseases of secondary immunological d e f i c i e n c i e s . In patients with Hodgkin's disease 50% of whom w i l l r e t a i n homo-grafts and even heterografts for longer than normal i n t e r v a l s (46) the incidence of anergy i s high (47). In association with these impaired In vivo cell-mediated Immune reactions there i s an accom-panying depression of the i n v i t r o PHA response (48). I t has also been reported that rubella v i r u s infections w i l l cause i n h i b i t i o n of the lymphocytes' Immune response i n v i t r o (49,50). With the knowledge of t h i s background information, studies were begun i n the laboratory to measure the i n v i t r o responses as a function of the immunological competence of the small lymphocyte i n normal and disease states. . SUBJECTS STUDIED 1. Normal controls - chosen at random from members of the h o s p i t a l s t a f f . 2. Tuberculous patients - chosen from the inpatient and outpatient 2. Continued.o. d i v i s i o n of the B r i t i s h Columbia Tuberculosis Control Unit. 3. Chronically i l l , bedridden, non-tuberculous patients. 4. Individuals suffering from acute upper respiratory i n f e c t i o n s . 5. Student nurses - chosen from volunteers who were i n t r a i n i n g at the Vancouver General Hospital. No individuals were used i n the study i f they had ingested ace-t y l s a l i c y l i c acid or sodium s a l i c y l a t e i n the previous 48 hours. METHODS AND MATERIALS 1. Procedure for Skin Testing Student nurses were skin tested by Miss E. Dorkin. A l l skin t e s t s were made intradermally by i n j e c t i n g 5 TU (0.0001 mg. i n 0.1 ml. of diluent) of tuberculin p u r i f i e d protein derivative (PPD) into the dorsal surface of the forearm. Although reactions to three tuber-c u l i n antigens (PPD-S, PPD-B and PPD-G) were recorded, the response was considered negative i f the area of induration and erythema was less than 6 mm. to PPD-S only. Those individuals who did not respond to 5 TU were retested with 250 TU (0.005 mg. i n 0.1 ml. of di l u e n t ) . I f the skin t e s t was s t i l l negative, vaccination with b a c i l l u s Calmette and Guerin (BCG) was administered. One-tenth of a m i l l i l i t r e containing 0.1 mg. of protein was Injected intracutan-eously into the upper l a t e r a l surface of the thigh. Six weeks following vaccination the student nurses were again skin tested. 2. Lymphocyte Culture Technique for Measuring PHA Induced Mitosis a) Preparation of Cultures Thirty m i l l i l i t r e s of blood obtained by venipuncture was divided a) Preparation o f Cultures Continued... eq u a l l y between two tubes each containing 400 u n i t s of phenol-free heparin and allowed to s e t t l e at 37°C at an angle o f 60° u n t i l the majority of the red c e l l s had sedimented out. This was u s u a l l y accomplished i n about 45 minutes to 1 hour. The leukocyte r i c h plasma was then removed, measured and pipetted into a s t e r i l e glass column packed with absorbent cotton which had previously been washed three times i n g l a s s - d i s t i l l e d water and dried at 37°C (51). The remainder of the blood was then spun at high speed and the c e l l - f r e e plasma removed and incubated f o r l a t e r use i n s e t t i n g up the c e l l c u l t u r e s . The column o f w h i t e - c e l l - r i c h plasma was incubated f o r 30 min-utes at 37°C and then eluted into s t e r i l e centrifuge tubes using twice the volume o f Eagle's minimal e s s e n t i a l medium (MEM), contain-ing 100 u n i t s of p e n i c i l l i n and 100 Mg. o f streptomycin per m i l l i -l i t r e . The eluate was centrifuged at 200g f o r 10 minutes and the c e l l s washed i n 10 ml. o f Eagle's MEM, then recentrifuged and r e -suspended i n 4 ml. of medium. The cultures contained 93-99% lymphocytes. The white c e l l s were counted and 4 ml. c u l t u r e s , each contain-ing 3 x 10^ lymphocytes, were set up i n Eagle's complete medium with 20%. autologous plasma. Where p o s s i b l e , 6 cultures were set up per i n d i v i d u a l , three acting as a c o n t r o l . Added t o the remaining three cultures was 0.01 ml. of Burroughs Wellcome phytohemagglutinin which had been rec o n s t i t u t e d with 5 ml. of d i s t i l l e d water. Once re c o n s t i t u t e d , the PHA was stored at 4°C and discarded a f t e r three weeks. A l l cultures were then incubated for 72 hours at 37°C. b) Determination of M i t o s i s M i t o s i s was blocked during the f i n a l four hours of incubation "by the addition o f 0.1 ml. of a 0.02% s o l u t i o n of c o l c h i c i n e . b) Determination of Mitosis Continued... Colchicine solutions, l i k e PHA, were stored at 4°C and discarded af t e r three weeks. The cultures were then pipetted into c e n t r i -fuge tubes and the c e l l s were resuspended by gentle manipulation. Following centrifugation at 200g for 10 minutes, the supernatant was removed except for about 0.5 ml. i n which the c e l l s were re-suspended. Two m i l l i l i t r e s of a 0.88% saline solution was then added to the suspension and spun at 160g for 10 minutes, a f t e r which the supernatant was removed. Two m i l l i l i t r e s of a ; 0.22% saline solution was then slowly added down the side of the tube and a f t e r four minutes at 37°C the suspension was spun again at 120g for 10 minutes. A l l the saline was removed and Carnoy's f i x a t i v e was added drop by drop to a volume of 2 ml. Cultures were then l e f t at room temperature for 30 minutes followed by 10 minutes centrifugation at 160g. Enough f i x a t i v e was removed to leave a fine granular suspension. Two s l i d e s were prepared per culture by pipetting onto the s l i d e a drop of the c e l l sus-pension. When the drop had spread to about the size of a quarter, drying was aided by gentle blowing. The prepared s l i d e s were then stained with Jenner-Giemsa. Two thousand c e l l s per culture were counted (1000 c e l l s per slid e ) i n such a manner that the entire s l i d e was covered. The number of mitotic figures was reported as an index per 1000 c e l l s As the number of mitotic figures r a r e l y exceeded 1 per 1000 i n control cultures these s l i d e s were only scanned. 3 3. Leukocyte Culture Technique for Measuring H-Thymidine Incorporation i n Stimulated C e l l s a) Preparation of Cultures S t e r i l e technique was used throughout. Blood was obtained by venipuncture and put into centrifuge tubes containing 400 unit a) Preparation of Cultures Continued... of phenol-free heparin for every 15 ml. of blood. The tubes were centrifuged for 10 minutes at 800g and the plasma and buffy coat transferred to another tube, resuspended, and placed i n an incub-ator at 37°C on a 60° angle. After the red c e l l s had sedimented (approximately one hour), the tubes were placed v e r t i c a l l y for a further 15 minutes to obtain a d i s t i n c t demarcation between red c e l l s and the w h i t e - c e l l - r i c h plasma. This supernatant was then transferred to another tube. From t h i s an aliquot was taken and the t o t a l leukocytes and t o t a l lymphocytes were counted i n a hemocytometer. The supernatant was 3 adjusted to contain between 500 and 550 lymphocytes per mm by the addition of Eagle's medium containing 100 uni t s of p e n i c i l l i n and 100 jug. of streptomycin per m i l l i l i t r e . In order to adjust the plasma concentration to between 10% and 20%, c e l l - f r e e autologous plasma was added i f the supernatant had been diluted more than 10 times with medium. The leukocyte cultures were incubated at 37°C i n Falcon 2001 or Falcon 3033 d i s -posable p l a s t i c tubes i n a t o t a l volume of 4 ml. Per i n d i v i d u a l , PHA cultures (0.01 ml. of Burroughs Wellcome PHA) and unstimulated control tubes were set up for a three day incubation period. Cultures stimulated with PPD, and unstimulated controls, were set up at the same time as the PHA cultures for a 7 day incubation period. The PPD o r i g i n a l l y used was Parke, Davis & Company PPD, 2nd Test Strength, Bio 489. Each tab l e t was diluted i n 0.5 ml. of Eagle's MEM just p r i o r to use and 0.25 ml. added to each culture (0.025 mg. PPD). As t h i s p a r t i c u l a r PPD was discontinued, Dr. S. Landi, of the Connaught Medical Research Laboratories, kindly supplied tuberculin PPD "CT68" and PPD-Battey No. .104 dissolved i n a s t e r i l e solution of M/30 Na2HP0^ containing no Tween 80 and no phenol, for the con-ti n u a t i o n of the study. The concentration of PPD used was the a) Preparation of Cultures Continued... same as used with the Parke, Davis & Company product. 3 b) Measurement of H-Thymidine Incorporation 3 At the end of the incubation period, 2 MCi of H-thymidine (New England Nuclear, s p e c i f i c a c t i v i t y 6.7 Ci/mmole) i n a volume of 0.1 ml. was added to each culture. The radioactive l a b e l was l e f t i n contact with PHA stimulated c e l l s and unstimulated controls for 2 hours. PPD cultures and t h e i r controls were incubated with 3 H-thymidine for 4 hours. 3 To stop the further incorporation of H-thymidine, 100 Mg. of "cold" thymidine ( N u t r i t i o n a l Biochemicals Corporation) i n 0.1 ml. physiological saline was added to each tube. The cultures were then spun down at 800g for 10 minutes and as much supernatant as possible removed without disturbing the c e l l button. Two ml. of 5% t r i c h l o r o a c e t i c acid (TCA) was added to each tube to precipitate the DNA. The precipitate was centrifuged and washed once with 2 ml. of TCA followed by a f i n a l 1 ml. wash. A l l f l u i d was removed and 0.5 ml. of hydroxide of Hyamine (Packard) was added to each tube. The tubes were then placed i n the dark u n t i l complete s o l u b i l -i z a t i o n had occurred. To count, the contents of each tube were transferred to l i q u i d s c i n t i l l a t i o n counting v i a l s (Picker Nuclear) along with two 0.3 ml. washings of absolute ethanol. Fifteen m i l l i l i t r e s of s c i n t i l l a t i o n solution (1 gallon dioxane - Fisher D - l l l ; 400 g. naphthalene - Eastman Organic Chemicals-168; 28 g. PPO - Packard; and 1.2 g. dimethyl P0P0P - Packard).was added to each v i a l . Counting was done i n a Picker Liquimat 220 s c i n t i l l a t i o n counter after overnight dark adaptation of the samples. A l l counts were corrected for quench using the external standard method. 4. Technique to Measure PPD Antibody T i t r e s (52) a) Preparation of Erythrocytes with Tannic Acid -done by Mrs. P. Aldred Sheep erythrocytes collected i n heparin were washed 6 times i n buffered saline (pH 7.2) and a 2.5% suspension prepared. One volume of t h i s c e l l suspension (5 ml.) was treated with an equal volume of 1/20,000 tannic acid (5 ml.). The tannic acid was d i l u t e d to t h i s concentration with buffered saline (pH 7.2) from a 1/100 stock solution just p r i o r to use. After t r e a t i n g the c e l l s for 10 minutes at 37°C, the mixture was then washed once i n 10 ml. of buffered saline (pH 7.2), centrifuged (int e r n a t i o n a l Centrifuge, Model K) at 1500 RPM for 10 minutes and resuspended i n one volume (5 ml.) of buffered sal i n e . b) Adsorption of Antigens by Erythrocytes To each volume of tannic acid treated erythrocytes i n buffer-ed saline was added 4 volumes of buffered saline (pH 6.4) contain-ing the appropriate concentration of antigen (0.2 mg. per ml. of c e l l s ) . Following incubation at room temperature for 15 minutes with frequent mixing, the c e l l s were centrifuged at 1500 RPM for 10 minutes and washed once i n 16 ml. of 1/100 normal rabbit serum (NRS) before resuspending i n 4 ml. of 1/100 NRS. c) Exposure of the Tanned, Sensitized C e l l s to the' Test Sera Doubling d i l u t i o n s of the test sera were made i n 1/100 NRS i n 0.5 ml. quantities. To each tube 0.05 ml. of the c e l l suspension was added. Controls consisted of (1) unsensitized c e l l s , (2) c e l l s treated with tannic acid only set up at the i n i t i a l l/4 d i l u t i o n of serum, (3) unsensitized tanned c e l l s and (4) sensitized tanned c e l l s set up against 1/100 NRS. 5. Serum Immunoglobulin Measurements Routine immunoglobulin measurements were carried out by the Vancouver General Hospital chemistry department. 6. Routine Hematology Hemoglobin l e v e l s , white c e l l counts, and d i f f e r e n t i a l s were kin d l y done by the Vancouver General Hospital hematology depart-ment. 7. Electron Microscopy . . . i ' C e l l cultures were prepared for electron microscope studies by Mrs. B. Z i n t e l of the B r i t i s h Columbia Cancer I n s t i t u t e . 8. Antibody T i t r e s T i t r e s to anti-A and anti-B were performed by standard t i t -r a t i o n methods by members of the Vancouver General Hospital blood bank s t a f f . Miss E. Wilcox determined the tetanus t i t r e . S. typhi and S_. paratyphi "H" and "0" antibody t i t r e s were determined by the Pr o v i n c i a l Health Laboratories. EXPERIMENTS AND RESULTS 1. • Lymphocyte Transformation by PHA i n the Tuberculous Patient (53) The immune response of the body to the tubercle b a c i l l u s i s apparently accomplished by both c i r c u l a t i n g humoral antibody ( 54 ) and by cell-mediated immune reactions (55) . With the accumulating e v i -dence that i t i s the small lymphocyte which' i s responsible for c e l l -mediated immune reactions and Gowan's work on the c i r c u l a t i o n of the 19. 