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1

ABBREVIATIONS USED µCi

Microcurie

µg

Microgram

µl

Micrilitre

µm

Micrometre

51

51

Cr

Cromium

BARC

Bhava Atomic Research Centre

BCR

B Cell receptors

BDI

Beck self rating depression inventory

Bmax

Maximum number of binding sites

BSA

Bovine Serum Albumin

CA

Catecholamine

CD3

Cluster of differentiation 3

cDNA

Complementary deoxyribonucleic acid

cm

Centimetre

COMT

Catechol-O-methyl transferase

Cpm

Counts per minute

CRH

Corticotrophin releasing hormone

DA

Dopamine

DAB

¶¶GLDPLQREHQ]LGine

DAT

Dopamine transporter

DNA

Deoxyribonucleic acid

dNTP

Deoxy nucleotide phosphate

DSM-IV

Diagnostic and statistical Manual for mental health

DTT

Dithiothritol

ECL

Enhanced chemilluminescence 2

EDTA

Ethylene dinitrilo tetra acetic acid

EGTA

Ethylene glycol bis-N, N, N, N-tetra acetic acid

ELISA

Enzyme linked Immuosorbent Assay

g/gm

Gram

GAD

General Anxiety Disorder

HBSS

+DQN¶VEDODQFHGVDOWVROXWLRQ

HIV

Human Immuno deficiency Virus

HPLC-ECD High Performance Liquid Chromatography with electrochemical detection HRP

Horse reddish peroxidase

Hrs

Hours

HRSD

Hamilton rating scale for depression

IFN-Ȗ

Interferon gamma

IgG

Immunoglobulin

IgM

Immunoglobulin M

Ki

Dissociation constant

l

Liter

m

Molar

mAb

Monoclonal antibody

MAO

Monoamine oxidase

mg

Milligram

mins

Minutes

ml

Milliliter

mM

Milimolar

MMLV

Mouse Murine Leukemia Virus

mol

Mole 3

MPTP

1-methyl-4phenyl1,2,3,6 tetrahydropyridine

mRNA

Messenger ribonucleic acid

n

Total number

ng

Nanogram

NK

Natural killer

nM

Nanomolar

PBMNCs

Peripheral blood mono nuclear cells

PBS

Phosphate Buffer Saline

PCR

Polymerase Chain Reaction

Pg

Picogram

PMSF

Phenyl methyl sulphonyl fluoride

POPOP

1, 4-bis[5-phenyl-2 oxazolyl] benzene

PPO

Diphenyloxazole

PVDF

Ploy vinyl difluoride

RDC

Research and diagnostic criteria

RPMI

Roswell Parker Memorial Institute

RT

Reverse transcription

RT-PCR

Reverse transcriptase polymerase chain reaction

SADS

Schedule for affective disorder and schizophrenia

SDS

Sodium dodecyl sulphate

SDS-PAGE Sodium dodecyl polyacrylamide gel electrophoresis SEM

Standard Error of Mean

TBS

Transfer buffer saline

TCR

T-cell receptor 4

TEMED

N,N,N,N tetramethylethylenediamine

TGF-ȕ

7UDQVIRUPLQJJURZWKIDFWRUȕ

TH

Tyrosine Hydroxylase

TTBS

Transfer buffer saline with Tween-20

V

Volts

Yrs

Years

CONTENTS

Page From

1. GENERAL INTRODUCTION

09

1.1. Conditions for different effects of stress on immune function

10

1.2. Anxiety Disorders

13

1.3. Depression

18

1.4. Psychosocial stressors and health consequences 21 1.5. Cancer caregivers

27

1.6. Effects of stress on immune system

28

CHAPTER-1 PLASMA AND INTRACELLULAR DOPAMINE CONTENT IN T&B CELLS OF NORMAL VOLUNTEERS AND CANCER CAREGIVERS

2. INTRODUCTION

33

3. MATERIALS AND METHODS

40

3.1. Selection of subjects and psychiatric assessment 40 3.2. Assay of dopamine in plasma and T 5

& B lymphocytes

42

3.3. Assay of tyrosine hydroxylase activity

60

3

61

3

3.5. Assay of [ H] DA uptake by B-lymphocytes

63

3.6. Statistical analysis

67

4. RESULTS

67

4.1. Plasma level of dopamine

67

4.2. Dopamine content of T lymphocytes

68

4.3. Dopamine content of B lymphocytes

71

3.4. Assay of [ H] DA uptake by T-lymphocytes

4.4. Tyrosine hydroxylase activity in T & B lymphocytes

74

4.5 Uptake of [3H] DA by T & B lymphocytes

75

5. DISCUSSION

78 CHAPTER-2

EFFECT OF PHYSIOLOGICAL CONCENTRATION OF DOPAMINE IN T & B CELLS FUNCTIONS 6. INTRODUCTION

86


90

7.1. T- cell proliferation assay

90

7.2. B- cell proliferation assay

92

7.3. T- cell cytokine assay (Assay of IL-2 and IFN-Ȗ

95


102

8. RESULTS

102

8.1. Effect of dopamine on proliferation of T & B lymphocytes

102 6

8.2. Effect of dopamine on differentiation (cytokine release) of T lymphocytes

108

9. DISCUSSION

113 CHAPTER-3

MOLECULAR MECHANISM OF DOPAMINE MEDIATED ACTION ON T&B CELL FUNCTIONS

10. INTRODUCTION

120


122

11.1. DNA fragmentation assay

122

11.2. Dopamine receptor assay

130

11.3. Semi-quantitative analysis of D2 r eceptor mRNA assay

135

11.4. Immunoblotting of Src family protein tyrosine kinase (p56 Lck & p59fyn) of T cells and (p53/56lyn & p55blk) in Bells

148


165

12. RESULTS

165

12.1. Effect of dopamine on apoptotic death of T & B cells

165

12.2. Effect of different classes of dopamine receptor antagonists on T & B cell functions 3

12.3. [ H] -Dopamine binding on T & B cells

167 168

12.4. Semi-quantitative mRNA expression of DA2 receptor from normal and cancer caregivers

174

12.5. Effect of dopamine induced intracellular signaling on 7

(p56 Lck & p59fyn) expression in T lymphocytes

180

12.6. Effect of dopamine induced intracellular signaling on (p53/56lyn & p55blk) expression in B lymphocytes

182


185

13. DISCUSSION

185

14. SUMMARY

196

15. REFERENCES

200

8

GENERAL INTRODUCTION 1. GENERAL INTRODUCTION: Stress "the nonspecific response of the body to any demand" is a process of adaptation which develops as a reaction to a stimulus (called the stressor) and is manifested through changes in hormones and catecholamines (CA) levels. It is nonspecific in its causation: it is a general response elicited by psychological, physical, or chemical agents. Activation of sympatheticadrenomedullary

system

resulting

in

the

secretion of CA and of pituitary adrenocortical system

resulting

in

the

secretion

of

glucocorticoids is classical indication of stress reaction. These hormones induce a number of' systemic modifications: the thymus and lymph node

involutes;

immune

and

inflammatory

reaction becomes evident. It has been proposed that nearly two-thirds of the human ailments are either induced by or related to stress. Stress is a major contributor to the development of many physiological disorder and psychiatric illness. Stress also increases the 9

susceptibility of' the body to infection, auto immune diseases and cancer (DaSilva JAP, 1999; Maddock & Pariante, 2001; Bryla, 1996; Anderson et al, 1994). The underlying mechanism of this stress mediated increased susceptibility to diseases was assigned to altered functional activity of different immune effector cells (Dobbs, 1995; Keller et al, 1998; Cohen et al, 2001; Dominguez- Gerpe et al, 2001 & Nagabhushan et al, 2001).

