BIOCHEMICAL AND IMMUNOLOGICAL STUDIES ON CIRCULATING AUTOANTIBODIES IN

DILATED CARDIOMYOPATHY

^ i - ' j » - * '

THESIS SUBMITTED FOR THE DEGREE OF

Bottor of $i)tlo£(opI)p

BIOCHEMISTRY

\ BY

NABIHA YUSUF ^

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£)r. Najmul Islam (Supervisor) ''vuy-

DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE

JAWAHARLAL NEHRU MEDICAL COLLEGE ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

1998

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(Htvtiiicnit

I certify that the work presented in the following pages has been

carried out by Ms. Nabiha Yusuf and is suitable for the award of Ph.D.

degree in Biochemistry of the Aligarh Muslim University, Aligarh.

•i^'d^/'

(Najmul Islam) Lecturer Department of Biochemistry Faculty of Medicine J.N. Medical College Aligarh Muslim University Aligarh-202002

D R A M I T BANERJEE MB MS MCh FNCCP FIACS

Professor of Cardiothoracic Surgery MAULANA AZAD MEDICAL COLLEGE AND ASSOCIATED G B PANT HOSPITAL Ex-Professor. JIPMER, Pondicherry Ex-Medical Officer. Intelligence Bureau and Police Training School, New Delhi Edifor, Indian Journal of

THORAaC AND CARDIOVASCULAR SURGERY

ROOM NO 102 ACADEMIC BLOCK

DEPARTMENT OF CARDIOTHORACIC SURGERY GoviND BALLABH PANT HOSPITAL

NEW DELHI 110 002 INDIA

Telephones: Office: 3234242/5102, 3234726

Residence: 6446522 Fax: 3239442

August 9,1996

TO W H O M S O E V E R !T MAY r O N C F R N

This is to certif\'that we have provided a human hear! from an autopsy rase to Ms Nabiha Ynsnfforher

research work She is involved in cardiovascular research in the Department of Biochemistry' in Medical

College, AMU, Aligarh Farlier she was a research associate in our institution

/ , 'L

7'.-Dr. Am if Baneriee

Dr. AN^T FAVERJEE

G I. : . ' • 'c-f i tal , .S. O b i

^n ianh vexmmhxnnce of muDAD

^ 0 my 'role mohel' my MOTHER

Sc my 'ancijor'

my BROTHER for tijetr lobe, encouragement an^ support

mo my SIR fottii

profounh gra t i tuhe

mijxs piece of foork is hehicateb to tl]e cause of tlte mi l l i ons suffer ing from carhiac

ailments (norlbioihe

ACKNOWLEDGEMENT

'He who goes in search of knowledge walks in the path of GOD'.

Perhaps, this is the most appropriate quote to sum up this quest. I take this

opportunity to thank the Almighty for His benevolent presence and invisible

prowess to guide me in His path It was my immense faith in Him which kept

me going undeterred and reach this destination. I pray to Him to keep my

faith strong and guide me with His guiding light After all 'Light dawns

after every darkness, believe in the divine'.

In this search for knowledge, my mind automatically goes to my

supervisor, Dr. Najmul Islam, who worked hand in glove with me to achieve

this mission. We were on no ordinary mission, it was a 'mission impossible'

and for us where the odds weighed heavily against the evens, mutual

understanding was the key words. He M'as willing to discuss my problems at

all levels, convince me and console me in moments of despair. I was allowed

to voice my opinions too and together we could knit this piece of work in its

present shape. I can never thank him enough for being a friend, philosopher

and guide to me Ms. Zeba Islam was also always around to put in a word

of encouragement.

I am grateful to Prof. Rashid AH for allow ing me to work m his

laboratory which was a great source of help in executing my experiments.

Without this, I could have never accomplished this task I am also thankful

for his constructive criticism often at the cost of his great discomfort and

inconvenience.

/ owe a deep sense of gratitude to Dr. Asif All, Chairman, who has

been a great source of help and encouragement during some of the trying

moments. Whenever, the going was tough, he did his best to remove every

hurdle that came my way. I am particularly grateful to him for his

affectionate ways and encouragement for this humble effort of mine.

Thanks are also due to Drs. Khushtar Salman, A.F. Faizy and

Moinuddin for their help from time to time. A special note of thanks to Dr.

M.U. Siddiqui, who always shielded me and gave a lot of moral support

whenever I needed it.

There have been noteworthy contributions from persons outside this

department who have always encouraged me to go ahead when the pace

slowed down and did their best whenever I approach them for help. This

includes Prof. W.A. Nizami, Deptt. of Zoology, A.M.U. and Dr. Saad Tayyab,

Reader, Interdisciplinary Biotechnology Unit.

This work would have remains a distant dream but for the love and

encouragement from my uncle. Dr. M. Khalilullah, Consultant in

Cardiology, Inderprastha Apollo Hospital, New Delhi. He has been a father

figure and source of inspiration to me. He was the one who was really keen

that I should undertake a study in the field of cardiology. He helped me

during the course of this study in every possible way. In his absence. Dr.

M. Ahmad, Cardiologist, J.N.M.C., Aligarh ably supported me with the

clinical details and also encouraged me to forge ahead. Thanks are also

due to Prof. Amit Bannerjee, Deptt. of Cardiothoracic Surgery, G.B. Pant

Hospital, New Delhi for providing a human heart which formed the pivotal

basis of this study.

My thanks are also due to my departmental colleagues, Drs. Aslam

and Sajid, Mr. Rizwan Ahmad, Waris, Deepak, Haseeb, Sharique, Humra,

Talat and Subia for their support at times. Here, I would like to mention a

special note of thanks to Dr. Rizwan Manzer whose incorrigible support

helped me to overcome the last and most crucial phase of this crusade.

My friends Drs. Shagufta and Farah, Rana, Shahroz, Farah, Lubna,

Minhaj, Salman and especially Saba and Jehangir, who stood with me

through thick and thin and gave me comfort whenever I needed it. I also

extend my thanks to my close friends and relatives who may be far but yet

near to encourage me.

I would also like to mention the relentless effort of Mr. Wajid and Mr.

Mansoor of Central Photographs. My genuine thanks are also due to Mr.

Saleemuddin, cartographer for his fine sketches.

The non-teaching staff of the department mainly Mr. Aleem, Mr.

Dabeer and Jumman who were everready to help me. The office staff mainly

Mr. Gautam and the staff of clinical lab^ Mr. Aslahuddin. The animal house

attendants also need a special mention for taking care of my animals.

Financial assistance from the U.G.C. Government of India in the form ofS.R.F. is

gratefully acknowledged.

Last but not the least, I would like to express my gratitude to Mr

Alaishya Hood for taking pains to print this manuscript in its present form.

Nabiha Yusuf

CONTENTS

PAGE NO

ABSTRACT

LIST OF FIGURES

LIST OF TABLES

ABBREVIATIONS

INTRODUCTION

EXPERIMENTAL

RESULTS

DISCUSSION

REFERENCES

1

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^bstvnti

p i l a t e d c a r d i o m y o p a t h y ( D C M ) is a c h r o n i c hear t

muscle disease of unknown etiology characterized by dilatation and

contracti le dysfunction of left and/or the right ventricle. In man,

myocarditis is known to be the precursor of DCM in many cases, al­

though clinical and histological diagnoses remain obscure. Since in

DCM, heart is the only affected organ, it fits into the criteria of organ

specific autoimmune disease. Autoimmune features in patients with

DCM include familial aggregation, a weak association with HLA-DR4

and with immunoglobulin genes, abnormal expression of HLA class II

on cardiac endothelium and increased levels of circulating cytokines.

Cardiac antibodies have been found against various antigens in

DCM patients. Multiplicity of parallel autoimmune reactions directed

towards a single organ may be due to spread sensitizatio n. Among

the various cardiac antigens, 150 kD C-protein has been found to in­

duce myocarditis leading to DCM in experimental animals. C-protein

plays an important role in contractile activity and is one of the major

constituent proteins which are essential to compete with natural anti­

gens at MHC antigen binding site.

In the present study, 150 kD human cardiac C-protein was

electroeluted and further purified by HPLC. C-protein was character­

ized by ultraviolet and circular dichroic spectroscopy andSDS-PAGE.

Due to identical molecular weights, IgG and C-protein gave a similar

pattern on 7.5% SDS-PAGE. C-protein is an intracellular member of

immunoglobulin superfamily and it shares some of its domains with

1 1

IgG. In order to further substantiate the similarity pattern between

the two, the above were subjected to urea denaturation. Both IgG and

C-protein were denatured to give two prominent bands on urea SDS-

PAGf using an acrylamide (5-20%) and a urea (1-lOM) gradient.

The thermodynamical data speculate the tremendous structural

stability exhibited by C-protein which is a highly stable dimeric pro­

tein molecule as evident from the observation that only 4.4 percent

had undergone thermal unfolding till TS^C. Fifty percent denaturation

was observed at around TQ.S^C. The negative AGj values above 79°C sug­

gests the transition of completely folded C-protein to the unfolding

state. In comparision to native DNA, our results are indicative for C-

protein to be topologically less constrained. The 150 kD C-protein

which is acting as an autoantigen in DCM was found to be equally

reactive with naturally occuring SLE autoantibodies in which DNA is

acting as an autoantigen. The antigenicity of electroeluted HPLC puri­

fied 150 kD cardiac C-protein was assessed by inducing the produc­

tion of polyclonal antibodies in rabbit and rats. The repertoire of

specificities of induced antibodies was ascertained by direct binding

and competition ELISA.

DNA-protein crosslinks have been produced by typical photo­

chemical reactions occuring in biological systems. The crosslinks have

been found to be covalent in nature and have been responsible for many

deleterious pathological processes. In this case, the HPLC purified C-

protein was photoadducted to commercially available calf thymus na-

I l l

tive DNA purified of protein and single stranded regions. Time course

kinetics revealed that the formation of photoadduct increased with

increase irradiation time. This was further reiterated by the amount

of bound lysine residues (from C-protein) to native DNA. Time course

kinetic study indicates that the formation of photoadduct obeys ap­

parent first order reaction.

The prima donna in the category of oxygen ^ e e radicals (OFR's)

are oxygen itself, superoxide, hydrogen peroxide, transition metal ions

and hydroxyl (OH) radicals, the first four of which interact to gener­

ate the last. These reactive oxygen species also produce DNA-protein

crosslinks in biological systems. In this study we have induced DNA-

C-protein crosslink by the action of H2O2 and OH. Among the two,

OH appears to form a better DNA-C-protein adduct in terms of recog­

nition with DCM, SLE and immune sera. The adducts/photoadducts

were characterized by nuclease SI sensitivity assay.

Autoantibodies found in DCM patients showed remarkable bind­

ing with HPLC purified C-protein. Competition ELISA results showed

inhibition in antibody activity ranging from 64.7% to 82.1%. More­

over, inhibition of antibody activity was achieved at a very low mag-

nitude of inhibitor concentration. The induced anti C-protein antibod­

ies exhibited much higher magnitude of binding. These results indi­

cate the major involvement of 150 kD C-protein as an autoantigen in

DCM.

Native DNA showed poor reactivity with sera of DCM patients.

On the contrary, mitochondrial DNA (mt DNA) showed appreciably

I V

higher binding with DCM autoant ibodies . On ROS modif ica t ion , the

magnitude of binding decreased in case of mt DNA whi le it increased

in case of ds DNA but a relat ively low magni tude of binding was

observed when nDNA was photoadducted with C-pro te in . Conversely ,

the photoadduct ion of nDNA-C-protein in p resence of OH exhibi ted a

high degree of b inding. This could be due to cumula t ive binding efect

of immunogenic epi topes on C-protein as well as somewhat s imilar

epi topes to the immunogen on ROS modified DNA and hence when

both ROS-DNA and C-protein were photoadduc ted , a h igher degree of

binding was observed. This finding suggests that p robab ly apart from

150kD C-protein, the photoadducted ROS-DNA-C-pro te in complex

could also act as an al ternate autoantigen for myoca rd i t i s /DCM.

The binding of induced anti C-protein an t ibod ie s with h is tones ,

ROS his tones , po ly-D- lys ine , poly-L-glutamate and poly (L+G) com­

plex was of low magni tude , thereby, suggest ing for the presence of a

minor subpopulation of epitopes responding to the anti-C-protein antibod­

ies. Similar binding patterns with DCM autoantibodies further substanti­

ates the finding.

In the present study, attempts were also made to probe the possible

involvement of 150kD cardiac C-protein which is an autoant igen in

myocarditis/DCM, in the induction of autoantibodies in SLE patients. As­

says of anti-DNA and anti-human heart antigens in the sera of DCM and

SLE patients were carried out by employing direct binding ELISA where as

specificity determination was accomplished by competition ELISA. Inter-

estingly, as evident from the binding results, human heart extract proteinic

antigen (s) reactive with DCM autoantibodies showed strong recognition of

SLE anti-DNA antibodies. This is suggestive for the possible involvement

of a common or somewhat similar trigger/epitope (s) for immune response

in autoimmune myocarditis/DCM and autoimmune SLE, although such in­

ferences could not lead to confirmity at such a preliminary level of investi­

gation. The mechanism or the role of myocarditis inducing 150kD C-pro-

tein in stimulation of immune system for production of antibodies in SLE

remains to be ascertained.

In conclusion, 150kD human cardiac C-protein has been found to be a

potent immunogen capable of inducing polyclonal antibodies in experimen­

tal animals. It is a thermodynamically stable molecule and shares some of

its domains with IgG as revealed by thermal denaturation and urea denatur-

ation studies. C-protein showed strong recognition with DCM sera as well

as immunoaffinity purified SLE anti-DNA autoantibodies. Out of the ad-

ducts/photoadducts formed by crosslinking C-protein with native DNA, the

one formed in presence of OH showed strongest recognition towards DCM

and anti C-protein autoantibodies, thereby indicating the possibility of ROS-

DNA-C-protein photoadduct as an alternate autoantigen in myocarditis/

DCM.

V I

LIST OF FIGURES

Figure 1

Figure 2

Figure 3

Piguie 4

Figuie 5

Figuie 6

Figuie 7

Figuie 8

Figuie 9

F-iguie 10

Figui e ] 1

Figuie 12

Fmuie 13

Eiec t rophore t ic pat tern of total human heart extract 76

(THHt ) in 5-20% gradient SDS-PAGE

Elution profile of purification of electroeluted 150 kD 77

human cardiac C-protein by HPLC

Agarose gel electrophoresis to show the absence of any 73

trace of DNA in THHE

Ciicular dichroic spectia of 150 kD human cardiac C- 80

protein

Thermal denaturation profile of HPLC purified 150 kD 81

human cardiac C-piotein

Electiophoretic pattein of C-piotein and IgG on urea SDS- 87

PAGE

Binding of anti C-piotein antibody in rabbit with C- 89

piotein

Binding of anti C-piotein antibodv in rat with C-piotein 89

Inhibition of anti C-piotein antibodv in rabbit with C- 90

piotein

Inhibition of anti C~piotein antibodv in lat with C-protein 90

Elution piofile of IgG on protein-A.-sepharose CL - 48 92

column

Binding of DC\1 and immune IgG with C-piotein 93

Inhibition of immune IgG activitv bv native and ROS C- 95

protein

V l l

Figure-14 Inhibition of immune IgG activity by nDNA C-protein 96

adducts/photoadducts.

Figure 15 Inhibition of immune IgG activity by native and ROS 97

histone.

Figure 16 Inhibition of immune IgG activity by Poly-L-lysine, Poly- 9 8

L-glutamate and Poly (L+G) complex.

Figure 17 Inhibition of immune IgG activity by ROS DNA and RNA. 99

Figure 18 Direct Binding ELISA of THHE with DCM autoantibodies. 102

Figure 19 Inhibition ELISA of DCM autoantibodies with C-protein lOa

as inhibitor.

Figure 20 Direct binding ELISA of DCM autoantibodies with 1 50 kD 105

C-protein.

Figure 21 Direct binding ELIS.A of DCM autoantibodies with native 106

DNA.

Figure 22 Inhibition of DCM IgG activity by native and ROS C- 108

protein.

Figure 23 : Inhibit ion of DCM IgG activity by nDNA C-protein 109

adducts/photoadducts.

Figure 24 Inhibition of DCM IgG activity by native and ROS llO

modified histone.

Figure 25 Inhibition of DCM IgG activity by Poly-D-lysine. Poly- 111

L-Glutamate and Poly (L+G) complex.

Figure 26 Inhibition of DCM IgG activity by ROS DNA and RNA. 112

Figure 27 Time course pattern of nDN.\-C-protein photoadducts us

formed by UV-B irradiation.

V l l l

Figure 28 UV absorption charactei is t ics of i rradiated nDNA-C- 116

protein

Figure 29 I'V absorption chractenstics of nDNA-C-protein and their ng

adducts after ROS modification

Figure 30 t lec t iophore t ic mobility of nDNA-C-protein and their ^21

adducts after ROS modification

Figure 31 Nuclease SI sensitivity assay of nDNA-C-protein adducts/ 122

photoadducts

Figure 32 Time course kinetic piofile of photoaddition of C-protein 124

to na tne D N ^

Figure 33 Standaid plot foi estimation of 1\ sine by T \ B S method 125

Figuie 34 kinetic profile of photobound 1\ sine residues (piesent in 126

C-piotein) to n D \ \

Figuie 35 'Xppaient first ordei kinetic profile of nD\A-C-protein 127

photoadducts formation at vanous inadiation times

Figure 36 Immunoaffinitv puiification of SLE IgG on nDNA poly 129

1\ s\ 1 sephaiose 4B column

Figure 37 Binding of n D \ ^ C-piotein photoadduct with DCM. SLE 131

and immune IgG

Figure 38 Binding of ROS D \ A C-piotein adduct with DCM. SLE 132

and immune IgG

Figuie 39 Binding of ROS D \ \ C-piotein photoadduct with DCM. 133

SI E and immune IgG

Figuie 40 Binding of SEE autoantibodies with ds DNA. ssDNA RNA134

and IHHE

IX

Figure 41 Inhibition ELISA of SLE autoantibodies with DNA, ssDNA 135

and RNA as inhibitors on plates coated with ds DNA.

Figure 42 Inhibition ELISA of SLE autoantibodies with THHE as i36

inhibitor on plates coated with ds DNA.

Figure 43 Inhibition ELISA of protein-A-sepharose isolated SLE IgG 137

with 150 kD C-protein on plates coated with ds DNA.

Figure 44 Inhibition ELISA of immunoaffinity purified SLE IgG with 138

150 kD C-protein on plates coated with ds DNA.

Figure 45 Direct binding ELISA of DCM autoantibodies with nativei40

and ROS mt DNA.

Figure 46 Inhibition ELISA of DCM autoantibodies with native ni t i4i

DNA as competitor.

Figure 47 Inhibition ELISA of DCM autoantibodies with ROS mti42

DNA as competitor.

Figure 48 Glucose le \e ls in sera of patients \\ith DCM. 143

Figure 49 Total serum cholesterol levels in patients with DCM. 144

Figure 50 Levels of uric acid is sera of patients with DCM. 145

Figure 51 C-reactive protein (CRP) level in sera of patients withl46

DCM.

LIST OF TABLES

Table 1 : Thermal denaturation of 150 kD human cardiac C-protein. 84

Table 2 : Thermal denaturation of IgG. 85

Table 3 : Antigenic specificity of anti C-protein antibodies. 100

Table 4 : Competition ELISA of 150 kD cardiac C-protein with DCM 104

autoantibodies.

Table 5 : Antigenic specificity of DCM autoantibodies. 113

Table 6 ; UV absorption characteristics of formation of nDNA C-protein 117

photoadduct.

Table 7 : UV absorption characteristics of formation of ROS DNA C- 120

protein adduct/photadduct.

Table 8 : Competition ELISA of mt DNA with DCM autoantibodies. 143

X I

ABBREVIATIONS

BSA : Bovine serum albumin

CD : Circular Dichroism

DCM : Dilated cardiomyopathy

DNA : Deoxyribonucleic acid

ds DNA : Double standard DNA

EDTA : Ethylene diamine tetra acetic acid

ELISA ; Enzyme linked immunosorbent assay

HPLC : High performance liquid chromatography

IgG : Imunoglobulin G

kD : Kilo Dalton

MHC : Major Histocompatibility complex

mt CM : Mitochondrial cardiomyopathy

mt DNA : Mitochondrial DNA

Oj" : Superoxide anion radical

°OH : Hydroxyl radical

RNS : Reactive Nitrogen species

ROS : Reactive Oxygen species

ss DNA : Single stranded DNA

SDS - PAGE : Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

SLE : Systemic lupus erythematosus

THHE : Total human heart extract

TNBS : Trinitrobenzene sulphonic acid

Tris : Tris (hydroxymethyl) aminomethane

TEMED : N, N, N , N- Tetramethylethylene diamine

UV : Ultraviolet

^1 : Microlitre

Jig : Microgram

(3lntr0buctt0n

^ i l a t e d Cardiomyopathy (DCM) is a chronic heart muscle

disease characterized by impaired systolic function of left or/and right

ventricle (Richardson et al., 1996). It is a major cause of severe

congestive heart failure in young people and the commonest cause of

transplantat ion worldwide. The heart is suscept ible to immune

mediated injury. The myocytes may be injured during or after immune

response to endogenous or exogenous cardiac antigens. Immune

mediated myocyte injury may be reversible and manifested as a

t rans ien t depression of ven t r i cu la r function and/or e lec t r i ca l

instability, or it may be irreversible, ultimately leading to myocyte

necrosis.

Mechanisms of immune mediated injury may be classified into

humoral and cellular types. Humoral immunity requires recognition

of an epitope by antibody molecules expressed by B-lymphocytes and

depends on circulating antibodies to initiate injury. Several cardiac

disorders like post myocardial infarction syndrome, idiopathic dilated

cardiomyopathy, viral myocarditis and doxorubicin cardiotoxicity are

accompanied by deposition of antibodies within the myocardium,

particularly on the sarcolemma (Rose et al., 1988). Moreover, 13-95%

patients with various myocardial diseases such as idiopathic dilated

cardiomyopathy, myocarditis and hypertrophic cardiomyopathy have

shown the presence of heart react ive antibodies in their serum

(Neumann et al., 1990). The major mechanism of cell injury mediated

by humoral immunity may be the activation of complement via the

classic pathway.

Cellular immunity also plays a role in the mechanism underlying

cardiac dysfunction. It is initiated by recognition of an immunogenic

epitope by antigen specific T-lymphocytes. This initial recognition

and activation are effected by helper T-cells and require presence of

histocompatible antigen presenting cells. Presentation of myocardial

antigen to T-lymphocytes by antigen presenting cells can result in the

clonal expansion and differentiation of cytotoxic T-cells, stimulated

by the release of immune cytokines. As a consequence, myocyte

necrosis occurs, resulting in fibrosis (Sanderson et al., 1985).

Extensive studies have provided appreciable evidences for the

presence of a wide spectrum of autoantibodies against cardiac tissue

in myocardial diseases (Maisch et al., 1983; Caforio et al., 1992). In

patients with DCM, circulating autoantibodies to distinct cardiac

autoantigens have been described providing evidences for autoimmune

involvement. Thus, autoimmune disorders result due to immunologic

reactions, humoral and/or cellular, directed against the individuals own

tissue components. The precise mechanisms underlying the trigger of

autoimmune reaction are not yet completely understood, however

certain factors are said to be involved.