1. Lymphocyte Transformation by PHA i n the Tuberculous Patient (53) Continued... lymphocyte as mentioned i n the introduction, the response of the lymphocyte i n vitro has been used as an Indicator of the immuno-logical responsiveness of the individual. . Active tuberculosis i s a frequent complication of Hodgkin's disease and i t has been demonstrated recently that the circulating lymphocytes are abnormal i n this disease (48). Furthermore, re-activation of tuberculosis following steroid therapy i s a common complication (56) and i t i s now well established that steroids are toxic to lymphocytes (57). With this background.evidence to suggest the importance of normal lymphocyte function In host resis-tance and the accidental finding of a poor lymphocyte response in a tuberculous patient, a study of the in v i t r o reaction of the lympho-cytes in tuberculous patients was undertaken. Four categories of tuberculous patients were studied: (l) newly diagnosed and hitherto untreated patients, (2) patients undergoing treatment with drugs, (3) patients with arrested disease not under treatment, and (4) patients with atypical mycobacterial disease. Figure 1 reveals a significantly lowered P H A mitotic index i n tuberculous patients with inactive disease, with active disease under treatment, and with newly diagnosed disease as compared to normal controls. Of the normal controls, eight were tuberculin positive and nineteen were tuberculin negative. The mean mitotic index of the former was 33.1 and of the la t t e r 36.3. This d i f f e r -ence was not s t a t i s t i c a l l y significant. It had been reported i n the literature (58) that acetylsali-c y l i c acid would depress the in vitro lymphocyte response. To confirm t h i s , normal individuals were given from 2.4 g. to 7.2 g. of acetylsalicylic acid ( A S A ) a day for 4 to 5 days. Figure 2 shows the drastic f a l l in the mitotic index after A S A ingestion. 60-\ 50 y 30 I— o _-3 10-0 -• • — a. • •> • • • • • — t • X s tt • - • • • • • • I • • • • r • •• - •• • • • ••• • t — • * • t • • • • Normal No Therapy Recently Active TB patients New Inactive PAS Untreated TB patients PAS INH INH only TB patients INH Strep MEAN = 35.9 MEAN= 24.8 MEAN= 21.1 MEAN= 25.7 MEAN= 13.8 MEAN= 19.8 P<01 p<01 P<01 p<01 p<01 F i g . 1. Phytohemagglutinin mitotic indices of lymphocytes from normal and tuberculous patients. 21. 1. Lymphocyte Transformation by PHA i n the Tuberculous Patient Continued. The mean pre-therapy mitotic index was 36.5 and the mean post-therapy index was 13.0 (p = 0.01). To exclude any depressed responses due sol e l y to the drug, no individuals were included i n the study had they taken ASA or sodium s a l i c y l a t e 48 hours p r i o r to bleeding. Bearing i n mind s a l i c y l a t e i n h i b i t i o n , the question was raised whether the lowered response i n patients on para-aminosalicylic acid (PAS) was a r e s u l t of t h e i r chemotherapy and not an l i n t r i n s i c defect i n t h e i r lymphocytes. In an attempt t o answer the question nine normal ind i v i d u a l s were studied, each of whom received 6, 9, and 12 g. of PAS on succeeding days. The mean mitotic index before therapy was 32.0 and after therapy 26.7. This drop i s not s i g n i f i -cant (p = 0.4) . The mitotic index of patients taken o f f PAS and INH (isonico-t i n l c acid hydrazine) therapy for one week before sputum culture i s shown i n Figure 3. The mean value for the mitotic index on therapy was 16.1 and o f f therapy was 21.5 (p = 0.5). To determine the influence of prolonged therapy on the mitotic index the patients were grouped according to duration of therapy (Table I ) . The four patients with active disease, s t i l l on therapy after 24 months, had a mean mitotic index of 11.5. TABLE I. Change of mitotic index with drug therapy. Number of patients Months on drug therapy Mean mitotic index 6 0 20 8 0 - 2 28 .2 0 - 4 20 6 4 - 1 2 19 3 1 2 - 2 4 18 4 24+ 11.5 23. F i g . 3. Repeat phytohemagglutinin mitotic indices of lymphocytes from patients on and o f f drugs for one week. 1. Lymphocyte Transformation by PHA i n the Tuberculous Patient Continued... Such chronic patients were compared with 10 bedridden, non-tuberculous, chronically i l l patients from a nursing home (Fi g . 4) who had a mean index of 14.3. Also presented i n F i g . 4 are the indices of 5 patients chronically infected with an a t y p i c a l myco-bacterium whose mean response was 8.2. Repeat indices on normals and patients are shown i n Figures 5 and 6. Although some v a r i a t i o n does occur both i n healthy and sick i n d i v i d u a l s the v a r i a t i o n can be seen to be independent of the time i n t e r v a l between cultures. In attempting t o correlate the PHA mito t i c index with the response to the disease several i n t e r e s t i n g factors emerged. When graded r a d i o l o g i c a l l y at the time of lymphocyte culture as minimal, moderately advanced, or far advanced, there was no s i g n i f -icant difference between the groups, the mito t i c index ranging from 19 to 24. Fifteen patients with one or more recurrences or with non-remitting disease had a mean mitotic index of 19. The e f f i c a c y of.modern chemotherapy has so altered the course of tuberculosis that i t i s d i f f i c u l t to evaluate what might have been the patient's response to the disease without drug therapy. Three physicians independently assessed a l l patients c l i n i c a l l y . They attempted to ascertain i f immunological impairment was impor-tant i n the a c q u i s i t i o n or the progression of the tuberculous i n f e c t i o n In each patient of the study group. Their c r i t e r i a of impairment were as follows: ( l ) a poor response to drug therapy with the presence of sensitive organisms and no anatomical obstruction; (2) the onset of disease i n the f i r s t three decades; (3) a large i n i t i a l l e s i o n ( r a d i o l o g i c a l l y ) and a rapid rate of progression of the l e s i o n ; (4) poor c a l c i f i - ' cation i n the le s i o n and (5) the presence of other diseases with •known immunological significance such as lymphoma, collagen-vascular 25. 60 - i 5 0 -x 4 0 -•= 3 0 -2 0 -10-Tuberculous Non Tuberculous mean ~ 8.2 mean = 14.3 Fig. 4. Phytohemagglutinin mitotic indices of lymphocytes from tuberculous (atypical mycobacteria Type 3) and non-tuberculous chronically i l l patients. 26. F i g . 5. Repeat phytohemagglutinin mitotic indices of lymphocytes from normal subjects. 1. Lymphocyte Transformation by PHA i n the Tuberculous Patient Continued. disease and atopy. With the exception of three patient's picked by one physician, there was no appreciable c o r r e l a t i o n . These three patients had a mean mitotic index of 10. Two of these were of Chinese extrac-t i o n , born and raised i n Hong Kong, and both had primary resistance to streptomycin. A review of the s i x Hong Kong Chinese i n the series revealed a mean index of 15. Only two of the s i x were streptomycin-resistant and f i v e were improving on therapy. The mitotic indices of patients with a h i s t o r y of massive exposure were not s i g n i f i c a n t l y d i f f e r e n t from the indices of patients with a h i s t o r y of minimal exposure. In order to determine whether the abnormal mi t o t i c indices i n tuberculous patients were due to abnormalities i n the lymphocytes, plasma, or both, the effect of culturing lymphocytes from tubercul-ous patients i n plasma from normal persons and normal lymphocytes i n tuberculous plasma was determined.(Table I I ) . TABLE I I . Effect of plasma on the mi t o t i c index of PHA stimulated lymphocytes. Lymphocyte cultures M i t o t i c index Normal c e l l s (GH) + normal plasma (GH) 31 Normal c e l l s (GH) + tuberculous plasma (ES) 37 Normal c e l l s (BS) + normal plasma (BS) 28 Normal c e l l s (BS) + tuberculous plasma (ES) 34 Tuberculous c e l l s (ES') + tuberculous plasma (ES) 21 Tuberculous c e l l s (ES) + normal plasma (GH) 21 The normal lymphocytes had a normal mi t o t i c index when grown i n tuberculous plasma, while the tuberculous lymphocytes remained abnormal when grown i n normal plasma. 1. Lymphocyte Transformation by PHA i n the Tuberculous Patient Continued... No cor r e l a t i o n was found between the mit o t i c Index and the absolute lymphocyte count (Table I I I ) . ' TABLE I I I . Relationship of the mit o t i c index to the absolute lymphocyte count. Absolute lymphocyte count Number of patients Mean mitotic index 0 - 1000 5 27 1000 - 1500 5 17 1500 - 2000 7 23 2000 - 2500 16 17 2500 - 3000 2 33 3000 - 3500 6 26 3500 - 4000 3 22 2. Lymphocyte Transformation by PHA i n Acute Upper Respiratory Infections (59) During the course of the tuberculous study, one of the in d i v -iduals used as a normal control came down with the " f l u " on the evening of the day her blood was taken. The PHA mit o t i c index was found to be severely depressed. To study the effect of such acute upper respiratory i n f e c t i o n s , presumably of v i r a l o r i g i n , mitotic indices were determined on blood samples taken from laboratory volunteers who f e l t they were coming down with a cold. A l l 13 volunteers refrained from taking a s p i r i n before being bled. A second mitotic index was determined on these in d i v i d u a l s 8-12 days l a t e r when they f e l t they had recovered. Where no objections were raised by the individuals concerned, add i t i o n a l blood samples were obtained for further study (Table IV) . TABLE I V . M i t o t i c indices during and a f t e r acute upper respiratory i n f e c t i o n s . Patient C l i n i c a l l y i l l C l i n i c a l l y recovered MP 22 18 19 31 GR 32 24 PC 32 45 GH 12 18 BA 19 31 27 JW 11 23 14 DM 30 30 KB 20 30 36 LM 12 33 25 14 N 19 40 16 5 32 BL " "'29 " 40 33 15 J J ' 17 21 29 R 26 32 32 Mean value Mean value 20.2 p = 0.001 29.2 When the patients f i r s t stated they were unwell, the mean Index was 20.2 and when subsequently they stated that they f e l t w e l l , the index was s i g n i f i c a n t l y raised to 29.2 (p = 0.001). These values may be compared to 33.8, the mean index of healthy normals done at a si m i l a r time. ; As found i n the tuberculous study, plasma interchange had no 2. Lymphocyte Transformation by PHA i n Acute Upper Respiratory Infections Continued, effect on the m i t o t i c index. 3. The In V i t r o Response to PHA and PPD Following BCG Vaccination The reaction of delayed hypersensitivity and the r e l a t i o n s h i p of t h i s reaction to immunity and tuberculosis has been a matter of study f o r many years. I t seems clear that the i n d i v i d u a l s i n the population that l a t e r develop tuberculosis are drawn largely from those members who have a positive skin t e s t while those with a negative skin t e s t have been found u n l i k e l y to develop tuberculosis to the point of not being required to have X-rays i n many instances. Tuberculin PPD was among the f i r s t antigens shown to be cap-able of transforming lymphocytes i n v i t r o (60,61). For antigenic response the i n d i v i d u a l must have had p r i o r s e n s i t i z a t i o n . Usually s e n s i t i v i t y i s determined by the development of delayed hypersens-i t i v i t y following the intradermal i n j e c t i o n of antigen. But i t has recently become apparent that even skin t e s t negative i n d i v i d -uals may respond to the antigen i n v_tro (62,63). Thus i t seems that from the lymphocytic immunological reactive point of view the population may be divided into three reactive groups: 1) The group who are skin t e s t negative and lymphocyte negative - t h i s group presumably have.never come In contact with the tubercle b a c i l l u s and are t r u l y negative. 2) The group of individuals with no i n vivo response while demonstrating a positive i n v i t r o response. I t i s suggested that t h i s group have developed immunity without hy p e r s e n s i t i v i t y . 3) The group demonstrating both i n vivo and i n v i t r o response to the antigen. The disease tuberculosis develops as a 3. The In V i t r o Response to PHA and PPD Following BCG Vaccination Continued... c a v i t y i n the lung as a re s u l t of tissue reaction and i s s i m i l a r to that seen i n a necrotic reaction of a highly sensitive i n d i v i d -u a l at the s i t e of skin t e s t i n g . Based on t h i s the disease tuberculosis may be equated to a hypersensitive response. A l l student nurses entering t r a i n i n g at the Vancouver General Hospital are skin tested and vaccinated when t h i s response i s negative. Consequently volunteers from among the nurses were studied to compare the i n vivo and i n v i t r o ' responses to tuberculin. a) Group I Student Nurses. These student nurses had a l l received t h e i r BCG vaccination while i n t r a i n i n g at the h o s p i t a l . None had been vaccinated or skin tested i n the three months p r i o r t o lymphocyte culture. The lymphocyte response to the tuberculin antigen i n v i t r o was compared to the i n vivo response. The skin t e s t s were performed immediately a f t e r bleeding. - - ' As the i n v i t r o response to both PHA and s p e c i f i c antigens was 3 to be determined, incorporation of H-thymidine into the DNA of stimulated c e l l s was used to measure the ind i v i d u a l ' s r e a c t i v i t y rather than the less sensitive mitotic index determination. A l l re s u l t s are the mean of t r i p l i c a t e . c u l t u r e s and represent counts per minute (CPM) i n stimulated cultures minus CPM i n control c u l t -ures. Parke, Davis & Company PPD was used as the antigen. 3 . Table V shows the re s u l t s of H-thymidine uptake by lympho-cytes following stimulation by PHA and PPD. The r e s u l t s are grouped according to the response of the i n d i v i d u a l to the skin t e s t . Analysis of these r e s u l t s showed that although the mean value of the tuberculin negative group i s lower than the tuberculin p o s i t i v e group the difference i s not s t a t i s t i c a l l y s i g n i f i c a n t . I t 3. The In V i t r o Response to PHA and PPD Following BCG Vaccination Continued... must be emphasized though that the grouping of the individuals into 2 reactive groups was based only on the- response to 5 TU and not to a larger dose of tuberculin (250 TU) to which the majority of the / negative group were p o s i t i v e . Also the negative responders had received more than one BCG vaccination. Repeat cultures were done on i n d i v i d u a l s C (tuberculin positive) and WW (tuberculin negative) one month a f t e r the i n i t i a l cultures to confirm the negative i n v i t r o responses to PPD. C at t h i s time showed a p o s i t i v e response of 250,000 CPM to PPD. WW however, while showing an increased response 4 times greater than her o r i g i n a l response was s t i l l w e l l below the mean response for the negative group. TABLE V. 3 H-Thymidine incorporation following PHA and PPD stimulation i n group I nurses. Donor Skin t e s t response CPM 5 TU PHA " PPD SC + • • 1,282,040 285,733 CL + • 493,597 164,463 HMc + 1,081,684 257,216 SB + 955,177 698,041 MC + 1,251,552 289,126 C + 734,432 10,932 LE + 872,707 182,862 JE + 1,536,702 591,179 DG + - 145,136 KR . + 855,566 311,743 Mean values 12.5 1,007,050 293,643 EB 1,350,674 151,258 SC - 508,512 107,764 LE - - 197,884 LG - 1,330,575 408,878 MH - 1,027,188 289,556 CL — 1,001,004 239,861 JS — 565,808 94,831 WW — 467,324 15,962 MY - 1,255,405 260,102 Mean values 0.7 938,311 196,232 Tuberculin positive vs. tuberculin negative: PHA p = 0.5 PPD p = 0.1 Of importance was the finding of a po s i t i v e c o r r e l a t i o n between the general response to PHA and the s p e c i f i c response to PPD (r .