1.1. CONDITIONS FOR DIFFERENTIAL EFFECTS

OF

STRESS

ON

IMMUNE

FUNCTION: A variety of dimensions may be of important in characterizing stressors (Cohen & Williamson, 1991; O'Leary, 1990; Herbert & Cohen, 1993). Among these dimensions, major ones arc acute and chronic stress which have different effect on immune system. Acute stress has been shown to affect numerous physiological parameters (Glavin et al, 1994). It activates

the

autonomic

nervous

system

(Kvetnasky, Fukuhara, Pacak, Cizza, Goldstein & Kopin, 1993) as well as the hypothalamic10

pituitary-adrenal axis (Dhabhar, McEwen, & Spencer, 1993; Dhabhar, Miller & Spencer, 1995a) resulting in the release of corticostreone and

catecholamines.

immune

Acute

enhancement

is

stress

induced

mediated

through

antigen presentation, effector cell function, antibody production and cytokine production. Support for this hypothesis comes from studies showing stress induced enhancement in vitro immune parameters such as mitogen induced lymphocyte proliferation (Lysle, Cunnick & Rabin, 1990; Rinner, Schauenstein,, Mangge, Porta & Kvetnsky, 1992; Wood, Kusnekov & Rabin, 1993; Shurin, Zhou, Kusnekov, Rassnick & Rabin, 1994; Wiegers, Reul, Holsboer, & De Kloet, 1994), macrophage phagocytic function (Lyte, Nelson, & Thomson, 1990), NK cell activity (Jain, Stevenson, 1991; Millar, Thomas, Pacheco, & Rollwagen, 1993), and IL-l, IL-2 and IFN-Ȗ production (Mekaouche et al, 1994; Petitto et al, 1994). Blecha et al (1982) have shown that acute stress enhances cutaneous delayed type hypersensitivity in mice and stress has been shown to accelerate antigen removal and to increase antigen specific antibody titers (Cocke et al & Ader, 1993; Wood et al, 1993; Persoons & Kraal, 1995). 11

Chronic stressors have different effects on immune system (Cohen and Williamson, 1990; Kiecolt-Glaser

and

Glaser,

1991;

O'Leary,

1990). Chronic stress suppressed immune system (Dhabhar FS & McEwen BS, 1997). Immune suppressive effects of chronic stress may be mediated by mechanisms involving inhibition of T-cell

activation,

antigen

presentation

and

inhibition of inflammatory mediator actions, activation of lipocortins, induction of lymphocyte apoptosis or suppression of effector cell function (Schleimer et al, 1989). The suppression of DTH may he related the overall chronicity of stressor exposure and or/ specifically to the inhibition of process involved in T cell activation during sensitization.

Immuno-suppression

observed

following repeated stress was a function of chronicity of stress, and not merely a function of the timing of stressor administration prior to sensitization because repeated stress administered for 7 a days before sensitization produced neither a suppression nor an enhancement of cutenous DTH (Dhabhar, 1996). The correlation between changes in corticosterone and catecholamines levels with chronicity of stressor exposure is indirect evidence that stressor duration is a critical factor determining the direction of changes in 12

immune reactivity following stressor exposure. In modern society majority of people are suffering from chronic stress due to increased psychosocial problem, arising out of present day social structure. Two expressive forms of chronic stresses are Anxiety and Depression which are discussed below -

1.2. ANXIETY DISORDERS: Normal A n x i e t y : Anxiety is a mood, usually unpleasant in nature, accompanied by bodily (Somatic) sensation and occurring with a subjective feeling of uncertainty and threat about the future. Most of the bodily changes seen in anxiety are caused by increased sympathetic adrenergic system discharges, i.e. Cannon's fight or flight reaction which results in the

release

of

adrenaline

and

other

catecholamines. We may merely experience such reactions when under stress in everyday life. The common mixed pictures of anxiety are as follows. Community surveys suggest about 25% 13

of the adult population are suffering from anxiety disorder at any time. About 15% of the patients attending GP surgeries are `anxious'. Women are affected more than men, upto ratio of 2:1 in some surveys. Anxiety is characterized by intense negative affect, associated with an undefined threat to one's physical or psychological status. It is additionally characterized by somatic, cognitive, behavioral, and perceptual symptoms. The somatic symptoms of anxiety are twitching tremors, hot and cold flashes,

increased

blood

pressure,

sweating,

palpitation, chest tightness, difficulty swallowing and nausea etc. Anxiety symptoms can results from numerous changes including endocrine, auto immune, metabolic and toxic disorders. The coexistance of anxiety symptoms and depression in major depressive disorder is substantial; symptoms such as anxious mood and irritability are seen in the majority of depressed patients.

Further division of stress related disorders were A) G e n e r a l i z e d A n x i e t y D i s o r d e r (GAD) :

14

This is also known as anxiety neurosis, anxiety state or anxiety reaction and is characterized by unrealistic or excessive anxiety and worry which is generalized and persistent and not restricted to particular environmental circumstances, i.e. it

is

`free

floating'.

Generalized

anxiety

disorder (GAD), which affects about 10 million global populations and is characterized by more or less constant state of tension. It should be noted, however that over half of those with GAD also

have

another

anxiety

disorder

or

depression. Given these conditions, a diagnosis of GAD is confirmed if three or more following symptoms are present (only one for children): feeling oh edge or very restless, feeling tired, having difficulty with concentration; feeling irritable; having muscle tension; experiencing sleep disturbances. Symptoms, should cause significant

distress

and

impair

normal

functioning and not to be due to medical condition or other mood disorder or psychosis. Anxiety is often manifested in individuals at various

times

during

cancer

screening,

diagnosis, treatment, or recurrence. Patients can experience moderate to severe anxiety while waiting for the results of diagnostic procedures 15

(Jenkins et al, 1991). Other symptoms of distress, and sleep disturbances and can be a major

factor

in

anticipatory,

nausea

and

vomiting. It has been shown that anxiety can lead to early death if untreated (Sirois F. 1993). Anxiety,

regardless

of

its

degree,

can

substantially interfere with the quality of life of patients with cancer and of their families and should be evaluated and treated (Davis ct al, 1993; Dahiquist et al, 1993 and Payne S. A. 1992).

B) Panic d i s o r d e r : Panic disorder is characterized by acute and unprovoked discrete periods of intense fear or discomfort (panic attacks) due to intense acute psychic and somatic anxiety symptoms which are unexpected and not triggered by situations.

C)

Mixed

anxiety

and

depressive

disorder: In this disorder symptoms of' anxiety and depression are both present but neither is clearly predominant.

Mixed

pictures

of

neurotic

disorders are much more common than discrete 16

entities, such as generalized anxiety disorder.

D) Phobic d i s o r d e r : Fear is a normal prudent situational anxiety, for instance if one is under threat of attack. A phobia is an inappropriate situational anxiety with avoidance. The degree of avoidance is a useful measure of the severity of the disorder. There are three main groups of phobic disorder: I) Specific phobias II) Agroraphobia III) Social phobias.

E) Obsessive-Compulsive disorder: Obsessive-Compulsive

disorder

is

a

non-

situational preoccupation in which there is subjective

compulsion

despite

conscious

resistance. Such preoccupations can be thoughts (ruminations or obsessions) or acts (rituals or compulsions).

F)

Post

Traumatic

Stress

Disorder

(PTSD): This

is

a

common

reaction

of

normal

individuals to an extreme trauma which is likely 17

to cause pervasive distress to almost anyone, such is natural or man-made disasters, combat, serious accident, witnessing the violent death of others, being the victim of the torture, terrorism, rape

of

other

crimes.