Shoenfeld and Isenberg (1989) described the wide spectrum of

autoimmune diseases as a mosaic of autoimmunity with many factors

leading to diverse diseases.

T-helper/T-suppresser cell imbalance:

The T-cell sub-sets control / regulate the immune response. In

autoimmune diseases, the ratio of T-helper (Th) to T-suppresser (Ts)

cells in peripheral blood tends to be nearly 10-15 : 1, particularly

during active or acute phase of the disease compared to 2 : 1 in normal

individuals (Deodhar, 1992).

Polyclonal B-cell activation:

Polyclonal B- and/or T-cell activation has been an initiating

mechanism. The proposition that polyclonal B-cell activators can

induce autoantibodies is predicted on the existence of non deleted

self reactive B-cells and/or developmentally arrested anergised B-cells

that might become active upon appropriate stimulation. A large number

of molecules, particularly of microbial origin have been found to act

as polyclonal B-cell activators and in the induction of autoantibodies.

Polyclonal stimulation of a large set of T-cells by bacterial/viral

surface antigens (SAgs) is another suggested scenario. T-cells that

react with MHC class II bound SAg on B-cells may mutually stimulate

the SAg displaying B-cells, thereby leading to production of polyclonal

immunoglobulins and in some instances, autoantibodies. Alternatively,

the activated T-cells themselves may induce tissue damage, through

cross reactions with self molecules (Theofilopoulos, 1995).

Genetic factors:

Gene t i c t ransmiss ion in Id iopa th ic DCM (IDC) has been

occasionally described in the past while importance of genetic factors

is a recent advance (Mestroni et al., 1990). In control studies.

familial IDC is detectable in over 20-25% of patients with a diagnosis

of I D C , with a prevalent autosomal dominant trait. A single dominant

locus was proposed and for the putative disease gene, a gene frequency

in the order of 10"* was estimated in overall population.

The candidate genes for familial IDC can be divided into two

main groups, genes that are responsible for normal heart function like

the ones encoding contractile proteins such as myosin light and heavy

chain, tropomyosin, troponins and actin. Other candidate genes are

genes coding for proteins involved in metabolic pathways: genes

encoding for atrial natriuretic factor and its receptor, phospho lamban,

G-proteins , p - adrenoceptors and Ca^' channels appear to be of

particular interest. According to autoimmune pathogenetic hypothesis

of IDC, the second group of genes are involved in immune function.

Immune dysfunctions in IDC involves cellular and humoral immunity

such as organ specif ic au toan t ibod ie s (Caforio et al . , 1994).

Association studies indicate a strong correlation between disease and

human leukocyte antigens (HLA). Associat ion between immune

response (Ir or MHC) and T-cell receptor (TCR) genes and the

development of immune mediated cardiac injury are not unexpected

since most autoimmune diseases are T-cell dependent and all T-cell

mediated responses are major histocompatibili ty complex (MHC)

restricted (Bach, 1995). MHC may alter predisposition by shaping of

TCR repertoire, antigenic peptide selection and presentation and

peptide transport. HLA-DR associations play a major role in patients

with DCM. HLA-DR positive patients were six times more likely to

have antibodies compared to those who did npt be long to this

phenotype. Antibodies against different cellular constituents may have

distinct immunogenetic associations. On the other hand, HLA-DR4

and HLA-DRl share common epitopes which may confer susceptibility

to autoimmunity in DCM.

In addi t ion , the e x i s t e n c e of an a s s o c i a t i o n b e t w e e n

autoantibodies, clinically overt disease and certain restriction fragment

length polymorphisms (RFLPs) of HLA-DRp and -DQa genes suggests

that subtypes of these molecules may have enhanced pathogenetic

significance (Limas, 1996).

Immune deficiency states:

The availability of severe combined immunodeficient (SCID)

mouse provides a potentially unique opportunity to study contributions

made by the immune system in induction of coxsackie virus B3 (CVB3)

induced myocarditis. Unlike congenitally athymic (nu/nu) mice, the

SCID mutation results in an absence of both B- and T- cell function,

due to an aberrant VDJ recombinase mechanism. While no functional

T- or B-cells are present in the SCID mouse, ant igen presenting

capabilities and natural killer cell activity are intact (Bosma and

Caroll, 1991).

In 1986, Cohen et al. described the first cases of rapidly fatal

DCM in three AIDS patients. Since then a number of prospective

clinical and echocardiography studies have shown that a subgroup of

HIV infected patients may be predisposed to the development of

clinically significant and progressive heart disease (Herskowitz et al.,

1992; Herskowitz & Baughman, 1994). This subgroup of patients

frequently present with class HI or IV congestive heart failure and

demonstrate rapid clinical deterioration. One emerging hypothesis to

explain the high frequency of dilated heart muscle disease in HIV

seropositive patients is the association between HIV related ventricular

dysfunction and myocarditis. The increased CD8+ T-lymphocytes and

sole induction of MHC class I in the HIV related myocard i t i s

popula t ion are l ikely due to the marked systemic decrease in

circulating CD4+ T-lymphocytes in AIDS patients (Levy et al., 1985).

Studies by Matsumori et al. (1994) have suggested alterations

in cytokine production in patients with dilated heart muscle disease.

Patients with HIV related myocarditis have elevated levels of TNF-a

and IL-6 compared to patients without myocarditis suggesting that

specific cytokines may play a role in the pathogenesis of HIV related

myocarditis (Herskowitz et al., 1995).

Molecular mimicry:

Most s tudies have addressed molecular mimicry as cross

reactions of antibodies with linear peptides shared by self and foreign

molecules (Theofilopoulos, 1995). The term 'molecular mimicry' was

i n i t i a l l y used in 1968 to exp la in pe r s i s t en t v i ra l i n f ec t i on .

Autoimmunity provided by molecular mimicry should occur only when

the microbial and host determinants are similar enough to cross react.

yet different enough to break immunologic tolerance. The induction

and breaking of tolerance at both the B- and T-cell levels have been

established in heterologous serum protein models , and the same

kinetics probably govern the establishment an4 breaking of tolerance

to microbial agents cross reacting to host proteins.

It is to be pointed out that an antibody reacts with the three

dimensional configuration of a molecule, the antibody could bind but

with a different affinity, to similar but not necessari ly identical

configurations. The multiple organ reactive antibodies recognized

either the same molecule present in more than one organ or different

molecules in multiple organs (Haspel et al., 1983). Molecular mimicry

is a common phenomenon. The role cross reacting antibodies play in

pathogenesis will depend on the nature and location of the autoantigens

as well as on the properties of the antibodies (Srinivasappa et al.,

1986). Homology by itself may not lead to a cross reacting immune

r e s p o n s e . Howeve r , unless the homology and subsequen t

immunological cross reactivity involve a host protein that can

precipitate disease, the autoimmune response is unlikely to lead to

autoimmune disease.

Infectious agents may participate in the induction of autoimmune

disease not only through the process of mimicry, but also through other

effects, including tissue damage and release of sequestered antigens,

availability of cryptic self determinants through increased MHC

expression, ec topic expression of molecules , redis t r ibut ion of

intracellular molecules to the cell surface, upregulation or shift in

the spectrum of cytokine production, immunological exhaustion and

bystander activation of T-cells (Theofilopoulos, 1995).

In humans, myocarditis is known to be the precursor of DCM in

some cases, although clinical and histological diagnosis remains

obscure (Aretz et al., 1985). The commonest cause of myocarditis is

said to be a virus infection, but majority of cases are of unknown

etiology (Woodruff, 1980). Pedigrees where myocarditis and DCM

were present in different family members have been repor ted

(O'Connell et al., 1983).

Since in DCM. the only affected organ is the heart muscle, to

prove autoimmune involvement, it is necessary that the criteria of an

organ specific autoimmune disease are fulfilled (Rose and Bona, 1991).

Autoimmune features in DCM include familial aggregation, a weak

association with HLA-DR4 and immunoglobulin genes, abnormal

expression of HLA class II on cardiac endothelium and increased levels

of circulating cytokines. The organ-specific antibodies produced a

diffuse cytoplasmic staining of myocytes but did not stain skeletal

muscle. Antibodies were classified as cross reactive 1, which exhibit

only partial heart specificity, gave a fine striational staining on cardiac

tissue and stained weakly skeletal muscle fibres. The entirely cross

reactive antibodies, classified as cross reactive 2. stained with a broad

striational pat tern both in heart and skele ta l muscle sec t ions .

Absorption studies with relevant tissues had confirmed the organ

specificity and cross reactivity of the antibody types (Caforio et al.,

1990).

Like other autoimmune conditions, DCM autoantibodies seem

to be produced against multiple antigen specificities. The multiplicity

of parallel autoimmune reactions directed towards a single organ could

be due to spread sensitization. Thus, a single, initial autoantigen would

give rise to the first attack of the target organ, which would result in

T-cell mediated inflammation or the release of degradation products.

These mechanisms would enhance autoantigen presentation or release

immunogenic cell components that would give rise to a secondary

autoimmune response that would be difficult to differentiate from the

primary one. Conversely, as suggested, the initial event is a target

cell abnormality or lesion that renders a set of immunogenic peptides

: only the peptide with a sufficient affinity for MHC molecules would

be recognized by the T-cell repertoire, leading to an autoimmune

response (Bach, 1995).

Several autoantigens which react with sera from patients with

DCM have been identif ied. Studies on t issue from pat ien ts of

myocardi t i s and DCM have identif ied increased express ion of

intracellular antigens such as adenine nucleotide translocator (ANT)

and branched chain ex-ketoacid dehydrogenase (BCKD) complex.

Antibodies against the intracellular mitochondrial ANT protein cross

react with myocyte sarcolemmal calcium ion (Ca^ ) channel proteins.

Binding of Ca' channel proteins by such cross reactive antibodies can

10

thus physiologically alter the metabolism of normal myocyte, possibly

leading to myocyte injury (Ansari et al., 1991). Since SR-Ca^* ATPase

constitutes the major calcium regulatory protein in the sarcoplamic

reticulum regulating myocardial contractility, autoimmune inhibition

of this enzyme may affect the functional outcome of the myocardium.

The time dependent association between SR-Ca^^ ATPase immunization

and development of severity of myocarditic lesions supports the

immunopathogenetic role of SR-Ca^' ATPase. The involvement of T-

cell immunity may contribute more to the generation of myonecrosis,

whereas the antibody mediated immunity may contribute more to

functional impairment and activation of complement (Sharaf et al.,

1994). The ADP/ATP carrier has organ specific antigenic determinants

although there is a partial identity among the carrier proteins from

heart, kidney and liver. The organ specificity of ADP/ATP carrier was

also conf i rmed by the organ specif ic r eac t ion of d i l a t ed

cardiomyopathy sera with this protein. The autoantibodies reacted with

antigenic determinants at the cardiac myocyte plasma membrane and

interacted with atleast one subunit of calcium channel. Therefore, the

autoantibody was also called the Ca-channel reactive antibody. A cross

reaction between antigenic determinants could be of pathogenic

significance because antibodies against the homologies were present

in pa t i en t s with m y o c a r d i t i s and DCM (Ansar i et a l . , 1 9 8 8 ;

Schwimmbeck et al., 1993).

After an au to immune response is in i t i a t ed , c i r c u l a t i n g

autoantibodies against the ADP/ATP carrier disturb myocardial energy

11

metabolism and cross react with myocyte sarcolemmal Ca^' channel

proteins. The antibodies were able to bind to the Ca^^ channel and

enhance calcium influx and calcium overload in cardiac myocytes,

finally resulting in cytotoxic damage (Liao, 1996).

The most important intracellular microorganisms associated with

viral disease include a variety of viruses (Woodruff, 1980), two

m e m b e r s of ch lamydiae (Chlamydia psittaci and Chlamydia

pneumoniae) (Kuo et al., 1993), several rickettsiae. Toxoplasma gondi

and Toxoplasma cruzi (Speirs et al., 1988). Trichinella spiralis is the

most prone worm to cause myocarditis. Myocarditis pathogenesis may

be complex and involve various immune mechanisms. In addition,

many gram-posi t ive and gram-negat ive bacter ia may persis t and

multiply intracellularly or extracellularly or both and cause damage

to the myocardium by various pathways . Some bacteria produce

exotoxins that may cause damage to the myocardium, whereas others

release endotoxins that may be harmful. The bacteria commonly

encountered in myocarditis are Beta-hemolytic streptococci (Putterman

et al., 1991), Corynebacterium diptheriac (Ua\a\daiT, 1992), Neisseria

meningitidis (Hardman and Earle. , 1969), Yersinia enierocolitica

(Zollner et al., 1992), Salmonella typhi or paratyphi and Borrelia

burgdorferi (Klein et al., 1991). Mycoplasma pneumoniae was

incriminated in 6% of consecutive military conscripts suffering from

unequivocal myocarditis, and in 1-2% of conscripts hospitalized with

mycoplasmal infections (Karjalainen, 1990). Trypanosoma cruzi

(Chagas ' disease) is a well recognized cause of myocarditis and

12

cardiomyopathy in both urban and rural areas of South America. The

African trypanosomes, T. gambiense and T. rhodesiense occasionally

cause myocarditis and cardiomyopathy (Tsala Mbala, 1988).

Coxsackie virion and other viral proteins share epitopes with

internal or plasma membrane proteins of normal cells (molecular

mimicry) and s t imulate immune responses which par t ic ipa te in

autoimmune reactions. These hypotheses may not be exclusive and

all may be operative in a single model. CVB3 or CVB4 particles share

epitopes with human cardiac myocyte sarcolemmal proteins, human

and mouse cardiac myos ins , s t reptococcal M-prote in , adenine-

translocator protein and an unidentified protein(s) on the plasma

membrane on no rma l mouse ca rd iac myocytes or f ib rob la s t s

(Srinivasappa et al., 1986).

This underlines the clearence of virus via rapid and other

immune responses significantly affects the outcome (acute resolved

versus acu te / ch ron ic m y o c a r d i t i s ) . Antecedent i n f e c t i o n s by

heterologous CVB3 serotypes can exacerbate or aneliorate in CVB3

induced myocarditic challenge mechanism by which shared epitopes

between viral cel lular prote ins can provide either protect ion or

increase severity of CVB3 induced myocarditis. Studies by Gauntt et

al. (1995) suggest that antibodies to only a limited numbers of epitopes

on the light meromyosin (LMM) rod portion of human cardiac myosin

(^M) are shared with CVB3m and participated in protection/enhanced

disease and antibodies to the majority of shared epitopes in LMM are

13

without consequence in CVBSm induced heart disease. Resistance to

this enterovirus and other intracellular infections partly depends on

natural kil ler cells (NK) which are vulnerable to the effects of

environmental pol lutants such as mercury, nickel, 8-tetrachloro-

dibenzo-p-dioxin and methyl mercury. The development of disease as

seen in the heart of CBS virus infected mice, can be severely affected

in different ways by different heavy metals . Furthermore, during

infection, the organ distribution of heavy metals may be changed in a

specific way for each compound. This may subsequently result in

altered infectious disease pathogenesis (Ilback et al., 1995).

The G-protein coupled membrane receptors are all members of

a superfamily of membrane proteins which structurally share the

overall bacteriorhodopsin architecture, seven a -helices, spanning

the membrane lipid bilayer and forming a hydrophobic pocket that

can serve as a pharmacophore. The seven helices are linked together

by three extracellular and three intracellular loops. The N-terminus

of the polypeptide is located in the extracellular space while the C-

terminus is located in the intracellular space. In order to induce an

immune r e s p o n s e aga ins t G-pro te in c o u p l e d r ecep to r s , two

requirements are necessary : firstly the receptor must be degraded by

proteolysis to small fragments and the fragments generated must be

able to form a complex with one of the MHC or HLA class II molecules

of the host.

The functionally important G-protein coupled receptors are (3 -

adrenoceptor and the M2 muscarinic receptor . Anti-p receptor

14

antibodies were present in 30-40% patients of dilated cardiomyopathy.

These antibodies were produced against P, peptide corresponding to

the second extracellular loop with little reactivity towards a P^ peptide

in patients with DCM. P,-adrenergic receptor has two antigenic

regions. Some of the sera recognize the first extracellular loop, other

sera the second extracellular loop of the P,-adrenoceptor. The

dominant epitope in the second loop is the cysteine-containing amino

acid sequence A-R-R-C-Y-N-D which forms a disulphide bridge with

the cysteine of first extracellular loop (Wallukat et al., 1995). The

immunoglobulin fraction isolated from serum samples of myocarditic

and cardiomyopathic patients contains stimulatory autoantibodies,

directed against P,-adrenoceptor which increase dose dependently,

the beating rate of the cultured neonatal heart myocytes. Recent

studies have pointed out the presence of anti-receptor autoantibodies

in DCM (Fu et al., 1994). Immunization of genetically predisposed

mice with cardiac myosin causes cardiac enlargement, the production

of heart-specific antibodies and a heart condition that resembles

human DCM. the autoantibodies in patients against human ventricular

myosin are directed against myosin heavy chain. The a - and P-myosin

heavy chains are relevant autoantigens by the heart specific antibodies

detected by immunofluorescence in DCM. In humans, the a -myosin

heavy chain isoform is expressed exclusively in atrial myocytes,

whereas the P -isoform is present both in ventricular myocytes and

slow skeletal muscle fibers (Caforio et al., 1992). Cardiac myosin

induced myocarditis is T-cell mediated and class II MHC restricted.

15

Myosin-reactive T-cells from normal myosin immunized donors

induced severe myocarditis in severe combined immunodeficiency

(SCID) mice. Potential myosin reactive T-cells, though lacking CD4

or CDS, still remain functional and hence CD4 or CDS molecules are

not essential for induction of autoimmune myocarditis (Pummerer et

al., 1995).

Myosin is an intracellular molecule. Viral infection or other

causes of tissue necrosis might lead to release or exposure of myosin

and trigger autoimmunity in individuals with a predisposing genetic

background. Virus may promote auto-sensi t izat ion to myosin by

molecular mimicry (Oldstone, 19S7; Srinivasappa, 19S6). Monoclonal

antibody to coxsackie VP-1 capsid protein has been demonstrated

which reacts with cardiac myosin heavy chain.

The mechanism by which myosin as well as other autoantigens

previously identified may trigger and perpetuate, although not directly

cause autoimmune disease is unknown. It also remains to be established

whether the antibodies found in DCM sera have a direct pathogenic

role or, like the anti-myosin antibodies found in murine autoimmune

myocarditis, are only markers of immune damage.

Role of 150 kD cardiac C-protein:

Cardiac C-protein is found to be a dominant antigen among many

types of cardiac pro te ins and is known to induce autoimmune

myocarditis as judged on the basis of experimental studies indicating

16

production of autoantibody by mouse specifically against 150 kD C-

protein on induction of myocarditis. This specific antibody is produced

inspite of immunization with whole cardiac tissue homogenate .

Purified C-protein also produced myocarditis and recombinant cardiac

C-protein (amino acid res idue 205-916) effect ively p roduced

myocarditis in some mice strains including SMA, DBA/IJ, SJL, and

02°/A, but not in A/J, AKR/J, or DBA/2J mice strains (Kasahara et

al., 1994). C-protein is a component of thick filament of skeletal and

cardiac muscles (Yamamoto and Moos, 1983). The function of C-

protein remains unknown, but it has been suggested that C-protein

may regulate thick filament assembly and length (Squire, 1981),

participate in thick filament structural support (Pepe et al., 1975),

maintain the structure and contribute to the radial elasticity of the

sarcomere, or regulate cross-bridge movement during contraction

(Magid et al., 1984).

C-protein has a number of properties that make particularly

attractive the suggestion that it plays a role in regulation of contractile

activity. For example, C-protein binds to both purified actin (Moos et

al., 1978) and myosin (Moos et al., 1975) and can alter the ability of

actin to stimulate myosin ATPase (Moos and Feng, 1980). The effects

of C-protein on ATPase activity are, however, complex . C-protein

inhibits skeletal muscle actomyosin ATPase, but stimulate cardiac

muscle actomyosin ATPase (Yamamoto and Moos, 1983). It has been

suggested that the physiological role of C-protein may involve a

calcium-regulated binding of C-protein to thin filaments, because C-

17

protein binding to native thin filaments occurs only in the presence

of micromolar concentrations of calcium (Moos, 1981). It has been

shown that C-protein in cardiac muscle becomes phosphorylated in

response to P-adrenergic agonists and dephosphorylated in response

to cholinergic agonists (Hartzell and Titus, 1982). The level of protein

phosphorylation correlates with the rate of relaxation of cardiac

contraction and it has been suggested that C-protein regulates twitch

relaxation in cardiac muscle. Electron microscopy shows C-protein

to be V-shaped. The other par t ic les could be seen as distorted,

stretched or collapsed V-shaped particles.

C-protein is one of the major constituent protein necessary to

compete with natural antigens such as invariant chain and MHC to

obtain the position at the MHC-antigen binding site (Demotz et al.,

1990; Harding and Unanue , 1990). Moreover, C-prote in is an

intracellular member of the immunoglobulin superfamily (Einheber

and Fischman, 1990). IgG like domains are conserved in most cell

surface immune recognition molecules and also TCR and MHC class

II have this domain (Williams and Barclay, 1988). The peptides of

recycled membrane proteins, class II HLA and invariant chain, have

been shown to be major self-antigens which are presented by class II

molecules. However, the HLA binding motifs are located in the

variable domain of this structure, not in this constant IgG like domain

(Rudensky et al., 1992; Baum et al., 1993). The question remains

whether or not these IgG like conformations work with each other at

cell surface similar to the directly bound MHC-TCR like superantigen

(Woodland and Blackman, 1993).

18

The findings of Kasahara et al. (1994) conflict with an earlier

report that myosin is the major antigen producing au to immune

myocarditis under H-2 genetic restriction (Neu et al., 1987). In SMA

mice, repeated injections of myosin-enriched heart extract with K 0 3

LPS still preferentially produced the autoantibody to C-protein. In

addition, myosin contamination was completely avoided by using the

fusion protein expressed in E. coli PI6-4, which is encoded by part of

the cDNA of C-protein. CB3 induced myocarditis which might further

trigger autoimmune reactions against myosin by a molecular mimicry

mechan ism, has been sugges ted to be a p o s s i b l e u n d e r l y i n g

pathogenesis in DCM (Noel et al., 1991). Kasahara et al. (1994)

suggested that the autoimmune triggering mechanism in C-protein

induced myocarditis may differ from a CB3-triggered or myosin-

triggered mechanism because cross-reactive epitopes between CBS and

C-protein have not been reported; contrary to the previous results that

A/J, C3H/He, Balb/c, DBA/2 strains have a high susceptibility to CB3

(Lawrence et al., 1991), the C-protein residue 205-916 hardly induced

myocarditis in these strains.

Fur ther assessment about the i nvo lvemen t of C-pro te in

t r iggered autoimmune myocardi t i s in DCM, immunoblot t ing was

employed with sera of 16 DCM patients against whole cardiac

proteins. Out of 16 DCM patients sera of 2 patients preferentially

recognized the C-proiein. On the contrary, none of the control sera

reacted specifically to C-protein (Kasahara et al., 1994).

20

crossreactivity. The maximum score possible for each peptide is

variable and is determined by its length and by the amino acids present,

rarer amino acids having a higher value. The scoring system used,

attaches the highest values to exact matching of rare amino acids. This

level of matching would be expected to lead to cross reactivity of

epitopes since far weaker matching occurs within conserved motifs of

peptide^ bound to class II major histocompatibility complex peptides.