= 0.59, p = 0.02) . • Immunoglobulin l e v e l s were determined by the ho s p i t a l chemistry department on serum obtained at the time of bleeding. These may be seen i n Table VI. The normal range of the immunoglobulins IgG, IgA, and IgM are 600-1200, 170-410, and 50-110 (mg.%) respectively. TABLE VI. Immunoglobulin l e v e l s of group I student nurses reported i n mg. %. Donor IgG IgA IgM MY 1450 360 ' 370 SC+ 800 235 220 MH 840 300 145 LG 920 175 109 KR 1050 205 235 SC- 820 68 99 HMc 1200 145 130 . EB 1200 135 215 CL+ 1200 160 130 SB 1200 79 160 JE 1100 230 370 CL- 840 250 230 LE- 840 69 310 MC 840 100 115 JS " 1000 235 85 DG • 900 350 165 C 700 140 200 LE + 1200 190 170 WW 1100 210 195 + represents skin t e s t p o s i t i v e i n d i v i d u a l . - represents skin t e s t negative i n d i v i d u a l . Correlation c o e f f i c i e n t s were determined between the immuno-globulin l e v e l s and the PHA response. Only between PHA and IgM was there the s l i g h t e s t indication of a po s i t i v e correlation ( r = 0.49, p = 0.05). Unfortunately only three in d i v i d u a l s had IgM l e v e l s that f e l l within the normal l i m i t s . The chemistry department considered the te s t to be t e c h n i c a l l y s a t i s f a c t o r y . 4. The In V i t r o Response to PHA and PPD Before and After BCG Vaccination a) Group I I Student Nurses. As group I nurses had been f i r s t vaccinated one year before 4. The In V i t r o Response to PHA and PPD Before and After BCG Vaccination a) Group I I Student Nurses. Continued... i n v i t r o studies were carried out, a new group of nurses were studied who were just entering t h e i r t r a i n i n g . This allowed in vivo and i n v i t r o studies of the development of the immunological response following BCG vaccination. Immediately preceeding i n i t i a l skin t e s t i n g by Miss E. Dorkin blood specimens were collected from 12 volunteers and cultured with PHA, PPD-S, and PPD-B. One week af t e r the i n i t i a l skin t e s t i n g the 9 g i r l s remaining negative to 250 TU were vaccinated with a freeze-dried Danish BCG having been bled f o r the second time just p r i o r to vaccination. Blood specimens were obtained at 2, 4, and 6 weeks post-BCG. The student nurses are routinely skin tested 6 weeks a f t e r the i n i t i a l vaccination to determine the rate of conversion. Individual i n vivo responses were concealed from the investigators u n t i l r e s u l t s of i n v i t r o cultures had been processed and tabulated to prevent any subjective influence on the interpretation of the r e s u l t s . Presented i n Tables V I I , V I I I , and IX are the i n v i t r o responses to PHA, PPD-S and PPD-B respectively. The response i s reported i n both CPM x 10 3 and as a per cent response of the mean for the p a r t i c -u l a r time of study. The l a t t e r became necessary due to the unusual responses i n a l l g i r l s at 4 and 6 weeks. At t h i s time a diminished response was observed rather than the expected increased response. On in v e s t i g a t i o n i t was found that the p a r t i c u l a r 3H-thymidine used at t h i s time although " f r e s h l y " prepared for the laboratory had come from an old supply. The radio-a c t i v i t y was s t i l l present when counted but decomposition of the 3H-thymidine had occurred so that although the l a b e l was s t i l l i n the solution i t was not present as part of the thymidine molecule. TABLE VII . PHA response i n student nurses (group II) before and af t e r vaccination with a standard dose of BCG (Danish). Donor Pre-BCG % Pre-BCG % 2 weeks % 4 weeks % 6 weeks % 12 weeks % Pre-Skin Res- Post-Skin Res- Post-BCG Res- Post-BCG* Res- Post-BCG* Res- Post-BCG Res-tes t „ ponse test „ ponse CPM x 10 3 ponse CPM x 10 ponse CPM x 10 3 ponse CPM x 10^ ponse CPM x 10 CPM x 10 DMc 552 78 310 67 277 62 188 117 92 71 1218 93 CB 863 123 533 116 538 '120 203 126 110 84 1487 113 CR 746 106 249 54 249 56 58 36 78 60' 641 49 C 839 119 719 156 120 27 254 158 146 113 1662 126 BT 878 125 309 67 824 184 • 204 127 191 148 1615 123 P 786. 112 . 437 95 522 117 102 64 138 107 1541 117 BD 692 98 679 147 565 126 114 71 145 112 1587 121 JF 587 83 367 80 552 123 169 106 176 136 1480 113 ND 392 56 540 117 379 85 149 93 , 87 67 585 44 * At 4 and 6 weeks post-BCG, the radioactive thymidine used had deteriorated during prolonged storage such that the results at t h i s time are not comparable. TABLE VIII. PPD-S response i n student nurses (group Ii) before and a f t e r vaccination with the standard dose of BCG (Danish). Donor Pre-BCG % Pre-BCG % 2 weeks % 4 weeks % 6 weeks % 12 weeks % Pre-Skin Res- Post-Skin Res- Post-BCG Res- Post-BCG* Res- Post-BCG* Res- Post-BCG Res-tes t „ ponse test „ ponse CPM x 10 ponse CPM x 10 s ponse CPM x 10 3 ponse CPM x 10 3 ponse DMc 20 146 11 40 8 22 12 33 13 28 388 92 CB 7 49 6 20 15 ' 40 9 26 33 72 939 222 CR 10 71 57 201 10 29 7 21 32 70 129 30 C -8 -60 30 106 28 79 63 175 63 138 395 93 BT 31 220 11 37 64 . 178 . 89 248 86 189 401 94 P 13 95 2 6 68 189 19 54 37 81 205 48 BD 17 124 121 430 81 227 39 108 65 142 418 99 JF 29 204 12 41 42 118 67 188 ' 55 121 688 162 ND 7 49 5 18 6 16 • 17 47 27 60 248 59 i At 4 and 6 weeks post-BCG, the radioactive thymidine used had deteriorated during prolonged storage such that the results at t h i s time are not comparable. I TABLE IX. PPD-B response i n student nurses (group Ii) before and after vaccination with the standard dose of BCG (Danish). Donor Pre-BCG % Pre-BCG % 2 weeks % 4 weeks % 6 weeks % Pre-Skin Res- Post-Skin Res- Post-BCG Res- Post-BCG* Res- Post-BCG* Res-test „ ponse test „ ponse CPM x 10 3 ponse CPM x 10 3 ponse CPM x 10 3 ponse CPM x 10 CPM x 10 DMc 18 169 4 11 8 . 2 9 8 39 16 64 233 81 CB 9 88 20 54 17 59 • 5 25 20 82 662 229 CR 3 28 34 92 5 19 5 26 23 93 32 11 C -8 -73 44 119 14 49 41 • 200 3 14 254 88 BT 21 194 13 33 73 257 59 287 37 149 324 112 P 18 172 19 51 53 187 3 16 32 128 204 71 BD 27 251 182 488 62 219 18 86 70 283 346 120 JF 2 21 . 14 • 38 20 70 37 179 3 12 ' 496 172 ND 5 49 4 11 3 10 • 9 41 19 74 • 44 15 " At 4 and 6 weeks post-BCG, the radioactive thymidine used had deteriorated during prolonged storage such that the results at t h i s time are not comparable. 12 weeks % Post-BCG Res-CPM x 10 3 ponse OS ! 4. The In V i t r o Response to PHA and PPD Before and Aft e r BCG Vaccination a) Group I I Student Nurses. Continued. Consequently, the radioactive l a b e l was not incorporated into the DNA of the c e l l . In order to report r e s u l t s i n CPM a further blood specimen was taken at 12 weeks post-BCG. The r i s e of the i n v i t r o response following vaccination i s graphically depicted i n Figures 7 and 8. As was seen i n group I nurses, a positive correlation was found between the PHA and PPD-S i n v i t r o responses. As only 9 g i r l s were included i n the second group of nurses, the re s u l t s of groups I and I I were combined (Fig . 9). A correla t i o n co-e f f i c i e n t of 0.61 was obtained which was s t a t i s t i c a l l y s i g n i f i c a n t (p = 0.01). 3 Table X gives the mean values of H-thymidine uptake for the three s t i m u l i used. 3 TABLE X. Mean values of H-thymidine incorporation i n group I I nurses following i n v i t r o stimulation 12 weeks after receiving a standard dose of Danish BCG vaccine. 3 Stimulus .•H-thymidine uptake - CPM PHA 1,313,246 PPD-S 423,465 PPD-B 288,272 I n i t i a l skin t e s t responses of these student nurses are shown i n Table XI. I t can be seen that except for 2 g i r l s who had a weak response to the a t y p i c a l mycobacterial antigens and 1 g i r l who reacted weakly both to a t y p i c a l mycobacteria and to 250 TU, i n vivo responses to Mycobacterium tuberculosis represented by PPD-S were completely negative. 500 -i 400-co o * 300-Q. U CO Q 200-CL. a. ^oo• 200-1 1 0 0 -PRE PPD-S CPM = MEAN OF 2 SAMPLES. SKIN TESTED BETWEEN SAMPLES. I SKIN TEST ' X , II • • BCG -1 0 WEEKS I I I I I I I I I I I I I -1 0 1 2 3 4 5 6 7 8 9 10 11 12 WEEKS F i g . 7. Effect of BCG vaccination as a function of time on the. i n v i t r o PPD-S response when measured by 3H-thymidine incorporation. Group IV = . Group I I = ... 500' 4 0 0 -o * 300' u Q 200 Q-a. 100-PRE PPD-B CPM = MEAN OF 2 SAMPLES. SKIN TESTED BETWEEN SAMPLES 200-100-0 y o-i - l o 1 i i I 2 3 4 5 } SKIN TEST -i o WEEKS 6 WEEKS T 9 I I I 10 11 12 F i g . 8 . Effect of BCG vaccination as a function of time on the i n v i t r o PPD-B response when measured by %-thymidine incorporation. Group IV . . Group -II = ... . F i g . 9. Correlation of the i n v i t r o PPD-S and PHA responses following BCG vaccination i n group I (.) and group I I (o) nurses measured by 3H-thymidine incor-poration. Dotted l i n e s represent - 1 S.D. 00 44. TABLE XI. I n i t i a l skin test response of group I I nurses to PPD-Sj PPD-B, PPD-G, and 250 TU measured i n mm. Donor PPD-S PPD-B PPD-G 250 DMc 0 0 0 0 CB 0 2 0 0 CR 0 0 0 0 C .0 0 0 0 BT 0 6 5 5 P 0 0 0 0 BD 0 5 3 0 JF 0 0 0 0 ND 0 0 0 0 Table XII shows the change i n the delayed hypersensitivity response s i x weeks af t e r vaccination. The mean values of the responses are as follows: PPD-S, 8.6 mm.; PPD-B, 7 mm.; and PPD-G, 11.1 mm. TABLE XII. Skin t e s t response of group I I nurses s i x weeks af t e r vaccination with a standard dose of BCG measured i n mm. Donor . PPD-S PPD-B . PPD-G 250 DMc 4 0 7 21 CB 5 4 8 22 CR 7 6 5 C 20 6 . 20 BT 7 12 20 P 7 5 12 BD 6 9 12 JF 11 13 10 ND 11 8 6 Skin t e s t i n g of the student nurses i s repeated at yearly i n t e r -vals throughout t h e i r t r a i n i n g i n order to ensure that t h e i r l e v e l of "immunity" i s maintained. Although i n v i t r o studies have not been carried t h i s f a r , the i n vivo response of group I I nurses one year after vaccination i s seen i n Table X I I I . Skin t e s t s were carried out only with 5 TU <PPD-S) or 250 TU. TABLE X I I I . Skin test response of group I I nurses one year following vaccination measured i n mm. Donor 5 TU 250 TU DMc CB CR C BT P BD JF 10 3 20 15 7 9 22 8 19 b) Group I I I Student Nurses. A number of investigators have studied the dose-response rel a t i o n s h i p of BCG vaccination as applied to the development of delayed hypersensitivity. There have been no attempts though to correlate the effect of dosage on the i n vivo res-ponse, as w e l l as the i n v i t r o response. Consequently 21 student nurses with negative skin t e s t s and thus e l i g i b l e for vaccination were bled and divided randomly into two groups. Nine g i r l s received one-fifth the regular dose of vaccine and 10 received twice the standard dose. Two nurses received the standard dose of vaccine by mistake. Routine PHA, PPD-S, and PPD-B cultures were set up at t h i s time. Six weeks l a t e r blood was again taken from a l l 21 g i r l s for culture and skin testing, was carried out. Due to the unusual skin t e s t responses at 6 weeks post-vaccination (8/9 small-dose recipients and 9/10 large-dose recipients were negative to 5 TU) and because of the unusually small area of s c a r i f i c a t i o n at the vaccination s i t e , the attending physicians concluded that the vaccine given had been unsatisfactory. As a r e s u l t , no attempt was made to correlate the i n v i t r o and i n vivo responses. Serum antibody t i t r e s to PPD were determined on pre- and post-vaccination blood specimens (Table XIV). Although minor TABLE XIV. Antibody t i t r e s to PPD i n a group (III) of student nurses before and after vaccination with BCG. Pre-BCG T i t r e s A n t i - Posit- Negat- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 body ive ive T i t r e Control Control 4. ++++ + + _ ++ ++ . ++ ++ + + + _ + + ++ + 8 +++ + + + + + + — 16 +++ + + + + + 32 ++ + + 64 + 128 + 256 + 512 . -4 _ tanned c e l l s + .+ + I + + ' +• + + untreated c e l l s Post-BCG T i t r e s 16 32 64 128 256 512 4 A + + ± ++ + ++ + + tanned c e l l s untreated c e l l s ++ ++ + + + ++ ++ ++ + + + + + .++ + + - +++ •++ ++ + + + ++ i + + + + 4. The In V i t r o Response to PHA and PPD Before and After BCG vaccination b) Group I I I Student Nurses. Continued... fluctuations occurred between pre- and post-BCG specimens, the variations were never more than one or two tubes and the direc-t i o n of change was not consistent, occasionally becoming more posi t i v e and at other times more negat'ive. A dosage effect cannot be detected. c) Group IV Student Nurses. The experimental design followed i n the study of group I I I nurses was modified s l i g h t l y for the fourth and f i n a l group of nurses. Blood specimens were taken from 30 volunteers before i n i t i a l skin t e s t i n g . Routine PHA, PPD-S, and PPD-B cultures were set up. Twelve g i r l s i n e l i g i b l e for vaccination due to positive skin t e s t s were replaced by new i n d i v i d u a l s at the time of the second pre-BCG bleeding. The freeze-dried vaccine administered to the 29 g i r l s f o l -lowing bleeding was a product of Connaught Laboratories. One g i r l received the International vaccine and was deleted from the study. Three groups were randomly formed and received either a standard dose, one-fifth standard dose, or twice the standard dose of vaccine. The i n v i t r o response was determined at 4 and 6 weeks post-BCG. After taking the 6-week specimen, skin t e s t i n g was carried out. The i n v i t r o responses to PHA, PPD-S, and PPD-B before and af t e r vaccination are shown i n Tables XV, XVI, and XVII. For comparison with the second group of nurses the response i s reported both i n CPM and as a percent response of the mean of a l l 28 nurses. Nurse number 29 was not included i n the study due to an abnormal response. Figures 7 and 8 show the r i s e of the i n v i t r o response with TABLE XV. PHA response i n a group (IV) of student nurses before and a f t e r vaccination with either a small, standard, or large dose of BCG (Connaught). Donor Pre-BCG, % Response Pre-BCG, % Response 3 weeks % Response 6.weeks % Response Pre-Skin a l l groups Post-Skin a l l groups Post-BCG a l l groups Post-BCG a l l groups test , test „ CPM x 10 . CPM x 10 3 CPM x 10 CPM x 10 Small Dose CA 1408 157 SG 481 54 SL 277 31 JMc 914 102 CMc 1023 . 