Clinical Significance of Adenosine Deaminase in Veterinary Medicine

Title

Clinical Significance of Adenosine Deaminase in Veterinary

_{Medicine( 本文(Fulltext) )}

Author(s)

HANKANGA, Careen

Report No.(Doctoral

Degree)

博士(獣医学) 甲第239号

Issue Date

2007-09-14

Type

博士論文

Version

publisher

URL

http://hdl.handle.net/20.500.12099/23184

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

Clinical

SignirICanCe

_{Of Adenosine}

I)eaminase

in Veterinary

Medicine

(獣医学領域におけるアデノシンデアミナーゼの

臨床診断的意義に関する研究)

2007

The

United

Graduate

School

_{of Veterinary}

Science?

Gifu

University

(Iwate

University)

TABLE

OF

CONTENTS

ABBREVIATIONS

CIIAPTER 1 GENERAL INTRODUCTION INTRODUCTION

ADENOSINE DEAMINASE GENERAL OI∋JECTIVES

CHAPTER 2 PLASMA ADENOSINE DEAMINASE IN NORMAL AND DISEASED DOGS AND CATS

INTRODUCTION OBJECTIVE

MATERIALS AND METHODS ANIMAL S

MEASUREMENT OF P-ADA ACTIVITY RESULTS

DISCUSSION

CHAPTER 3 INVESTIGATION OF THE CLINICAL VALUE OF ADA IN CANINE LYMPHOMA

INTRODUCTION OBJECTIVE

MATERIALS AND METHODS RESULTS 4 6 6 8 ll 12 12 13 13 13 13 14 15 24 24 25 25 26

CIIAPTER 4 INVESTIGATION OF ADA ACTIVITY

IN LYMPHOCYTES AND NEUTROPHILS IN NORMAL AND DISEASED ANIMALS

INTRODUCTION OBJECTIVE

MATERIALS AND METHODS ANIMAL S

CELL SEPARATION

MEASUREMENT OF L-ADA AND N-ADA ACTIVITY RESULTS

CANINE LYMPHOCYTE/NEUTROPHIL ADA ACTIVITY FELINE LYMPHOCYTE/NEUTROPHIL ADA ACTIVITY DISCUSSION

CHAPTER 5 INVESTIGATION OF ADA IN T AND B LYMPHOCYTES

INTRODUCTION

EXPERIMENT 5A INVESTIGATION OF T AND B LYMPHOCYTE AI)A ACTIVITY IN DOGS AND CATS OBJECTIVE

MATERIALS AND METHODS ANIMAL S

P-ADA ACTIVITY MEASUREMENT

T AND B LYMPHOCYTES SEPARATION USING NYLON

33 33 34 35 35 35 36 36 36 37 38 48 48 48 48 49

WOOL RESULTS DISCUSSION

CHAPTER 5B PROLIFERATION ASSAY ANDAI)A mRNA MEASUREMENT IN CANINE LYMPHOCYTES AND TUMOR CELLS OBJEC TIVE

MATERIALS AND METHODS ANIMAL S

TUMOR CELLS CELL PROLIFERATION MTT MEASUREMENT

RNA EXTRACTION AND _CDNA SYNTHESIS P RIMERS

SEMI-QUANTITATIVE

RT PCR CELLULAR ADA MEASUREMENT RESULTS

DISCUSSION

CHAPTER 6 GENERAL CONCLUSION ACKNOWLEDGEMENTS REFERENCES 65 65 65 65 65 66 66 67 67 68 68 68 70 81 86 87

AC ACP Ado ADA AIDS ALT ALP AMV ARC CAMP CDNA ConA CPA CRF dAdo DXR D-PBS EHNA FAIDS FBS FIV GGT GLDH Hl.077 ABBREVIATIONS Asymptomatic _carrier Acid _phospbatase Adenosine Adenosine deaminase

Acquired immune de員ciency _syndrome Alanine _{aminotransferase}

Alkaline _pbosphatase Avian _{myoblastosis virus} AIDS-related _complex

Cyclic _{adenosine monophospbate}

Complementary deoxyribonucleic _acid Concanavarin A

Cyc lopbospbamide Chronic _{renal failure} 2､ deoxyadenosine Doxorubicin Dulbeccos'PBS

Erythro-9-

_{(hydroxyl-3-nonyl)}

_adenine Feline AIDS

Petal bovine semm

Feline immunodeficiency _virus Gamma _glutamyl transferase Glutamate dehydrogenase Histopaque l･077

Hl.119 HIV L-ADA L-ASP LDH LPS mRNA MTT N-ADA P-ADA PBMC PBS PCR PGL PMNC Pred. RNA RT-PCR SCID SI T-ADA Tm VRC Histopaque 1.1 19