Differences between individuals in MHC class II may influence the

selection of particular hsp epitope and the corresponding target antigen

that gives rise to an autoimmune disease (Jones et al., 1991).

The amino acid sequences/peptides of autoantigen region 384-

396 and 451-470 of hsp share homology with the amino acid sequences/

peptides of myosin heavy chain 136-148 and 253-272 with a similarity

score of 65 and 64, respectively, as seen in coxsackie myocarditis

(Jones et al., 1991). Latif et al. (1993) have reported antibodies against

a 60 kD band in 85% DCM patients by Western blotting. Two

dimensional gel analysis and protein sequencing revealed 100%

homology of this peptide with hsp 60. The presence of circulating

antibodies against hsp 60 in 85% of DCM patients is clear evidence

of a damaged myocardium. This suggests that myocyte injury induces

the expression of hsp60 or other stress proteins that may have immuno­

modulatory activities and could influence the development and course

of autoimmune recognition of the myocardium. The exact pattern and

distribution of hsp60 in normal and diseased myocardium remains to

be established.

21

DNA-protein photoadducts/photointeractions:

A variety of chemicals and ionizing and ul traviolet (UV)

radiations are known to produce DNA-protein crosslinks (Oleinick et

al., 1986). Several reports indicate that the bonds formed between DNA

and proteins are of covalent nature (Oleinick et al., 1986; Cress and

Bowden, 1983). Proteins and nucleic acids are the two most important

classes of functional biomolecules in cel ls . These "co lour less"

macromolecules absorb in the UV region of spectrum. Typical

photochemical reactions have been identified that are relevant to

biological damage induced by exposure of organisms to radiation.

The crosslinking of nucleic acids to protein is one of the lesions

produced in biological systems by UV light, which has been observed

in bacteria (Smith, 1962; Alexander and Moroson, 1962; Bridges et

al., 1967) and in mammalian cells (Alexander and Moroson, 1962;

Habazin and Han, 1970; Han et al., 1975; Zimmernan et al., 1972).

The photobinding of serum albumin to DNA (Maukovitz, 1972) and

gene-5-protein to bacteriophage IdDNA (Anderson et al., 1975)

provides direct proof at a molecular level for the crossl inking

p h e n o m e n o n . The role of DNA-pro t e in c ros s l inks in ag ing ,

carcinogenesis and radiation biology has been well documented

(Smith, 1975).

The recognit ion between nucleic acids and proteins is of

fundamental importance since it triggers most steps of nucleic acid

metabolism. This recognition, presumably, first involves electrostatic

22

interactions of phosphate groups with positively charged regions in

proteins; then, more specific interactions take place which involve

possible interactions between nucleic acids bases (or other structural

elements) and amino acids side chains in proteins. A more extended

scope of examination reveals the possible recognition of secondary

or tertiary structures of nucleic acids by proteins. In either cases,

recognition may be dependent of nucleotide sequence (Duguet, 1981).

The initial isolation of a mixed photoproduct of thymine and

cysteine (5-S-cysteine-6-hydrothymine) from in vitro UV irradiation

of a solution of thymine and cysteine has served as a model for

crosslinking phenomenon. Fifteen amino acids have been found to

react photochemically with DNA. Cysteine, lysine, phenylalanine,

tryptophan and tyrosine were the most reactive; alanine, aspartic acid,

glutamic acid, serine and threonine are unreactive (Shetlar et al.,

1985). It has been suggested that the resistance of Micrococcus

radiodurans, one of the most radiation resistant organisms known, is

due to its extraordinary ability to repair pyrimidine dimers, but what

ultimately kills the organism is damage that involves both DNA and

protein. The crosslinking of DNA and protein may constitute one type

of such damage (Shetlar, 1980).

A different type of photochemisty arises when protein and DNA

are irradiated together as compared to when they are irradiated

separately. Since, DNA and proteins do not exist in cells as pure

solutions of the separate molecules but are in intimate contact with

23

each other, it is suggested that photochemical interaction of DNA and

protein would play a significant role in the inactivation of UV

irradiated cells under certain conditions.

Hydroxyl (OH) radicals produced from water by ionizing

radiation appeared to be responsible for the formation of ionizing

radiation induced DNA-protein crosslinks iil isolated chromatin and

in intact cells (Oleinick et al., 1986; Mee and Adelstein, 1981). The

nucleosomal core of the histones as well as non-histone proteins has

been found to be involved in the formation of DNA-protein crosslinks

in isolated chromatin exposed to ionizing radiation (Mee and

Adelstein, 1981). The hydroxyl radical induced formation of DNA-

protein crosslink between methyl group of a Thy moiety and the C-3

position of a Tyr moiety in calf thymus nucleohistone in aqueous

solution has been described (Dizdaroglu et al., 1989).

DNA-protein crosslinking in normal and solar UV sensitive ICR

2A frog cell lines exposed to solar UV radiation has been observed

(Rosenstein et al., 1989). Induction of DNA-protein crosslinks in these

mutant cell lines may help in examining the role played by this type

of DNA damage in the hypersensitivity to solar UV radiation which

are exhibited by these mutant cells. These crosslinks might be lethal

to the cell if they interfere with normal gene functions.

Role of free radicals:

A free radical can be defined as a chemical species possessing

an unpaired electron and is capable of independent existence. Free

24

radicals can be formed by:

i) Hemolytic cleavage of a covalent bond of a normal

molecule, with each fragment containing one of the

unpaired electrons

ii) By the loss of a single electron from a normal molecule

iii) By the addition of a single electron to a normal

molecule.

The prima donna in the biochemistry of oxygen free radicals

are oxygen itself, superoxide, hydrogen peroxide, transition metal ions

and the hydroxyl radical, the first four of which conspire by a variety

of interactions to generate the last (Halliwell and Gutteridge, 1990).

Under normal circumstances, the major source of free radicals in cells

is electron 'leakage' from electron transport chains. These can be

mediated by the action of enzymes or non-enzymatically, often through

the red-ox chemistry of transition metal ions (Cheeseman and Slater,

1993). Free radical production can also be increased by exogenous

sources like toxic foreign compounds and ionizing radiations.

Although there are several sites of ROS generation in cells, the main

source of O^'and its stoichiometric product, H^O^ is the auto oxidation

of the reduced components of the mitochondrial electron transport

chain (Haiku et al, 1993). In the presence of trace amounts of transition

metal (M) ions (i.e. Fe^^ Cu" ') found in biological systems (Halliwell

and Gutteridge, 1985), H^O^ can participate readily in Fenton-like

reactions (Fenton, 1894; Cohen, 1985) resulting in the production of

OH as shown.

25

Mn+ + H ^ O ^ ^ M(" ')+ OH + OH

Hydroxyl radicals have been implicated in the dele ter ious

processes such as gene mutation (Okada, 1970), cell transformation

(Borek, 1985) and cell death (Painter, 1980). Nitric oxide (NO') is an

important physiological free radical produced by phagocytes in which

the unpaired electron is delocalized between two atoms. Nitric oxide

or its derivative acts upon smooth muscle cells in vessel walls to

produce relaxation, besides acting as a neurotransmitter (Hirono et

al., 1997).

DNA damage by ROS:

DNA is an important target for free radicals since it is clearly

of major significance for cell function as well as its susceptibility of

being damaged by oxidizing radicals (Wiseman and Halliwell, 1996).

The most important reac t ions seems to be based on hydrogen

abstraction to form carbon-centered radicals followed by oxygen

addition to form peroxyl radicals that subsequently decay. Addition

of OH to double bonds is also an important mechanism. Both the

nucleobases and the sugar phosphates offer potent ia l targets for

hydroxyl radicals (*0H). The consequences of oxidat ive radical

damage to DNA are highly serious which inc ludes cell death,

reproductive cell death, mutation and transformation into cancer cells

(Feig et al., 1994). Oxidative damage to DNA includes a range of

specifically oxidized purines and pyrimidines as well as alkali labile

sites and strand breaks formed directly or by repair processes

26

.(Dizdaroglu, 1991; Dizdaroglu, 1994; Breen, 1995). The apparently

most abundant and certainly the most studied oxidative modification

of DNA bases involves the C-8 hydroxylation of guanine, frequently

est imated as the oxidized deoxynucleos ide , 8-oxo-7,8-dihydro-

2'deoxyguanosine (8-oxodG). Oxidation of guanine can also lead to

the ring opened product of 2,6-diamino-4-hydroxy-5-formamido

pyrimidine (Dizdaroglu, 1991). Other abundant oxidatively modified

purines and pyrimidines include 8-oxoadenine, 2-hydroxyadenine,

Fapy adenine, 5-hydroxy methyluracil, 5-hydroxycytosine, cytosine

glycol and thymine glycol (Tg). In addition, a large number of other

modifications of bases and sugars have been identified (Dizdaroglu,

1991; Dizdaroglu, 1994). All these modifications are present in DNA,

and the level can be increased in vivo or in vitro by systems generating

ROS. The yie ld of the individual DNA modificat ions is highly

dependent on which ROS are involved. Thus, whereas, singlet oxygen

induces preferentially 8-oxodG (Epe, 1991), superoxide has low

reactivity to induce this modification at all. Apriori to the above,

hydroxyl radicals can cause almost any modification (Dizdaroglu,

1991). In addition, the target molecule conditions, such as oxygen

tension, chelation of transition metals and the presence of reductants

may influence the yield and nature of oxidative DNA modifications

(Dizdaroglu, 1994).

Oxidative modifications of DNA bases can lead to mutations if

left unpaired or repaired with errors before replication. From studies

on bacteriophage and plasmid DNA, it appears that although radicals

21

generated by iojiizing radiations damage all four bases, mutations are

usually related to modifications of GC base pairs in contrast to AT

base pairs. Thus, AT base pair perturbations/modifications rarely lead

to mutations . The mutations are clustered in hotspots and are mainly

base pair substitutions, whereas, base deletions, large deletions, and

insertions are less frequent. Hydroxyl radicals generated from ionizing

radiation induce mainly GC to CG or AT base pair substi tutions,

depending on DNA expression system, whereas, hydrogen radicals

preferentially induce GC to AT substitutions (Retil et al., 1993).

Singlet oxygen and 1,2-dioxetanes preferentially induce 8-oxodG and

GC to AT base pair substitutions (Epe, 1991; Retel et al., 1993; Emmert

et al., 1995). In agreement, modified guanine such as 8-oxoguanine

and apurinic sites are mispaired mainly with adenine, at a frequency

dependent on the adjacent base sequences and the involved polymerase

(Kamiya et al., 1995). Majority of ROS induced mutations appear to

involve modification of guanine, in particular 8-oxoguanine, causing

G to T transversions. In addition to the mutations related to mispairing

to oxidised bases, ROS may also cause reduced fidelity of DNA J3

polymerase (Feig and Loeb, 1993) and alter the methylation of

cytosine and thus gene control (Weitzman et al., 1994; Loft and

Poulsen, 1996).

Protein damage by ROS:

Most proteins are susceptible to modification by OH or by OH

+ O " (+ Oj). The modification includes altered molecular weight

28

(aggregation or fragmentation), altered net electrical charge (+ or - ) ,

loss of tryptophan, and production of bityrosine. Since Oj 'alone has

no measurable effect on any of these parameters, it has been suggested

that OH is the initiating species. Oxygen and/or O2" appear to modify

significantly the damage induced by OH. Protein fragmentation has

been reported following exposure to OH + O^-*- Oj (Garrison et al.,

1962; Schuessler and Schilling, 1984). This process involves hydrogen

abstraction by OH from amino acid a-carbon atoms, followed by

reaction with O^to produce peroxyl species. Decomposition of carbon

peroxides was proposed as the mechanism for protein fragmentation

(Garrison et al., 1962).

All amino acids in the protein are susceptible to modification

by OH or by OH + 0 , ( + O^). In contrast, O^"alone appeared only to

reduce cysteine residues. In agreement with known rate constants for

reaction of free amino acids with H, tryptophan, histidine and cysteine

are more vulnerable than most other residues. Tyrosine is formed as a

simple addition product of OH + phenylalanine. The selectivity of

OH for tryptophan, tyrosine, histidine and cysteine is less than might

expected from reactions with free amino acids (Dorfman and Adams,

1973). The p rocess of aggregat ion by OH appears to involve

intermolecular bityrosine formation. It is highly unlikely, however,

that bityrosine is the only (non-disulfide) covalent modification.

Essentially any amino acid radical formed within a peptide chain could

crosslink with an amino acid radical in another protein. Alternatively,

some radicals may crosslink with amino acid molecules. Protein

29

denaturation also preceded increases in proteolytic suscept ibi l i ty

following exposure to OH + O^. Thus d e n a t u r a t i o n / i n c r e a s e d

hydrophobic i ty is a pos s ib l e cause for enhanced p r o t e o l y s i s .

Oxidatively denatured proteins are degraded by novel ATP and calcium

independent, soluble proteolytic systems (Davies et al., 1987).

ROS in SLE:

Antibodies to DNA in SLE have been extensively studied and

may be specific for ssDNA, reactive with both ssDNA and dsDNA or

specific for dsDNA (Worrall et al., 1990; Stollar, 1979). However, it

is ant ibodies to nat ive dsDNA that r epresen t a c h a r a c t e r i s t i c

serological finding in these patients and are of important diagnostic

and clinical value. It has been shown that modification of DNA can

render it more immunogenic (Ali et al., 1985). Native dsDNA has been

reported to be a weak immunogen (Tan and Stoughton, 1969) and yet

in certain autoimmune conditions like SLE, autoantibodies to multiple

nuclear antigens, including DNA and histones are formed. Blount et

al, (1991) postulated that DNA damaged by ROS may affect the

development of autoimmune diseases, like SLE. The process by which

DNA is damaged in such cases may involve the release of ROS

inteiinediates during the characteristic respiratory burst of activated

neutrophils, denature DNA in such a way that SLE serum anti-DNA

antibodies bind better to it (Blount et al., 1989). ROS denaturation

exposes base residues in the DNA backbone and minor regions of

ssDNA. Lymphocytes i so la ted from pat ien ts with SLE contain

30

increased levels of 8-oxodG in the DNA. In urine, the SLE patients

did not secrete 8-oxodG. A study on cultivated blood monocytes

isolated from SLE patients showed reduced capacity for repair of 8-

oxodG induced by incubation with hydrogen peroxide. In DNA isolated

from immune complexes precipitated from plasma, 8-oxodg was a 100

fold more abundant in SLE patients compared to usual values of

nuclear DNA. This oxidatively modified DNA may be a source of DNA

antibodies characteristic for SLE. The SLE patients suffer from an

increased rate of oxidative DNA damage and also from deficient repair.

This may contribute to the pathogenesis and even to increased risk of

malignant diseases in these patients (Lunec et al., 1994; Bashir et al.,

1993). Hydroxyl free-radical mediated in vitro modification of calf

thymus DNA showed single strand breaks, decrease in melt ing

temperature and structural alteration of purine and pyrimidine bases

leading to polyspecificity of induced antibodies (Alam et al., 1993,

Ara and Ali, 1993). Antibodies against free radical modified DNA

recognize B-conformation (Ara et al., 1992). ROS modified DNA

promises to be a more discriminating antigen than native DNA for

binding of SLE autoantibodies (Ara and Ali, 1993) and the binding

increased considerably with increase in DNA fragment size (Ara and

Ali, 1992).

ROS/RNS in autoimmune myocarditis/DCM:

Oxygen derived free radicals and their metabolites have been

implicated to participate in various types of myocardial injury. These

31

radicals and metabolites are thought to mainly consist of superoxide

anion (O^'" ) hydrogen peroxide (H^O^) and hydroxyl radical (OH),

however, the culprit playing the most important role is still

undetermined. The involvement of HjO^ generated through dismutation

of 02'caused severe myocardial damage (Kloner et al., 1989). Oxygen

derived free radicals and their metabolites can cause enzyme

inactivation either directly or indirectly (Miki et al., 1988). Thus,

functional deterioration may be linked to ATP reduction by inactivation

of enzymes necessary for glycosylation and oxidative phosphorylation

(Goldhaker et al., 1989). On the other hand, calcium transport in the

sarcoplasmic reticulum has been demonstrated to be decreased by

exposure to oxygen radicals (Hess et al., 1984). *0H, on the other

hand, is produced by H^Oj through iron catalysed Haber Weiss reaction

(Haber and Weiss, 1934) and is highly reactive. It causes lipid

peroxidation of the biological membranes (Meerson et al., 1982)

resulting in changes in membrane permeability and fluidity, and finally

results in loss of membrane integrity. The plasma membrane and

mitochondria are more damaged in the heart exposed to higher OH

levels. The combination of these changes may have resulted in

decreased contractility in hearts exposed to OH (Takemura et al.,

1994). Experimental studies by Fukuchi et al. (1991) have indicated

the possible involvement of free radicals and antioxidants in the early

stages of the development of cardiomyopathy in BIO 14.6 Syrian

hamster.

32

Recent studies on the molecular genetics of the myocardium have

revealed that a certain percentage of patients with DCM possess

mutation in the mitochondrial (mt) DNA of the myocardium. These

patients can be regarded as microchondrial cardiomyopathy (mtCM),

a new clinical entity. The cumulative increase in oxygen free radical

damage in human heart mt DNA closely correlates with deletions

associated with age and with mtCM. This led us to the ' redox

mechanism of ageing and degenerative disease ' in which the oxygen

free radical damage in mt DNA results in a cumulative increase in

somatic mutations, leading to bioenergetic deficit, cell death, ageing

and degenerative diseases. The accumulation of hydroxyl radical

adducts in mt DNA hydrolysate, such as 8-hydroxydeoxyguanosine

(8-OH-dG) in place of deoxyguanosine (dG), could satisfy this

prerequisite to the deletion (Hayakawa et al., 1992). The mt DNA,

having no protective protein, is located inside the inner mt membrane.

During the evolution from yeast to mammals, mt DNA reduced to one

fifth of its size by loss of introns. Thus, human mt DNA more easily

acquires oxygen free radical damage than single celled organisms. The

revelation that human mt DNA is highly susceptible to hydroxyl attack

resulting in breakdown into hundreds of mini circles suggests that the

major target of the active cell death machinery would be mt DNA.

The cumulative and synergistic increase in oxygen damage and

deletions in mt DNA associated with age implies that normal ageing

and development seem to be the two sides of a coin. The cell death

machinery could be easily imbalanced by the point mutat ions.

33

especially (syn) , in the mt DNA of patients with mtCM, leading to

their premature ageing (Ozawa, 1995).

In recent years, nitric oxide (NO) has been implicated in a wide

variety of immunologically mediated disorders. Pathologic release of

NO is considered to be one of the probable cause of diminished

myocardial contractility such as septic shock (Brady et al., 1992),

cardiac allograft rejection (Yang et al., 1994) and some form of

idiopathic dilated cardiomyopathy (Winlaw et al., 1994). Human

myocarditis varies greatly in origin and is generally a self limiting

process; acute congestive heart failure may develop during the active

phase, but cardiac function improves significantly in most cases as

myocarditis resolves histologically (Mason et al., 1995). It is possible

that the excessive production of NO by inducible nitric oxide synthase

(iNOS) is responsible for the development of myocardial organic

lesions as well as for functional changes in myocardial contractility

during the active phase of the disease.

Physiological actions of NO are mediated through the activation

of soluble guanyla te cyclase and the c o n s e q u e n t e levat ion of

intracellular cGMP level (Moncada and Higg, 1993). Studies suggest

that NO modulates myocardial contractility through an increase in

myocardial cGMP. It is reported that cGMP produces inhibition of

Ca^* influx into myocytes (Levi et al., 1989) or attenuation of the

positive inotropic effect of p-adrenergic stimulation (Watanabe and

Besch Jr., 1975). It is also possible that excessive and prolonged

34

production of NO by iNOS in infiltrating inflammatory cells modulates

the mitochondrial respiration in surrounding viable myocytes, thus

NO synthase inhibition in the early phase of viral myocarditis may

alter the adequate immune response to invading cardiotropic virus,

thus preventing the elimination of virus from cardiac tissue (Hirono

et al., 1997).

Systemic lupus erythematosus:

Systemic lupus erythematosus (SLE) is a multisystem prototype

auto immune disease (Isenberg and Shoenfeld , 1987) which is

characterized by the appearance of circulating of autoantibodies that

bind to numerous cellular and macromolecular antigens of self and

exogenous origin. Although there is a distinct correlation between SLE

and immune system, SLE continues to remain as a disease of unknown

etiology and origin. It has been postulated that immune responses

against host antigens could resul t from genetic p red ispos i t ion ,

exaggerated B-cell activity, cross reactivity between foreign and host

antigens or modification of host antigen as a consequence of infection,

inflammation, drug administration etc.

A lot of literature is available on the production and assay of

antibodies to DNA, structural analysis of specific antibody-nucleic

acid interaction and their application in biomedical research (Stollar,

1992). A central paradox of the pathogenesis of SLE is the origin of

anti-DNA antibodies. DNA to which most of the antibodies are

detected in SLE and other autoimmune disorders, is no longer regarded

35

as the inciting antigen because immunization with native DNA does

not induce SLE like disease. Animals immunized with covalently/non

covalently modified DNA induced high titre antibodies, which are

almost exclusively directed towards modified structures (Stollar,

1989). Genes encoding anti-DNA antibodies, however, are not

restricted to SLE and are part of normal B-cell repertoire (Cairns et

al., 1989; Stewart et al., 1993). The possible role of immunogenic

DNA, DNA-psoralen crosslink, hydroxyl radical, positively charged

amino acids and P-estradiol in the pathogenesis of SLE has also been

reported (Pisetsky et al., 1990; Hasan et al., 1991; Moinuddin and

Ali, 1994; Alam et al., 1993; Ara and Ali, 1993; Ara and Ali, 1992;

Alam et al., 1992; Ara et al., 1992).

Anti-DNA antibodies have been shown to cross react with

polynucleotides (Lafer et al., 1981), polynegatively charged structures

such as phospholipids (Smeenk et al., 1987), proteoglycans (Faaber

et al., 1986), cardiolipin (Eilat et al., 1986), dextran sulphate (Wollina,

1985), membrane associated proteins (Jacob et al., 1984), intermediate

filament of cytoskeleton (Andre - Schwartz et al., 1984), histones

(Gohill and Fritzler, 1987), bacterial products (Carroll et al., 1985),

platelets (Zouali et al., 1988), hyaluronic acid and chondroitin sulphate

(Faaber et al., 1984). Monoclonal anti-DNA antibodies of both human

and murine origin showed cross reactivity with antigens other than

DNA (Shoenfeld et al., 1983). There are many compounds in the

vicinity of DNA which may react upon irradiation. These compounds

include polyamines, histones and nuclear matrix elements. Polybasic

36

molecules found in cells have great affinity for acidic-constituents

such as DNA (Held and Awad, 1991) and their interaction may play a

role in the pathogenes is of SLE. Thus , the po lyspec i f i c i ty of

monoclonal anti-DNA antibodies and synthetic polypeptide antigens

suggests that antigens other than normal self ant igens serve as

immunogenic triggers.

SLE is a non organ specific autoimmune disease and as seen

earlier, a large panoply of autoantibodies have been identified in the

serum of lupus patients in varying frequency. The clinical picture of

SLE shows malar and discoid rashes, photosensitivity, oral ulcers, non-

eros ive a r t h r i t i s , p l e u r i t i s , p e r i c a r d i t i s , h e m o l y t i c anaemia ,

lymphopenia and thrombocytopenia.