114 KMc 861 96 MM 694 77 FS 1083 121 MS 659 73 TV 1338 149 Standard  Dose CB - 879 98 LB GC ND 1377 153 1030 163 950 328 52 939 86 14 444 396 63 . 592 539 85 985' 480 76 845 694 110 993 412 ' 6 5 806 429 68 989 737 117 1067 814 129 1233 206 33 684 754 120 1052 729 116 1285 101 1133 148 100 ; 1243 163 48 38 9 51 63 770 101 105 1112 ' 146 90 799 105 106 1001 • 131 86 794 104 105 690 90 114 924 121 132 1063 139 73 409 53 112 704 , 92 137 888 116 Continued... TABLE XV. Continued. Donor Pre-BCG, Pre-Skin t e s t r cm x 10c % Response a l l groups Pre-BCG, Post-Skin test „ CPM x 10 % Response a l l groups 3 weeks Post-BCG CPM x 10 3 % Response a l l groups 6 weeks Post-BCG CPM x 10 3 % Response a l l groups Standard Dose IF KG VS CW RW 369 991 41 110 334 975 134 780 935 53 155 21 124 148 683 695 230 842 1621 73 74 25 90 173 327 943 270 901 507 43 123 35 118 66 Large Dose MB PD LA CL NR GF GH DA WK 1137 559 1090 1011 127 62 121 113 861 605 501 1265 652 997 1234 494 881 137 96 79 201 103 158 196 78' 140 1082 488 1146 1195 1010 1416 1461 1386 1064 115 52 122 128 JL08 151 156 148 114 965 523 206 638 833 950 1365 709 1082 126 68 27 84 109 124 179 93 142 TABLE XVI. PPD-S- response i n a group (IV) of student nurses before and a f t e r vaccination with either'a small, standard, or large dose of BCG (Connaught). Donor Pre-BCG, % Response Pre-BCG, % Response 3 weeks % Response 6 weeks % Response Pre-Skin a l l groups Post-Skin a l l groups Post-BCG a l l groups Post-BCG a l l groups tes t _ test „ CPM x 10 3 CPM x 10 3 CPM x 10 CPM x 10 Small Dose • ' CA 197 141 32 63 43 28 498 124 SG 14 10 3 5 40 26 65 16 SL 19 13 29 57 29 18 129 32 JMc 39 28 -. .119 -0.2 10 7 263 66 CMC 98 70 • 49 97 184 118 821 205 KMc 635 456 ' 77 153 296 . ' 189 753 188 MM 9 6 44 88 71 46 356 89 FS 59 42 13 26 65 ' 41 50 12 MS 18 13 39 78 184 117 535 133 TV 361 259 245 484 272 173 : 588 147 Standard Dose CB LB GC ND 13 262 188 0.6 -3.8 20 11 1 -7 40 60 97 225 157 38 62 144 100 501 268 95 1105 125 67 24 275 Continued.. TABLE XVI. Continued. Donor Pre-BCG, % Response Pre-BCG, % Response 3 weeks % Response 6 weeks % Response Pre-Skin a l l groups Post-Skin a l l groups Post-BCG a l l groups Post-BCG a l l groups test „ CPM x 10 3 CPM x 10 3 test „ CPM x 10 CPM x 10 Standard . . Dose ' . • • . IF -21 -41 138 88 379 94 KG 88 174 398 • 254 581 145 VS 41 30 10 20 26 17 38 10 CW 186 368 95 60 879 219 RW 169 121 55 109 236 150 205 51 Large Dose MB FD LA CL NR GF GH DA WK 44 10 234 284 31 7 168 204 ' 4 26 118 101 9 81 143 32 87 52 233 200 17 161 283 63 171 113 23 284 219 98 180 189 259 84 72 15 181 139 63 115 121 165 53 497 260 103 369 142 183 510 634 156 124 65 26 92 35 46 127 158 39 TABLE XVII. PPD-B response i n a group (IV) of student nurses before and a f t e r vaccination with either a small, standard, or large dose of BCG (Connaught). Donor Pre-BCG, % Response Pre-BCG, % Response 3 weeks % Response 6 weeks % Response Pre-Skin a l l groups Post-Skin a l l groups Post-BCG a l l groups Post-BCG a l l groups test „ test CPM x 10 CPM x 10 3 CPM x 10 CPM x 10 Small  Dose CA SG SL JMc CMc KMc MM FS MS TV Standard  Dose CB LB GC ND 163 5 10 207 97 561 138 48 39 371 -7 427 104 3 6 131 61 356 88 30 25 235 -4 271 27 1 33 15 37 176 67 -1. 53 •175 17 -12 5 145 36 1 44 20 49 233 88 -2 70 231 23 -16 7 191 46 11 40 16 93 349 47 91 94 152 97 44 70 126 42 10 36 14 85 316 42 83 85 138 88 40 64 114 260 70 129 440 380 1069 242 82 250 369 205 146 83 1355 86 23 43 146 126 355-80 27 83 122 68 48 27 450 Continued... TABLE XVII. Continued.. Donor Pre-BCG, % Response Pre-BCG, % Response 3 weeks % Response 6 weeks % Response Pre-Skin a l l groups Post-Skin a l l groups Post-BCG a l l groups Post-BCG a l l groups test o test o CPM x 10 3 CPM x 10 3 CPM x 10 CPM x 10 Standard  Dose IF -16 -22 60 55 161 54 KG 86 113 294 266 126 42 VS 14 9 8 11 • 16 15 14 5 CW 184 244 68 61 399 132 RW 115 73 81' . 107 106 . , • 96 73 24 Large . . Dose . • MB 84 53 7 9 115 104 • 569 189 PD 7 5 34 45 31 28 60 20 LA 239 152 86 113 156 141 89 30 CL 319 202 158 208 148 134 375. . 124 NR . 4 5 60 103 94 56 19 GF 169 223 113 102 98 33 GH 172 227 96 87 315 105 DA . 65 85 187 169 219 72 WK • 82 108 73 66 111 37 4. The In V i t r o Response to PHA and PPD Before and After BCG vaccination c) Group IV Student Nurses. Continued... time. Each point on the graph represents the mean of a l l nurses i n the p a r t i c u l a r group. The i n v i t r o response attained by nurses when measured by the incorporation of H-thymidine was si m i l a r i n both groups. What was variable though was the length of time required to achieve the s i m i l a r i t y . Twelve wejeks had elapsed before those g i r l s receiving the Danish vaccine' had reached the l e v e l of response shown 6 weeks after Connaught BCG was given. In both groups the response to PPD-S was larger than the response to PPD-B.' As the graph depicts the i n v i t r o res-ponse to BCG, the pre-BCG response was an average of the i n i t i a l blood sample and the second blood sample taken before BCG but aft e r having been skin tested. The i n d i v i d u a l samples are shown i n the inserts of Figures 7 and 8. Disturbing also i s the wide d i s p a r i t y between the pre-BCG response of the two groups. From previous studies i n the labor-atory the mean response of group IV nurses would represent a positive response while the response i n the second group i s i n keeping with responses obtained from tuberculin negative respon-ders. What effect does dosage v a r i a t i o n have on the i n v i t r o response to the three stimuli? Table XVIII presents the mean values of i n v i t r o responses i n the three subgroups 6 weeks after BCG. As expected, there was no difference between the response to PHA as determined by an analysis of variance (p = O.l). PHA, being a general mitotic agent, should not be influenced by the development of s p e c i f i c immunity. 3 TABLE XVIII. Mean values of H-thymidine uptake i n group IV nurses following i n v i t r o stimulation 6 weeks a f t e r vaccination with either a small, standard, or large dose of BCG (Connaught). Stimulus Dosage S i g n i f i c a n t Small Standard Large difference PHA 885,303 667,995 807,740 0, p = 0.1 PPD-S 405,734 450,055 317,276 0, p = 0.1 PPD-B 320,114 284,646 210,218 0, p = 0.1 Prime interest though l i e s i n the response to a n t i g e n i c s t i m u l i . By analysis of variance, no s t a t i s t i c a l l y s i g n i f i c a n t difference could be observed among the subgroup's i n v i t r o response to either PPD-S or PPD-B (p = 0.1). The v a r i a t i o n between the responses i n the small and standard groups are quite s i m i l a r to those seen within t r i p l i c a t e cultures of one i n d i v i d u a l . There i s the impression though that the large dose of vaccine i s beginning t o induce a state of p a r t i a l unresponsiveness ( p a r t i a l tolerance). In a l l three groups the in v i t r o response to PPD-S was higher than the response to PPD-B. The effect of dosage on i n vivo, responses follows a quite different pattern i l l u s t r a t i n g the dichotomy between the two methods of measuring the immune capacity. Table XIX presents the pre-BCG skin t e s t response of the 28 subjects studied. Only 4 individuals showed any response to the skin t e s t s and was i n a l l cases a response to the at y p i c a l mycobacteria. The i n vivo response after BCG i s shown i n Table XX. Only 8/28 g i r l s were negative to 5 TU. I f compared to the t h i r d group of nurses where 17/19 individuals were negative to 5 TU i t appears that the decision to discontinue that experiment was correct. The mean responses of the groups to the skin test antigens PPD-S, PPD-B and PPD-G may be seen In Table XXI. Not unexpectedly the individuals receiving the regular dose of vaccine showed the TABLE XIX. I n i t i a l skin t e s t response of group IV nurses to PPD-S, PPD-B, PPD-G and 250 TU measured i n mm. Donor PPD-S PPD-B PPD-G 250 BCG-small dose CA SG SL JMc CMc KMc MM FS MS TV 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 BCG-standard dose CB LB GC ND IF KG VS CW RW 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 BCG-large dose LA DA .. MB PD GF GH WK CL NR 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 4 0 3 0 0 0 0 7 0 0 0 0 0 0 0 0 0 0 0 0 TABLE XX. Skin t e s t response of group IV nurses 6 weeks aft e r vaccination with either a small, standard, or large dose of BCG (Connaught) measured i n mm. •Donor PPD-S PPD-B PPD-G 250 BCG-small dose CA SG SL JMc CMc KMc MM FS MS TV 6 0 0 0 7 6 8 0 13 0 0 3 0 0 5 8 2 0 0 0 10 3 0 11 8 9 10 2 9 8 17 12 9 25 23 BCG-standard dose CB LB GC ND IF KG VS CW RW 9 10 14 11 10 3 5 10 12 8 6 6 8 7 0 0 10 7 12 13 9 19 13 6 5 12 14 24 11 BCG-large dose LA DA MB PD GF GH , WK CL NR 7 11 12 14 6 2 .0 7 7 13 4 3 3 6 5 12 18 17 12 10 6 8 12 2 17 4. The In V i t r o Response to PHA and PPD Before and After BGG vaccination c) Group IV Student Nurses. Continued... ~ best immunity when judged by the development of delayed hyper-s e n s i t i v i t y . TABLE XXI. Mean values of skin t e s t responses i n group IV nurses 6 weeks following vaccination with the smail, standard, or large dose of BCG (Connaught) measured i n mm. Stimulus Small Dosage  Regular Large PPD-S PPD-B PPD-G 4 p = o.Ol 9.3 8.5 p = 0.025 1.8—p = 0.02—5.7 5.3 ___ p = 0.05 7—p = 0.05 11.4 10.7 In contrast to the i n v i t r o r e s u l t s though an analysis of variance showed a s t a t i s t i c a l l y s i g n i f i c a n t difference between the small and regular dose response to PPD-S, PPD-B, and PPD-G (p = 0.01, p = 0.02, and p = 0.05 re s p e c t i v e l y ) . Although no s t a t i s t i c a l difference between the regular and large dose was observed, there i s the impression, as there was i n the i n v i t r o response, that a state of diminished responsiveness had been produced by the massive dose of antigen. The response i n vivo showed a higher l e v e l of r e a c t i v i t y to PPD-S than to PPD-B which p a r a l l e l e d the i n v i t r o response. When the PPD-S i n v i t r o response was compared to the PHA t e s t tube response no correla t i o n was observed when the 4. The In V i t r o Response to PHA and PPD Before and After BCG vaccination c) Group IV Student Nurses. Continued... determination of t h i s c o e f f i c i e n t was based on the responses of a l l 28 i n d i v i d u a l s . But, a s i g n i f i c a n t c o r r e l a t i o n (r = 0.66, p = 0.001) was found between the i n v i t r o PPD-S and PHA responses i n the subgroup receiving the regular BCG dose confirming what was already found i n groups I and I I . There was no correlation between the PPD-B response and the PHA response i n either groups I I or IV or within the subgroups of IV. The co r r e l a t i o n between the PPD-B and the PPD-S i n v i t r o response was excellent ( F i g . 10), when calculated on a l l group IV indiv i d u a l s (r = 0.76, p = 0.001). On a subgroup basis the regular group shows the best correlation (r - 0.83, p = 0.01) The group receiving the small dose was s i g n i f i c a n t at the 0.05% l e v e l with r = 0.68. While the responses of the large dose group show a cor r e l a t i o n c o e f f i c i e n t of 0.65, t h i s just escapes being s i g n i f i c a n t (p greater than 0.05). The s i g n i f i c a n t p o s i t -ive c o r r e l a t i o n p a r t i c u l a r l y i n the regular subgroup i s substan-t i a t e d by the response i n group I I where r = 0.96 (p = 0.001). In vivo correlations were also found ( F i g . 11). A r e l a t i o n -ship between the i n vivo response to PPD-B and PPD-S was estab-l i s h e d (a) when groups I I and IV were combined. This gave an r value of 0.57 (p = 0.001). (b) Group IV by i t s e l f showed a high co r r e l a t i o n c o e f f i c i e n t (r = 0.67, p = 0.001). Because dosage resulted i n a s t a t i s t i c a l difference between the small dose group and the other two dosage groups a correlation was determined (c) between the re s u l t s of the large and regular group r e s u l t i n g i n a regression l i n e having an r value of 0.77 (p = 0.001). Sig n i f i c a n t correlations were also found between the i n vivo responses to PPD-G and PPD-S (Fig. 12). Groups I I +. IV showed an 1400n PPD-B C P M x l O 3 F i g . 10. Correlation of the i n v i t r o PPD-B and PPD-S response as measured by 3H-thymidine incorporation i n student nurses (group IV) 6 weeks after vaccination with either a small, standard, or large dose of BCG (Connaught). Upper and lower l i n e s represent i 1 S.D. 6 1 . 20- • 19-18-17-16-PPD-S 15-14- o TO 13-[mm) 12-Z 11-* BO • o 10- o o s o INDURA1 9-8-A 0 ' • i. A i — to 7 * 1 / • LU 1— 6-< • ^ ^ • SKIN 5-' 4 -3-2-\ / / •> y< m yS i 1 G R O U P II > yS R E G U L A R D O S E yS G R O U P iv ySA S M A L L D O S E • • yS S T A N D A R D D O S E O 1 r = . 7 7 r - . 6 7 r = . 5 7 1- S L A R G E D O S E A J p < . 0 0 1 p < . 0 0 1 p < . 0 0 1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 SKIN TEST INDURATION ( mm) TO PPD-B •Fig. 11. Correlation of the i n vivo skin t e s t response to PPD-S and PPD-B following BCG vaccination i n groups I I and IV student nurses. Upper and lower l i n e s represent - 1 S.D. -Fig. 12. Correlation of the i n vivo skin t e s t response to PPD-S and PPD-G following BCG vaccination i n groups I I and IV student nurses. Upper and lower l i n e s represent i ' l S.D. 4. The In V i t r o Response to PHA and PPD Before and After BCG vaccination c) Group IV Student Nurses. Continued. r value of 0.62 (p = 0.001). Group IV alone showed a c o e f f i c -ient of correlation of 0.66 (p = 0.001). The large and regular subgroups combined resulted i n an r value of 0.61 (p = 0.01). When the relationship between the i n vivo responses to the a t y p i c a l mycobacteria was calculated, as i l l u s t r a t e d i n Figure 13, correlation c o e f f i c i e n t s of the various groups and subgroups were s t a t i s t i c a l l y s i g n i f i c a n t (p not more than 0.025) with the c o r r e l a t i o n c o e f f i c i e n t varying between 0.51 and 0.55. No c o r r e l a t i o n was found between in vivo and i n v i t r o res-ponses to the same st i m u l i (PPD-S or PPD-B) whether calculated on a group basis or on a subgroup basis. There was also no relationship between the i n vivo response to either PPD-S or PPD-B when correlated to the i n v i t r o PHA response. ^ — ^ The absolute lymphocyte count correlated neither with the i n v i t r o response nor with the i n vivo response. 