The

syndrome

is

characterized by I) Experience of a major trauma II) Intrusive recollections in the form of thoughts, nightmares or flashback III) Sense of numbness IV) Increased arousal and hyper vigilance and insomnia.

1.3. DEPRESSION: The term depression has been used to describe an emotional state, a syndrome, and a group of specific disorders. When seen as part of a syndrome

or

disorder.

autonomic,

visceral,

Depression

emotional,

has

perceptual,

cognitive, and behavioral manifestation. The sex ratio is unequal, with depressive episodes being more common in females. The incidence of depressive episodes is between 80200 new cases per 1,00000 of the population per year in man and between 250-7,800 new cases per 1,00000 of the population per year in women.

The

point 18

prevalence

in

Western

countries is between 1.8-3.2% for man and 920% in women. It has a higher incidence in those who are not married, including who are divorced or separated. The rise in the rate of depression is consistent with the findings of other recent studies reviewed by Singh, 1979 and by Nandi et al, 1992. In a recent study of both

Indian

and

Western

patients

with

depression (Anath et al, 1993), it was found that 62% (74 out of 1 19) of the Indian patients suffered from guilt as compared with 84% of the Western patients (96 out of 114). Studies conducted in the 1960s and 1970s reported a low prevalence (varying between 5.3% and 26.7%) of guilt feeling in Indian patients with depression (Murphy et al, 1964; Venkoba Rao, 1966). Carstairs & Kapur, 1976, in their survey of a rural community in Karnataka, India, found that the rate of depression was 30.0 per 1000; Nandi et al, 1992 reported that the rate of depression in a village in West Bengal rose from 37.7 per 1000 to 53.3 pert 000 after an interval of 10 years.

B i o l o g i c a l S y m p t o m s in d e p r e s s i o n : xReduced appetite 19

xReduced weight xConstipation xEarly morning wakening (terminal insomnia) xDiurnal variation xReduced libido xAmenorrhoea Two major diagnostic schemes have been used to

detect

depression:

Research

Diagnostic

Criteria (RDC) (Spitzer, Endicott, & Robins, 1978) and the diagnostic criteria from the Diagnostic and Statistical Manual of Mental Disorders

[DSM-IV]

(SLID;

First

Spitzer,

Williams, & Gibbons, 1995) RDC diagnoses are typically generated from a structured interview which is outlined in the schedule of Affective Disorders and Schizophrenia (SADS) (Endicott and Spitzer, 1978). The RDC diagnostic group most commonly utilized in the studies reported below is major depressive Disorders (MDD). For DSM-IV diagnoses, a semi structured interview which

focuses

on

specific 20

symptoms

and

symptom clusters is typically employed. The DSM-IV diagnosis essentially equivalent to MDD is major depression (MD). There is considerable overlap between these two sets of diagnostic criteria, although some differences exist.

1.4. PSYCHOSOCIAL STRESSORS AND HEALTH CONSEQUENCES: Many

patients

emotional

stress.

attribute The

their loss

of

illness

to

important

relationship, e.g. a spouse, by death or divorce or other adverse life events has been associated with the subsequent onset or exacerbation of many types of illness (Anderson et al, 1995; Glaser et al, 1985; Winza et al, 1991; and Rahe, 1994) and with increased mortality (Iverson et al, 1987; Moser et a], 1987; Bullman and Kang, 1994). The results of some case control studies have been interpreted as showing that stressful life events contribute to the onset of cancer, a mutational disease (Scherg and Blomke, 1988; Forsen 1991; Kune et al, 1991; Courtney et al, 1993;

Chen

et

al, 1995).

The prevailing

hypothesis for an association is that which in turn predispose to the initiation and progression 21

of

various

patho-physiological

processes,

including infectious, allergic, autoimmune and neoplastic diseases (Ader et al, 1995). A recent example

that

provides

evidence

for

the

existence of such a pathway is impaired wound healing in persons who are earring for a relative with Alzheimer's disease (Kiecolt-Glaser et al. 1995). Increased cancer incidence has been observed in patients with various well defined immunodeficiency states (Rabkin & Yellin, 1994; Birkeland et al, 1995). Major stress with disease particular to the cancer and depression are major cause of mortality and morbidity in the Western world (Maddock C & Pariente CM, 2001). There is evidence to link stress with the onset of major depression and with a poorer prognosis of cancer. Infectious diseases, cancer and autoimmune disorders are associated with the development of behavioral symptoms, similar to those seen in the chronic stress and major depression (Raison CL & miller All, 2001). Chronic stress may increase vulnerability to infectious disease (Kerney ME & Gruenewald TL, 1999). Stress, anxiety and depressive states are associated with enhanced frequency of 22

tumors (Lissoni P et al, 2001). Depression is associated with two important processes for carcinogenesis: poorer repair of damaged DNA and alteration of apoptosis (Kiecolt-Glaser & Glaser R, 1999).Chronic stress associated with familial cancer risk have negative health consequences

through

psychobiological

reactivity

changes

in

(Valdimarsdottir

HB et al, 2002). Association

between

stress

and

increased

illness behavior and evidence was found for a similar

association

between

stress

and

infectious pathology. Introverts, isolates and persons lacking social skills may also be at increased

risk

both

illness,

behaviors

and

pathology (Cohen S & Williamson GM, 1991). Psychological stress seems able to alter the susceptibility of animals and man to infectious agents, influencing the onset, course and outcome of certain infectious pathologies (Biondi M & Zannino LG. 1997). This type of study has already led some authors to propose experimental protocols

of

psychological

intervention

or

psychoimmunotherapy in pathologies such as tuberculosis, HSV, or HIV infections.

23

Chronic stress of an individual, which aims out of caregiving rendered to an ill spouse, is also an important example of health consequences of stress. Principally financial as well as due to different psychosocial factors, a caregiver is usually exposed to chronic stress. Caregivers of some major illness including caregiving have also showed important evidences of physiological impact of stress on their health profile most of which

are

at

the

level

of

immunological

resistance to opportunistic infections. Cancer caregivers

experience

increased

emotional

distress, regardless the amount care provided, when limited in their ability to participate in valued activities and interests. Caregivers with less than high school education experience more emotional distress (Cameron JI et al, 2002). Intrusive thoughts about cancer, often identified as `cancer specific worries' or `cancer specific distress' have been postulated to be associated with dysfunction with women at increased risk of developing breast or ovarian cancer (Trask PC et al, 2001). There

is

mediated

evidence

linking

immunological

psychosocially

alterations

with

cancer, infectious illness and HIV progression 24

(Kiecolt-Glaser JK & Glaser R, 1995). The relationship

between

stress

and

the

development of breast cancer that investigates the immune system as a possible mediator (Bryla CM, 1996). Effects of stress on pathogenesis of all these diseases are highly relevant to assessment of biological

importance

of

the

immune

impairments that have been associated with stress (Peterson PK et al, 1991). The life styles of Indian people are changing very rapidly. In modern

society

depression

the

are

level

increasing

of

anxiety due

to

and job

responsibility, family conflicts and illness of long term diseases. Among these psychosocial factors, health consequences of individuals who care for their spouses, suffering from diseases requiring, long term treatment, has now been a major issue in public health in National and as well in International context. At National level, the

numbers

of

cancer

caregivers

are

significantly higher than the caregivers of other well know long term sufferings like Parkinson's disease, Schizophrenia etc. The underlying reason has been suggested to be increased incidence rate of cancer along with increased 25

longevity of the cancer patients due to the significant advancement in the field of early cancer diagnosis and treatment. Therefore the present work is aimed to evaluate the physiological significance of plasma

dopamine

level

in

increased

anxiety

and

depression model of Indian patients. In the present

investigation

physiological

concentration of plasma DA in anxiety and depression as observed in cancer caregivers have

been

studied

in

terms

of

health

consequences at the level of immune functions since

these

individuals

have

significantly

increased incidence of infectious and other pathological conditions. Till now little information is available on the health consequences due to stress and anxiety of Indian cancer caregivers in relation to their immune

functions

and

its

underlying

mechanisms. Information on these aspects are surely to be National importance in terms of upliftment of social health. Therefore the present study is aimed to evaluate the immune status of Indian cancer care givers following exposure to stress and anxiety and alteration, if 26

any its underlying mechanisms at the level of plasma dopamine.