Anti-DNA antibodies play a major role in the development of

lupus nephritis in MRL/lpr mice. Although anti - dsDNA antibodies

are considered to be the hallmarks of human lupus nephritis, most of

the anti-DNA antibodies in the murine lesions also bind to ssDNA

(Gavalchin et al., 1987). Before the onset of disease, the production

of anti-DNA antibodies shifts markedly from IgM to IgG isotype

(Papoian et al., 1974). This sw^itch is also associated with an increased

cationic charge of the anti-DNA antibody production (Batta et al.,

1987). The majority of anti - DNA antibodies in the renal lesions are

of lgG2a and IgG2b subclasses (Gavalchin et al., 1987).

Purified forms of nucleic acids are poor immunogens (StoUar,

1973). Modification of DNA, for example by ultraviolet light exposure

37

(Davis et al., 1976) or the presentation to the immune system of DNA

fragments (Lafer et al., 1981) has induced antibodies which bind to

immunogen but not to unmodified B-DNA.

The anti-DNA antibody response of lupus mice resembles an

antigen driven response that is composed of antibodies that are

heterogeneous in their DNA binding specificity. More compelling

evidence for the role of DNA as a selecting antigen has been obtained

from the sequencing of panels of anti-DNA antibodies isolated from

individual lupus mice (Schlomchik et al., 1990). Since the antibodies

in these panels all bind to DNA and show clonal restriction, it may be

concluded that DNA functions in vivo as a selecting antigen. A microbe

possessing both foreign epitopes and epitopes that mimic autoantigens

has the potential to elicit an autoimmune response (Rekvig, 1995).

Nucleic acids complexed with proteins have also been considered as

potential immunogens. Many human monoclonal anti-DNA antibodies

of both isotypes use VH genes which are members of fetally expressed

repertoire. It is possible that these genes contain CDR sequences which

lend themselves to formation of a DNA binding site. Comparison of

sequences of large numbers of anti-DNA antibodies enables us to

identify motifs which might contribute to such binding properties.

Comparison of the sequence of antibody cDNA with that of the

germline gene from which it is derived enables us to identify the

positions and nature of somatic mutations which may likewise

contribute to antigen binding (Isenberg et al, 1997).

38

Cardiovascular manifestations of SLE:

The cardiovascular manifestations of SLE have become more

apparent with prolonged survival and improvement in diagnost ic

modalities. While most patients still die of infection or renal failure,

the incidence of cardiovascular morbidity and mortality in SLE is

rising, and is now the third leading cause of death in SLE (Urowitz et

al., 1976). Whether there is a cardiomyopathy directly attributable to

lupus is unresolved. Most series cite a high incidence of heart failure

but attribute it to multiple problems, including; fever, infections,

anaemia , u raemia , h y p e r t e n s i o n , s t e ro ids and a c c e l e r a t e d

atherosclerosis. Studies on systolic time interval in SLE patients

indicate a shorter left ventricular ejection time and a longer pre-

ejection period than control subjects, suggesting left ventricular

systolic dysfunction, an indicator for primary lupus cardiomyopathy

(delRio et al., 1978). Haemodynamic studies have revealed that lupus

cardiomyopathy may exist even without clinical signs or may represent

abnormalities of the intrinsic contractile and relaxation properties of

the myocardium (Strauer et al., 1976). Various etiologies have been

proposed for lupus cardiomyopathy. Das and Cassidy (1973) detected

anti heart antibodies in 20 of 32 patients with SLE and proposed that

these antibodies might be involved in the genesis of myocardial

dysfunction in lupus. Bidani et al (1980) demonstrated immune

complexes in the myocardium in 9 of 10 necropsies. They proposed

that immune complex deposi t ion led to complement act ivation,

inflammation and myocardial damage. Either mechanism (anti heart

39

antibodies or immune complex deposition) may provoke the small

vessel vasculitis, focal myocarditis, fibrosis and myocardial necrosis

that is seen at autopsy in many SLE hearts. Further evidence is needed,

however, to establish the link between these pathologic findings,

immunologic abnormalities and cardiac dysfunction in v/vo (Doherty

and Siegel, 1985). Frustaci et al. (1996) have recently reported a case

of lupus myoca rd i t i s caus ing left v e n t r i c u l a r a n e u r y s m . An

immunologic mechanism of myocardial damage in this patient is

suggested by the results of serologic studies (increased anti-nuclear

antibodies, increased anti-DNA, decreased C3-4, increased ant i -

sarcolemmal antibodies) by the normal coronary arteriogram and by

the cardiac biopsy which showed active myocarditis and fibrinous

e n d o c a r d i t i s . Pos t -mor tem s t u d i e s have r evea led m y o c a r d i a l

inflammatory lesions in upto 70% of victims of SLE. It remains to be

seen if improved diagnosis and t reatment of the cardiovascular

manifestations of SLE can enhance survival.

Objectives of the present s tudy:

Organ specific au to immune d isease are charac ter ized by

production of autoantibodies which react with autoantigens unique to

that organ. Since in DCM, the only affected organ is the heart muscle,

it is necessary that the criteria of an organ specific autoimmune disease

is fulfil led. This includes gene t ic p red i spos i t ion , presence of

inflammation and abnormal HLA molecule expression in target organ,

reproduction of human disease in susceptible animals with relevant

40

autoantigens and detection of organ and disease specific antibodies

in affected patients and symptom free relatives. Although there is

evidence that autoantibodies of DCM patients react to several different

proteins, the epitopes for experimental autoimmune myocarditis and

DCM still remain limited. Therefore, the cardiac muscle protein

responsible for autoimmune myocarditis causing DCM must be

identified.

In the present investigation, a systematic study was carried out

to characterize the 150 kD C-protein autoantigen which is known to

induce autoimmune myocarditis leading to DCM. The autoantigen, 150

kD human cardiac C-protein was characterized by ultraviolet

spectroscopy, circular dichroism, thermal denaturation, SDS-PAGE and

urea denaturation studies. The immunogenicity of C-protein was as

certained by inducing polyclonal antibodies in experimental animals.

The repertoire of specificities of induced antibodies was assessed by

direct binding and competition ELISA. Commercially available calf

thymus native DNA was purified free of protein and single standed

regions and adducted to C-protein is presence of ROS and/or UV-B

irradiation. The DNA C-protein adduct/photoadduct was characterized

by nuclease SI sensitivity assay. Kinetic behaviour of addition of

lysine residues (present in C-protein) at various irradiation time was

studied in case of DNA C-protein photoadduct. The recognition of C-

protein and DNA C-protein adducts/photoadducts by DCM, SLE and

induced anti C-protein antibodies was evaluated by direct binding and

competition ELISA and band shift assay.

"^xptvxxntninl

41

Materials:

Calf Thymus DNA, Histone, Poly - D - lysine. Poly - L -

glutamate, Picryl sulfonic acid, bovine serum albumin, agarose NA,

anti - human / anti - rabbit IgG alkaline phosphatase, anti - rat IgG

horseradish peroxidase conjugate, ethidium bromide, coomassie

brilliant blue G - 250 and R - 250, standard protein markers, sodium

dodecyl sulphate, freund's complete and incomplete adjuvant and

Tween - 20 were purchased from Sigma Chemical Company, U.S.A.

Protein - A - Sepharose CL - 4B and Ficoll 400 were obtained

from Pharmacia Fine Chemica l , Sweden. Tris (hydroxymethyl )

methylamine, EDTA (disodium salt), chloroform, isoamyl alcohol and

h y d r o g e n p e r o x i d e were p u r c h a s e d from Q u a l i g e n s , India .

Immunod iagnos t i c ki ts were purchased from St ' n g e n , India.

Biochemical assay kits were purchased from Bayer i: gnistics.

Polystyrene microtitre flat bottomed plates were purchased from

Nunc, Denmark, p-nitrophenyl phosphate and Folin - Ciocalteau

reagent were obtained from Centre for Biochemical Technology, New

Delhi. Acrylamide, ammonium persulphate, bisacrylamide, N, N, N,'

N - tetra ethyl methylene diamine (TEMED) were from Bio - Rad

Laboratories, U.S.A. Diphenylamine and ethanol were chemically pure.

All other chemicals were of highest analytical grade available.

Equipment:

Shimadzu UV - 240 s p e c t r o p h o t o m e t e r e q u i p p e d with

thermoprogrammer and controller unit, ELISA microplate reader MR

42

- 600 (Dynatech, U.S.A..), Beckman DU - 640 spectrophotometer with

densitomt'ter, Avanti 30 table top high speed refrigerated centrifuge

(Beckman, U.S.A.), Ultraviolet lamp (Vilber Lourmat, France), agarose

gel electrophoresis assembly (GNA - 100), Po lyacry lamide gel

electrophoresis assembly (Bio - Rad, U.S.A.), gradient former model

385 (Bio - Rad, U.S.A.), electroeluter (Bio - Rad, U.S.A.) HPLC

(Beckman System Gold) and ELICO pH meter LI - 120 were the major

equipments used in this study.

Collection of sera:

Normal human sera were collected from healthy subjects without

evidence of DCM / SLE or other autoimmune disease. Sera from

patients with dilated cardiomyopathy (DCM) and systemic lupus

erythematosus (SLE) were obtained from the patients admitted to the

Medical College Hospital of A.M.U. and from outdoor Cardiology and

Medicine c l i n i c s , GB Pan t Hosp i t a l and AIIMS, New D e l h i ,

respectively. DCM patients were clinically diagnosed based on the

definition and classification of cardiomyopathies (Richardson et al.,

1996). The SLE s^ra were obtained from the patients who had elevated

levels of anti-DNA antibodies and met the revised criteria for the

American Rheumatism Association (ARA) for the diagnosis of the

d isease (Arnett et al . , 1988) . All serum samples were hea t

decomplemented at 56 °C for 30 min, and were subsequently stored at

-20 °C with 0.02% sodium azide as preservative.

43

DNA estimation:

Colorimetric estimation of DNA was carried out according to

Burton (1956) using diphenylamine reagent.

a) Diphenylamine reagent:

The reagent was prepared immediately before use by dissolving

750 mg of recrystallized diphenylamine in 50 ml of glacial acetic

acid containing 0.75 ml of concentrated sulphuric acid.

b) Assay procedure:

Varying amount of DNA adjusted to 1.0 ml with Phosphate

Buffer Saline (PBS), pH 7.4, was mixed with IN perchloric acid

and incubated at 70 °C in a water bath for 15 minutes. One

hundred microliter of 5.43 mM acetaldehyde was added followed

by 2.0 ml of diphenylamine reagent. The tubes were vortexed

and allowed to stand at room temperature for 16-20 hr for colour

development. Absorbance was recorded at 600 nm. The DNA

concentration in the unknown sample was determined from the

standard plot constructed by employing O-IOO ig of purified calf

thymus DNA.

Protein Estimation:

Protein was estimated by the method of Lowry et al. (1951) and

Bradford (1976).

44

A. Protein estimatian by Folin-Phenol reagent:

This method of protein estimation utilises alkali (to maintain

high pH), Cu^ ions (to chelate proteins) and tartarate (to keep

the Cu^^ions at high pH).

a) Folin-ciocalteau reagent:

The reagent was purchased from Cent re for Biochemical

Technology, New Delhi (India). It was diluted with distilled

water in the ratio of 1 : 4 before use.

b) Alkaline copper reagent:

The components pf alkaline copper reagent were:

i) Two percent sodium carbonate in 0.1 M sodium hydroxide,

ii) 0.5 percent copper sulphate in 1.0 percent sodium potassium

tartarate.

The working reagent was prepared afresh by mixing components

(i) and (ii) in the ratio of 50 : 1.

c) Procedure:

To 1.0 ml of protein sample was added 5.0 ml of alkaline copper

reagent, the contents were mixed and left at room temperature

for 10 min. Then 1.0 ml of Folin-ciocalteau reagent was added

and after 30 min. the absorbance was monitored at 660 nm. The

protein content of unknown samples were determined from a

standard plot constructed against bovine serum albupiin.

45

B. Protein estimation by Bradford method:

This colorimetric assay is performed under acidic condition. The

colour change occurs when Coomassie Brilliant Blue G-250

binds strongly to protein hydrophobical ly and at posi t ively

charged groups. In the environment of these positively charged

groups, protonation is suppressed and a blue colour is observed.

Binding of dye protein causes a shift in absorption maximum of

the dye from 465 to 595 nm and it is the increase in absorption

which is read at 595 nm which is monitored.

a) Dye preparation:

One hundred milligram of Coomassie Brilliant Blue G-250 was

dissolved thoroughly in 50 ml of 95% double distilled ethanol.

To this solution was added 100 ml of 85% (v/v) orthophosphoric

acid. The resulting solution was diluted to a final volume of

one litre. It was filtered before use.

b) Protein assay:

Solution containing 0-100 ^g protein in a volume of upto 0.1

ml was pipetted into test tubes. The volume was adjusted to 1.0

ml with appropriate buffer. Five milliliter of dye solution was

added and the contents were vortexed. The absorbance was read

at 595 nm within an hour against a reagent blank prepared from

1.0 ml of buffer and 5.0 ml of dye solution.

46

Lysine est imat ion by TNBS method:

Lysine estimation of the samples was determined by using

trinitrobenzene sulphonic acid (TNBS) as described by Habeeb (1966).

Varying concentrations of lysine (0-100 [ig) were diluted with

distilled water to a final volume of 1.0 ml. To this was added 1.0 ml

of 4% N a H C 0 3 , pH 8.5 and 1.0 ml of 0 . 1 % TNBS solution. The

reaction mixture was incubated for 2 hrs at 37 °C. Subsequently, 1.0

ml of 10% SDS was added to solubilize the protein and to prevent its

precipitation on addition of 0.5 ml of IN HCl. The colour intensity

was recorded at 335 nm against a blank treated in the same manner

except that it contained 1.0 ml of water instead of lysine solution.

The lysine content in the unknown samples was determined from the

standard plot constructed against L-lysine.

Isolation of 150 kD C-protein from human heart:

Acetone powder of human heart was prepared by the method of

Horecker et al. (1955). Seventy five grams of frozen left ventric 1

was pulverized and homogenized in 5 volumes of chilled acetone. The

material was filtered through suction on a Buchner funnel using

Whatman filter paper No. 1. The semi-dry filter cake was again

homogenized as described above and was subsequently washed several

times with chi l led acetone and dried at room temperature. The

connective tissue was screened out and the remaining acetone powder

was stored at -20 °C.

4 7

Extraction of acetone powder was carried out as described by

Kurata and Tan (1976). The acetone powder was extracted with 10

volumes of PBS, pH 7.4 for 4 hrs at 4 °C with slow continuous stirring.

The solution was centrifuged at 10,000 rpm for 15 min. and the

supernatant was stored in small aliquots at -20 °C.

Gel e lectrophores is :

Polyacrylamide slab gel electrophoresis:

Polyacrylamide slab gel electrophoresis was performed under

denaturing conditions as described by Laemmli (1970). The following

stock solutions were prepared :

i) Stacking gel buffer 0 5 M Tris-HCl, pH 6 8

ii) Resolving gel buffer 3 0 M Tris-HCl, pH 8 8

iii) Reservoir buffer 0 025 M Tris, 0 192 M glycine, pH 8 3

iv) Acrylamide-Bisacrylamide (30 0 8)

Thirty grams acrylamide and 0.8 g bisacrylamide were dissolved

in distilled water to a total volume of 100 ml. The solution was stored

at 4°C in an amber coloured bottle.

v) Sample buffer

a) Six gram Tris was dissolved in 80 ml distilled water and pH

adjusted to 6.8 with o-phosphoric acid. The final volume was

brought to 100 ml.

b) To 12.5 ml of the above solution was added 1 mg bromophenol

blue and 12.5 ml glycerol. P-mercaptoethanol was added just

before use.

48

Procedure:

The glass plates (19 cm x 16 cm) soaked in chromic acid were

thoroughly washed with tap water followed by final rinsing with

distilled water and ethanol. Finally, the plates were dried and sealed

with 1% agarose with 1.5 mm thick spacers.

Recipe for 5 - 2 0 % Gradient Gel

(Total volume: 24 ml)

Solution

Acrylamide-Bisacrylamide (30 : 0.8)

Resolving gel buffer

10% SDS

1.5% ammonium persulphate

TEMED

esolving

5%

2 ml

1.5 ml

0.12 ml

0.6 ml

0.006 ml

gel

20%

8 ml

1.5 ml

0.12 ml

0.6 ml

0.006 ml

Final volume raised to 12 ml each with distilled water.

2.5%) Stacking Gel

(Total volume: 10 ml)

Acrylamide-Bisacrylamide (30 : 0.8)

Stacking gel buffer

10% SDS

1.5% ammonium persulphate

TEMED

1.25 ml

2.5 ml

0.1 ml

0.5 ml

0.0075 ml

Final volume raised to 10 ml with distilled water.

49

Recipe for 7.5% SDS-PAGE

(Total volume: 10 ml)

Acrylamide-Bisacrylamide (30 : 0.8)

Resolving gel buffer

10% SDS

1.5% ammonium persulphate

TEMED

7.5 ml

3.75 ml

0.3 ml

1.5 ml

0.015 ml

Final volume made upto 30 ml with distilled water.

The reagents were mixed and poured between glass plates. The

resolving gel was allowed to polymerize at room temperature. Later

stacking gel was layered on top followed by comb insertion and the

gel was left to polymerize at room temperature. In case of gradient

gels, a gradient was formed with the help of a gradient former (Bio-

RAD, model 385) in case of resolving gel. After ensuring complete

polymerization, the protein samples (25-50 mg) in one fourth volume

of sample buffer were electrophoresed at 70 volts for 6-8 hrs at room

temperature. The gel was stained by 0.25% Coomassie Brilliant Blue

R-250 or with silver stain reagent.

Silver staining was done by the method of Merril et al (1983).

Briefly, the gel was incubated in 40% methanol, 12% acetic acid for

45 min. followed by incubation in 50% ethanol for 30 min. Next the

gel was treated with 0.02% Hypo (sodium thiosulfate) for one min.

After washing with distilled water, the gel was placed in 0.2% silver

50

nitrate (with 0 .05% v/v formaldehyde). After subsequent washing with

distilled water, the gel was transferred to a 6% solution of sodium

carbonate (with 0 .15% v/v formaldehyde). After colour development,

the gel was washed with distilled water and the reaction was arrested

by treating the gel with 3% v/v acetic acid and 5% v/v methanol. All

the reagents used in this procedure were freshly prepared.

Gel dens i tometry:

The electrophoresed stained gels having an array of protein

bands , r ang ing from low to high m o l e c u l a r weights were

densitometrically scanned on a DU-640 spectrophotometer equipped

with a densitometer at a fixed wavelength of 550 nm.

Electroelution:

The electrophoresed 150 kD C-protein band from human heart

was carefu l ly s l i ced off from the gel and was subjec ted to

electrophoretic transfer in an electroelution chamber equipped with

BT-1 and BT-2 membranes (BIO-RAD). The electroelution was carried

out in TBS (150 mM Tris-HCl, 175 mM NaCl), pH 7.4, containing

20% methanol) at 150 volts for 5 hours at room temperature. The

electroeluted 150 kD C-protein was dialyzed against distilled water

and stored at -20 °C. Prior to storage for further s tudies , i ts

homogeneity was reassessed by PAGE.

51

CD measurements:

Circular dichroism measurements were carried out at 25°C on a

Jasco spectropolarimeter model J - 720 using a SEKONIC X - Y plotter

(model SPL - 430 A), NESLAB water bath model RTE 110 and quartz

cell of path length 0.1 cm or 1.0 cm. Other experimental conditions

were, sensitivity 5 mdeg, response time 0.5 s e c , scan speed 20 nm /

min. and aresolution of 1.0 nm. The mean residue ellipticity [9] of a

protein was calculated using the expression.

[0] = 0 / ( 1 0 n X Cp X 1)

Where 0 is the observed ellipticity in mdeg. n the number of

aminoacid residues in the protein whose molar concentration is Cp

and 1 is the path length in om.

High performance liquid chromatography (HPLC):

The electoreluted 150 KD C protein (from human heart) was

further purified by HPLC using a sephader G - 200 column. The

running buffer was PBS, pH 7.4.

Agarose gel electrophoresis of DNA:

Agarose gel was prepared by bringing 1% agarose NA to molten

state in electrophoresis buffer (0.04 M Tris acetate, pH 8.0 containing

0.002 M EDTA). Molten agarose was poured on the gel tray and

allowed to solidify for one hour at room temperature. The DNA

samples in one tenth volume of stop-mix (30% ficoll, 0 .025% xylene

52

cyanole FF and 500 mM EDTA in 10 times concentrated TAE buffer)

were electrophoresed for 2-4 hrs at 30 mA in the same buffer. The gel

was stained with ethidium bromide (0.5 \ig/m\).

Preparation of DNA - C-protein adducts:

a) Preparation of DNA - C protein photoadducts:

Calf thymus DNA, free of histones and single stranded regions

was mixed with HPLC purified C protein in the ratio of 1:2 in

PBS, pH7.4 and incubated at 2hrs. at 37°C. The complex was

subjected to UV-B irradiat ion for 30 min. at 254 nm. The

temperature during irradiation was kept constant at 25 + 1°C by

water curculation. Native DNA, unirradiated DNA - C protein

complex, unirradiated C protein, irradiated DNA and irradiated

C protein were used as controls.

b) P repa ra t ion of DNA - C - protein adduc ts in presence of

reactive oxygen species (ROS):

ROS induced photoupling of C protein to the genetic material

(DNA) was carried out in the presence of hydroxyl radical ( OH)

generated by hydrogen peroxide (1.51 M) in presence of UV - B

irradiation (254 nm) DNA - C protein complex was also formed

only in the presence of hydrogen peroxide (1.51M). Purified calf

thymus DNA and C protein were mixed in the ratio of 1:1 in

PBS, pH 7.4. Similar amounts of native DNA, unirradiated C

protein, irradiated DNA and irradiated C protein were used as

controls.

53

All the treated samples were extensively dialyzed in PBS, pH

7.4. The modif ied and unmodified samples were subjected to

electrophoresis on 1% agarose using Tris acetate EDTA buffer, pH 8.0

for 2 hrs. at 30 mA. The gel was stained with ethidium bromide (0.5

^g / ml) and was photographed under UV illumination.

Time course kinetic stsudy of formation of nDNA - C - protein

photoadduct:

The kinetic behaviour of photoaddition was assessed in order to

determine the frequency of C protein photo cross linked to DNA. The

DNA - C protein complex was subjected to UV - B irradiation (254

nm) for various time intervals (10 - 30 min). Unirradiated DNA - C

protein complex was used as control. The DNA - C protein photo

adducts were precipi ta ted in two volumes of cold ethanol . The

precipi ta te was col lec ted by centrifugation and dissolved in I.O

ml of PBS, pH 7.4. The bound prote in was e s t ima ted in the

photoadducts (Bradford et al, 1976). The lys ine con ten t in the

s amp le s was a s s e s s e d by c o l o r i m e t r i c me thod u s ing TNBS

(Habeeb, 1966).

Kinetic analysis of the photoaddition reaction was performed

by the method of Warren et al (1974) using first order rate equation.

-In ( V a - Vt) - a + kt

Where Vaand Vt are the lysine molecules photobound at infinity

(30 min.) and at a particular time t respectively. The -In (Va - Vt)

54

values were plotted against the irradiation time t and the slope yielded

the apparent first order rate constant k for the photoaddition process.