5. Case Report of an Abnormal In V i t r o Response As the nurses used i n these studies were assumed to be normal healthy individuals whose responses could be considered representative of a s i m i l a r l y aged population, the 29th nurse (SR) was deleted from the study due to her abnormal PHA response. SR w i l l i n g l y gave her blood on a number of further occasions i n order that a more complete picture of her immunological status could be developed. I t was hoped that a more complete workup would define s p e c i f i c a l l y where the abnormality l a y . The studies carried out are shown i n Table XXII. o r I I I I I I I I I O 1 2 3 4 5 6 7 8 9 10 11 12 13 14 SKIN TEST INDURAT ION (mm) T O PPD-B Correlation of the i n vivo skin t e s t response to PPD-B and PPD-G following BCG vaccination i n groups I I and IV student nurses. . Upper and lower l i n e s represent 1 1 S.D. TABLE XXII. Case report of an abnormal i n v i t r o PHA response. A. . In v i t r o response to st i m u l i - CPM Time PHA PPD-S PPD-B SR Group IV Mean SR Group IV Mean SR Group IV Mean Pre-BCG 3,225 769,752 -6,253 94,972 . 45,291 117,280 3 weeks post-BCG 256 936,970 471,828 145,617 291,300 104,045 6 weeks post-BCG 21,046 760,789 667,727 391,547 600,040 290,862 Normal Normal Normal 11 weeks post-BCG 314,280 1,467,380 1,215,847 723,198 765,465 522,043 12 weeks post-BCG 445,235 1,634,276 — — — — 14 weeks post-BCG 31,974 1,349,208 - - - • B. In vivo skin t e s t response - mm Time • s. " " ^ B G .250 SR Group IV Mean SR Group IV Mean SR Group IV Mean SR ' Group IV Mean Pre-BCG 0 0 0 0.6 0 . 0.5 0 0 6 weeks post-BCG 0 7 2 4 8 10 15 17 C. Effe c t of plasma on i n v i t r o PHA response - CPM "Normal" c e l l s + autologous plasma 1,634,276 "Normal" c e l l s + "abnormal" plasma 123,382 "Abnormal" c e l l s + autologous plasma 445,235 "Abnormal" c e l l s + "normal" plasma 2,278,621 Continued... TABLE XXII. Continued... Immunoglobulin l e v e l s of "abnormal" individual - mg. % IgG 1250 normal range IgA 115 normal range IgM 130 normal range - 700 - 1500 - 125 - 425 - 50 - 150 Serum protein electrophoresis - gm. % albumin alpha_ globulin alpha2 globulin beta globulin / gamma globulin 4.88 (normal 3.5 - 5.5) 0.14 (normal 0.1 - 0.4} -0-..60 (normal 0.3 - 1.0) 0.43 (normal 0.5 - 1.3) 1.15 (normal 0.7 - 1.6) complement 0.115 (normal range 0.123 - 0.167) Antibody t i t r e s S. typhi "H" 1/40 S. paratyphi "H" 1/80 Tetanus 1/3200 Anti-A and Anti-B 1/128 5 . Case Report of an Abnormal In V i t r o Response Continued... Her PHA response as seen i n part A of the table and com-pared to the mean response of the other 28 nurses done at the same time shows an almost complete lack of r e a c t i v i t y . The p o s s i b i l i t y that PHA was not added to her cultures i s most u n l i k e l y , p a r t i c u l a r l y as the cultures were set up i n t r i p -l i c a t e and on di f f e r e n t days. At 6 weeks post-BCG, her PHA response had increased to 21,000 CPM. This magnitude of response may be seen i n cancer patients p a r t i c u l a r l y 3 weeks following i r r a d i a t i o n therapy and i s probably associated with the leukopenia and lymphopenia which develop (64). At a l l times studied, SR's hematological picture was e n t i r e l y normal. In contrast to the PHA response, the response to both PPD-S and PPD-B at 3 and 6 weeks post-BCG was excellent and at a l l times was much higher than the mean response of the other 28 nurses. Blood specimens were also taken at 11, 12, and 14 weeks post-BCG for leukocyte culture. Set up at the same time was a normal tuberculin positive i n d i v i d u a l as a t e c h n i c a l control. The PHA response had increased to 300,000-400,000 CPM which although much improved must be considered w e l l below the normal response. The PHA response at 14 weeks had once again shifted into a more negative range. The response to PPD-S and PPD-B at 11 weeks post-BCG was s t i l l increasing. Electron micrographs of PHA stimulated c e l l s of both a normal and SR showed a lack of large transformed lymphocytes i n SR's preparation as compared to the normal. There was a high percentage of polymorphonuclear c e l l s i n SR's preparation. The appearance of SR's PPD stimulated c e l l s was markedly different than her PHA c e l l s . They appeared to be more immature and 5. Case Report of an Abnormal In V i t r o Response Continued... b l a s t - l i k e i n appearance than the normal and these immature c e l l s were present i n large numbers. • Although showing a greater transformation to PPD than the normal (as judged by the presence of the b l a s t - l i k e c e l l s ) , the number of mono-cytes was much lower than the normal. Part B of the table shows SR's skin t e s t response as compared to the other 28 nurses. SR was o r i g i n a l l y given the large dose of BCG. Only one other g i r l who had received t h i s dosage showed a zero reaction to PPD-S. Although con-version occurred as shown by the po s i t i v e 250 TU response, the response of SR i s more i n keeping with the responses shown by recipients of the small dose of vaccine. At 12 weeks post-BCG, PHA cultures were set up to deter-mine whether there was any i n h i b i t o r y factor present i n SR's plasma (part C, Table XXII). ' U n t i l now there had been no evidence i n the laboratory to confirm reports,(65) of c i r c u l a t -ing i n h i b i t o r y factors. The effect of "normal" plasma on SR's c e l l s ("abnormal") was to increase the response f i v e times from 445,235 CPM to 2,278,621 CPM. Conversely SR's plasma markedly decreased the response of normal lymphocytes. Immunoglobulin levels of serum taken at 6 weeks post-BCG were a l l within the normal range (part D, Table XXII). Albumin, alpha, beta and gamma globulins were a l l normal. Complement l e v e l s were s l i g h t l y below normal. SR showed a weakly reactive t i t r e to S. ty p h i "H" antigen (1/40) and a reactive t i t r e of 1/80 to S. paratyphi B "H" antigen. There was no response to either S. ty p h i or _S. para-typhi B "0" antigens. Anti-A and anti-B t i t r e s were both normal (1/128). Antibody t i t r e s t o tetanus were positive (l/32 69. 5. Case Report of an Abnormal In V i t r o Response Continued... In summary, SR i s an i n d i v i d u a l who i s able to respond i n v i t r o to s p e c i f i c antigen, who i s .able to develop a humoral response as judged by the various antibody t i t r e s which were positive and yet who i s unresponsive to the gen-e r a l mitotic stimulant, PHA. DISCUSSION The r e s u l t s of the tuberculous study show t h a t , irrespec-t i v e of whether the tuberculosis i s active or healed the mean mitot i c index i s s i g n i f i c a n t l y lower i n tuberculous patients. These r e s u l t s are s i m i l a r to those reported by Rauch (66). The fact that the response of tuberculin positive normal con-t r o l s did not d i f f e r s i g n i f i c a n t l y from tuberculin negative controls indicates that a primary tuberculosis i n f e c t i o n does not a l t e r the responsiveness of the host lymphocyte. I t remains to be established whether a lowered mitogenic response to PHA i s analogous to a lowered immune c a p a b i l i t y and also whether the lowered immune response i n the tuberculous patient i s on a congenital or acquired basis. I f the low lymphocyte response was present congenitally the i n d i v i d u a l might be more susceptible to progressive disease. The incidence of tuberculosis i s high i n Indian and Eskimo populations. A survey of the i n v i t r o lymphocyte response i n these populations would perhaps answer the question of whether there was a correlation between acquired disease and a low PHA response congenitally acquired. Environmental influences could of course attribute to the increased s u s c e p t i b i l i t y . Recently i n the laboratory a small number of tuberculous Eskimos 3 were studied. The PHA response, when measured by H-thymidine incorporation, was approximately h a l f that seen i n normal Caucas-ian controls. This depressed response was not s i g n i f i c a n t l y d i f f e r e n t from that occurring i n Eskimos hospitalized with non-tuberculous respiratory Infections. Unfortunately, no healthy Eskimo controls were available for study. I f , however, the lowered index i s on an acquired basis i t may be secondary to the disease or any chronic i l l n e s s . As was seen i n Table I , 4 patients with active disease s t i l l on therapy a f t e r 24 months had a mean mitotic index of 11.5. A l -though only 5 patients with a t y p i c a l mycobacterial infections were studied, t h e i r mitotic responses were extremely low despite t h e i r r e l a t i v e l y good c l i n i c a l condition. The duration of active disease i n these patients was 1, 7, 11, and 12 years but one patient had no a c t i v i t y for two years. In view of the s i m i l a r l y low mitotic indices i n a group of chronically i l l , bedridden non-tuberculous patients, i t could not be determined whether the lowered indices i n patients with a t y p i c a l mycobacterial infections represent a f a i l u r e 'pf primary lymphocyte response or the effects of chronic active disease. In Figure 1 also, the low mitotic index of patients on INH may only be the r e s u l t of chronic i l l n e s s because two of the very low r e s u l t s were i n in-patients who have had unremitting tuber-cu l o s i s for over 15 years. I t i s common knowledge that with increasing age there i s a deterioration i n the immune response i n both the a b i l i t y to ward o f f infections and the a b i l i t y to .manifest delayed hypersensit-i v i t y reactions ( 6 7 ) . Pisciotta( et al. (68 ) found the mitotic index and percent blast transformation frequently lower i n e l d -e r l y Individuals than i n younger i n d i v i d u a l s . Could age account for the very low mitotic indices i n the chronically i l l bed-ridden patients? This i s u n l i k e l y as P i s c i o t t a et a l . found a mean index of 22 i n individuals 90 years of age which Is much higher than the indices found i n the chronic patients. S i m i l a r l y normal male controls over the age of 50 i n our laboratory show a lymphocyte response only two-thirds of younger normals when measured by the incorporation of ^H-thymidine into DNA. The response i s no lower than that shown i n tuberculous Eskimos with non-chronic disease. The type of response which develops ( c e l l mediated versus humoral) i n response to an antigen i s Influenced by the dose of the antigen. The response to Mycobacterium tuberculosis i s primarily cell-mediated. The fact that there was no s i g n i f i -cant difference between mitotic indices i n patients having a h i s t o r y of massive exposure as compared to minimal exposure suggests that at l e a s t beyond a l e v e l leading to c l i n i c a l d i s -ease the lymphocytes have an inherent capacity to respond to a stimulus whether i t be large or small. Because of reports (65,69) on the i n h i b i t o r y effect of tuberculous plasma on the response of normal lymphocytes to PPD s p e c i f i c a l l y and non-specifically to PHA by the action of a cytotoxic f a c t o r , plasma interchange was carried out on a num-ber of normals and patients. Contrary to the above reports there was no non-specific t o x i c factor present i n the plasma of the tuberculous patients studied. I t i s possible though that the disease i n these patients was not severe enough to l i b e r a t e such factors from the c e l l s into the serum. Both Heilman and McFarland (65) and Hirschhorn (69) found that the tuberculous patient's response was normal to PPD i f the c e l l s were cultured i n normal/plasma indicating that the lowered response i n tuberculous individuals did not represent a malfunction of the c e l l . Although no PPD cultures were set up i n our tuberculous study, the recent experiments on the tuberculous Eskimos showed a marked response to PPD indicating that there was no s p e c i f i c antibody present i n these patients' plasma which would depress the response as a l l cultures con-tained autologous plasma. I t i s f e l t that t h i s confirms and i s o l a t e s the primary defect at the c e l l u l a r l e v e l . The observation that the mitotic index was independent of the absolute lymphocyte count (Table I I I ) i s not surprising as a l l cultures were set up to contain 3 x 10^ lymphocytes. But, on a s t r i c t l y numerical basis i t seems reasonable to assume that those individuals having a higher absolute count could respond better to infections due to a greater number of antigen sensitive c e l l s present and/or a larger depot of c e l l s which could be recruited into active duty. I t i s evident that there i s a s i g n i f i c a n t l y depressed mit o t i c index during the acute phase of most upper respiratory infections (Table IV). The degree of suppression i n t h i s series i s comparable to that seen i n patients with acute tuber-culosis at the time of diagnosis. After c l i n i c a l recovery from the acute respiratory i n f e c -t i o n s , the patients' mean mitotic index bf 29.2 i s s t i l l some-what below the mean mitotic index of 33.8 i n normal individuals i n the laboratory. The presumption i s that they had not a l l recovered completely at the time the repeat indices were deter-mined. Of p a r t i c u l a r interest were cases N and LM. In v i t r o r e s u l t s when N f i r s t stated she f e l t w e l l confirmed her c l i n i c a l response. The mitotic index had r i s e n from 19 to 40. A repeat specimen taken.one week after.recovery however