1.5. CANCER CAREGIVERS: The definition of caregiver provided by (Miller & Keane, 1992)- "a lay individual who assumes responsibility for the physical and emotional needs of another who is incapable of self care". Caregivers attributes of poor health, lower socioeconomic status and less education were found to be related to increased perceptions of harm or loss and threat, which in turn, led to increased caregivers load (Oberst, Thomas, Gass & Ward, 1989). The diagnosis of cancer has not only a single impact on the affected persons, but also on their families, and cause emotional shock, doubt, anxiety and depression (Given et al., 1992; Kurtz et al, 1995). With cancer rapidly developing into a continuous care problem because of increasing incidence rates, longer survival times, and a trend toward outpatient

treatment,

providing

support

and

managing care has placed added responsibilities on caregivers. However, the consequences for these caregivers still are unclear.

27

The immunological decrements associated with the stress of caregiving are of particular concern because older individuals already have age related reduction in cellular immune function with important

health

consequences;

respiratory

infection such as influenza and pneumonia remain major cause of morbidity and mortality among older adults (Patriarca PA, 1994; McElhancy JE, 1994; Burns EA & Goodwin GS, 1990). Adults who show poorer responses to vaccines and other antigenic challenges also experience higher rates of clinical illness, including virus infection (Burns EA & Goodwin GS, 1990; Gravenstein S et al, 1994).

1.6. EFFECTS OF STRESS ON IMMUNE SYSTEM: After exposure to stressors, suppression of a wide variety

of

immunological

parameters

is

consistently reported in response to intense stressors or prolonged exposure to stressors (Bonncau, 1991; Dobbs, 1996; Fuchs, 1993; Han, 1995

and

antibody

Zwilling, responses

activity (Frcier,

1990). (Dobbs,

1994) and

Suppression 1996),

of

NK .cell

the number

of

lymphocytes in the spleen and thymus (Pruett, 28

2000 and Tarcic, 1998) are typically noted following intense and long term stress. Stressor induced production of neuropeptides and hormones interferes with antigen uptake, antigen

processing,

antigen

presentation,

T

lymphocyte recognition, cytokine production, or B

lymphocytes

proliferation,

antibody

production may be impaired. Studying these events, which occur within organized lymphatic tissue, is obviously not going to be easy in the human system. However if the ability of CD8+ lymphocyte to kill a virally infected cell is important to recover from viral infection or a NK cell lymphocyte killing a virally infected cell, such interactions and mechanisms may be susceptible to experimental study. Thus a basic understanding of immune system function is important in devising approaches to study the mechanism by which a stressor alters immune system functions. Although catecholamine, endogenous opiates and

a

variety

of

other

hormones

and

neurotransmitters can affect the immune system, much less is known about quantitative aspects of their effects than glucocorticoids. In the case of 29

catecholamines, it is very important to know the role about the immune system. The same applies to most other stress related mediators that are released rapidly (within several seconds) after stress

(Sapolsky,

2000).

However,

studies

involving administration of exogenous mediators or antagonists have indicated a role for several of them in stress induced immuno-suppression. In a particularly instructive study, Marotti et al found that different immunological parameters are suppressed by activation of the hypothalamic pituitary

adrenal

axis,

opiates

and

other

mediators like catecholamines during the same response to restraint stress in mice. Increased

concentration

of

corticotrophin

releasing factors (CRF) in the CSF has been reported

in

However

the

both

anxiety

release of

and

depression.

other peptides

or

hormones of the HPA axis is regulated differently in the two disorders. Anxiety is characterized by hypocortisolemia, dexamethosone

supersuppression and

increased

number

after of

glucocorticoids receptors, whereas depression is characterized nonsuppression

by after

hypercortisolemia dexamethasone

and

decreased number of glucocorticoids receptors. 30

(Boyer P, 2000). Dopaminergic overactivity is compared with major and minor depression (Pitchot et al, 1992). CRH influences the immune system indirectly, through

activation

of

the

HPA

axis

and

sympathetic system and directly through local modulatory actions of peripheral (immune) CRH. Both glucocorticoids and catecholamines, the end products of the stress system may selectively suppress cellular immunity and favour humoral immune responses. However, little is known about significance of increased plasma dopamine during anxiety and depression and also in immune depression in individual. The relationship of increased plasma dopamine levels to severity of depression and psychomotor retardation seems to play a critical role in patients who met criteria for major depressive episode. Plasma norepinephrin (NE) demonstrated

a

trend

toward

a

negative

correlation with the HRSD total score (Hammer MB & Diamond BI, 1996). Based on these observations, it has been suggested

that

other 31

pathways

including

sympathetic nervous system may be important in mediating stress induced changes in immune function and were not correlated with changes in corticosterone, or occurred in adrenalectomized animals (Keller SE et al, 1983; Esterling B Rabin BS, 1987; Blecha F, Kelley KW & Satterlee DG, 1982). Although results so far have

been

discussed

which

showed

corticosterone as one of the important mediators of stress mediated immune function. Beside establish corticosterone, recent in vitro results suggested that catecholamine, especially DA can significantly inhibit the functional activity of T & B cells (Cook-mills et al, 1995; Offen et al, 1995; Joseffson et al, 1996; Basu & Dasgupta, 2000

and

Bergquuist

et

al,

2000).

Other

pathways including the sympathetic nervous system, may be important in mediating stress induced

changes

in

immune

alteration.

Psyhological stressors are known to stimulate the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system resulting in the release of catecholamines including dopamine that can affect various immune parameters and can alter overall immune competence of the individual.

32

CHAPTER - 1 Plasma and intracellular dopamine content in T & B cells of normal volunteers and cancer caregivers. 2. INTRODUCTION: The people who provide health care to the patients of long term diseases are recognized as caregivers. The majority of studies regarding the health consequences of long term disease are family

members

neurological

of

disorders

patients like

with

some

Schizophrenia,

Parkinson's disease and dementia (Pot and van Dyck, 1995 and Schulz et al, 1995). In addition now a days patient with cancer live longer because of more effective and modern disease treatment and they need for their physical and psychosocial care have also increased (Stctz et al, 1987). Some information are available which showed that cancer caregivers suffered from anxiety and depression due to caregiving stress and become vulnerable to various diseases due to impairment of immune system (Leonard BE et al, 1996; Kieeolt-Glaser JK, 1995).