Characterization of adduct by nuclease SI digestion:

The formation of DNA - 150 KD C - pro te in adduct was

characterized by SI nuclease digestion followed by gel electrophoresis.

a) One percent agarose:

Agarose (.3g) in 30 mix TAE buffer, pH 8.0 was brought to

molter state and after cooling to 50° C was poured onto a

horizontal gel tray of GNA - 100 electrophoresis apparahis

(Pharmacia Sweden) and was left at room temperature for

complete solidificatin.

b) Electro buffer:

40 mM Iris - acetate and 2mM EDTA, pH 8.0 (TAE buffer)

c) Treatment of adduct with SI nuclease:

One jig of DNA - 150 kD C -protein adduct and riative DNA

were treated with SI nuclease (20 units of S1 nuclease per jig of

adduct) in acetate buffer for 30 minutes at 37°C. The reaction

was stopped by the addition of one tenth volume of 0.2 M EDTA,

pH 8.0.

d) Sample p repara t ion :

The treated and untreated samples in low ionic strength and at

concentrations of less than 1 jig / ml were loaded. The sample

55

contained one tenth volume of 30% Ficoll (Pharmacia), 0.025%

bromophenol blue, 0.5 M EDTA and lOX electrophoresis buffer.

e) Running conditions:

30 mA for 2 hours.

f) Staining:

The electrophoresed gel was stained with ethidium bromide (0.5

M-g / ml).

Urea - SDS - PAGE-:

Urea - SDS - PAGE was performed according to the method of

Swank and Munkres, (1971). The following stock solutions were

prepared :

i) Acrylamide : Bisacrylamide : 12.5 g acrylamide

(10 : 1) 1.25 g bisacrylamide

dissolved and made upto 50

ml with distilled water.

ii) Gel buffer stock 1% SDS

1.0 M H^PO^ (adjusted to

pH 5.0 with Tris base)

iii) Reservoir buffer stock 0 .1% SDS

0.3M H,PO,

(adjusted to pH 6.8 with

Tris base)

56

iv) Sample buffer 1% SDS

8 M Urea

1% 2-mercaptoethanol

O.IM H,PO,

(adjusted to pH 6.8 with Tris

base) Bromophenol blue

added to 0.002% (w/v)final

concentration.

RECIPE

12.5% Resolving gel

Acrylamide : Bisacrylamide (1:10) 15.0 ml

Gel buffer stock 3.0 ml

Urea 14.4 g

6% ammonium persulfate 0.3 ml

(freshly prepared)

TEMED 0.02 ml

The volume is made upto 30 ml with distilled water.

3.75% Stacking gel

Acrylamide : Bisacrylamide (1:10) 1.5 ml

Gel buffer stock 0.3 ml

Urea 1.44 g

6% ammonium persulfate 0.03 ml

(freshly prepared)

57

TEMED 0.002 ml

The final volume is made upto 10 ml with distilled water.

Procedure:

A low concentration gel of 3.75% was layered over 12.5% gel,

with the same buffer composition. This is done to avoid breakage of

sample well divis ions which become very br i t t le due to high

crosslinking of resolving gel. The gels were run for 6-8 h at 70 volts.

Stainer:

After electrophoresis, the gels were stained for one hour in:

methanol : water : glacial acetic acid in ratio of 5 : 5 : 1

Coomassie Brilliant Blue R 250

Destainer:

Destaining is by diffusion in

12.5% isopfopanol

10% acetic acid.

Urea SDS-PAGE was also pe r fo rmed by doing va r ious

alterations.

UREA - SDS-PAGE

(with varying concentrat ions of urea)

A gradient gel of 1 M to 10 M urea was polymerized

5b

RECIPE

Resolving gel (12.5%)

Acrylamide : Bisacrylamide (1:10)

Gel buffer stock

Urea

6% ammonium persulfate

(freshly prepared)

TEMED

1 M Urea

: 2 ml

0.4 ml

0.24 g

0.04 ml

0.0027 ml

10 M Urea

2 ml

0.4 ml

2.4 g

0.04 ml

0.0027 ml

The final volume is made upto 4 ml each with distilled water.

Stacking gel (3.75%)

Acrylamide : Bisacrylamide (1:10)

Gel buffer stock

Urea

6% ammonium persulfate

(freshly prepared)

TEMED

0.3 ml

0.06 ml

0.36 g

0.02 ml

0.0016 ml

The final volume is made upto 2 ml with distilled water.

UREA - SDS-PAGE

(with varying concentrations of acrylamide : bisacrylamide)

A 5-20% gradient gel of acrylamide : b i sacry lamide was

polymerized.

Gel buffer stock

Urea (8 M)

6% ammonium persulfate

(freshly prepared)

TEMED

0.4 ml

1.92 g

0.04 ml

0.0027 ml

59

RECIPE

Resolving gel (5-20%)

5% 20%

Acrylamide : Bisacrylamide(l:10) 0.6 ml 2.4 ml

0.4 ml

1.92 g

0.04 ml

0.0027 ml

The final volume is made upto 4 ml each with distilled water.

Stacking gel (3.5%)

Acrylamide : Bisacrylamide : 0.3 ml

Gel buffer stock : 0.06 ml

Urea : 0.36 g

6% ammonium persulfate : 0.02 ml

TEMED : 0.0016 ml

The final volume is made upto 2 ml with distilled water.

Absorption - temperature scan:

Thermal native to coil transition analysis of native Cardiac C

protein and native IgG was carried out in order to assess / probe their

comparative thermal stability in view of the established fact that both

of them possess similar domains.

Native C pro te in and nat ive IgG were sub jec ted to heat

denaturation on a Shimadzu UV-240 spectrophotometer coupled with

60

a temperature programme and controller assembly (Hasan and Ali,

1990). The samples were melted from 30°C to 100°C at a rate of 1°C/

min. Prior to scanning, the samples were temperature-equilibrated at

30°C for 10 minutes. The change in absorbance at 280 nm was recorded

with increasing temperature. Percent denaturation was calculated using

the following equation :

% denaturation = AT-ASO X 100 Amax-A30

where.

AT = Absorbance at a temperature T°C

Amax = Final maximum absorbance on the completion of

denaturation

A30 = Initial absorbance at 30 °C.

Thermodynamic analysis of 150 kD cardiac protein as well as

native IgG:

The thermal transition of proteins from native to denatured state

was characterized as a single variable, fo, the fraction of protein in

the denatured state. The percent loss in absorbance was used as

experimental variable to follow the transition. At any given point along

the transition curve, the observed percent change in absorbance was

related to the fraction of protein in the denatured state, by the

following expression

fo = (A)obs - (A)N ( A ) D - (A)N

61

where, (A)obs, (A)N and (A)D represent the percent loss in

absorbance in any observed, native and fully denatured s ta tes ,

respectively. Since each experimental value of (A)obs will give a

unique value of (A)D, the later was used to construct transition curves

for the thermal denaturation of proteins.

Since the unfolding/denaturation of proteins is a more than one

step process due to various factors, the initial and final states of

thermal denaturation of proteins were related to apparent equilibrium

constant, defined as

Kapp = (A)obs - (A)N ( A ) D - (A)obs

The free energy change, AG^, for the thermal reaction (native

coil conformation) was calculated by using the relationship

AGD = - RT InKapp

where, Kapp is the equilibrium constant, R is the gas constant

(8.314 J/mol/deg) and T is the absolute temperature.

Immunization schedule:

Heal thy female rabbi t and ra t s were immunized with

electrophoretically pure 150 kD C-protein antigen emulsified in an

equal volume of Freund's complete/incomplete adjuvant as described

earlier (Hasan and Ali, 1990). Injection dose of 100 \ig and 50 ng 150

kD C-protein was given to rabbit and rats, respectively.

62

The first injection eliciting primary response was given in

Freund's complete adjuvant, followed by three successive boosters

which were given after two weeks at weekly intervals in Freund's

incomplete adjuvant. The volume of the emulsion to be injected was

kept as 1.0 ml and 0.5 ml for rabbit and rats, respectively.

Blood was drawn from experimental animals by cardiac puncture

after the third booster and the antibody activity in the immune serum

was evaluated by ELISA . Prior to immunization, pre-immune serum

was c o l l e c t e d for employ ing it as c o i r e s p o n d i n g con t ro l in

immunological studies.

Isolation of IgG by affinity chromatography:

Protein A Sepharose CL-4B (1.5 g) was employed as affinity

matrix. First ly, it was swelled in 0.01 M PBS, pH 7.4 at room

temperature for 12 hrs. The swelled matrix (about 5.0 ml) was washed

with PBS and packed in a column of 0.9 cm x 15 cm. The packed

column was washed with 0.1 M sodium citrate, pH 3.0 in order to

elute any bound material. This was followed by equilibration of the

packed matrix with 5 volumes PBS, pH 7.4. Serum diluted with equal

volume of PBS, pH 7.4 was loaded onto the column and allowed to

bind at a flow rate of 20 ml/hr. Unbound protein was eliminated with

PBS and absorbance of effluent monitored till a negative absorbance

was obtained at 280 nm The bound IgG was eluted with 0.58% acetic

acid in 0.85% sodium chloride. To prevent the effect of acidic elution

63

buffer on IgG, fractions were collected in 1 M Tris-HCl, pH 8.5. The

fractions containing IgG were monitored at 251, 260, 278 and 280

nm. The fractions with the appropriate ratio for mammalian IgG (2.5-

3.0) were dialyzed in 0.01 M PBS, pH 7.4.

To check the purity of IgG, the samples were subjected to 7.5%

SDS-PAGE.

Immunoaffinity purification of ant ibodies:

a) CNBr activation of Sepharose 4B:

15 ml of supplied Sepharose 4B was suspended in distilled water,

and then washed with 300 ml of cold distilled water on a sintered

glass funnel (porosity G-2). Moist gel (about 10 g) in 10 ml of

2.0 M sodium carbonate was kept in ice - NaCl bath placed on a

magnet ic s t i rrer . One gram cyanogen bromide in 0.8 ml

acetonitrile was slowly added to the gel with gentle stirring.

The reaction was allowed for 12 min. and after that the reaction

product was filtered on a sintered glass funnel, washed with 400

ml of cold 0.01 M sodium bicarbonate and resuspended in 10 ml

of the same buffer. The unreacted CNBr was treated with ferrous

sulphate to convert it into harmless ferrocyanide. All steps were

carried out in a fumehood chamber (Ali, 1984).

b) Poly-L-lysine coupling to activated Sepharose 4B:

The method descr ibed by Wilchek (1973) was fo l lowed.

Immediately after CNBr activation of gel, equal volume of Poly-

64

L-lysine (100 mg in 10 ml of 0.1 M sodium bicarbonate) was

added to the activated gel and kept at 4 °C for 12 hrs with slow

stirring. The buffer was drained out and the gel was washed

successively with 100 ml each of (i) cold distilled water, (ii)

0.1 N HCl, (iii) 0.1 M sodium bicarbonate and (iv) distilled water

till neutral. Finally the gel was suspended in 40 ml of 0.15 M

acetate buffer, pH 4.5. The extent of Poly-L-lysine depletion

due to its covalent coupling with activated Sepharose 4B was

calculated by TNBS estimation of lysine in the drained effluent

as described by Habeeb (1966).

c) Affinity purifiction of ant ibodies:

DNA-[polylysyl-Sepharose 4B] was prepared as described by

Nicotara et al. (1982) with a slight modification. Previously

prepared 20 ml po ly ly sy l -Sepha rose 4B was packed and

equilibrated with 25 ml acetate buffer in a mini column (1.5 cm

X 5 cm). Purified DNA (12.5 ml of 100 \ig/m\) in acetate buffer

was passed through the gel and unbound material was eliminated

with PBS, pH 7.4. Heat decomplemented SLE serum dialyzed

against and diluted (1 : 10) with PBS was passed through the

column. Unbound proteins were washed with PBS and the bound

antibodies were eluted with linear ionic strength gradient of

0.15-3.0 M sodium chloride in 0.01 M phosphate buffer. pH 7.4.

The fractions were monitored at 251 nm. 260 nm, 278 nm and

280 nm.

65

d) Regeneration of affinity column:

The column was r e g e n e r a t e d severa l t imes by wash ing

successively with 50 ml each of the following (i) distilled water,

(ii) 0.1 N HCl, (iii) 0.1 M sodium bicarbonate, and (iv) distilled

water till neutral.

Enzyme linked immunosorbent assay (ELISA):

The antibodies were detected and quantitated by ELISA using

polystyrene flat bottom microtiter plates as solid phase (Alam and Ali,

1992) . The method described by Ali et al. (1985) and Arif et al. (1994)

were followed for the assay.

Buffers:

i) Tris buffered saline (TBS), pH 7.4 :

10 mM Tris, 150 mM NaCl, pH 7.4

ii) Tris buffered saline Tween-20 (TBS-T), pH 7.4 :

20 mM Tris, 144 mM NaCl, 2.68 mM KCl and 500 ^1 Tween-20,

pH 7.4.

iii) Bicarbonate buffer, pH 9.6 :

15 mM sodium carbonate, 35 mM sodium bicarbonate, pH 9.6.

iv) a) Substrate buffer, pH 9.6 :

(for a n t i - human / anti rabbit IgG alkaline phosphate conjugate)

15 mM sodium carbonate, 35 mM sodium bicarbonate and 2 mM

magnesium chloride, pH 9.6.

substrate : 0.5 mg/ml of p-nitrophenyl phosphate

66

b) Substrate buffer, pH 5.0 :

(for anti rat) IgG horse radish peroxidase conjugate)

50 mM citric acid, 50 mM di-sodium hydrogen orthophosphate

dihydrate (Na2HP0^.2H20)

substrate : 0.5 mg/ml of o-phenylene diamine.

Procedure:

Polystyrene microtiter plates were incubated with 100 |ul of

protein antigen (50 ng/ml in carbonate/bicarbonate buffer, pH 9.6) for

two hours at room temperature followed by overnight incubation at

4°C. The plates were washed thrice with TBS-T and the unoccupied

sites were blocked by 150 pi of 1.5% BSA in TBS for four hours at

room temperature. Varying dilutions of sera diluted in TBS were added

to antigen coated as well as control wells. The ant igen-ant ibody

interaction was allowed to proceed for two hours at room temperature

followed by overnight incubation at 4°C and subsequently the plates

were washed four times with TBS-T in order to remove the uninteracted

antibodies. Bound antibodies were assayed by means of appropriate

anti-immunoglobulin alkaline phosphatase / anti-immunoglobulin horse

radish peroxidase conjugate using p-ni t rophenyl p h o s p h a t e / o-

phenylene diamine as substrate, respectively. The reaction was stopped

by 3N NaOH / 5 N H^SO_j, respectively and the absorbance of each

well was monitored at 410/490 nm on an ELISA microplate reader.

Each sample was coated in duplicate and the results were expressed

as a mean of Atest - Acontrol.

67

For nucleic acid antigen, the polystyrene microtiter plates were

preincubated with 100 |il of poly D-lysine solution (50 ^g/ml in

dist i l led water) for 30 minutes at RT so as to increase antigen

immobilization (Arif et al., 1994). The plates were washed thrice with

TBS and coa ted with 100 fil of nuc le ic acid a n t i g e n ( s ) at a

concentration of 2.5 ng/ml in TBS (pH 7.4) for two hours at room

temperature followed by overnight incubation at 4°C. After eliminating

the unbound antigen by washing thrice with TBS-T, the unoccupied

positively charged polylysine molecules were neutralized by saturating

the plates with 100 1 of negatively charged poly-L-glutamate (50 \ig/

ml in TBS, pH 7.4) for two hours at room temperature. The rest of the

steps were same as described above.

Inhibit ion ELISA:

The antigen binding specificity of antibody was determined by

inhibition experiments (Hasan et al., 1991). Varying concentration of

inh ib i tors (0-20 ^g/ml) were mixed with a constant amount of

antiserum/IgG. The mixture was incubated for two hours at 37°C

followed by overnight incubation at 4°C. The resulting immune

complex was employed in the immunoassay instead of serum. The rest

of ihe steps were as in Direct binding ELISA. The resul ts were

expressed as percent inhibition

% Inhibition - 1 - A inhibited x 100 A uninhibited

68

Band shift assay:

The antibody binding to ROS modified and unmodified DNA -

C p ro t e in a d d u c t s / p h o t o a d d u c t s were ana lyzed by a l t e red

electrophoretic mobility on agarose gel (Sanford et. al., 1988). A

constant amount of antigen (1.0 ng) was allowed to interact with

varying amounts of antibody (0-150 ng). The interaction was allowed

to proceed for 2 hrs at 37°C and overnight at 4°C. This resulted in the

formation of immune complex (IC). Two microliter dye solution was

added to each assay tube and the complex was electrophoresed on 1%

agarose gel for 2 hrs at 30 mA using Tris-acetate EDTA buffer, pH

8.0. The electrophoresed gels were stained with ethidium bromide (0.5

^g/ml) and were visualized and photographed under UV illuminator.

Appropriate controls were also run simultaneously.

Estimation of glucose in serum of DCM patients:

Autopak reagent kit was used for in vitro quantitative estimation

of glucose in serum (Trinder, 1969).

In this method, glucose is oxidised by glucose oxidase (GOD)

into gluconic acid and hydrogen peroxide.

Glucose + 0 , > Gluconic acid +UXf^.

Hydrogen peroxide in presence of peroxidase oxidizes the

chromogen

4- amino phenazone/H^O, + Phenolic compound + 4 amino

phena zone > Red compound + 2H O Phenolic compound to a red

69

coloured compound . The in tens i ty of red co lour p roduced is

proportional to the glucose concentration.

b) Assay procedure:

One ml of working solution was added to 10 //I of sample volume

and incubated for 15 minutes at 37°C. The samples were read at 505nm

against reagent blank.

a) Reagents:

i) Tablets: Buffer / enzymes (GOD, POD) / Chromogen

(4 aminophenazone, phenolic compound)

ii) Standard: Glucose lOOmg/dl

One tablet was dissolved in 50ml of distilled / deionized water

to prepare the working solution.

Estimation of Cholesterol in Serum of DCM Patients:

Autopak r eagen t kit was used for in v i t ro quan t i t a t i ve

determination of cholesterol in serum (Tartuttor and Gunter, 1974).

In this method, cholesterol esters are hydrolysed by cholesterol

ester hydrolase to free cholesterol and fatty acids. The free cholesterol

produced and pre existing one are oxidised by cholesterol oxidase to

A - cholestenone and hydrogen peroxide.

CEH Cholesterol esters > cholesterol + fatty acids .

Cholesterol + 0 , > A cholestenone + H ,0 , .

70

Hydrogen peroxide in the presence of peroxidases, oxidize the

chromogen (4 aminophenazone / phenol) to a red coloured compound.

H2O2+ 4 aminophenazone / phenol > Red couloured compound.

a) Reagents:

i) Buffer / Enzymes / Chromogen

ii) Phenol

iii) Standard : Cholesterol - 200mg / dl

Reagent (i) and (ii) were mixed in the ratio of 1:1 (V/V) to

prepare working solution which was stored in a dark bottle at 2 - 8° C

b) Assay procedure:

One ml of working solution is added to 10 //I of serum and the

reaction mixture was incubated for 5 min. at 3 7'^C. The samples were

read at 510 nm against reagent blank.

Estimation of uric acid in serum of DCM patients:

Autopak reagent kit was used for in vitro quantitative estimation

of uric acid in serum (Fossati et al., 1980).

In this method, uric acid is converted by uricase into allantoin

and hydrogen peroxide which in presence of peroxidase (POD)

oxidizes the chromogen (4 - aminophenazone / 3,5 dichloro hydroxy

benzene sulphonic acid) to a red coloured compound.

uricase Uric acid + O2+ H^O > allantoin +C0^+ H^O^

2H2O2+ 4 - aminophenazone + DHBS > Red coloured + H2O

compound

71

a) Reagents:

i) Buffer / Enzymes / 4 - aminophenazone / Potassium ferrocyanide.

ii) 3,5 - Dichloro - 2 - hydroxy benzene sulphonic acid / surfactant,

iii) Standard: Uric acid - 6 mg / dl

The working solution was prepared by mixing 10ml of reagent

(ii) to one vial of reagent (i) and this was stored at 2 - 8°C.

b) Assay procedure:

One ml of working solution was added to 30 ^1 of serum. The

reaction mixture was incubated at room temperature for 15 min and

read at 505 nm against reagent blank.

Estimation of C reactive protein (CRP) in serum of DCM patients:

Stangen C - PROTEST kit was used for in vitro quantitative

and semi - quantitative determination of C - reactive protein in serum

samples in the acute phase of infection (Shire et al., 1981).

Stangen C - PROTEST is a rapid latex agglutination procedure

for the direct detection and semiquantitation on slide of C - reactive

protein in human serum samples. The reagent, a smooth suspension of

latex par t i c les coated with speci f ic an t ihuman CRP ant ibody,

agglutinates in the presence of CRP in the patients serum, a positive

result is indicated by the presence of agglutination and vice versa.

72

Reagents:

i) CRP latex reagent

ii) Positive control

iii) Negative control

Assay procedure:

A) Qualitative Test:

One drop of each control and 50 |il of test serum was placed

onto separate circles on the slide. One drop of CRP reagent was

added to each sample and mixed well over the entire encircled

area by mixing sticks. The slide was rotated gently and slowly

and after 2 min., it was observed for agglutination.

The presence of a visible agglutination on or before 2 min.

indicate a content of CRP > 6mg / L. The sample positive for

CRP was titred by semiquantitative method.

B) Semi quantitative test:

Serial two fold dilutions of positive sample were prepared in

normal saline . The rest of the procedure was same as in

qualitative test (A).

The serum titer was defined as the highest dilution showing

visible agglutination of latex particles. The approximate CRP

level present in the sample was obtained by the formula:

CRP (mg / L) - S x D

73

S = Limit of sensitivity i.e. .6 mg / L

D = Highest dilution of serum sample showing agglutination.

Detection of rheumatoid factor (RF) in serum of DCM patients:

Stangen RHEUMA TEST kit was used for in vitro quantitative

detection of Rheumatoid factor in serum samples (Linker and Williams

Jr, 1985)

RHEUMATEST Kit c o n s i s t s of a smooth s u s p e n s i o n of

polystyrene la tex pa r t i c l e s coa ted with a pur i f ied human IgG

molecules. The pre diluted serum sample to be tested was mixed with

the latex particles and allowed to react. The human IgG forms complex

with rheumatoid factor present in serum and results in an agglutination,

an indicator for positive result and vice versa.

a) Reagents:

i) RF latex reagent

ii) Positive control (prediluted)

iii) Negative control (prediluted)

iv) Specimen diluent

Assay procedure:

Quantitative test:

To 50 \i\ of specimen diluent , 10 ^1 of test serum was added

and mixed well on the slide. One drop of latex reagent was added to

the test samples as well to drop each of positive and negative control.

The reaction mixture was mixed well on be entire circle and the slide

74

was rotated gently for observation of agglutination at the end of 2

min. Agglutination observed on or before 2 min. showed presence of

Rheumatoid factors in the serum.

JR^suIts

75

Isolation of 150 kD C- protein:

¥ rotein antigens of different molecular weights in the total human

heart extract (THHE) were separated on a 5 - 20% gradient SDS - PAGE.