showed a mitotic index of only 16. N reported that the following day she had developed marked c l i n i c a l manifestations of recurrence or a new upper respiratory i n f e c t i o n . This suggests that the altered lymphocyte response occurs before the onset of c l i n i c a l symptoms. An i d e n t i c a l h i s t o r y developed i n patient LM whose blood was taken at the same time as N's. The i n i t i a l "sick" mitotic index of 12 had increased to 33 with the f i r s t symptoms of well-being. A blood specimen one week l a t e r though showed an index of only 25. This difference i s f e l t to be just barely at the level, of s i g n i f i -cance. After obtaining t h i s specimen, LM reported that she had again come down with an Infection. One week l a t e r LM's blood was resampled as she f e l t she had recovered. Her i n v i t r o res-ponse negated her c l i n i c a l f e elings. In fact the mitotic index was down to a l e v e l comparable with her f i r s t blood specimen taken when she f e l t i l l . This would perhaps .suggest that i n v i t r o recovery, following a second closely spaced i n f e c t i o n requires a longer i n t e r v a l of time than the i n i t i a l i n f e c t i o n . I t i s unfortunate that a f i f t h specimen was not taken from LM when N Ts l a s t specimen was obtained as N did not show i n v i t r o recovery u n t i l t h i s time. . What remains to be ascertained i s the c l i n i c a l s i g n i f i -cance of t h i s depression of lymphocyte response to PHA. A comparable effect on the PHA response has been demonstrated during rubella infections (49,50). The depressed response remains u n t i l the vir u s has been eliminated from the body. In infectious mononucleosis (70) " c i r c u l a t i n g lympho-cytes maintain an active RNA synthesis. DNA r e p l i c a t i n g c e l l s can be isolated from the c i r c u l a t i o n and therefore the usual reaction t o PHA would be subnormal not because of a functional capacity but because the c e l l s are already a c t i v -ated by i n vivo stimulus" (71). I t would seem possible that the depressed response to PHA r e f l e c t s a general i n h i b i t i o n of lymphocyte immune res-ponse which allows the tuberculous i n f e c t i o n to extend. In a s i m i l a r manner repeated upper respiratory infections may re s u l t i n reactivation or spread of tuberculosis. I t i s also suggested that a depressed PHA response may predispose to the secondary b a c t e r i a l infections which follow acute respiratory infections (72). This explanation would also be i n agreement with the established observations that large doses of adrenal steroids are known to be lymphocyto-; t o x i c and that patients on such therapy are subject to. b a c t e r i a l infections as w e l l as reactivation of old tubercul-ous lesions. The lowered mitotic index may be due to the increase i n the number of mitotic breaks which have been reported i n v i r a l infections (73,74). S i m i l a r l y the limited p r o l i f e r a t -ive function of lymphocytes with increasing age may be the res u l t of acquired errors i n c e l l d i v i s i o n . The i n v i t r o PHA lymphocyte response has also been shown to be depressed i n human hereditary immunological abnormalit-i e s a f f e c t i n g the lymphocytic series. Such patients are known to be highly susceptible t o v i r a l and fungal diseases as w e l l as being unable to reject skin transplants (12). From the data presented and discussed so far and from the l i t e r a t u r e c i t e d i t becomes apparent that the determination of the i n v i t r o PHA response i s one method of appraising an indi v i d u a l ' s general immunological c a p a b i l i t i e s . With the knowledge that s p e c i f i c antigens r e s u l t i n lymphocyte transfor-mation, i t has also become possible to measure s p e c i f i c respon-ses i n v i t r o t o such antigens as PPD. Before the refinement of i n v i t r o techniques, the develop-ment of delayed hypersensitivity to a s p e c i f i c antigen was taken as a measure of the individual's immunological responsive-ness. Consequently many attempts have been made to correlate the i n v i t r o response to PPD with i t s i n vivo counterpart. Whether measured by blast transformation, mitosis, or thymidine incorporation, i n vitro 7responses have been c o r r e l -ated either q u a l i t a t i v e l y or quantitatively with in vivo skin t e s t indurations (38,61,62,63,66,75,76,77). But i n the major-i t y of these studies there are always a few unexplainable false positive and/or false negative individuals.? A number of factors may account for discrepancies between the various laboratories. Many studies attempt to show the correlations i n tuberculous patients, or i n normal tuberculin positive i n d i v i d u a l s . The immediate problem a r i s i n g here and one which could account for many of the differences revolves around the causative organism: ( l ) Is i t the same organism i n a l l cases? (2) Is there a difference i n virulence? (3) Was exposure intimate? (4) Was exposure of long or short duration? I t i s true that i n the tuberculous patients studied here the amount of exposure, whether' massive or minimal, did not seem to a ffect the mitotic index. But i t i s possible that t h i s influence could only be detected at the l e v e l of the developing delayed hypersensitivity response. Perhaps exposure whether massive or minimal i s intimately associated with duration of contact. To circumvent t h i s variable influence student nurses were chosen to study as a l l negative tuberculin reactors receive BCG. Palmer and Nissen-Meyer (78) f e e l that 50% of a l l variance i s b i o l o g i c a l . Twenty-five percent of t h i s i s of f a m i l i a l o r i g i n and 25% due to i n d i v i d u a l factors. I f true and i f tech-n i c a l v a r i a t i o n s have been cut to a minimum what we are measuring and comparing are the host factors which control the response to a standardized dose of antigen. The development of tuberculin s e n s i t i v i t y as measured by delayed hypersensitivity i s used to indicate either i n f e c t i o n or an e f f e c t i v e vaccine. What i s meant by delayed hy p e r s e n s i t i v i t y (79)? Following the intradermal i n j e c t i o n of an antigen such as PPD no macroscopic changes at the i n j e c t i o n s i t e occur during the f i r s t 5-24 hours. This i s followed by erythema and induration which reach a peak at 72 hours after which v i s i b l e signs of res-ponse gradually disappear. • H i s t o l o g i c a l l y (79) there i s d i l a t i o n of c a p i l l a r i e s accoun-t i n g for the erythema^which develops and a c e l l u l a r i n f i l t r a t e across the walls of venules Into the s i t e of the antigen deposit-ion r e s u l t i n g i n induration. The predominant c e l l s at the onset are polymorphonuclear c e l l s but at ,24 hours t h i s dominance has been replaced by a mononuclear c e l l i n f i l t r a t e . Where do these i n f i l t r a t i n g c e l l s come from? Evidence has accumulated that they are not tissue macrophages but c e l l s a r i s -ing from the c i r c u l a t i n g pool. From the work by Lubaroff and Waksman (80) the o r i g i n of the c i r c u l a t i n g c e l l s which i n f i l t r a t e i n t o the t e s t s i t e i s believed to be bone marrow. I t has also been shown that 80% of the i n f i l t r a t i n g c e l l s belong to the monocyte/macrophage class which had been s e l e c t i v e l y concen-trated at the s i t e (81). These c e l l s represent the non-s p e c i f i c element of the response which arises at the t e s t s i t e following the i n i t i a l Interaction between antigen and a few sensitized c e l l s . The need for only a few sensitized c e l l s has been confirmed i n vivo (82) and i n comparable i n v i t r o experiments (83). The general feeling i s that t h i s i n i t i a l i n t eraction r e s u l t s i n the release of both s p e c i f i c and non-specific soluble factors which r e s u l t i n accumulation or recruitment i of non-sensitized c e l l s at the t e s t s i t e . These soluble factors have not as yet been conclusively proven to occur i n delayed hypersensitivity reactions but i n v i t r o a c t i v i t y implicates them strongly. These factors include: ( l ) lymph node permeability factor (LNPF), (2) blastogenic f a c t o r , (3) potentiating factor, (4) cytotoxin, and (5) migration i n -h i b i t i o n factor (MIF). The LNPF (84,85,86) has been isolated from supernatants of sensitized c e l l s with or without the addition of antigen. When t h i s supernatant i s injected into the skin of animals an increased permeability r e s u l t s at t h i s s i t e . I f released i n vivo such inflammatory reagents could account for the erythema of the reaction. Schilds and Willoughby (86) also found that the LNPF content of the skin and the i n t e n s i t y of the tuberculin reaction ran a p a r a l l e l course. Blastogenic factor i s so c a l l e d because i t induces mitosis and i s also released into the supernatants of lymphocyte cultures (87,88). I t i s easy to v i s u a l i z e that following i t s i n i t i a l release from the few sensitized c e l l s that have responded to the s p e c i f i c antigen i t may affect non-sensitized c e l l s that are present and that are passing by i n such a way that they remain at the t e s t s i t e to respond to the antigen. This could explain why, i n non-immunized animals injected with l a b e l l e d sensitized c e l l s the majority of c e l l s at the.reaction s i t e are non-sensitized host c e l l s . Blastogenic factor may be obtained also from c e l l - f r e e supernatants of long term lymphocyte cultures (89). I r r a d i a t i o n does not i n h i b i t i t s production (90). Potentiating factor (91,92) d i f f e r s from the above i n that i t does not cause blastogenesis but only augments the mitotic response of lymphocytes to antigen. Is i t possible that those individuals who show a greater response to antigen as measured by the incorporation of 3H-thymidine are able to produce more potentiating factor and/or blastogenic factor? Are t h e i r responses l i m i t e d not by t h e i r a b i l i t y to undergo transformation following antigen recognition but by t h e i r a b i l i t y to produce these non-specific factors which by t h e i r a c t i v i t y r e c r u i t non-sensitized c e l l s ? The development of delayed hypersensitivity at prepared t e s t s i t e s , that i s , at s i t e s where non-specific inflammation has been induced, develops extremely rapidly (93) which also argues strongly against s p e c i f i c a t t r a c t i o n of reactive c e l l s into the reaction s i t e . The release of a cytotoxic factor has been correlated with i n vivo delayed hypersensitivity reactions. Williams and Granger (94) have described a lymphotoxin which i s released from c e l l s either by s p e c i f i c antigen or PHA r e s u l t i n g i n non-s p e c i f i c k i l l of target c e l l s and which they f e e l could be an effector mechanism i n cell-mediated immune responses involving t i s s u e destruction. Ruddle and Waksman (95) also found a close c o r r e l a t i o n between the i n v i t r o c y t o t o x i c i t y and cutan-eous delayed hypersensitivity. Because the i n i t i a l turning on of the c e l l i s c a r r i e r s p e c i f i c , they f e e l that the phenomenon i s a true correlate of delayed hypersensitivity. But evidence from our studies indicates that the correlation of c y t o t o x i c i t y .with delayed hypersensitivity i s not so clear-cut. This though may only be a question of semantics. I f by delayed hyper-s e n s i t i v i t y we mean the erythematous indurated area that develops a f t e r intradermal i n j e c t i o n of antigen then the correl a t i o n i s not so d i s t i n c t . One normal i n d i v i d u a l from our laboratory whose i n vivo response to PPD was negative responded i n v i t r o to PPD. These stimulated c e l l s were also able to destroy "L" s t r a i n f i b r o b l a s t s . The a b i l i t y to k i l l therefore depends on the a b i l i t y of the c e l l to be activated. I t has been shown that i n an i n v i t r o heterograft and.allo-graft r e j e c t i o n system using PHA induced c y t o t o x i c i t y a lowered transformation and mitotic index response to PHA as occurs i n patients with Hodgkin's disease, lymphocytic leukemia and chronic disease usually r e s u l t s i n a lessened c y t o t o x i c i t y to the monolayer culture (96). Probably the most important soluble factor produced i n v i t r o by sensitized lymphocytes i s migration i n h i b i t i o n factor or MIF. The development of t h i s assay i s based on the obser-vation of Rich and Lewis (97) who observed that i f one took explants of spleen c e l l s or lymph node c e l l s from animals who had been immunized with PPD and placed I t i n t i s s u e culture, the addition of the s p e c i f i c antigen would prevent the explants from migrating normally. There was no antigen induced migrat-ion i n h i b i t i o n of normal explants. In 1962, George and Vaughan (98) further refined the tech-nique. I f peritoneal exudate c e l l s from immunized animals were placed i n small c a p i l l a r y tubes and fixed into chambers, the addition of antigen-containing media prevented the migration bf the c e l l s from the c a p i l l a r y when compared to the migration of non-sensitized c e l l s or sensitized c e l l s i n antigen-free media. By placing the c e l l s i n small c a p i l l a r y tubes the method became quantitative such that the area of migration could be measured. Dr. J. David has further modified t h i s technique and has done much to define chemically the nature of the i n h i b i t i o n (99). 