33

As a consequence of long term caregiving to ill patients,

anxiety

developed.

and

Alterations

chronic in

depression

resting

plasma

catecholamine (CA) concentration have been described in depression (Roy et al, 1985; Rudorfer et al, 1985). Beside corticosterone pathway which has been suggested as an important mediator of stress mediated immune suppression, recent several in vivo and in vitro results suggest catecholamine induced immune suppression (Joscffson et a], 1996; Basu & Dasgupta, 2000 and Bergquist et al, 2000). This suggests the significance of sympathetic nervous system in mediating stress induced changes in immune function (Glaser et al, 1998; Bauer et al, 2000; Glaser et al, 2001; Scanlan et al, 2001; Kiecolt-Glaser et al. 2001; Dominguez- Gerpe et al, 2001; Nagabhusan et al, 2001 and Murray et al, 2001) which has been corroborated well in several studies. Therefore psychological

stressors

hypothalamic-pituitary-adrenal

which axis

stimulate and

the

sympathetic nervous system resulting in the release of catecholamine may affect the functions of both T and B cells.

34

DA has recently been shown as an important endogenous

regulator

of

neural-immune

communication (Basu and Dasgupta, 2000). Some observations from our laboratory and from others have shown that brain DA depletion by MPTP, which selectively and irreversibly destroys central dopaminergic pathways, resulted in significant alterations of proliferation of T cells and tumor cell killing ability of cytotoxic T cells and NK (natural killer) cells (Jankovic and Marie, 1990; Basu et al, 1995a). Since central DA cannot cross the blood brain barrier, its direct involvement in this immune alteration cannot be ascertained. However the possibility of an alternative indirect way by which this monoamine neurotransmitter can exert its influence on functions of peripheral immune effector cells has been suggested (Basu & Dasgupta, 1995a). Surprisingly little knowledge is available on the effect of circulating plasma concentration of DA on the functional activity of immune cells. Interestingly, though in normal individuals, only minute quantities of DA is present in the plasma, its level shoots up significantly following exposure to stress and chronic depression during various psychosocial factors like bereavement, separation and long term caregiving. In cancer caregivers, suppression of 35

functional activity of major immune effector cells like T & B lymphocytes have been reported (Oya et al, 2000 & Rozlog et al, 1999). However, a correlation between stress and immune

alterations,

which

may

leads

to

favorable for susceptible to diseases have been reported (Keller et al, 1998; Cohen et al, 2001; Dominguez-Gerpc et al, 2001 & Nagabhushan et al, 2001). Moreover, from the previous preliminary experiments, the degree of plasma DA alteration in cancer caregivers in relation to age and sex was not reported. Therefore it appeared to be rational to critically analyze the plasma DA level in cancer caregivers and also to evaluate the effect of this altered plasma DA, if any, on the functional activity of T and B cells, which serves a major arm of the immune response against cancer and long term diseases. The circulating DA from peripheral origin is less

understood.

Adrenal

medulla

and

mesenteric organs (spleen, gastrointestinal tract and pancreas) appear to be the major source of plasma DA (Clark et al, 1985; Eisenhofer et al, 1997),

the

unknown.

contribution Moreover,

of

recent

each

remains

studies

by

Bergquist et al 1998, Marino et al, 1999 and 36

Basu and Dasupta, 2000, have shown that there is the existence of specific DA receptors in human mononuclear cells. Recently Bergquist et al, 2000 has shown that this inhibitory effect of dopamine is mediated through suppression of NF-Kȕ, a transcriptional factor. Pharmacological inhibition of Tyrosine hydroxylase, the enzyme for synthesis of dopamine, and Monoamine oxidase (MAO), the enzyme for breakdown of DA, profoundly affect intracellular DA and its metabolites, indicating that these cells are able to synthesize and breakdown DA. Based on these findings, a possible role of dopaminergic autoregulatory mechanisms in modulation of lymphocyte functions in relation to immune response has been suggested (Bergquist et al, 1994). Experimental evidences which showed endogenous synthesis (Bergquist et al, 1994; Musso ct al, 1996), transport (Bondy et al, 1992; Basu et al, 1993) as well as uptake (Faraj et al, 1991 and 1994; Krieger, 1998) of DA by the

lymphocytes,

suggesting

its

possible

influence in alteration of the plasma pool of DA. However direct evidences are not available. Since T cells constitute more than 85% of the circulating lymphocyte population (Hokland et al, 1994), determination of the intracellular DA 37

content in T and B lymphocyte is deserved to be understood. Uptake of [ 3 H] DA by T and B cells along with the activity of Tyrosine hydroxylase, the rate limiting enzyme of DA synthesis, will definitely be relevant to this study, in order to evaluate the contribution of synthesized DA of these cells. Therefore,

all

importance

of

these

facts

studying

emphasize

the

synthesis

the of

endogenous DA in T cells, uptake of this monoamine along with reactivity of the rate limiting enzyme of Tyrosine Hydroxylase (TH) in cancer caregivers and controls (normal individuals). To our knowledge, no specific information regarding the status of peripheral DA in cancer caregivers is yet available. So the findings will help us to collect conclusive evidences regarding alteration, if any, in the level of peripheral DA during depression, in relation

to

its

correlation

with

functional

activity of immune effector cells. We estimated DA within plasma and in T and B cells by the method of high performance liquid chromatography with electrochemical detectors (HPLC-ECD). The method, HPLC with ECD, 38

though little expensive, has higher sensitivity, reproducibility and selectivity (Davies and Molyneux, 1982). The HPLC with ECD has provided

an

accurate

means

to

measure

picogram (pg) quantities of this monoamine and has also enabled determination in very small volume of body fluids and individual cells. Moreover no modification of catecholamine structure prior to analysis is required. Hence this assay method is widely used and has gained considerable acceptance as a suitable method for routine DA analysis (Davies and Molyneux, 1982; Cosentino et al, 2000). Recently another method, Capillary electrophoresis has been used in

different

laboratories

as

an

effective

analytical tool for separation and detection of neurotransmitters

at

single

cell

level.

Successful use of this instrument has enabled different laboratories to quantitate single celllevel of DA in lymphocytes, neutrophils and macrophages (Bergquist et al, 1998). However the running cost of this instrument is very high and so cannot be used for routine analysis of experimental and clinical samples. Therefore, considering all the advantages with respect to reproducibility

and

cost

effectiveness,

the

HPLC with ECD method has been utilized to 39

evaluate the DA content in plasma, T and B cells. The Tyrosine hydroxylase, the rate limiting enzyme for dopamine synthesis is assayed by immunocytochemistry.

Since

this

method

employs the antigen antibody reaction, the result

obtained

is

more

specific

than

biochemical assay procedures. This method also enables localization of the site of enzyme activity

precisely.

Moreover

it

enables

determination of enzyme activity in individual defined cells lying within a complex histology. Conventional biochemistry, on the other hand requires the tissue to be homogenized and the enzyme to be isolated into a foreign medium, which

may

alter

the

enzyme

activity

considerably (Chayen and Bitensky, 1994). Hence the immunocytochemical method was utilized

to

determine

Tyrosine

hydroxylase

activity within the T and B cells.

3. MATERIALS AND METHODS: 3.1.

SELECTION

OF

SUBJECTS

PSYCHIATRIC ASSESSMENT:

40

&

Cancer caregivers and normal controls were divided into two separate groups. Each cancer caregivers and his or her age and sex--paired control received an initial clinical review by a staff psychiatrist. A structured interview, the schedule

for

affective

disorders

and

schizophrenia (SADS) (Endicott & Spitzer, 1978), then was held with each subject by two psychiatrists together, and on the basis of SADS results patients were classified according to the Research Diagnostic Criteria (RDC) (Endicott & Spitzer, 1979). The Hamilton Depression Rating Scale (HDRS) and Beck self rating Depression Inventory (BDI) also were completed for each patient (Lechin et al, 1982). The psychiatrists examining each subject agreed on 92% of their diagnoses. Cancer

caregivers

were

recruited

among

relatives and spouse of the patients. All were well, according to physical; all were taking no medication, had normal chest X-ray and ECG, and showed routine blood test results within the normal range. Neither the patients nor the control

subjects

showed

present

or

past

psychiatric illness. We used a modified 18 item HDRS, with scores ranging from 17 to 58, and a 41

21-item BDl which ranged from 21-63 points (Lechin et al, 1982). Patients and controls were assessed in both tests on the same day, and scales were administered under conditions as constant as possible.