The electrophoresed results depicted in Fig.l inidicated a wide spectrum of

protein antigens ranging from low to high molecular weights. In order to

localize and isolate the 150 kD C protein which is known to induce

myocarditis, the above electrophoresed SDS - polyacrylamide gel was sub­

jected to densitometric scanning. The densitometric scanned gels showing

prominent bands of 150 kD C - protein were sliced off and subsequently

cut into small pieces and thereafter subjected to e lec t roelut ion. The

electroeluted pure 150 kD C - protein was further ascertained for its homo­

geneity by PAGE. The single band of 150 kD C - protein in PAGE revealed

the inducing 150 kD C -protein antigen. In order to minimize or eliminate

the chances or probability of undetectable impurities in the isolated pro­

tein antigen, the visibly pure electroeluted 150 kD C - protein was sub­

jected to HPLC (Fig.2). The electroeluted and subsequently HPLC purified

150 kD cardiac C - protein was employed in the binding studies.

Ethidium bromide (Et - Br) intercalat ion assay:

The possibility of traces of DNA present in the total human heart ex­

tract (THHE) could not be ruled out. Thus, in order to ascertain and there­

after eliminate the possible presence of traces of DNA in THHE which could

interfere in the binding studies, agarose gel electrophoresis was performed.

Calf thymus dsDNA as a control along with THHE were loaded onto the

wells and the gel was electrophoresed. As evident from Fig. 3, visualized

76

1^0 kP Iret-n

Fig. I Electrophoretic pattern of total human heart extract (THHE) on 5 20% gradient SDS - PAGE

77

1-8

1.6 E «= 14 o OD «N 1.2 h-

^ 1.0 UJ o 2 0.8 < OQ CE 0-6 o in ^ . (O OA <

0.2

0.0 <

-

_

—

—

I j \ 1 \ 1 1 j I / 1

/ 1 / I

/ 1 / 1 1 1

1 \ / 1 j \ / 1

/ 1 1 I

/ I 1 I / 1

f / / f -^-^ ' T—^—-t--t--r-2 10 n 12 13 U 15

TIME (min.) 16 17 18

Fig. 2 Elution profile of purification of electroeluted 150 kD C protein by HPLC

78

^

Fig. 3 Agarose gel electrophoresis to show the absence of anv trace of DNA in THHE. Lane 1 (native DNA) Lane 2 (THHE)

79

after staining with Et - Br, only lane containing DNA as control showed

fluorescence upon Et - Br intercalation, whereas no fluorescence was ob­

served in the lane containing THHE, thereby ruling out the possibility for

the presence of trace amount of DNA in human heart extract.

Ultraviolet and circular dichroic spectral analysis of 150 kD C - protein:

Prior to immunological characterization, the purified 150 kD cardiac

protein was subjected to ultraviolet and circular dichroic spectroscopy. The

spectral curves showed a X max at 280 nm along with a shoulder at around

289 nm. The Xmin was observed at 248 nm. The A26O / A295 ratio was

computed out to be 1.49. It showed the characteristics of a typical dimeric

protein (Fig. 4) .

Thermal native to coil transition or unfolding study of 150 kD C - protein:

Thermal denaturation of 150 kD C - protein and IgG as control were

investigated in between 30°C and 95°C. The process was characterized by

determining the percent fraction of protein in the denatured state at various

temperatures. Increase in UV absorption at 280 nm was taken as a measure

of thermal unfolding. As evident from Fig. 5, till 75° C thermal unfolding

was found to be of a very low magnitude. Only 4.4 percent of the total 150

kD C - protein underwent thermal unfolding till 75°C. Nearly half of the

150 kD C - protein (i.e. 50%) was observed to have undergone thermal un­

folding at around 79.5°C. Further supplementation of thermal energy as

evident from Fig. 5, exhibited a sharp transition in the partially unfolded C

- protein to the fully unfolded state. Complete thermal unfolding was found

1 0 -

80

8 -

-10

I _L _L 210 220 230 2A0 250

WAVELENGTH(nm) 260

Fig. 4 Circular dichroic spectra of 150 kD C protein

81

80 70 60 50 TEMPERATURE(°C)

40 30

Fig: 5(A) Thermal unfolding profile of HPLC purified 150 kD human cardiac C-pro-tein.

82

1 0 0 -

z o 1-< (T TJ 1-< Z UJ

o z ixi

u a: UJ 0 .

80

60

40

20

30 50 60 70 TEMPERATURE(^C)

Fig. 5(B) Percent thermal native -> coil transi­tion profile (% Denaturation) of HPLC purified 150 kD human cardiac C-protein.

83

to be achieved at 87°C. Thus, as evident from the thermal unfolding curves,

significant onset of unfolding was at around 77°C (around 9.19 percent

unfolding) which spanned till 87°C i.e. the temperature at which 100%

unfolding was obtained. Thus, the results indicated the effective thermal

unfolding transition breath to the degree of lO^C (i.e. between 77°C to 87°

C). Furthermore, the above data is indicative for the tremendous structural

stability of the 150kD cardiac C-protein. Similar observation were also made

for IgG (data not shown) which was used as control in view of the estab­

lished fact that both IgG as well as 150 kD C - protein have somewhat simi­

lar domains. It is to be pointed out that further supplementation of thermal

energy above 87°C exhibited a decrease in absorbance at 280 nm. As evi­

dent from Fig.5, the absorbance at 87°C was 1.217 and that it was found to

decrease gradually above 87°C. As evident, the A28O at 95°C was observed

to be 0.565. Thus, at 95°C, nearly more than 50 percent of the absorbance

recorded at 280 nm for 150 kD C - protein in the fully unfolded state (i.e. at

87°C) was found to be lost. Thus a sharp hypochromic effect was observed

at temperatures above the unfolded state of 150 kD C - protein.

Thermodynamical analysis of 150kD C - protein:

The high molecular weight cardiac C -protein was also characterized

by means of thermodynamic analysis. The free energy of unfolding/dena-

turation (AG^) for 150kD C - protein was computed to be 12.363 K. cal.

deg -• at 303°K (30°C) i.e. prior to thermal unfolding. As evident from the

results, the free energy of denaturation / unfolding decreased linearly from

30°C (303OK) to 78°C (351°K). The AG^ at 78°C was computed to be 2.332

84

TABLE 1

THERMAL DENATURATION OFlSOkD CARDIAC CPROTEIN

TEMPERATURE

°C

30 35 40 42 45 48 50 52 55 63 65 70 72 75 77 78 79 80 81 82 83 84 85 86 87 89 90 91 92 94 95

OK

303 308 313 315 318 321 323 325 328 336 338 343 345 348 350 351 352 353 354 355 356 357 358 359 360 362 363 364 365 367 368

^D

0 000 0 008 0 022 0 036 0 038 0 041 0 046 0 048 0 051 0 059 0 067 0 076 0 086 0 122 0 254 0 689 1 222 1 619 2 029 2 216 2 468 2 665 2 743 2 754 2 762 2 110 1 792 1 570 1 343 1 132 1 132

K, App

0 000 0 008 0 022 0 030 0 039 0 042 0 048 0 051 0 054 0 063 0 072 0 082 0 095 0 138 0 341 2 217 -9 222 -2 616 -1 971 -1 822 -1 681 -1 601 -1 574 -1 570 -1 567 -1 900 -2 263 -2,754 -3 913 -8 551 0 370

AG„ = - RT In K, D App

(Kcal.deg')

12 363 9 932 9 184 8 576 8 460 7 984 8 041 7 960 7 724 7 393 7 132 6 752 5 731 3 131 2 322 -6 502 -2 823 -1 998 -1 767 -1 536 -1 397 -1 351 -1 346 -1 343 -1 932 -2 469 -3 074 -4 139 -6 547 3 041

85

TABLE 2

THERMAL DENATURATION OF IgG

TEMPERATURE

»C •'K

30 303 35 308 40 313 45 318 50 323 55 328 60 333 65 338 70 343 75 348 77 350 78 351 79 352 80 353 81 354 82 355 83 356 84 357 85 358 86 359 87 360 88 361 89 362 90 363 91 364 92 365 93 366 94 367 95 368 96 369 97 370 98 371 99 372 100 373

fo

0 000

-0 003 -0 010 -0 013 -0 020 -0 017 0 014 0 024 0 034 0 089 0 166 0 279 0 393 0 521 0 669 0 776 0 883 0 948 1 010 1 028 1 048 1 066 1 083 1 089 1 086 1 082 1 079 1 062 1 052 1 038 1 000

App

0 000

-0 003 -0 010 -0 014 -0 020 -0 017 0 014 0 025 0 036 0 098 0 198 0 388 0 648 1 086 2 021 3 462 7 529 18 333 -97 667 -37 250 -21 714 -16 263 -13 083 -12 154 -12 600 -13 083 -13 609 -17 111 -20 333 -27 364 0 290

AG„ = RT In K, D App

(K cal deg-1)

15 607

12 564

11 818

10 993

11 620

12 351

10 734

9 700

6 798

4 752

2 787

1 281

-0 246

-2 089

-3 696

-6 026

-8 707

-13 752

-10 887

-9 289

-8 440

-7 802

-7 600

-7 730

-7 867

-8 009

-8 73 5

-9 291

-10 234

1 449

86

k.cal. deg '. The large positive AG^ values obtained at the initial stages of

thermal unfolding which decreased linearly till 78°C is indicative for the

folding of C - protein by ionic and hydrophobic interactions with probably

intermittent presence of disulfide bonds. Furthermore, the results indicate

that C - protein having domains similar to IgG is not that so thermodynami-

cally compact molecule as is native DNA, where due to stable stacking of

the base pairs, a negative AG^is observed in the initial stages of thermal

melting. Further elevation of thermal energy showed negative AGjj values

for 150 kD C -protein . The AG^ values from 79«C to 94°C (i.e. 352°K -

367°K) were computed to be - 6.502 K.cal. deg ' and -6.547 K.cal. deg "'.

The AGp values above 79''C decreased linearly till 89°C (362°C) followed by

again an increase in the AG^ values till 94°C. The results are indicative for

the complete thermal unfolding of the 150 kD C - protein at around 78°C or

79°C. Due to the reason that 150 kD C - protein is having some what simi­

lar domains to IgG, comparative thermodynamical analysis between the two

as also carried out. As evident from the results, IgG was found to be a

thermodynamically more compact molecule than 150 kD C - protein. These

thermodynamic parameters are depicted in Tables - 1 & 2.

Urea denaturation studies:

The 150 kD cardiac C protein and IgG were subjected to urea SDS-

PAGE at constant concentration of acrylamide (12.5%) and urea (8M), us­

ing an acrylamide (5-20%) and urea (I-IOM) gradient. A more or less simi­

lar picture emerged with both denaturing process where two prominent bands

of more of less same molecular weights (Fig. 6) were observed.

87

(A)

pi' n 1 r I

Fig. 6 Electrophoretic pattern of C protein and IgG on urea-SDS-PAGE (A) 12.5% urea SDS - PAGE with 8M urea (B) 12.5% urea SDS - PAGE using a linear gradient of 1-10 M urea. (C) 5 - 20% urea SDS - PAGE using a linear gradient gel of 8M urea.

Lane 1 (C protein) Lane 2 (IgG)

88

Antigenicity of 150 kD C -protein:

Antibodies against native 150 kD C-protein were raised in rabbit and

rats. The titre of antibodies was ascertained by direct binding ELISA wheras

inhibition ELISA was performed to probe the antigenic specificity of these

antibodies.

a) Evaluation of antibody response:

Direct binding ELISA was used to characterize the immune response

in rabbit and rats following immunization with 150kD C - protein.

The serum showed a high titre of greater than 1:12800 (Fig. 7 & 8).

Pre immune serum served as negative control as it did not show any

appreciable binding to 150 kD C - protein.

b) Competition ELISA for the antibodies raised against human heart

150 kD C - protein:

The antigenic specificity of the induced antibodies in rabbit and rats

against human heart 150 kD C - protein was probed by competition

ELISA. In rabbit, a maximum inhibition of 98.7 percent was obtained

in immune serum at an inhibition concentration of 10 ng/ml (Fig. 9).

Whereas the immune serum of rat exhibited a maximum inhibition of

88.1 percent at inhibitor concentration of 10 |ig/ml (Fig. 10). Fifty per­

cent inhibition was achieved at inhibitor concentration of 0.0016 \xgl

ml with rabbit serum compared to 0.07 fig/ml with rat serum.

89

1.4

1.2

E 1-0 c o

»- 0.8

u

S 0.6 a o to ** 0.A

a2

•

- \

-

^ g 9 ^ =£;^=

•^•^^..^^

= 9 u_ 2.0 2.3 2.6 2.9 3.2 3.5

-LOGSERUMDILUTION 3.8 A.I

Fig. 7 Binding of anti - C protein antibody in rabbit with C protein. Immune serum (#—# ) Preimmune serum(C>-0)

2.9 3.2 3.5

LOG SERUM DILUTION

3.8 4.1

Fig. 8 Binding of anti - C protein antibody in rat with C protein. Immune serum ( # - ) Preimmune serum ( O O )

90

0.01 0.1 1.0 10,0

INHIBITOR CONCENTRATION [Mgln>\)

Fig. 9 Inhibition of anti - C protein antibody activity in rabbit. Antigen : C protein Competitor : C protein

0.01 0.1 1.0 INHIBITOR CONCENTRATION ( >j g / m I )

10.0

Fig. 10 Inhibition of anti - C protein antibody acti\il> in rat by C protein Antigen : C protein Competitor : C protein

91

c) Purification of characterization of immune IgG:

Immune IgG was isolated from serum by Protein - A - Sepharose 4B

affinity chromatography. The purity of IgG was ascertained by SDS -

PAGE under non - reducing condition (Fig. 11) with the IgG moving

as a single band.

Direct binding ELISA of the purified IgG showed strong reactivity

with the immunogen (Fig. 12). Pre immune IgG as negative control

showed negligible binding to 150 kD C - protein.

Antigenic specificities of antibodies induced against human heart 150

kD C - protein:

The fine antigenic specificity of antibodies induced against 150 kD C

- protein was ascertained by competition ELISA. The plates coated with 150

kD C protein exhibited varying degrees of inhibition against a variety of

inhibitors. C - protein exhibited maximum inhibition of 98.7 percent at con­

centration of 10 ng/ml when used as an inhibitor. Fifty percent inhibition

was achieved at a concentration of 0.0016 i^g/ml of inhibition. On ROS

modification there was a decrease in inhibition of the antibody activity that

is, 38.7 percent at an inhibitor concentration 10 |Ag/ml. Fifty percent inhi­

bition was not achieved in this case (Fig. 13). Among the three photoadducts

formed by the interaction of 150 kD C -protein and nDNA, the one formed

in the presence of OH (ROS DNA-C-protein photoadduct) gave maximum

inhibition to the tune of 99.2 percent. This was followed by the photoadduct

formed in the presence of H2O2 (ROS DNA-C-protein adduct) that is 87.8 per-

92

2 3 ^ 5 6 FRACTION NUMBER 1 2 3 A 5 6 7

FRACTION NUMBER

3 A

FRACTION NUMBER

Fig. 11 Elution profile of IgG with 0.58% acetic acid and 0.85% Nacl from Protein A sepharose CL - 4B column, a) DCM IgG b) SLE IgG c) Immune IgG Inset shows single band of IgG on 7.5% SDS PAGE

93

2 /. 6 8 10 CONCENTRATION OF I g G ( ; u g / m l )

Fig. 12 Binding of rabbit immune IgG with C protein Immune IgG ( • - • ) Preimmune IgG ( Q - O )

10 20 30 AO 50

CONCENTRATION OF I g G ( ; i j g / m l )

60 70

Fig. 12 Binding of DCM IgG with C protein DCM IgG ( - ) Normal human IgG ( C M D )

94

cent was observed when the photoadduct was formed only in the presence

of ultraviolet radiation (UV-B). In all these, the maximum inhibitor concen­

tration was 10 ^g/ml. The same pattern was observed when the concentra­

tion for fifty percent inhibition was taken into account. The photoadduct

formed in the presence of OH gave fifty percent inhibition at an inhibitor

concentration of 0.09 \ig/ml followed by the adduct formed in presence of

H202(3.4 |ag/ml) and photoadduct formed in presence of UV - B irradiation

(7ng/ml) (Fig. 14). To check the cross reactivity of the induced antibodies,

various other inhibitors were also used. Out of these histone showed maxi­

mum inhibition of 7.3 percent at 20 [ig/ml which increased to 38.7 percent

on ROS modification (Fig 15). Poly - D - lysine gave a maximum inhibition

of 9.9 percent at 20 ixg/ml as compared to Poly L - Glutamate which showed

inhibition of 30.1 percent. The Poly (L + G) compelx formed after incuba­

tion of Poly - D - lysine and Poly L Glutamate at 37°C for 2 hrs. showed

maximum inhibition of 25.2 percent. Fifty percent inhibition was not

achieved in all these cases (Fig. 16). In case of nucleic acids, on ROS

modification of DNA, extent of inhibition was 15.4 percent at 20 (ig/ml. RNA

gave an inhibition of 5.2 percent at 20 ng/ml. Fifty percent inhibition was

not achieved in these two cases (Fig. 17). Percent relative affinity was calcu­

lated for inhibitiors in which fifty percent inhibition was achieved. Out of

the various inhibitors, photoadduct formed in the presence of OH showed

highest relative affinity of 1.8 percent followed by photoadduct in pres­

ence of H2 02(0.04 %) and photoadduct in presence of UV - B irradiation

(0.02%). The results have been summarized in Table 3.

95

0,001 0.01 0.1 1.0

INHIBITOR CONCENTRATION ( > j g / m l )

10.0

Fig. 13 Inhibition of rabbit immune IgG acitivity by native and ROS C pro­tein. Antigen : C protein Competitors : C protein ( O O )

ROS C protein ( 0 - )

96

0

c 20

z o

5 /loj z 1 -

S 60 u CE UJ

80

S ...

-

u

•

1

^ ^ \ . *

1

N. O

0O1 0.1 1.0 10.0

INHIBITOR CONCENTRATION ( j ug /m l )

100

Fig. 14 Inhibition of rabbit immune IgG activity by nDNA - C protein ad-ducts Antigen : C protein Competitors: nDNA - C protein photoadduct

ROS-DNA - C protein adduct ROS-DNA - C protein photoadduct

(O-O)

97

z i*J 6 0 « j

tLl

a.

80

J_ COl 0.1 1.0 10.0

INHIBITOR CONCENTRATION ( > j g / f D l )

100

Fig. 15 Inhibition of rabbit immune IgG activity by native and ROS Histone Antigen : C protein Competitors: Histone ( Q - O )

: ROS Histone ( # - )

98

20 z g H^

E9 AO I z »-z uj AO u Q: UJ

a 80

k TM—' ^

^

•

1 1 1

0.01 0 1 1.0 10.0

INHIBITOR CONCENTRATION (> jg /m l )

100

Fig. 16 Inhibition of rabbit immune IgG acitivity by poly D lysine, poly L. glutamate and poly (L+G) complex. Antigen : C protein Competitors: Poly D - Lysine (O-O)

Poly - L Glutamate ( - ) Poly (L+G) complex (O-Q)

99

04

20 z g P S AO I z »-z iJ 60 CJ cr UJ CL

80

^

_

—

_ _ _ _ _ _ ( J > u

1

"U < X ^

0.01 0.1 1.0 10.0

INHIBITOR CONCENTRATION (jjglmi)

100

Fig. 17 Inhibition of rabbit immune IgG activity by ROS-DNAand RNA Antigen : C protein Competitors: ROS DNA ( # - # )

RNA ( O O )

100

TABLE 3

ANTIGENIC SPECIFICITY OF ANTI C-PROTEIN ANTIBODIES

Inhibitors 50% Inhibition Maximum Inhibition % Relative (Hg/ml) (at 20 Hg/ml) (%) Affinity

C-Protein 0.0016 98.7" 100

ROS C-protein a 38.7''

nDNA C-protein 7.0 52.2'' 0.02 photoadduct

ROS-DNA-C-protein 3.4 87.8 0.04 adduct

ROS-DNA-C-protein 0.09 99.2'' 1.8 photoadduct

Histone a 7.3

ROS Histone a 38.7

Poly-D-Lysine a 9.9

Poly-L-Glutamate a 30.1

Poly (L-G) Complex a 25.2

ROS DNA a 15.4

RNA a 5.2

a = Fifty percent inhibition not achieved,

b = Maximum inhibition at 10 jig/ml.

101

Binding of total human heart extract (THHE) by DCM autoantibodies:

Out of the 15 DCM sera tested, most of them showed appreciable bind­

ing with total human heart extract (50 pig/ml). Sera from 9 DCM patients

gave a titre of more than > 1:6400. Three sera gave a titre of 1:3200 whereas

two gave a titre of 1:1600 (Fig. 18).

Recognition of human heart ISO kD C - protein by DCM autoantibodies:

The sera from DCM patients showed appreciable recognition towards

gel electroeluted, followed by HPLC purified human heart 150 kD C-pro-

tein. Of the 8 sera tested, 6 sera gave a titre of >r .6400 whereas two sera

gave a titre of 1:3200 (Fig. 19). The antigenic specificity of autoantibodies

against 150 kD C-protein was ascertained by competition/inhibition ELISA.

The 6 sera showing a titre of > 1:6400 were selected for inhibition studies

(Fig. 20). The degree of inhibition varied from 67.4 percent to 81.2 percent

at inhibitor concentration of 20 ng/ml. The order of specificity of these DCM

sera was ascertained by evaluation of concentration for fifty percent inhibi­

tion. The concentration for fifty percent inhibition varied from 0.02 to 2.0

|ig/ml of inhibitor (Table 4).

Binding of native DNA with DCM autoantibodies:

Native ds DNA which is the main trigger for SLE autoantibodies giv­

ing an antibody titer of >1:6400 showed poor recognition with DCM au­

toantibodies. Of the 7 DCM sera tested, one gave a titer of 1:800, followed

by 1:400 by three sera andtKree sera gave a titre of 1:200 (Fig. 21).