80. Two c e l l types were necessary - the sensitized lymphocyte and the indicator c e l l or macrophage. I t was also found (100, 101) that i f sensitized c e l l s were incubated with antigen a factor (MIF) would be released into the supernatant which could then be assayed on normal guinea pig peritoneal macro-phages. Recently, Thor and co-workers (102) adapted t h i s technique t o man using human peripheral blood lymphocytes as the source of MIF and detecting i t by i t s i n h i b i t o r y effect bn guinea pig macrophages. The macrophage i n h i b i t i o n assay has been shown to cor-r e l a t e with delayed hypersensitivity (98,103). Recently, Rocklin et a l . (104) have attempted to assess the lymphocyte function of several patients with immunologic deficiency syndromes by means of the i n h i b i t i o n assay. They showed that normal thymic function i s necessary for production of MIF just as i t i s for the development of such cell-mediated immune res-ponses as delayed hypersensitivity. Consequently they found patients with DiGeorge's syndrome unable to make MIF. Unfortunately as with a l l other techniques of measuring delayed h y p e r s e n s i t i v i t y , t h i s method does not give the f i n a l answer. There i s still^some part of the puzzle missing. Rocklin and co-workers reported at the F i f t h Leukocyte Culture Conference (105) that i n some cases there does occur a dissoc-i a t i o n between the p r o l i f e r a t i v e lymphocyte response to antigen and the production of MIF. In 10/34 patients studied skin t e s t responses and MIF production were negative while incorpor-ation of °H-thymidine by blood lymphocytes was p o s i t i v e . From t h i s i t could be assumed that MIF production was s t r i c t l y assoc-iated with the development of delayed h y p e r s e n s i t i v i t y and that these individuals had developed immunity without hy p e r s e n s i t i v i t y . But they also found 4 patients with negative skin t e s t s and posi-t i v e MIF production. Could t h i s be due to non-specific anergy (106)? > Correspondingly, experimental work i n animals (107),.has shown further discrepancies. Following s e n s i t i z a t i o n to PPD the skin induration reached a peak very early i n the course of the study and then plateaued o f f whereas MIF pro-duction continued to increase. From t h i s evidence, Salvin (107) feels there i s some l i m i t i n g factor present i n the delayed response. Supporting t h i s would be t h e i r finding i n newborn animals. Here, the a b i l i t y to show hypersensitivity had not as yet developed but MIF was produced. Following the introduction of isol a t e d MIF intradermally, monocytes accumulate at t h i s s i t e within 6-8 hours (108). This lends support to the idea that MIF production by the lymphocytes at the t e s t s i t e accounts for the immobilization of monocytes i n the area. I t remains to be established what i s lacking i n the patients who f a i l to show delayed hypersen-s i t i v i t y even though there i s no impairment of the production of MIF. I t appears, therefore, that while the lymphocyte response to PPD i s necessary for delayed hyp e r s e n s i t i v i t y other factors may be equally Important. I t has been shown that the degree of tuberculin s e n s i t i v i t y induced by BCG vaccine i s not always related to the immunity conferred by the vaccination (109). Similar r e s u l t s i n animal experiments have been reported. Vaccinated animals (110) irrespective of t h e i r degree of a l l e r handled labelled tubercle b a c i l l u s i d e n t i c a l l y . At t h i s h o s p i t a l those g i r l s that f a i l to develop sensit-i v i t y following vaccination are revaccinated and again skin tested. This procedure may be repeated. Is i t r e a l l y neces sary though i f immunity i s not necessarily associated with sen s i t i v i t y and i f there i s no difference i n the handling of infectious organisms i n skin t e s t positive i n d i v i d u a l s as com-pared to individuals showing no allergy following one or more vaccinations? Palmer and Nissen-Meyer (78) f e e l that revac-cination i s not necessary i f the f a i l u r e to develop allergy resides i n an inherent lack of allergy-producing capacity and not due merely to technical f a i l u r e s . I t i s not an uncommon finding for tuberculin positive in d i v i d u a l s t o suddenly revert to a tuberculin negative status. Does t h i s represent a loss of immunity? Strom (110) has studied 53 lymphogranulomatous patients who l o s t t h e i r tuberculin s e n s i t i v i t y with progression of t h e i r disease. The re s u l t s indicated that tagged BCG reacted quite independently of the skin t e s t . His conclusion was that immunity was i n -t a c t . The i n v i t r o r e s u l t s i n group I nurses would tend to support the suggestion that revaccination to a t t a i n s e n s i t i v i t y i s not necessary. Although there was a s i g n i f i c a n t difference between the g i r l s that had converted and those that had not when based on the degree of allergy that developed, there was no difference i n v i t r o . The incorporation of %-thymidine following antigenic stimulation was s i m i l a r i n both groups. Based on the i n v i t r o r e s u l t s one could not predict the degree of skin s e n s i t i v i t y that would develop. I t i s suggested that there i s some unknown^factor necessary for the development of delayed hypersensitivity and which although present i s not present i n s u f f i c i e n t quantities to r e s u l t i n an a l l e r g i c res-ponse following the i n j e c t i o n of 5 TU. Perhaps 250 TU i s able t o activate the c e l l s more consequently r e s u l t i n g i n the production of more factor "X" - possibly MIF. There have been no reports of individuals able to respond to an antigen i n vivo while lacking the a b i l i t y to respond i n v i t r o . Because student C (group I nurses) showed t h i s unusual response a repeat culture was carried out which showed a good response to PPD. Two p o s s i b i l i t i e s could account for the i n i t i a l low response. I f i t was not the r e s u l t of some tech-n i c a l error i t i s possible that there was present i n C's plasma a s p e c i f i c but t r a n s i t o r y i n h i b i t o r . As no further studies were carried out on C the l a t t e r p o s s i b i l i t y cannot be ruled out. i t i s d i f f i c u l t to separate precisely responders from non-responders. The repeat response of WW (group I nurse) was d e f i n i t e l y higher than her o r i g i n a l response but s t i l l w e l l below the group mean. Because of t h i s she must be consid-ered a true negative but not i n the sense that she had never met the organism before. Tuberculin s e n s i t i v i t y did develop even i f only to 250 TU which suggests the presence of at least a few sensitized c e l l s . I f recruitment of other c e l l s takes place v i a the l i b e r a t i o n of soluble factors such as blastogenic and potentiating factor from the sensitized c e l l s i n i t i a l l y present there i s the p o s s i b i l i t y that WW lacks the a b i l i t y to produce them. I t i s extremely d i f f i c u l t to come to any d e f i n i t e con-clusion as far as the immunoglobulin l e v e l s of group I nurses are concerned. Although a correlation s i g n i f i c a n t at the 95% l e v e l was found between the PHA response and the IgM l e v e l s i s i t worthy of further exploration considering the unusually high l e v e l s within the normal range a l l belonged to the tuberculin negative group with the lowest responses to PPD, excluding the response of WW. I f IgM i s associated with the humoral res-ponse to PPD and subsequently to the tubercle b a c i l l u s i t seems unusual that there was no co r r e l a t i o n to the PPD response p a r t i c u l a r l y as the PPD response correlated to the PHA response. As the student nurses are representative of normal healthy i n d i v i d u a l s , the IgA levels obtained are perhaps also question-able as 8/19 g i r l s were below normal values set by the chemistry department. Here there did not appear to be any group specif-i c i t y as 3 of the individuals were negative responders and 5 Because of the s e n s i t i v i t y of the ^H-thymidine method The three g i r l s that had IgM positive responders. The IgG l e v e l s with one exception were normal. The one exception, MY, showed an elevated IgG l e v e l and also had the highest IgA and IgM l e v e l s . .In Rauch's study of BCG vaccinated in d i v i d u a l s (66) 6/7 showed positive hemagglutination t i t r e s following vaccination which she f e l t correlated with the p o s i t i v e leukocyte cultures. Contrary to t h i s , Hirschhorn (69) could find no correlation between the l e v e l s of antibody and the i n v i t r o response. S i m i l a r l y Daniel and Baum ( i l l ) found no difference i n antibody t i t r e s i n controls when compared to tuberculous patients. From our findings (Table XIV) i t seems obvious that both pre- and post-vaccination sera must be studied i n order t o judge the responses i n true perspective. Rauch (66) did not study pre-vaccination sera and cannot say that the hemagglutination t i t r e s above 1/16 were s i g n i f i c a n t . In f a c t , Freedman's study (112) of 51 healthy individuals suggests a t i t r e of l/80 as the l i m i t for negative reactions. The r e s u l t s obtained with group I I I nurses however do not exclude the p o s s i b i l i t y that s i g n i f i c a n t differences before and after vaccination may occur under the experimental conditions used providing a sa t i s f a c t o r y BCG i s used i n the vaccination program. The r e s u l t s pf^group I nurses suggest there i s no iri v i t r o difference between reactors and non-reactors as judged by the development of tuberculin s e n s i t i v i t y . I t was of interest to follow the development of immunological a c t i v i t y following BCG vaccination. I t has been determined by a number of i n v e s t i g -ators that the i n v i t r o response always precedes the conversion of the skin t e s t (15,63). Rauch (66) found evidence of i n  v i t r o s e n s i t i v i t y 3 weeks after BCG administration. Gotoff's r e s u l t s (15) suggested 2 weeks. Based on t h i s data group I I nurses were studied at 2, 4, 6, and 12 weeks post-BCG. As BCG dosage had no effect on the i n v i t r o response to PPD, i t i s not unreasonable to compare the development of the response i n v i t r o i n groups I I and IV.. For p r a c t i c a l pur-poses the 28 g i r l s i n group IV were studied only at 3 and 6 weeks post-BCG. To obtain a good negative baseline 2 pre-BCG samples were taken. One of these samples had been taken one week af t e r the i n i t i a l 5 and 250 TU skin t e s t s . Reports i n the l i t e r a t u r e have suggested that skin t e s t i n g may affect the i n v i t r o response to the p a r t i c u l a r antigen used. K e l l y and co-workers (113) found skin t e s t negative lymphocytes became positive a f t e r intradermal t e s t i n g with no marked increase i n the percent bla s t s u n t i l a f t e r 2 months. They also found the response of skin t e s t positive individuals enhanced. Matsan-i o t i s and others (63) also f e e l that i n tuberculin positive individuals as w e l l as tuberculin negative individuals increased a c t i v i t y may be due to skin t e s t i n g . Kerby found a transient but s i g n i f i c a n t r i s e i n the i n v i t r o transformation response to tuberculin a f t e r a positive skin reaction which lasted only 2 weeks (75). The insets of Figures 7 and 8 show a s l i g h t increase i n the response of group I I nurses to PPD-S and to PPD-B one week after skin t e s t i n g . I t i s doubtful whether t h i s increase i s s i g n i f i -cant and i s i n a l l j p r j p b a b i l i t y due to technical variations and not the re s u l t of skin t e s t i n g . The average of the pre-BCG responses to PPD-S and PPD-B (21,000 and 24,000 respectively) represents a t y p i c a l negative response. Group IV on the other hand showed a marked drop i n the i n v i t r o response following skin t e s t i n g . Although the average of the two responses was used as the pre-BCG l e v e l i t i s most l i k e l y that the second sample t r u l y represents the negative l e v e l and would be much more i n l i n e with the pre-BCG average of group I I . There was no difference i n the batch of PPD used f o r stimulating and a v a r i a t i o n here could not be held respon-s i b l e for the high i n i t i a l response. The i n v i t r o development of s e n s i t i v i t y following vac-cination occurs quite d i f f e r e n t l y i n the two groups when the response i s considered as a function of time (Figs. 7 and 8). I t i s suggested that t h i s difference i s not due to technical or i n d i v i d u a l variations but to v a r i a t i o n i n the immunizing capacity of the vaccines used (114). I t has been reported (115) that there are variations between the BCG vaccines produced by different centers. Group I I nurses received a Danish vaccine while group IV nurses received Connaught vaccine. The viable count of the vaccines used has also been shown to affect the degree of tuberculin s e n s i t i v i t y which develops (109,116) with vaccines of high viable count r e s u l t i n g i n a greater degree of s e n s i t i v i t y . On t h i s basis i t i s sug-gested that the Connaught vaccine was a more potent vaccine because of s t r a i n v a r i a t i o n and/or a higher viable count. Supporting t h i s i s the finding that the g i r l s receiving the regular dose of Connaught BCG had a higher mean induration to PPD-S than did group I I nurses. Whether the type of vaccine used would effect the ultimate i n v i t r o response to PPD cannot be concluded from the experiments. I t i s not known whether the i n v i t r o response at 6 weeks following the administration of Connaught BCG isjna-ximum such that the response l e v e l s o f f or whether i t continues to increase. I t would be most i n t e r -esting to see whether the f i n a l s e n s i t i v i t y attained In v i t r o i s present for a longer period of time i n the g i r l s receiving the Connaught vaccine when compared to the Danish re c i p i e n t s . Skin t e s t r e s u l t s one year post-BCG suggest that by t h i s para-meter the Danish recipients are maintaining t h e i r s e n s i t i v i t y (Table X I I I ) . Studying the effect of vaccination dosage on the tuber-c u l i n s e n s i t i v i t y i s not a new idea. What i s new and e x c i t -ing i s the effect that dosage was found to have on the i n v i t r o response when compared to i t s i n vivo e f