3.2. ASSAY OF DA IN PLASMA, T AND B LYMPHOCYTES:

3.2.1. Sources of chemicals: All chemicals were purchased obtained from Sigma Chemicals, F. Merck, Germany; Life Technologies, USA; Bengal Chemicals, India and Glaxo laboratories, India SRL, SD-fine, India. Milli Q water was used throughout all the protocol,

obtained

from

Central

Research

Instrumentation Facility (CRIF) of our Institute.

3.2.2. Collection of blood sample for analysis:

Materials: Blood was collected in a heparinised glass vial, 10 of Heparin solution [Sigma, USA] was used for l0 ml blood.

42

Methods: 1. Blood was collected from subjects (cancer caregivers

and

heparinised

glass

normal vial.

volunteers) Blood

was

in

a

drawn

carefully to avoid hemolysis. 2. The blood was then mixed with chilled 10 microlitre (µl) of anticoagulant by thoroughly mixing several times and was then placed immediately in an ice bath.

3.2.3. Separation of plasma and storage for analysis: Blood thus collected was centrifuged in a refrigerated centrifuge at 300g for 15 min and plasma

was

separated.

The

plasma

was

collected by Pasteur pipette and kept in eppendorf tube for future use. The plasma was stored at -70 °C until assayed for DA level. 3 . 2 . 4 . S e p a r a t i o n of T & B l y m p h o c y t e s :

3 . 2 . 4 . a) I s o l a t i o n of p e r i p h e r a l b l o o d m o n o n u c l e a r c e l l s (PBMNCs):

43

Materials: (1) Normal physiological saline (0.9% NaCl) (2) Ficoll hypaque [Sigma, St Louis, USA]; (3) Phosphate buffered saline (PBS) comprise of (1 lit): 0.23 gm NaH2PO 4 , 1.15 gm Na 2 HPO 4 and 9 gm NaCl added to 900 ml water, the pH adjusted to 7.2-7.4 using 1(M) NaOH or 1(M) HC1 and total volume made upto 1 litre.

Methods: 1. Blood collected was mixed with equal volume of normal saline. 2. 5 ml of the mixture was carefully layered onto 3 ml of Ficoll Hypaque in a 15 ml glass centrifuge tube so that interface was maintained intact. 3. The tube was centrifuged at 400g for 15 minute at room temperature 4. Peripheral blood mononuclear cells were carefully harvested from the interface using a Pasteur pipette avoiding contamination.

44

5. Presence of Ficoll Paque was washed with 4 5 volume of PBS and mixed well to disperse the remaining Ficoll hypaque. 6. This washing was performed three times by PBS. 7. The mixture was centrifuged at 300g for 15 mins. 8. PBMNCs were resuspended in PBS.

3.2.4. b) Isolation of T & B lymphocytes from PBMNCs by Nylon wool column: Principle: PBMNCs

consist

of

populations: 60 - 70 % T lymphocytes 5-10% B lymphocytes, 5 - 15% monocytes 5 - 15% NK cells 45

the

following

cell

Passage of PBMNCs over a nylon wool column (Berke, G et al 1972; Julius. M. 11. et al, 1973) depletes monocytes

the

adherent

and

B

lymphocytes,

macrophages

and

a

cell

population containing mainly T lymphocytes is obtained. Hence this method was employed as an effective means for obtaining T cell enriched cell population.

Materials: (1) Uni-SorbT & B cell enrichment column (Product description): Uni-Sorb T & B are sterile, ready to use nylon wool columns prepacked in uniquely designed polypropylene tubes. While the method of use is the same as in standard practice. The design of the column allows aseptic loading and elution of the sample. The sample was to be loaded via a silicon rubber septum by injection and eluted via a simple control valve at the other end of the

column)

[NYCOMED

PHARMA

Division Diagnostica, Oslo, Norway]

Methods:

46

AS,

1. The control valve was opened and the tube inverted so that the valve was at the top. 2. The tab was ripped off at the septum, leaving the metal collar in place. 3. A suspension of PBMNCs (1X 10 8 cells in 5 ml) was slowly injected into the column so that there was even distribution of the sample without air bubbles. 4. The valve was closed and the column was incubated at 37° C for 60 min. 5. After incubation the non-adherent T cells were washed through the column (now facing right side up) with two or three volumes of buffer,

introduced

via

the

septum

by

hypodermic syringe, first opening the control valve. 6. Wash off the adherent 13-cells by removing the septum and plunge the column with a piston from a syringe in a separate centrifuge tube. 7. T & B cell suspension (separately) was centrifuged

at

200g 47

for

12

minutes

and

resuspended in RPMI- 1640 media.

P u r i t y and V i a b i l i t y : The described method has found to be rapid, simple and reliable and gives excellent results with

human

mononuclear

cell

population

isolated on ficoll-hypaquc centrifugation. The recovery of T cells has found to be 50-90% with availability greater than 92% that was found by trypan blue dye exclusion method.

3 . 2 . 4 . c) HPLC a s s a y p r o c e d u r e : 1. Principle: High

performance

liquid

chromatography

(HPLC) coupled with electrochemical detection (HPLC-ECD) is a technique which has found increasing

implication

in

neuro-chemical

research. Interest in the technique has centered on the advantages it possess for the analysis of biogenic amines and their metabolities. A sample is introduced into the system via an injector from which it is forced by a flowing stream of solvent, called mobile phase, through a narrow bore transport tube into a 48

column

containing small particles [ 5 µm ] known as stationary phase. The sample mixture separates as a result of different components adhering to or diffusing into the packing particles and various zones of sample components called bands are formed. These bands continue to migrate

through

the

chromatographic

bed,

eventually pass out of the column by elution, and pass through the electrochemical detectors. In

the

electrochemical

cell

the

controller

(potentiostat) maintains the potential of the working

electrode

(relative

to

a

reference

electrode) at a value which caused DA, if present, to electrolyze. At the surface of the electrode DA undergoes oxidation from its hydroquinone form to its O quinone form and two electrons and two protons are released as a result. The release of these electrons, i.e. electron transfer produce a reaction current which is amplified by the potentiostat and signals are recorded in a strip chart recorder. The intensity of the response is proportional to the concentration

of

electrochemically

active

substance present on the electrode. The recorder tracing from the elution of a single band is called a peak and it is identified by the retention time, which

is

the

time 49

required

to

elute

the

corresponding band from the column (Hashimoto et al, 1983).

2. Reagents: i) Sodium acetate, ii) Sodium citrate iii) Glacial acetic acid, iv) Sodium hydroxide v) Methanol, vi) Sodium-octane 1-sulphonic acid, vii) Sodium metabisulphite, viii) Dopamine, ix) Perchloric acid, x) Sodium bisulphite, xi) Tris, xii) HC1, xiii) Alumina (activated by heating on dry oven for 1 hour).