1 0 2

DIRECT BINDING ELISA OF THHE WITH DCM AUTOANTIBODIES

6 6 7 8 9 10 11 Number of serum samples

Fig. 18

12 13 14 16

a

b

1 : 1 0 0

1 :200

c d e f

g

1:400 1:800 1: 1600

1:3200

1:6400

103

0.01 0.1 1.0 10

INHIBITOR CONCENTRATION {pglm\)

0.01 0.1 1.0 10.0

INHIBITOR CONCENTRATION ( p g / m l )

Fig. 19 Inhibition ELISA of DCM autoantibodies with C protein as inhibi­tor. Antigen : C protein Competitor : C protein Antibody : Serum 1 ( Q - O ) Serum 4 ( O O )

Serum 2 (O-O) Serum 5 0 - 3 ) Serums ( - ) Serum 6 ( - )

104

TABLE 4

COMPETITION ELISA OF 150 kD CARDIAC C-PROTEIN

WITH DCM AUTOANTIBODIES

DCM Sera 50% Inhibition Maximum Inhibition (Hg/ml) (at 20 Jlg/ml) (%)

1 0.5 75.8

2. 0.03 78.6

3. 0.24 81.2

4. 0.02 71.9

5. 2.0 67.4

6. 0.6 73.6

105

DIRECT BINDING ELISA OF DCM AUTOANTI­BODIES WITH 150 KD C-PROTEIN

c o

•H

r-l •H

o O

to 2 3 4 6 6

Number of serum samples 8

"r <

F i g . 20 i DCM Sera NHS I

106

DIRECT BINDING ELISA OF DCM AUTO ANTIBODIES WITH NATIVE DNA

0.6

0.4

c 0-3 o

p 0.2 rH •H

O 0.1 O

4J NHS

i • ^ 1 2 3 4 5 Number of serum samples

DCM sera ^ ^ NHS

Fig. 21

107

Antigenic specificity of DCM autoantibodies:

DCM autoantibodies showed variation in percent inhibition with a

wide spectrum of inhibitors. In all the cases, the coating antigen was hu­

man heart 150 kD C -protein (50 [ig /ml in COj"/ HCO 3- buffer pH 9.6). A

maximum inhibition of 83.9 percent was obtained at 20 |xg/ml when C pro­

tein was used as inhibitor and fifty percent inhibition was observed at in­

hibit concentration of 0.9 ng/ml. The same on ROS modification showed a

lesser extent of inhibition which was found to decrease drastically upto 25.8

percent at 20 jxg/ml. Fifty percent inhibition was not achieved in this cases

(Fig. 22). The degree of inhibition among the nDNA - C protein photoadducts

varied in accordance with the agents used in their formation. A maximum

inhibition to the extent of 90 percent was formed at 20 |xg/ml in case of

photoadduct formed in presence of OH as compared to inhibition of 88.6

percent at 20 jxg/ml in case of photoadduct formed in presence of H2 O2.

Fifty percent inhibition was achieved at 1.0 |ig/ml in latter. The lowest

amount of inhibition of 41.7 percent was seen in the photoadduct formed

only in presence of UV - B irradiation (Fig. 23). To check the cross reactivity

of these antibodies various inhibitors were used. When histone was used as

an inhibitor, a maximum of 9.2 percent inhibition was achieved at inhibi­

tion concentration of 20 |ig/ml . On ROS modification of the same there

was an increase of upto 33.9 percent at 20 iig/ml (Fig. 24). Poly - D - Lysine

and Poly - L - Glutamate gave an inhibition of 6.6 and 40 percent at an

inhibitor concentration of 20 )ig/ml. The Poly (L + G) complex formed by

incubation of equimolar concentration of Poly D - Lysine and Poly L -

Glutamate (for 2hrs. at 37°C) gave an inhibition of 34.2 percent at 20 ng/ml

108

0.01 0.1 1.0 10.0

INHIBITOR CONCENTRATION {jjglm\)

Fig. 22 Inhibition of DCM IgG activity by native and ROS C protein Competitors: C protein (CKD) ROS C protein ( - )

109

0,01 0.1 1.0 10.0 INHIBITOR CONCENTRATION ( ; u g / m l )

Fig 2.3 Inhibition of DCM IgG activity by nDN.A - C protein adducts Antigen : C protein Competitors : nDNA- C protein photoadduct ( # - # )

ROS-DNA - C protein adduct ( ( ^ - 3 ) ROS-DNA - C protein photoadduct (Q-O)

110

z o 1 -

00

I z

z UJ a cr Q.

AO

60

80

JL 0.01 01 1.0

INHIBITOR CONCENTRATION ( ;ug /m l ) 10

Fig. 24 Inhibition of DCM IgG activity by native and ROS modified histone. Antigen : C protein Competitors: Histone ( # - # )

ROS Histone ( O O )

I l l

"i 20

g

2 40 X 2

z lu 60 u cr a

80

k

^ - - - - _ _ _ •

—

/> VP

U

1

/^ \M

#

1

f l <» -J^

• s r

1

0.01 0.1 1.0 10

INHIBITOR CONCENTRATION ( > i g / m l )

Fig. 25 Inhibition of DCM IgG antivity by poly -D-lysine, poly-L-glutamate and poly (L+G) complex. Antigen C protein Competitors: Poly D lysine ((f_o )

Poly L Glutamate (O-O ) Poly (L+G) complex (^-9) '

112

0.01 0-1 1.0 10

INHIBITOR CONCENTRATION ( > j g / m l )

Fig. 26 Inhibition nDNA of DCM IgG activity by ROS DNA and RNA. Antigen : C-protein Competitors: ROS-DNA(#-# )

RNA ( O O )

113

TABLE 5

ANTIGENIC SPECIFICITY OF DCM AUTOANTIBODIES

Inhibitors 50% Inhibition Maximum Inhibition % Relative (Hg/ml) (at 20 \lg/m\) (%) Affinity

C-Protein 0.9 83.9 100

ROS C-protein a 25.8

nDNA-C-protein a 41.7 photoadduct

ROS-DNA-C-protein 1.2 88.6 75 adduct

ROS-DNA-C-protein 1.0 90.0 90 photoadduct

Histone a 9.2

ROS Histone a 33.9

Poly-D-Lysine a 6.6

Poly-L-Glutamate a 40.0

Poly (L-G) Complex a 34.2

ROS DNA a 15.9

RNA J .-•>

a = Fifty percent inhibition not achieved.

114

(Fig. 25). The extent of inhibition was seen to be 15.9 percent on ROS modi­

fication of nDNA. On the other hand, RNA gave an inhibition of 3.3 per­

cent at 20 ^ig/ml (Fig. 26). The percent relative affinity was calculated for

inhibition in which fifty percent inhibition was achieved. Among the

photoadducts, the one formed in presence of OH showed elimination in an

antibody activity as high as to the extent of 90 percent followed by the

photoadduct formed in presence of H2O2 which showed 75 percent relative

affinity. In rest of the inhibitors fifty percent inhibition was not achieved.

Formation of cardiac 150 kD C protein native DNA photoadduct:

The photocoupling of purified calf thymus native DNA and HPLC

purified 150 kD C - protein (human heart) was carried out in the presence

of UV - B irradiation at 254 nm. The photocoupling was allowed to proceed

at different time intervals of exposure to UV - B radiation (Fig. 27). The

irradiated samples were extensively dialyzed against PBS, pH 7.4 and

subjected to ultraviolet spectroscopical analysis. Irradiated DNA showed a

substantial decrease in absorbance i.e. hypochromism was observed in the

entire spectral range. Ultraviolet irradiation of C-protein also exhibited

substantial hypochromism at maxima and minima (Fig. 28) . The native

DNA - C - protein photoadduct showed relative increase in hypochromicity

with increase in time of exposure to ultraviolet irradiation . Moreover, there

was a shift in maxima and minima of the photoadduct as compared to native

DNA. The maxima showed a shift from 260 nm to 255 nm whereas the shift

in minima from 230 nm to 235 nm relative to the ultraviolet absorption

characteristics of native DNA. Also, a sharp decrease in the ratio of

115

Ui O z <

o in CD <

075 -

200 300

WAVELENGTH ( n m )

400

Fig. 27 Time course kinetic profile of photoaddition of C protein to native DNA. Irradiation time (min) were. a) 10; b) 15 c) 20 and d) 30.

116

(A) '

u u z < 01 a. o 1/1 10

<

0.75

(B) 0.5

UJ u z < " 0.25 (r o (/) <

300

WAVELENGTH (nm)

CO 05

iVOO 2 00

200 300 WAVELENGTH(nm)

Fig. 28 UV absorption characteristics of .A<j native nDNA ( )

300

WAVELENGTH (nm)

400

AOO

native C protein ( d DNA ( ) ^®)UV irradiated C protein (

) UV irradiateu ui^r-\. y j - u v mauiai^u ^ pi^n-m v, -/

CO Difference UV spectra of nDNA, C protein and nDNA - C protein photoadduct

native DNA ( ) native C protein ( ) nDNA - C protein photoadduct (-»—)

117

TABLE 6

UV ABSORPTION CHARACTERSTICS OF FORMATION

OF nDNA - C PROTEIN PHOTOADDUCTS

SAMPLES

native DNA

(control)

native DNA

(Irradiated)

C protein

(Control)

C protein

(Irradiated)

nDNA - C protein

Photoadduct

Control ( 0 min)

(10 min)

(15 min)

(20min)

(30min)

ABSORBANCE 260 nm

1.044

0.901

0.082

0.069

1.071

0.920

0.926

0.946

0 978

295nm

0.091

0.082

0.055

0.052

0.146

0.112

0.116

0.122

0.128

ABSORBANCE RATIO 260/295

11.47

10.99

1.49

1.33

7.34

8.21

7.98

7.75

7.64

118

260/295 was observed for the photoadduct. This decrease was relative to

increase in the time of exposure to ultraviolet irradiation. Similarly, native

DNA and 150 kD C protein individually showed a decrease in 260 / 295

ratio on ultraviolet irradiation (Table 6 ).

Format ion of nat ive DNA 150 kD cardiac C - prote in adduct s /

photoadducts in presence of ROS:

Electroeluted and electrophoretically pure 150 kD human cardiac C -

protein was covalently crosslinked to purified calf thymus native DNA in

presence of ROS i.e. U^O^and OH (generated by H^Ojin presence of UV -

B irradiation) (Fig. 29). The samples were extensively dialyzed in PBS, pH

7.4 and subjected to UV spectroscopical analysis (Fig. 30). A substantial de­

crease in absorbance i.e. hypochromism was observed in the case of

photoadduct in presence of OH. A sharp decrease in the ratio of 260/295

was observed in this case. ROS-DNA also exhibited hypchromism but of a

lower magnitude than the former with reference to native DNA. The nDNA

- C protein adduct formed in presence of H2O2 exhibited high degree of

hypochromism with an increase in 260/295 ratio . Furthermore, as evident

from the (Fig. 30), ROS-C - p io t e in exhibi ted a cer ta in degree of

hyperchromism at 280 nm with reference to native C - protein. The ultra­

violet spectroscopical characteristics of modified and unmodified samples

are depicted in table 7.

De tec t i on of DNA - C - p ro t e in adduc t format ion by a g a r o s e gel

electrophoresis :

Agarose gel electrophoresis was carried out to detect the adduction

119

200 300 WAVELENGTH ( n m )

400

300 NWAVELENOTH ( n m )

600

Fig. 29 UV absorption characteristics of: native C protein( ) rgxnDNA ROS C protein (---- ) ^ ^ROS DNA

r -v ROS-DNA - C protein adduct ( ) ROS-DNA - C protein photoadduct ( )

CA) (___

120

TABLE 7

UV ABSORPTION CHARACTERISTICS OF FORMATION OF

ADDUCT/PHOTOADDUCT IN PRESENCE OF ROS.

SAMPLE

native DNA

ROS-DNA

C protein

ROS-C-protein

ROS-DNA - C

adduct

ROS-DNA - C

photoadduct

protein

protein

ABSORBANCE 260nin

1.944

1.244

0.038

0.066

1.999

1.115

295nni

0.196

0.201

0.023

0.033

0.328

0.140

ABSORBANCE RATIO 1 260/295

9.92

6.19

1,65

2.00

6.10

7.96

121

M 3

Fig. 30 Electrophoretic mobility of samples (1.0 [xg each) after ROS modi­fication. The samples were run on 1% agarose gel at 30 mA for 2 hrs. Lane 1 (native DNA) Lane 2 (ROSDNA) Lane 3 (DNA - C pro­tein + H^O^) Lane 4 (DNA - C protein + H^^^ U V )

122

C^) \ ^

iu 1 7, i Z

CC)

Fig. 31 Nuclease SI sensitivitiy of DNA - C protein adducts. DNA - C pro­tein adducts (1.0 pg) were treated with nuclease SI (20 u/mg of DNA) a) nDNA - C protein photoadduct b) ROS-DNA - C protein adduct c) ROS-DNA - C protein photoadduct

Lane 1 (control) Lane 2 (treated samples)

123

of DNA to C protein. The adducts were treated with SI nuclease (20 units/

Hg adduct) and the digested sample were subjected to electrophoresis . The

results showed enhanced mobility of SI treated adducts (Fig. 31). This was

seen in the case of adducts formed in the presence of ROS which include

HjO^and OH. On the other hand photoadduct formed in the presence of UV

- B irradiation alone did not show any change in mobility. The untreated

adducts were used as controls. The results indicated the generation of single

stranded portions in DNA - C- protein a d d u c t photoadduct formed by H2O2

and OH as recognised by single strand specific enzyme SI nuclease.

Kinetic of lysine photoaddition to native DNA:

The nature of photointeraction between lysine residues of C - protein

and DNA was also investigated by kinetic study. The irradiated samples

(10-30 min) were dialyzed against PBS, pH 7.4 and their UV spectra re­

corded. The results showed a constant increase in absorbance at 260 nm

with the increase in irradiation time. There was a linear increase in percent

hyperchromicity with the increase in radiant energy (Fig. 32). The number

of lysine residues in the photoadduct C - protein was determined as a result

of varying period of UV irradiation (Fig. 33). One lysine molecule of the C-

protein was in the photobound state in between 7.1, 6.3, 5.4 and 3.6 nucle­

otide base pairs of DNA on irradiation for 10, 15, 20 and 30 min. respec­

tively (Fig. 34). The results obtained indicates a progressive increase in the

photoaddition of lysine molecules (present in C-protein) to DNA with the

increase in irradiation time. A linear regression plot of hi (Va - Vt) vs. the

irradiation time 't' gives a straight line which is characteristic of apparent

first order reaction mechanism (Fig. 35).

124

1 ^ 3 J^ S'

Fig. 32 Time course pattern of DNA - C protein photoadducts formed by UV - B irradiation . The samples (1,0 |ig each) were electrophore-sed on 1% agarose gel at 30mA for 2 hrs. Lane 1 ( O min.) Lane 2 (lOmin.) Lane 3 (15 min.) Lane 4 (20 min.) Lane 5 (30 min.)

125

0 10 20 30 40 50 60 70 80 90 100

CONCENTRATION OF L-LYSINE ( j j g / m l )

Fig. 33 Standard plot for estimation of lysine by TNBS method.

126

10 20 30

IRRADIATION TIME (min.)

Fig. 34(A) Concentration of photobound lysine at various irradiation times in native DNA - C protein photoadduct.

10

10 2 0 30 IRRADIATION TIME (min )

Fig. 34 (B) Kinetic profile of photobound lysine residues to native DNA at various irradiation t imes.

127

5 10 15 IRRADIATION TIME(min.)

Fig 35 Apparent first order profile of nDNA-C-protein photoadduct forma­tion at various irradiation times. The values were least square fitted and the apparent first orderate constant K was computed from the

slope.

128

Isolation of DCM and SLE IgG with protein A - sepharose:

Protein A binds IgG from most mammalian species (Kronvall et al,

1970; Langone, 1978). Although IgG is the major reactive human

immunoglobulin, some other types have also demonstrated to bind with

Protein A. The interaction of Protein A with IgG occurs via Fc portion of

IgG molecules from many species.

Protein A sepharose purified IgG's from normal, DCM and SLE sera

were each found to elute in a single symmetrical peak. The absorbance

ratio (A^^g/ A^j,) of the peak fraction was found to be 2.5, typical of mam­

malian IgG. The homogeneity of the peak fraction was assessed by its

single band movement in SDS - PAGE under non reducing conditions

(Fig. 10)

Affinity purification of SLE IgG on native DNA - polylysyl sepharose 4B

column:

Poly-L - lysine sepharose 4B is an amphoteric derivative of sepharose

4R which is a group specific absorbent for purification of dsDNA. Poly - L

- lysine sepharose 4B is prepared by coumpling poly - L - lysine to sepharose

4B by the cyanogen bromide method. The IgG fractions were collected us­

ing a linear gradient of ionic strength 0.15 - 3.0 M sodium chloride in 0.01

M phosphate buffer, pH 7.4 (Fig. 36). Affinity purified SLE IgG was found

to be nearly free from contamination by other classes of immunoglobulins

as judged by their homogenous movement in SDS PAGE under non reducing

conditions.

1 2 9

E c o 00

o < GO a o en CD <

1.0 -

0.8-

0.6-

0.^-

0.2-

T r 1 1 1 1 1 r-HI—T—*-nr

2 ^ 6 8 10 12 U 16 23 25

FRACTION NUMBER

Fig. 36 Imraunoaffinity purification of SLE IgG on nDNA-[polylysyl sepharose 4B] column

130

Band - shift assay:

The binding of DCM, SLE and induced 150 kD C - protein antibodies

with adducts/photoadducts was reiterated by band, shift assay. One micro­

gram each of nDNA - C - protein photoadduct, ROS - nDNA - C protein

adduct ROS DNA - C - protein photoadduct was incubated with varying

amounts of DCM, SLE and immune IgG for 2 hrs at 37°C. The immune com­

plex thus formed was kept overnight at 4°C and later electrophoresed on

1% agarose at 30mA for 2 hrs. in tris acetate EDTA (pH 8 0) . The wells in

the gel showed increase in the amount of immune complex on increase in

IgG concentration. A retardation in mobility was also observed indicating

the strong interaction between the DNA - C - protein adducts and DCM,

SLE and immune IgG. (Figs. 37, 38, 39).

Recognition of human cardiac antigens by SLE autoant ibodies:

The recognitiion of antigens ranging from nucleic acids to myocarditis

autoantigen (s) by naturally occuring SLE autoantibodies by direct binding

and competition ELISA irect binding ELISA on plates coated with nucleic

acid antigens i.e. ds DNA, SS DNA and RNA as well as THHE exhibited

appreciable reactivity with SLE autoantibodies (Fig. 40). Competit ion

inhibition ELISA exhibited inhibition in antibody activity with ds DNA, ss

DNA, RNA (Fig. 41) and THHE (Fig. 42) to a tune of 75.2%, 75%, 10.5%

and 75% respectively. Fifty percent inhibition varied except in case of RNA

where it could not be achieved. Protein A-sephorose purified SLE IgG with

C-protein exhibited inhibition of 79.3% with fifty percent inhibition at 1.8

Mg/ml (Fig. 43) was employed to further ascertain the specificity. This

131

i 9. 3 "^

Fig 37(A) Binding of DCM IgG by DNA - C protein photoadduct as analyzed by gel retardation assay nDNA - C pro­tein photoadduct (1 0 ng) was incu­bated with buffer ( l ane l ) and with various concentrat ions of DCM IgG (25 (ig, lane 2, 50 (ig. lane 3 and 75 Mg. lane 4 The complex was electro-phoresed for 2 hrs on 1% agarose at 30 mA

Av '-) ^

Fig. 37(B) Binding of SLE IgG by DNA - C pro­tein photoadduct as analyzed by ge! retardation assa\ nDNA - C protein photoadduct (1 0 fjg) was incubated with buffer ( l ane l ) and with various concent ra t ions of SLE IgG (25 |ig. lane 2, 50 (jg. lane 3, 75 (ig, lane 4

The complex was electrophoresed for 2 hrs on 1% aga­rose at 30 iiiA

1 ^ 3 ^ 5~

fig 3 7 ( 0 Binding of anti C" proicin Immune IgG b\ nDNA - C protein photoadduct as analyzed hy gel retardation assav nDNA - C protein photoadduct (1 0 Mg) was inci'bated with buffer (lane 1 ) and with \c loiis concentrations of anti - (" protein immune IgG (25^ig. Lane 2. 50 pg, 1 anc 3, ""5 g Lane 4 and 150 g. Lane 5) The complex was electrophoresed for 2 hrs on l°o aga­rose at 30 m \

1 7. W^ f

Fig 38(B) Binding of SLE IgG by ROS - D \ A, -C protein adduct and analyzed b\ gel retardation assay ROS-DN A - C pro­tein adduct (1 0 ng) was incubated with buffei (lane 1) and with \ a r ious concent ra t ions of SLE IgG (25 pg Iane2 50 ng lanel 75 pg lane and 150 (.ig ) The complex was electro-phoresed for 2 hrs on l%agaros t at iO mA

' 3 ;i 1 A ^ 0

Fig 38(A) Binding of DCM IgG by ROS - 1 ; \ A -C protein adduct and inalyzed by gel retardation assay ROS-DNA - C pro­tein adduct ( I 0 ng) was incubated with buffer ( lane 1) and with \a r ious concentrat ions of DCM IgG (25 ng lane2, 50 pg lane"? and 1'^ \x% lane 4) The complex was electrophoresed for 2 hrs on 1% agarose at 30 mA

Fig i8(C) Binding of anti - C protein / Immune IgG b \ ROS-DN^ - C protein adduct as anaKzed bj gel retardation assay ROS-DN^ - C p r o t e i n adduct (1 0 lag) was incubated with buffer (lane 1) and with \ar ious concentrations of anti - C protein / immune IgG (25 iig lane2' '50Lig lane3''75Mg lane 4 150 Hg lane 5) The complex was electro­phoresed for 2 hr on 1% agarose at 30 mA

133

1 2. ;3 ^ 5-

Fig 39(A)Binding of DCM IgG by ROS DNA -C protein phoioadduct as analyzed by gel retardation assay ROS DNA - C protein photoadduct (1 Ong) was in­cubated with buffer (lane 1) and with various concentrations of DCM IgG (25 Jig, lane2, 50 ^^g lane 3, 75 ng, lane 4 and 150 |ig, l aneS) The complexed was electrophoresed for 2 hrs on 1% agarose at 30 mA

i ^ 3 ^ 5'

"w' Wp, jfKt^.m^-^^

Fig 39(B) Binding of SLE IgG by ROS DNA - C protein photoadduct as analyzed by gel retardation assay ROS DNA - C prote in photoadduct (1 O^g) was in­cubated with buffer (lane 1) and with various concentrations of SLE IgG (25 Hg lane2 50 )ig, lane 3, 75 ng, lane 4 and 150 fig lane'>) The complexed was elec'.rophorestd for 2 hrs on 1% agarose at ' O mA

1 ^ 5 ^ r Fig 39(C) Binding of anti - C protein / immune

IgG by ROS - DNA - C p r o t e i n photoadduct as analyzed b> gel retar­dation assa\ ROS DNA - C protein photoadduct (1 0 \ig) was incubated with buffer (lane 1) and with \ a r ious concentration^, of anti - ( protein / immune IgG (25 g lane2 50g lane3 75 g lane 4 and 150 g lane 5) The complex was electrophoresed for 2 hr on P/o agarose at 30 mA

134

e o

< UJ o z < CD o: o (/) CO

<

1:100 1:200 1:400 1:800 1:1600

-LOG SERUM DILUTION

1:3200 1:6400

Fig. 40 Binding of SLE auto antibodies with ds DNA, ssDNA, RNA and THHE dsDNA iA-k) ss DNA ( S - ^ ) RNA ( A ^ ) THHE ( © ^ ) NHS {O-O}

135

5 lO 15

NHIBITOR CONCENTRATION,jug/ml

Fig. 41 Inhibition ELISA of SLE autoantibodies on plates coated with na­tive DNA Competi tors: native DNA(O-O) ss DNA (A- t^)

RNA ( § . # )

136

100 5 10 15 C INHIBITORS,;jg/ml

20

Fig. 42 Inhibition ELISA of SLE autoantibodies on plates coated with ds DNA Competitor : THHE ( • - • )

137

100 5 10 15 CiNHlBlTORa,Aig/ml

20

Fig. 43 Inhibition ELISA of protein A sepharose isolated SLE IgG on plates coated with ds DNA Competitor : THHE ( • - • )

138

5 10 15

CINHIBIT0R3,>ig/ml

Fig. 44 Inhibition ELISA of i Competitor : C protein

immunoaffinity purified SLE IgG

139

revealed inhibition of 76% and fifty percent inhibition was achieved at 1.5

^g/ml (Fig. 44).