f e c t . Edwards and Gelting observed an increased induration with 4 times the regular dose of BCG (117). These workers also found that small doses of vaccine decreased the size of induration but even a f t e r 1/256 of standard strength BCG more than 60% of the children tested reacted with .at least 5 mm. of indur-ation (118). Table XXI shows the mean skin t e s t response of the varying doses. In agreement with Edwards and Gelting (118) there i s a decreased skin t e s t response following the small dose of vaccine although the vaccine does not seem to confer the same degree of immunity (or allergy) as did t h e i r BCG. Contrary to t h e i r r e s u l t s the large dose of BCG used i n t h i s study decreased the skin t e s t response. I t i s true that the decrease i s not s t a t i s t i c a l l y s i g n i f i c a n t when compared to the response obtained with the regular dose but there i s the i n d i c a t i o n of a decreased response. The fact that a comparable decrease occurred i n v i t r o as w e l l suggests that i t i s not just a chance occurrence. A state of tolerance i s known to develop following the introduction of large doses of antigen which, l i k e a c t i v a t i o n , involves the same c e l l s . Dr. Braun (119) fe e l s that bypass of the primary antLgen/handling c e l l (macrophage), such that the antigen encounters the T c e l l s d i r e c t l y , may r e s u l t i n a state.of unresponsiveness. That i s , antigen i s normally modified by the macrophage which when attached to the lympho-cyte t r i g g e r s a response. By bypassing the macrophage though and attaching to the lymphocyte d i r e c t l y blockage ensues as the antigen has not been changed to an activated state by the macrophage. I t i s suggested that t h i s could account f o r the decreased response In these studies. Because stimulation did occur i t i s believed that some antigen had become processed and attached t o a few remaining unblocked receptor s i t e s . Had a larger dose of antigen been used perhaps a greater degree of non-respon-siveness would have been observed. Dosage had no s t a t i s t i c a l l y s i g n i f i c a n t effect on the i n v i t r o response to either PPD-S or PPD-B (Table XVIII). This dichotomy between responses i n vivo, and i n v i t r o could explain why there was no difference i n the mitotic index of tuberculous patients who acquired t h e i r disease following a massive or minimal exposure to the tubercle b a c i l l u s . I f MIF i s intimately associated with the delayed hypersensitivity response rather than the i n v i t r o response t h i s could be the factor which i s affected primarily by dosage. I f repeat vaccinations are given to non-responders and i f t h i s can be equated to an increased dosage i t seems l i k e l y that no further i n v i t r o stimulation would r e s u l t and repeat vaccinations should be eliminated providing the individuals have the capac-i t y to respond i n the test tube. Accepting that the PHA response measures the general immunological c a p a b i l i t y , i t i s not surprising that a positive correl a t i o n was found between the PHA response and the PPD-S response i n v i t r o i n groups I , I I , and IV. As the correla t i o n i n group IV occurred only i n the subgroup that had received the standard dose of BCG^dosage may have some effect on the i n v i t r o response which was perhaps missed because of the small sample numbers. No correla t i o n was found between PPD-B and PHA i n v i t r o responses suggesting that the vaccination induces s e n s i t i v i t y primarily to i t s tuberculin counterpart, PPD-S. This does not undermine the importance of using the at y p i c a l antigens i n routine t e s t i n g though as in f e c t i o n with these organisms does induce non-specific cross reactions (114,120) which could lead to false conclusions when using only PPD-S to detect s e n s i t i v i t y . That BCG does affect the response to a t y p i c a l mycobacteria though i s obvious from the excellent correlation i n v i t r o between PPD-S and PPD-B i n groups I I and IV. I t s s p e c i f i c i t y may be accounted for by the generally higher response to PPD-S. Here dosage did not seem to affect the correlation as i t was positive i n a l l 28-girls even though the subgroup receiving the regular dose did show the best correlation. I f the large dose subgroup were larger the r value of 0.65 would perhaps be s i g n i f i c a n t . Similar i n vivo correlations were seen between the t y p i c a l and a t y p i c a l antigens used as w e l l as between the a t y p i c a l antigens themselves. The correlation between the PPD-S and either PPD-B or PPD-G was s l i g h t l y poorer sugges-t i n g l e s s cross r e a c t i v i t y within the atypicals. The most s i g n i f i c a n t finding was the lack of correlation between i n vivo and i n v i t r o responses suggesting that these two parameters are measuring something e n t i r e l y d i f f e r e n t . I t would also indicate the need for a much closer study of the a l l e r g i c response as an indicator of immunity p a r t i c u l a r l y the role played by such soluble factors as MIF i n the develop-ment of delayed hypersensitivity. One patient has been reported (121) with chronic mucocut-aneous candidiasis whose lymphocytes were positive to Candida antigen i n v i t r o but who f a i l e d to develop delayed hypersensit-i v i t y and who f a i l e d r o produce MIF. I t was suggested that her i n a b i l i t y to handle the i n f e c t i o n was a r e s u l t of her i n a b i l i t y to develop delayed hypersensitivity so that here delayed hypersensitivity rather than being deleterious as sug-gested at the beginning of the thesis would perhaps be bene-f i c i a l . I ndividual SR with a very low PHA response and a very high PPD response presents as a most interesting and unusual case. Rubin (71) suggests the following as.causes of reduced PHA trans-formation: (1) humoral factors; (2) drugs such as steroids, a s p i r i n , or a l k a l o i d s ; (3) i n vivo activation of lymphocytes; (4) abnormal lymphocyte architecture associated with lympho-penia and a l t e r a t i o n of lymphocyte c i r c u l a t i o n ; (5) i n t r i n -s i c abnormalities of c i r c u l a t i n g lymphocytes. Drugs are not the cause of SR's abnormality for two reasons: (1) drugs would interfere with a l l i n v i t r o responses; (2) verbal v e r i f i c a t i o n from SR that she had not been under any s p e c i f i c medication and had not taken any a s p i r i n or s i m i l a r drugs p r i o r t o bleeding. Her apparent well-being with no previous h i s t o r y of major i l l n e s s e s would tend to rule out i n vivo ac t i v a t i o n of lymphocytes. Her normal hematological picture would discount suggestions of abnormal lymphocyte architecture associated with lymphopenia and a l t e r a t i o n of lymphocyte c i r c u l a t i o n . As was the case with drugs, these reasons could not account for the one-sided depression. Two p o s s i b i l i t i e s s t i l l remain from Rubin's suggestions. In considering an i n t r i n s i c abnormality of c i r c u l a t i n g lympho-cytes i t must be remembered that there was a good i n v i t r o response to PPD. As i t i s l i k e l y that lymphocytes are i n v o l -ved i n some fashion i n the production of antibody (presumably by plasma c e l l s ) , and as SR could manifest a humoral response (when based on her antibody t i t r e s ) t h i s cause may be ruled out. " ^ . This leaves only the presence of a humoral factor as the cause of the low response. SR's plasma, when used as a supplement for normal c e l l s , markedly inhi b i t e d t h e i r response to PHA. S i m i l a r l y "normal" plasma allowed a vigorous PHA response of SR's c e l l s . In addition to Heilman and McFarland's work on a s p e c i f i c i n h i b i t o r (65) , there i s a recent report i n the l i t e r a t u r e of a factor present i n s y p h i l i t i c patients which i n h i b i t s the PHA response of normal controls (122). The i s o -l a t i o n of an alpha globulin from normal lymphocytes has been described (123) which was suggested to function as a normal c i r c u l a t i n g immunosuppressive agent. A si m i l a r factor i n SR's plasma would not explain why i t affected only the PHA response. I t i s suggested though that SR has a c i r c u l a t i n g factor, possibly an antibody, s p e c i f i c for PHA. Attempts are being made to develop a rabbit-anti-PHA antibody to act as a positive control when te s t i n g for an anti-PHA antibody i n SR's plasma. I t w i l l be necessary also t o t e s t various concentrations of PHA i n an attempt to overcome the effect of the i n h i b i t o r on SR's lymphocytes. One further p o s s i b i l i t y remains i n r e l a t i o n to the naturally occurring feedback mechanism. A lymph node ex-t r a c t has been isolated which i s suggested to control m i t o t i c homeostasis (124). This extract most e f f e c t i v e l y blocked PHA induced transformation when added 2 days after the start of the cultures. When added on the l a s t day of culture, the i n h i b i t i o n was just s i g n i f i c a n t l y lower than control cultures which would suggest that perhaps i t had not been i n contact long enough to exert i t s e f f e c t . But the extract, i f added on the f i r s t day of culture, did not cause as severe a depres-sion as when added on the second day suggesting that i t s blocking effect i s not permanent. As the severest blockage occurs at day 2 when/t-he c e l l s are just entering DNA synthesis i t i s possible that a si m i l a r factor i s present i n SR's plasma. This would perhaps explain SR's extremely high PPD response. The factor has blocked (temporarily) transformation. When i t loses i t s e f f e c t , the c e l l s may be more or less synchronized as a resul t of the blockage res u l t i n g i n a burst of a c t i v i t y occurring at one time - i n t h i s case 7 days a f t e r i n i t i a t i o n o,f the cultures. I t remains to be established whether SR lacks the a b i l i t y to produce MIF which could be hypothesized from her t o t a l lack of response to 5 TU after receiving a large dose of BCG. I t i s true that she responds to 250 TU but Palmer feels t h i s only represents i n f e c t i o n with a different organism - i n t h i s case to PPD-B and PPD-G. From the experiments that have been conducted, from the re s u l t s that were obtained and from the r e s u l t i n g discussion i t i s obvious that there are l i m i t l e s s areas for exploration. The major questions s t i l l unanswered are numerous. Is sus-c e p t i b i l i t y t o tuberculosis acquired or congenital? What role do the so-called soluble factors (blastogenic factor, potentiating factor, migration i n h i b i t i o n factor) play i n the development of the in vivo and i n v i t r o response i f indeed they play any role at a l l ? Are they instrumental i n r e c r u i t -ment of non-sensitized c e l l s ? Are these the factors which are l i m i t i n g the developing response rather than an i n a b i l i t y to activate the sensitized c e l l s ? Does delayed hypersensit-i v i t y have a b e n e f i c i a l or deleterious effect on the disease process? Is the i n v i t r o response to antigen enough? I f the answer to the l a t t e r question i s affirmative serious thought must be given to revaccination programs i n individuals who either f a i l to convert or lose t h e i r h y persensitivity. For i n v i t r o t e s t i n g to be even feasible on a large scale a sat i s f a c t o r y microtechnique would have to be developed. F i n a l l y , i t would be necessary to determine the eff e c t both i n v i t r o and i n vivo of the varioi^s^ vaccines used, t h e i r potency and t h e i r e f f e c t i v e i n t e r v a l of action. I t was called by Jerne "waiting for the end". CONCLUSIONS 1. The mean mitotic response to phytohemagglutinin of lymphocytes from people with inactive and active tuberculosis i s s i g n i f i -cantly lower than the mean of the normal population. 2. Chronic i l l n e s s has a severe depressant effect on the mitotic index. 3 . The mito t i c response to PHA i s depressed i n individuals suffer-ing from acute upper respiratory infections. 4. Non-responsiveness i n vivo to the antigen PPD following BCG vaccination cannot be detected i n v i t r o by the methods employed i n the study. 5. The i n v i t r o and i n vivo response that develops following BCG vaccination i s dependent on the p a r t i c u l a r vaccine used. 6. The indiv i d u a l ' s PHA" response i s an in d i c a t i o n of the response that can be mobilized to the antigen PPD-S following BCG vaccination. 7. The in v i t r o response's^which develop following vaccination with either a small, standard, or large dose of BCG do so regardless of the dose of vaccine. 8. The dose of vaccine used plays a s i g n i f i c a n t role i n the e l i c i t -ation of the delayed hypersensitivity response. 9. The lack of a quantitative correlation between i n v i t r o and i n vivo responses suggests that one i s measuring two r e l a t i v e l y unrelated immunological events. ACKNOWLEDGEMENTS I t i s a pleasure to express my sincere gratitude to my supervisor, Dr. J.W. Thomas, for h i s continuous encourage-ment and guidance throughout the course of the study and during the preparation of the manuscript. The student nurses of the Vancouver General Hospital deserve special thanks for t h e i r willingness to volunteer t h e i r blood, especially SR who has raised no objections to the frequent samplings during the past 7 months. Credit must also go to Mrs. R. Burge, R.N., who so ably managed to co-ordinate the a c t i v i t i e s of the trainees such that they were always at the appointed place at the specified time. I would also l i k e to thank the g i r l s of laboratory 1 at the B r i t i s h Columbia Cancer I n s t i t u t e who helped me c o l l e c t the blood samples. In p a r t i c u l a r I express thanks to Mrs. A. Yuen who came back to work many evenings to help prepare cultures. The help and_advice of Mrs. M.E.J. Young and Miss E. Swait was invaluable during the standardization of the "Liquimat 220". 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