3 . B u f f e r s and i n t e r n a l s t a n d a r d s : A ) Acetate citrate buffer (pH-5.2): 1 liter containing: 5.75g sodium citrate, 6.80g sodium acetate. 1.05 gm glacial acetic acid and 2.40gm sodium hydroxide. B) Mobile phase buffer: 1 litre containing 780 ml acetate citrate buffer (pH-5.2), 220m1 methanol and sodium-l- octane sulphonic acid (5mM final concentration). This buffer was prepared just before the day of running. The solvent was pre-filtered through a 0.45im (Millipore, London, Great Britain) and degassed 50

prior to use. C) Internal standard: 0.1 micromolar (µ M) solution of 3,4-dihydroxybenzylamine in 0.1 M perchloric acid (containing 400µ M sodium metabisulphite). D) Alumina washing buffer: 100 ml double distilled water with l00µ 1 of 1M sodium bisulphite and 1 ml 0.5M Tris-HCl buffer (pH8.6). E) Alumina eluting solution: 0.6M perchloric acid (50µ l containing 400µ 1 of 1 M sodium metabisulphite /l).

4. Equipments: The liquid chromatograph comprised of a pump, injection valve fitted with a 2 (4 ' 1 sample loop and a 25 cm x 4.6mm I.D. stainless steel analytical column packed with 5pm diameter Ultrasphere IP particles. The analytical column was fitted with a 5 centimetre (cm) x 4.6 millimetre (mm) ID. precolumn packed with 3038µ m diameter Co Pell O DS . All components were supplied by the Waters, USA. 51

5. Methods: The methods of Davies and Molyneux, 1982 was followed,

i) Isolation of catecholamine from plasma and cell lysates: 1. The protein in the sample was first denatured by storage in the frozen state and separated by centrifugation at 800g at 4°C. 2. 2 ml of the soup was taken in a 15 ml capacity centrifuge tube and treated with 200µ l of internal standard solution, 400µ l of 0.5 M Tris-HCI, pH 8.6 was added. 3. This was followed by addition of 20 mg of activated alumina. 4. The contents of the tube were then shaken gently for 15mins in a spiral mixer. 5. The tube was centrifuged at 600g for 2min. 6. The supernatant was removed and the alumina was washed three times with alumina wash by 52

buffer solution, centrifuging each time at 600g for 2mins. 7. DA was eluted from the alumina into 50µ l of alumina eluting solution containing 400 µM NaHS0 3 in 0.6 M perchloric acid. 8. The elute was centrifuged at 800g for 3 mins. 9. 20 µl of the supernatants were collected and injected into the chromatograph.

ii) Co l l e c t i o n of d a t a : ( a ) 20µl of the supernatant was injected into the chromatograph. (b)Resolution

and

sensitivity

of

the

chromatographic system was determined by injection of 20µ l aliquot of a DA [10ng/ ml] reference solution. (c) A complete separation of the individual components of the catecholamine mixture was obtained and a sample running time of 4 mins recorded.

53

(d)

The

linearity

of

and

the

procedure

both

the

extraction

detector

response

(determined from peak area) was verified for DA over the anticipated range of assay. The former was investigated by assaying pooled plasma to which known amount of DA was added and determining the peak area ratios (sample vs. internal standard). The calibration curve constructed showed a linear relationship between catecholamine concentration and peak area

ratio,

over

the

concentration

ranges

studied.

3 . 2 . 4 . d) D e t e r m i n a t i o n of t h e p u r i t y of the

T

lymphocyte

population

by

Immunoperoxidase staining using the avidin-biotin me t hod :

Principle: Immunoperoxidase staining is a method of detecting molecular components within cells by microscopy

using

specific

antigen-antibody

reactions. Antigen specific antibody is used to detect cellular antigens and the antibody's site within cells with enzymes, fluorochromes or visible particles (Janossy et al, 1987). For a 54

specific antigen it can be determined the type of cells producing this substance and levels of the substance produced. It involves use of a primary antibody specific for the antigen to be localized, a biotinylated secondary antibody capable of binding to the primary antibody, a complex of peroxidase conjugated biotin and avidin. Avidin due to its high affinity for biotin, has its free sites bound to the biotin of the secondary antibody. A mixture of peroxidase substrate

and

chromogen

(3,3'-

diaminobenzidine or DAB) is added. The biotin cojugated peroxidase enzyme, in presence of a small amount of hydrogen peroxide catalyses a reaction with DAB to produce an insoluble golden

brown

precipitate.

The

peroxidase

antigen, and therefore the original antibody is thus visualized (Osborn, 1994),

Materials: The peroxidase staining has been carried out using the Goat

ABC

Staining

System

(Santa

Biotechnology, Inc., USA).

1. C o n t e n t s of t h e a s s a y s y s t e m :

55

Cruz

(i) 1.0 ml normal blocking serum, (ii) 250 microgran (µg) biotinylated secondary antibody, (iii) 0.5 ml each of avidin and biotinylated horse radish peroxidase (AB reagents), (iv) 1.0 ml 50x peroxidase substrate, (v) 1.0 ml 50x DAB chromogen, (vi) 3.0 ml 10x substrate buffer.

2. Phosphate buffer saline (PBS) 3.

Hydrogen

peroxide

solution:

0.1-1%

hydrogen peroxide diluted in PBS. 4. Primary antibody: It is a goat polyclonal antibody specific for CD3 of human origin obtained from Santa Cruz Biotechnology, Inc., USA. T cell receptors (TCR) are directly associated with CD3 (Cluster of differentiation 3) which is a multi subunit complex of proteins. So anti-CD3 antibodies react with the CD3 antigen and thus enable recognition of T cells. 5 . C e l l a t t a c h m e n t a g e n t : Poly-L-Lysine solution (0.1% w/v in water) used for attaching suspension cells (T lymphocytes) to slides. 6 . F i x a t i v e : Methanol. 56

7. M o u n t i n g m e d i u m : DPX.

Methods: 1. Preparation of working solutions: i) Blocking serum (1.5%): 750 normal blocking serum mixed with 5 ml PBS. ii) Primary antibody: 5µg/ml diluted in 1.5% blocking serum in PBS. iii) Biotinylated secondary antibody: 75 µl normal blocking serum stock and 25µ1 biotinylated secondary antibody stock in 5 ml PBS. iv) AB enzyme reagent: 5 0 µl reagent A (avidin) and 50µl reagent B (Biotinylated HRP) in 2.5 ml PBS. v) Peroxidase substrate: 1.6 ml water combined with 5 drops 10x substrate buffer, 1 drop 50x DAB chromogen and l drop 50x peroxidase substrate.

2. Attachment of cells to slides:

57

i) Glass slides were cleaned and coated with Poly-L-lysine solution by incubating in this solution for 15 min. ii) T lymphocytes were suspended in PBS at a concentration of l05 cells/ml. iii) Cells were added to the slide and smeared and incubated for 10 mills at room temperature after which they were ready for handling.

3. Fixation: i) Samples were incubated with chilled methanol for 5 mins kept at -20°C. ii) Samples were washed with PBS and were then ready for staining.

4. Staining procedure: i)

After

cell

adherence,

slides

were

incubated for 5-10 mins in hydrogen peroxide

solution

to

quench

any

endogenous peroxidase activity. This was followed by washing with 2 changes of PBS for 5 mins each. 58

ii) Then cells were incubated with primary antibody solution for 30 mins at room temperature.

This

was

followed

by

washing with 3 changes of PBS for 5 mins each. iii) Next, cells were incubated with biotinylated secondary antibody solution for 30 mins. This was followed by washing with 3 changes of PBS for 5 mins each. iv) After that cells were incubated with AB enzyme reagent for 30 mins. This was followed by washing with 3 changes of PBS for 5 mins each. v)

In the next step, cells were incubated for

1-3 drops of peroxidase substrate for 30 seconds to 10 rains until desired stain intensity developed. After that cells were washed in deionized water for 5 mins, vi) Immediately 1-2 drops of DPX were added and covered with glass coverslip. Cells were observed by light microscopy.