Probing mitochondrial DNA damage in DCM patients:

The sera from DCM patients showed preferentially higher binding with

native mitochondrial DNA (mt DNA) as compared to its ROS modified

conformer (Fig. 45). The specifity was ascertained by inhibition ELISA. The

six DCM sera tested showed inhibition in the antibody activity, ranging from

64.5 percent to 73.1 percent in case of native mt DNA (Fig. 46) as compared

to inhibition varying from 54.6 percent to 63.8 percent in case of ROS mt

DNA (Fig. 47) at maximum inhibitor concentration of 20 pig/ml. The range of

inhibitor concentration for fifty percent inhibito varied from 0.15 to 4.0

jxg/ml in native mt DNA. In ROS mt DNA, fifty percent inhibition was

achieved in the range of 2.0 jig/ml to 10.0 [ig/ml in the six tested DCM

sera. The results have been summarized in Table 9.

Glucose, cholesterol, uric acid estimation and evaluation of CRP and RF

content in patients with dilated cardiomyopathy (DCM):

Elevated levels of glucose that is 146.1, 115.3 and 115 mg/dl were

found in 3 of 15 DCM patients under taken in this study (Fig. 48). Amongst

them only one showed higher level of serum cholesterol that is 248 mg/dl

(Fig. 49). An increase in uric acid content in serum was found in 7 of the 9

female patients, in the range of 4.5 to 24.9 mg/dl. The males showed a varia­

tion from 4.3 to 11.2 mg/dl (Fig. 50). The C reactive protein (CRP) latex

agglutination test revealed positive agglutination (>6mg/dl) in 9 out of 15

140

DIRECT BINDING ELISA OF DCM AUTO ANTI­BODIES WITH NATIVE AND ROS mt DNA

3 4 6 6 Number of serum samples

[HID Native mt DNA ROS mt DNA

Fig. 46

141

0.01 0.1 1.0 10.0

INHIBITOR CONCENTRATION ( / j g / m l )

0.01 0.1 1.0 1 0 0

INHIBITOR CONCENTRATION {^glmi)

Fig. 46 Inhibition ELISA of DCM auto antibodies with native mt DNA as inhibitor

Competitor: native mt DNA (O-O ) Serum 4 ( o - o ) ( o - O ) Serum 5 ( O-O) ( ©-€)) Serum 6 ( • - • )

Antigen : native mt DNA Antibody : Serum 1

Serum 2 Serum 3

142

0.01 0.1 1,0 10.0 INHIBITOR CONCENTRATION ( ; j g / m l )

O.Ol 0.1 1.0 10.0

INHIBITOR CONCENTRATION C<ug/ml)

Fig. 47 Inhibition ELISA of DCM auto antibodies with ROS mt DNA as inhibitor. Antigen : ROS mt DNA Antibody : Serum 1

Serum 2 Serum 3

Competitor : (a—O) Serum 4 {9—0) Serum 5 (•—•) Serum 6

ROS mt DNA

{o-o) (•-») ( — )

143

TABLE 8

COMPETITION ELISA OF MITOCHONDRIAL DNA (mtDNA) WITH

DCM AUTOANTIBODIES

DCM Sera

50% In {\lg/m\)

1 1 6

2 1 1

3 1 0

4 0 15

5 4 0

6 2 4

Antigen-Inhibitoi

hibition

mt DNA r-mt DNA

Maximum Inhibition (at 20 ng/ml)%

67 6

64 5

73 1

64 9

65 6

65 2

50%

Antigen -In

In (^ig/ml)

3 0

2 4

2 2

2 0

100

5 0

ihibitor

hibition

ROS mt DNA - ROS mt DNA

Maximum Inhibition (at 20 p,g/ml)%

63 8

61 0

63 2

58 2

54 6

56 8

144

GLUCOSE LEVELS IN SERA OF PATIENTS WITH DILATED CARDIOMYOPATHY

160 Y[

E

tn O O :s 1—1

F i g . 48

4 6 6 7 8 9 10 11 12 13 14 16 Number of serum samples

Normal range-. 70-100 mg/dl

145

TOTAL SERUM CHOLESTEROL LEVELS IN PATIENTS WITH DILATED CARDIOMYOPATHY

S

c <u •p c o o

o

« r-l o x: u

1 2 3 4 6 6 7 8 9 10 11 12 13 14 16 Number of serum samples

F i g . 49 Normal range: 130.0-220.0 mg/dl

146

LEVELS OF URIC ACID IN SERA OF PATIENTS WITH DILATED CARDIOMYOPATHY

s

T3 •H

u

u

e D

0)

Fig . 50

5 6 7 8 9 10 11 12 13 14 Number of serum samples

Normal range :-Males: 3.4 - 7.0mg/dl

Females: 2.4 - 6.7 mg / dl

147

C - REACTIVE PROTEIN (CRP) LEVELS IN SERA OF PATIENTS WITH DILATED CARDIO­

MYOPATHY

•p c Q) ^J C O

o a,

200

160

100

60

T 1 r

6 6 7 8 9 10 11 12 13 14 16 Number of serum samples

F i g . 51 Normal CRP content < 6mg/L

148

patients in qualitative estimation. Semiquantitative analysis of the positive

samples showed variations in CRP contents from 12 mg/dl in 4 sera, 24 mg/

1 in 2 sera to 48 , 96 and 192 mg/1 in one serum each of DCM patients

(Fig. 51). Qualitative analysis of Rheumatoid factor (RF) by RF latex agglu­

tination test did not show any agglutination in any of the tested DCM sera.

B xscuBsion

149

A utoimmune diseases are characterized by the presence of circu­

lating autoantibodies which are not necessarily pathogenic but represent

markers of immune mediated injury (Rosa, R and Bona, C, 1991). Autoim­

mune diseases result due to humoral and / or cellular immunological reac­

tions against the self components of an individual. The triggering mecha­

nisms of autoimmune reaction have not yet been completely understood.

Shoenfeld and Isenberg (1989) described the Avide spectrum of autoimmune

diseases as mosaic of autoimmunity with many factors leading to various

diseases.

Dilated cardiomyopathy (DCM) is a chronic heart muscle disease of

unknown etiology characterized by dilatation and contractile dysfunction

of left and / or the right ventricle. In man, myocarditis is known to be the

precursor of DCM in many cases, although clinical and histological diag­

nosis remain problematic. Since in DCM, the only affected organ is heart, it

fits into the criteria of organ specific autoimmune disease. Autoimmune

features in patients with DCM include familial aggregation (Keeling et al. ,

1995), a weak association with HLA DR4 (Carlq uist et al., 1991) and im­

munoglobulin genes ( Martinetti et al., 1992), abnormal expression of HLA

class II on cardiac endothelium (Caforio et al., 1990), and increased levels

of circulating cytokines (Limas et al., 1995).

Several groups have found cardiac antibodies to various antigens in

DCM (Caforio et al . , 1992; Schultheiss et al., 1990; Limas et al., 1995).

Multiplicity of parallel autoimmune reactions directed towards a single or­

gan may be due to spread sensitization (Bach, 1995). Among the various

150

cardiac antigens, 150 kD C protein has been found to be a potent antigen

causing autoimmune myocarditis in experimental animals. C protein plays

an important role in contractile activity. It is one of the major constituent

proteins which are essential to compete with natural antigens at MHC - an­

tigen bindings site (Demotz et al., 1990; Harding and Unanue, 1990). More­

over C protein is an intracellular member of immunoglubin superfamily

(Einheber and Fischman, 1990). Due to identical molecular weights IgG,

and C protein gave a similar pattern on 7.5% SDS - PAGE. Urea denatur-

ation studies also showed more or less similar patterns in Urea - SDS -

PAGE. When SDS -PAGE was performed with a urea gradient (1-10 M urea)

as well as acrylamide gradient (5-20%), both IgG and C protein were dena­

tured to give two prominent bands each. This further gives an insight into

this theory. The HLA binding motifs are located in the variable domain and

not the constant IgG like domain (Rudensky et al., 1992; Baum et al., 1993).

The thermal behaviour of 150 kD C - protein and human IgG as con­

trol displaying Tm values of unfolding at around 79.5°C is typical of a highly

stable dimeric protein molecule. The highly stable conformational state of

150 kD C - protein was evident from the observation that only 4.4% had

undergone thermal unfolding till 75°C. Thus our observation reveals for

the tremendous structural stability of the 150 kD C - protein auto antigen.

Additional evidence for the structural changes in native 150 kD C-

protein as a result of thermal energy supplementation was revealed by ana­

lyzing the data and computation of thermodynamic parameters (Table 1 &

2). The thermodynamical data speculate the tremendous structural stability

151

exhibited by 150 kD C - protein. Disruption of the hydrophobic and ionic

interaction as well as disulfide bonds in the 150 kD C - protein which

ultimately resulted in unfolding was inferred from the shift in the scales of

the AGJJ values. The negative AG^ values above 79°C suggests the transi­

tions of completely folded 150kD C - protein to the unfolded state. Further­

more, in comparison to the already established thermodynamic behaviour of

native DNA, our results are indicative for the 150 kD C - protein to be topo-

logically less constrained than nucleic acids. This comparative analysis has

been pointed out due to the important factor of DNA acting as auto antigen

in SLE whereas 150 kD - C-protein acting as autoantigen in DCM /

myocarditis and that as per our observations, the 150 kD C - protein which

acts as autoantigen in myocarditis was found to be equally reactive with

naturally occuring SLE autoantibodies.

Photochemical reaction have been found to produce DNA - protein

crosslinks in biological systems. Proteins and nucleic acids are colourless

macromolecules which absorb in the UV range of spectrum. These two

biomolecules coexist in vivo and their interaction results in biological dam­

age in organisms. The crosslinking between DNA and protein has been found

to be covalent in nature. (Oleinick et al., 1986). In this study , we have

formed photoadducts between native DNA and C - protein in UV light (254

nm). Time course kinetics revealed that the formation of photoadduct in­

creased with increase in duration of exposure to UV irradiation. This was

further reiterated by the amount of total protein bound to DNA. The protein

content increased remarkably with the increase in period of exposure. The

amount of lysine residues present in C-protein were estimated to assess their

152

binding to native DNA. Time course kinetic study indicates that the forma­

tion of photoadduct obeys apparent first order reaction.

Oxygen free radicals (OFR's) occupy a prime position in the world of

free radical biochemistry . The prima donna in this category are oxygen

itself, superoxide, hydrogen peroxide, transition metal ions and hydroxyl

radicals, where the first four interact in many ways to produce the last

(Halliwell and Gutteridge, 1990). Exogenous sources like toxic foreign com­

pounds and ionizing radiations are known to increase the production of free

radicals. There are several sites for generation of reactive oxygen species

(ROS) in cells, but the main source is the autooxidation of reduced compo­

nents of mitochondrial transport chain (Haiku et al., 1993).

Reactive oxygen species produce DNA - protein crosslinks in biologi­

cal systems. Here we have taken two entities, hydrogen peroxide (H^

O^) and hydroxyl radical (OH) to produce crosslinks and photoadduct for­

mation between native DNA and C - protein. Among the two, hydroxyl radi­

cal (OH) appeared to be a better agent to induce DNA - C- protein crosslinks.

The formation of photoadduct in the above cases causes single strand breaks

in DNA as revealed by nuclease SI sensitivity assay. On the contrary, the

control and treated samples of nDNA C-protein photoadduct showed the

same pattern. It signifies that in the photoadduct, interaction may have taken

place via lysine residues. The other aminoacids in the protein can also un­

dergo photointeraction to form protein-protein photoadduct. Thus, crosslinks

are again formed between nDNA C-protein and C-protein C-prote in

photoadduct which is responsible for such a pattern.

153

The recognition between nucleic acid and proteins is of great signifi­

cance as it triggers most important steps of nucleic acid metabolism. This

recognition involves electrostatic interactions between phosphate groups and

positively charged groups in protein. Later more specific interactions be­

tween nucleic acid bases (or other structural elements) and amino acid side

chains in proteins take place. A closer look reveals the possible recognition

of secondary structure of nucleic acids by proteins, either way recognition

is dependant of nucleotide sequence (Duguet, 1981).

A different chemistry arises when protein and DNA are irradiated to­

gether or separately. Since DNA and protein are in intimate contact with

each other in vivo, it is suggested that photochemical interaction of DNA

and protein would play a significant role in the inactivation of UV irradi­

ated cells under certain conditions.

The photoadducts formed showed appreciable reco. gnition with DCM,

SLE and immune sera. The reactivity of DCM and immune sera was maxi­

mum with photoadduct formed by hydroxyl radical (OH) followed by the

one formed only in the presence of UVB irradiation showed least reactivity

as shown by band shift assay and completion ELISA studies. This indicates

the effectiveness of hydroxyl radical(-OH) to generate DNA protein crosslinks

which produce pathological changes.

The sera of patients with dilated cardiomyopathy (DCM) showed a

typical picture in the biochemical investigations . Quantitative determina­

tion of serum cholesterol gave normal values except for one patient. The

determination of serum cholesterol is considered to be significant mostly

154

in coronary artery diseases, hyperlipoproteinemas, hypothyroidism,

nephrosis, diabetes mellitus, and various liver diseases, and has very little

role to play in dilated cardiomyopathy (DCM). Similarly patients with DCM

rarely show an increase of serum glucose. Only two of the total fifteen cases

showed a moderate increase in glucose levels. The moderate rise is associ­

ated with infectious diseases, intracranial diseases and haemorrhage. There

was a significant increase in uric acid levels in serum. Serum or plasma

uric acid determination has an important diagnostic values in gout and kid­

ney failure. It is also increased in acute stages of infectious diseases, se­

vere uremia, toxemia of pregnancy and leukemia. DCM is a end stage car­

diac disorder in which there is active degeneration of cardiac tissue leading

to heart failure. This chronic inflammatory process may be responsible for

increase in uric acid levels in DCM patients.

A significant rise was observed in the C - reactive protein (CRP) lev­

els in sera of DCM patients. A majority of patients gave a high titre on semi

- quantitative determination of CRP in serum. CRP an acute phase reactant

is considered to be the most sensitive marker in the acute phase and there­

fore, a universal early indicator for inflammatory, necrotic and neoplastic

diseases. CRP is a non - glycosylated cyclic pentamer which increases in

concentration in a fixed and phased sequence during inflammation and / or

active tissue infections. It is important in protection against development

of autoimmunity. It acts by clearing cell debris autoantigens recognised by

auto antibodies. The rise in CRP levels in DCM patients can be attributed

to this factor. Rheumatoid factor (RF) was absent in all the sera of DCM

155

patients. RF are credible markers in inflammatory diseases of the joints .

Hence their absence rules out the possibility of any such factor in DCM.

Autoantibodies found in patients with dilated cardiomyopathy (DCM)

exhibited remarkable binding with the HPLC purified 150kD human C-pro-

tein as is evident from competition inhibition ELISA results. Nearly all the

sera of patients with DCM showed inhibition in the antibody activity by

150 kD C-protein in the range of 64 .7% to 82.1%. Furthermore, an appre­

ciable number of DCM sera exhibited inhibition in their antibody activity

by a very low magnitude of inhibitor concentration, which in turn, is fur­

ther substantiative/or indicative for the remarkably high specificity of the

DCM autoantibodies towards 150kD C-protein. However, the binding to­

wards 150kD C-protein was of higher magnitude with the induced anti 150

kD C-protein antibodies in comparison to the binding with DCM autoanti­

bodies. The binding results are suggestive for the major involvement of

150kD C-protein as autoantigen in autoimmune DCM/myocarditis.

It is to be pointed out that native DNA was found to be of low reactiv­

ity with all the sera of patients with DCM. But interestingly, mitochondrial

DNA (mt DNA) on the other hand, exhibited appreciable binding with DCM

autoantibodies, although the magnitude of binding was lower than with

150kD C-protein. Moreover, surprisingly, modification of mt DNA with ROS

showed decrease in binding with DCM autoantibodies, whereas conversely,

nDNA which was unreactive towards DCM autoantibodies, showed a bind­

ing of low magnitude when modified with ROS. The results are suggestive

for the probable presence of conformational epitopes on mtDNA and to a

156

lower extent on ROS DNA which are somewhat similar to the ones present

on 150 kD C-protein.

The highly immunogenic nature of native 150kD C-protein was evi­

dent from the competition results, where around 98 percent of the antibody

activity was inhibited by the immunogen. However, when native 150kD C-

protein was ROS modified, the antibody binding was lost to the extent of

more than fifty percent. The results suggest for the damage or loss of sig­

nificant number of immunodominant epitopes on 150kD C-protein upon ROS

modification.

Although the induced anti-150kD C-protein antibodies were unreactive

with native DNA, a low magnitude of binding was observed when nDNA

was photoadducted with C-protein, whereas, surprisingly, ROS-DNA-C-Pro-

tein photoadduct exhibited a high degree of binding which was even higher

than the binding observed with the immunogen i.e. 150kD C-protein. This

could be attributed to the cumulative binding effect of immunogenic epitopes

on C-protein as well as somewhat similar epitopes to the immunogen on the

ROS modified DNA and hence when both ROS-DNA and C-protein were

photoadducted, a higher binding was observed. The results suggest that prob­

ably apart from 150kD C-protein, the photoadducted ROS-DNA C-protein

complex could also act as an alternate autoantigen for myocarditis/DCM.

The above possibility could not be ruled out because of the fact that reac­

tive oxygen species (ROS) are formed in vivo which modifies the nucleic

acid macromolecule and that the possibililty of photoconjugation of ROS-

DNA with C-protein exists. Thus, our findings could possibly throw some

light on the pathogenesis and etiology of autoimmune myocarditis/DCM.

157

The binding of induced anti-C-protein antibodies with histones, ROS-

histone, poly-D-lysine, poly-L-glutamate and poly (lysine-glutamate) com­

plex was of low magnitudes thereby suggesting for the presence of a minor

sub-population of epitopes responding to the anti-C-protein antibodies. Simi­

lar binding results were also observed with DCM autoantibodies against C-

protein, ROS C-protein ROS-DNA, ROS-DNA photoadducted to C-protein,

ROS-histone, histones, poly-D-lysine, poly-D-glutamate and poly (L+G)

complex respectively. The similarity in binding results further substanti­

ates the above argument.

Apart from the above the systematic probe to see the correlation be­

tween myocarditis autoantigen (C-protein) and autoimmune SLE autoanti­

bodies gave encouraging results.

Systemic lupus erythematosus (SLE) is a multisystem prototype au­

toimmune disease (StoUar, 1981) characterized by circulating autoantibod­

ies that bind to cellular antigens of self and exogenous origin. Although the

correlation of SLE with immune system is well documented, neither the

origin of these autoantibodies nor the etiology of the disease is known. Re­

cent studies point out that native DNA might actually be a cross reactive

antigen whereas some other structures like modified nucleic acids (Alan et

al., 1993; Ara and AH; 1993; Ara and Ali, 1992), polypeptides (Arif et al.,

1994) and phospholipids (Smeenk et al., 1987), etc. stimulate the antibody

response. Moreover, the polyspecificity of autoantibodies with a variety of

heteroantigens might be the result of immune response to substance (s) other

than native DNA and interplay of some environmental factors.

158

In the present study attempts were also made -to probe the possible

involvement of 150 kD C - protein isolated from human heart which is known

as potential autoantigen in myocarditis and DCM in humans , in autoimmune

SLE. The protein is known to have similar domain as to those of IgG and

has been implicated in the induction of autoantibodies in patients with SLE.

The 150 kD C- protein, which is a component of thick filament of skeletal

and cardiac muscles, plays an important role in the contractile activity.

Assays of anti - DNA and anti - human heart antigens in the sera of patients

with SLE and DCM respectively were carried out by employing direct

binding ELISA whereas specificity determination was accomplished by

competition ELISA. The antigen binding characteris t ics of anti - DNA

autoantibodies towards native and modified nucleic acids were in accordance

with earlier findings (Losman et al., 1993).

The most striking finding of the present study is the appreciable

recognition of THHE as well as 150 kD C - protein (human heart) by SLE

autoantibodies. As revealed by competition inhibit ion studies, the SLE

autoantibodies expressing specificity towards dsDNA, ssDNA, DNA-lysine

photoadduct and Br - DNA also showed almost similar recognition of human

heart proteinic antigen (s) . Interestingly, as evident from the binding results

the human heart extract antigens reactive with the DCM autoantibodies

showed strong recognition of SLE anti-DNA autoantibodies whereas on the

contrary DNA was found to be less reactive with DCM autoantibodies. This

is suggestive for the possible involvement of a common or somewhat similar

trigger/epitope (s) for immune response in autoimmune myocarditis/DCM

and autoimmune SLE, although such inferences could not lead to confirmity

at such a preliminary stage of investigation. Moreover, the high binding

159

Specificity of immunoaffinity purified anti - DNA antibodies to 150 kD C -

protein from human heart is strongly suggestive for the presence of

immunodominant epitopes on 150 kD C- protein for SLE anti - DNA anti­

body binding. The binding results are indicative for the presence of unique

conformational epitopes on the 150 kD C - protein whose domains are some­

what similar to IgG. It is to be pointed out that recently nucleosome spe­

cific antibodies have been indicated in lupus prone mice. The monoclonal

antibody was found to react with nucleosome core particle but not histone-

histone and histone-DNA complexes. The core particles having ordered as­

sociation of histones and DNA were found to express a unique conforma-

tional autotpitope (s). Only the pathogenic T - helper cells of lupus prone

mice responded to nucelosomal antigens and their stimulation was specific

(Chandra et al., 1993). Therefore, it appears that pathogenic anti - DNA

autoantibodies are generated through some conformational epitope (s) of

nucleic acids or proteins / polypeptides.

Thus, in view of the above argument, it appears from our data that

150 kD C-protein having domains similar to those of IgG and being involved

in the induction of autoimmune myocarditis may provide some unique con­

formational epitopes which could possibly act as an alternate autoantigen

or trigger in the production of anti-DNA autoantibodies in SLE. Our pre­

liminary investigation is supportive for the above argument. The mecha­

nism or the role of myocarditis inducing 150 kD human heart C-protein in

the stimulation of immune system for the production of antibodies in SLE

remains to be ascertained.

160

In conclusion, it would be inferred from this study.that:

(1) 150kD C-protein is having somewhat similar domains to human IgG

as indicated by urea SDS-PAGE.

(2) 150kD C-protein was thermodynamically more stable than human IgG

although both of them had somewhat similar domains.

(3) The C-protein is a potent immunogen inducing hightiter polyclonal

antibodies with major reactivity towards HPLC purified immunogen.

(4) Nearly all the DCM sera tested exhibited elevated C-reactive protein

(CRP) and uric acid levels and that all of them showed remarkable

recognition towards electroeluted.followed by HPLC purified 150kD

C-protein.

(5) The kinetics of formation of DNA-C-protein photoadduct at various

irradiation time obeyed the apparent first order reaction.

(6) Out of the various adducts/photoadducts formed by crosslinking na­

tive DNA with C-protein, the one formed in the presence of'OH (ROS

-DNA-C-protein photoadduct) showed highest reactivity with DCM,

SLE and immune IgG.

(7) Nearly all the DCM sera tested, showed preferentially higher binding

with native mitochondrial DNA than its ROS modified conformer.

(8) The most striking finding is the appreciable recognition of THHE as

well as 150 kD cardiac C-protein by immunoaffinity purified anti-DNA

SLE autoantibodies.

'^titxtntts

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