Human immunodeficiency _virus Lymphocyte ADA

しAsparaglnaSe

Lactate dehydrogenase Lipopolysaccharide Messenger RNA

2-

[4,5-dimethylthiazol-2-y]-2,5-diphenyl

tetrazolium bromide Neutrophil ADA

Plasma ADA

Peripheral blood _{mononuclear cells} Phosphate-buffered _saline

Polymerase _{chain reaction}

Persistent _generalized lympbadenopatby Polymorpbonuclear _{neutrophil cells} Prednisolone

Ribonucleic _acid

Reverse transcrlptaSe PCR

Severe Combined Immunodeficiency Disease Stimulation indexes

T cell ADA

Melting temperature Vincristine

CHAPTER 1

GENERAL

INTRODUCTION

1. 1 INTRODUCTION

Enzymes are _proteins that have _{catalytlC Properties that} include _specific activation of their respective substrates･ Much emphasis is placed on _{the application}

of plasma enzymes as _{markers of}

organ damage,with many enzymes used in toxicologlCal studies to measure _{cellular Injury, enZyme} induction _{and activation} or

inhibition _{of enzymes･} The distribution _{of enzymes} in different tissues _varies between tissues, _and therefore influences _{their diagnostic valueinparticular species･ The tissue} distribution _of an _enzyme can be affected by age and sex _{and may vary} ln _{the different}

celltypeswithin an _organ

(Braun

_etal,

_1983).

The intracellular distribution _{of enzymes also varies and the proportions may} be _such that an _enzyme can _{be regarded} as _{relatively specific} to a _{particulartype of}

organelle･ Several enzymes are _{cytosolic, for example} LDH, _{whilst other enzymes} are

located in _{organelles such} as GLDH in _mitochondria or ACP in lysosomes･

Cytoplasmic _enzymes are _{usually soluble, easily released, and readily pass血ough}

the cell membrane

(even

when it appears microscopically

intact).

This _{propertymakes} them sensitive diagnostic皿arkers･ Some _enzymes occur both in mitocbondria _and in

the cytosol, whereas other enzymes may be largely membrane bound e･g･ GGT･ Membrane-bound _{enzymes are not soluble and} are _{firmly attached} to _{the cell}

membrane and may be shed a鮎r severe damage･

When _{there is tissue injury there is increased release of some enzymes.}

_Injury

from

alterations without microscoplCally visible cell changes｡ Hence, cells need not die to

release their enzymes･ A _{short period of hypoxia is enough} to disrupt _{the integrityof} the cell membrane and potentially allow soluble cytosolic enzymes to _escape or leak into _{their surrounding matrix} to be drained _away in lymph

_(Lemasters

_{et al,}

_1983).

Hypoxic or toxic lnJury results in exocytosis or formation _{of membrane} blebs _where

their cytosoiic enzymes are _{released into the surrounding plasma}

_(Gores

_{et al,}

_1990).

Plasma _{enzymes may} be _classified as

(i)

Plasma _specific

enzymes,

(ii)

other secreted enzymes, e･g･ amylase, and

(iii)

intracellular enzymes. Plasma enzymes include those _enzymes that are _secreted by some _{organs and} have a direct action in the

plasma, for example coagulation enzymes･

For _{several enzyme} measurements, 1t is preferable to use _{plasma rather than}

serum because _{of the release of erythrocytlC enZymeS} during _{the clottlng Process}

(Korsrud

and Trick,

1973)･

Some enzymes are _present at _relatively high

concentrations in erythrocytes compared to _{plasma and therefore may}

Interfere with

the measurements

(Czerwek

_and Bleuel, 198

_1).

There are a _{number of enzymes that} are _established as diagnostic _enzymes in small animal medicine including'ALP which has been as an _indicator

of hepatic Injury Since the twenties･ This enzyme is found pnmarily ln intestine, kidney, _liver

and bone･ ALT is a _{cytoplasmic enzyme that is of great} importance in diagnosis _of

liver disease _{in small animals.} LDH _{is contained in various tissues and is elevated} during tissue damage･ Desplte the _{existence of many enzymes} that are _available fわr

clinical diagnosis in small animals, investlgations into new _{enzymes of clinical}

significance is

justified

in order to improve or _compliment the diagnostic _abilityof

1.2. ADENOSINE DEAMINASE

ADA

_{(EC 3･5･4･4)}

isan important _{enzyme of purine catabolism. It catalyses}

the deamination of Ado and dAdo to_produce inosine _{and 2､deoxylnOSine respectively.} The importance _{of the enzyme for vertebrate organisms} stems in part &om the physiologlCal impact of its substrates. ADA has in the past been thought to be _purely cytosolic but has been also fourl-do_n_ _{the cell surfTace of lymphocytes}

_(Aran

et _ai,

1991)･

There isrecerl-t _{evidence about} a _{specific role of ecto-ADA, which} is different

from that of intracellular ADA. Apart from degrading extracellular Ado or dAdo,

ecto-ADA has an _{extraenzymatic} function _{via its interaction with} CD26. CD26 is a

sialoglycoprotein whose pbysiologlCal role seems to be related to _{cell activation.} The

ADA-CD26 interaction _{results in co-stimulatory signals in T cells}

_(Franco

et _al,

_1998).