5. Counting of cells:

59

500 cells/ slide were scored. The percent of positive cells were calculated. The isolated T cell populations showed 95% purity.

6. Preparation of cell lysates: T cell lysates were prepared following the method of Keller et al, 1976. For cell rupture, a suspension of 10 6 cells/ ml in PBS was subjected to sonication (Kontes #K-88140 with 4 inch probe) for 10 mins, while keeping on ice to prevent warming. It was then centrifuged at 8,000 RPMI at 4°C for 15 mins for removal of cellular debris. The soup was stored in aliquots at 70°C until assayed for DA level.

3.3. ASSAY OF TYROSINE HYDROXYLASE ACTIVITY: Tyrosine Hydroxylase (TH) activity in T & B lymphocytes

was

determined

by

immunoperoxidase staining using the avidinbiotin method. The principle and methods have already been described in details in section 3.3.4.d. only with the following differences: 1. The primary antibody used was Tyrosine 60

Hydroxylase -an affinity purified goat polyclonal antibody raised against a peptide mapping at the carboxy terminus of tyrosine hydroxylase of human

origin

(obtained

from

Santa

Cruz

Biotechnology, Inc., USA). 2. Number of cells with high and low staining intensities was scored following the procedures of Dasgupta and Lahiri, 1987. 3.4. ASSAY OF [ 3 H] DA UPTAKE BY T LYMPHOCYTES: 1. Materials: 1)

RPMI

medium

1640

powder

with

L-

Glutamine (GIBCO BRL, USA) 2) NaHCO 3 , 3) Penicillin, 4) Streptomycin, 5) Gentamycin, 6) Fungizone, 7) Trypan Blue dye, 8) CD3 antibody USA),

(Santa 9)

Cruz

Human

Biotechnology,

Inc.,

Recombinant

IL-2

(Amersham, UK), 10) [ 3 H] DA (Amersham, UK, specific activity 67 millicurie (mCi) /m mole), 11) Scintillation fluid comprise of: 3.84g 2,5, diphenyl oxazole (PPO) and 80mg 1,4-bis [5phenyl-2-oxazolyli-benzene(POPOP)

dissolved

in 1 litre Toluene. 12) Glass fiber discs 61

(Retention capacity >2 µM , 2.5cm circles).

2. Methods: i) Preparation of culture medium: Milli Q water (900m1) was taken in a conical 1000ml flask. Powdered medium was added to the water at room temperature with gentle stirring. The inside of the packet was rinsed out to remove all traces of powder. 2g. of NaHC0 3 was added per litre of medium. The pH was adjusted to 0.2-0.3 below the final working pH (7.2-7.4) by adding 1 (N) NaOH or I (N) HCI. Antibiotics

( 5 0 µg

penicillin-streptamycin,

0.125 µg fungizone/ml and 10µ g Gentamycin/ ml) were added to the medium. The total volume was adjusted to l litre by adding required quantity of water. The medium was immediately sterilized by membrane filtration, by positive pressure.

ii) Cell counting: After separation T lymphocytes were counted manually by using an improved Neubauer hemocytometer.

Cell 62

viability,

tested

with

trypan Blue dye exclusion method was found to be 90-95%.

iii) Cell Culture: Anti-CD3 mAb was immobilized in plastic plates by incubation of 96 well flat bottomed plates (Tarson) with 100µl of anti-CD3 mAb (5µg/ml) for 1 hour at 37 ºC and used after washing with PBS. Cell concentration was adjusted to 106 /ml in complete medium. 100 µl (105 cells) of cell suspension was added to the appropriate wells of a flat bottom micro titer plate. Plates were incubated in a humidified 37 ºC, 5% CO2 incubator for 24 hours, the time at which DNA replication initiates (Alberola-Ila et al, 1997). iv) Incubation of T cells with [3H] DA: [3H] DA stock was diluted in PBS and 10 µl of the diluted solution was added to the culture wells. Reaction was stopped by adding ice- cold PBS. Experimental conditions in different aspects were varied as follows: 1. Incubation time varied from 0 min to 60 mins. 2. Final concentration of the isotope varied from 0.01 to 1 (nM). 3. Temperature range varied from 4 ºC to 37 ºC 3.5Assay of [3H] DA uptake by B lymphocytes: 1. Materials: 63

1. RPMI medium 1640 powder with L-Glutamine (GIBCO BRL, USA) 2. NAHCO3, 3. Penicillin 4. Streptomycin 5. Gentamycin, 6. Fungi zone 7. Trypan Blue dye 8. Anti-IgM (Santacruz Biotecnology, Inc, USA) 9. [3H] DA (Amersham, UK) (Specific activity 67 millicurie (mCi)/ mmol), 10.Scintillization fluid comprise of : 3.84g 2,5, diphenyl oxazole (PPO) and 80mg I ,4-bis[5phenyl-2-oxazolyl]-benzene(POPOP) dissolved in l litre Toluene, 11. Glass fiber discs (Retention capacity 2.4µ M, 2.5cm circles).

2. Methods: i) Preparation of culture medium: Milli Q water (900ml) was taken in a conical 1000ml flask Powdered medium was added to the water at room temperature with gentle stirring, The inside of the packet was rinsed out to remove all traces of powder. 2g. of NaHCO 3 64

was added per litre of medium. The pH was adjusted to 0.2-0.3 below the final working pH (7.2-7.4) by adding IN NaOH or IN HC1. Antibiotics

(50µ g

penicillin-streptamycin,

0.125µ g

fungizone/ml

and

10µ g

Gentamycin/ml) were added to the medium. The total volume was adjusted to l litre by adding required quantity of water. The medium was immediately sterilized by membrane filtration, by positive pressure.

ii) Cell counting: After separation B lymphocytes were counted manually by using an improved Neubauer hemocytometer.

Cell

viability,

tested

with

Trypan Blue dye exclusion method was found to be 90-95%.

iii) Cell culture: Anti- Ig M was immobilized in plastic plates by incubation of 96 well flat bottomed plates (Tarson) with 100µl of anti-IgM mAb (l5µg/ml) for 1 hour at 37°C and used after washing with PBS. Cell concentration was adjusted to 10 6 /ml in complete medium. 100µl (10 5 cells) of cell 65

suspension was added to the appropriate wells of a flat bottom microtitre plate was added. Plates were incubated in a humidified 37°C, 5% CO 2 , incubator for 24 hours, the time at which DNA replication initiates. iv) Incubation of B cells with [ 3 H] DA: [ 3 H] DA stock was diluted in PBS and 10µl of the diluted solution was added to the culture wells. Reaction was stopped by adding ice-cold PBS.

Experimental

conditions

in

different

aspects were varied as follows: 1)

Incubation time varied from 0 min to 60

mins. 2)

Final concentration of the isotope

varied from 0.01 to 1 (nM). 3)

Temperature range varied from

4°C to 37°C.

v) Counting of Radioactivity: Cells were harvested on the glass fiber filter discs and washed several times with PBS in a 66

millipore manifold. Each disc was placed in a vial containing 10m1 of the scintillation fluid. Radioactivity was counted by a computerized LKB liquid scintillation counter (LKB-1610).

3 .6 . STATISTICAL ANALYSIS: Results of plasma DA level, DA content in T lymphocytes, DA contents in B-lymphocytes percentage of cells showing different intensities of reactions for Tyrosine Hydroxylase and uptake of [ 3 H] DA by T cells were expressed as mean ± SEM. Data was analyzed and compared between the groups using student's `t' test and statistical

significance

was

assigned

when

p