ADA _{is present in all celltypes} but _the amount _{of enzyme} differs _widely

amongst tissues･ The highest ADA levels in humans are _{fわund in lymphoid tissues}

(Hirschhom

et al,

1978)･

In animals ADA has been shown to have bigber _activity in organs such as _spleen, lymph _{nodes and} thymus in most species

(Tanabe,

1993).

In human, _{the activity ofADA} has been _shown to be greatest in lympbocytes _and higher in T than in B _{cells･ One report} has documented _{the levels ofT-ADA activity} to be ten _{times that of B cells. As} ADA

_activityis

increased _{in plasma} or body fluids in

diseases _{where cell mediated}

_immunityis

_stimulated, it has been _considered a _marker

of T cell activation

(Kose

et al, 2001, Hoviet al,

1976).

In addition, ADA has been reported as a _{marker of cell-mediated} immunityin human

(Baganha

_{et al,}

_1990).

Tbe _{enzyme plays} a _{vital role in the ma山ration of the immunologlCal system}

because _congenital deficiency _{of this enzyme in erythrocytes and} lymphocytes in human _{is associatedwith} SCID. The _{patients usually present} in infancywith recurrent

hypoglobulinemia, _and an _{inability to mount speci丘c antibody responses}

_(Bollinger

_et

al,

1996).

This condition is characterized by both T- and B- celifunction impairment.

Several theories exist as to how deficiency ln a _{Purine catabolic enzyme} can cause

lympbopenia. Most _{evidence suggests} that accumulation of ADA substrates is detrimental to lymphocyte development _{and survival}

_(Aldrich

et al,

2000).

Another theory lS that elevated Ado levels couldalso trlgger _aberrant Ado _{receptor signaling.}

Ado transduces extracellular slgnals by binding to G-protein-coupled Ado _receptors that can _regulate intracellular _{CAMP and calcium} levels. Ado _{receptor engagement}

can thus lead to _{elevation of CAMP} levels _that can lead to _{thymocyte apoptosis and}

developmental a汀eSt

(McConkey

et al,

1990).

Like Ado, _elevated dAdo _{is tbougbt} to

inhibit S-adenosylhomocysteine hydrolase, an _{enzyme critical} to _cellular

transmethylation reactions, resulting cell death and apoptosis mediated by the latter

(且atter

et al,

1996).

Most _{of the clinical signs of} the disease in humans, can be

attributed to _{the existlng} T, B _and NK _{cell 1ymphopenia, other abnormalities} include bepatic _{pathology, costochondral}

_junction

_{abnomalities, 1nCreaSed asthma} incidence and neurological abnomalities

(Aldrich

et _{al, 2000,} Hirschhorn,

_1995).

_{In animals,}

some _studies have _shown that ADA deficient mice die perinatally with marked liver-cell degeneration

(Migchielsen

et _{al, 1995 and} Wakamiya et _al,

_1995).

_{In addition,}

other studies, in ADA deficient mice, have showed marked metabolic and immunologlCal _{abnomalities such} as lymphopenia, _{elevated plasma} Ado, severe liver

impalrment, _{Pulmonary Insufficiency and elevated adenosine} levels _{in plasma and}

some _organs

(Blackbum

etal,

1996).

HoⅥ′ever, a _s山dy by Tax _and Veerkamp

(1978)

reported that ADA activityievels in horse lymphocytes were _comparable to _those in lymphocytes _of human _{patients with} SCID _{associated with} ADA deficiency.

not be necessary for normal lymphocyte function in horses because low ADA activity

was fわund in lympbocytes _of both healthy _adults and fわals with combined immunodefic iency.

ADA has two _prlnCIPal isozymes, ADAl _and ADA2, _which have different optimal pH, Michaelis constants _{and relative substrate specificitypatterns}

(Ungerer

et

ai,

1992)c

ADA 1_activlty!S inhibited by EHNA while ADA2 isnot･ ADAl is found

in most body cells, particularly lymphocytes

(Shibagaki

et al,

1996),

where it is present not only ln _{the cytosol} but _{also as the ecto-form} on the cell membrane

attached to CD26. This isozyme is critically Important in lymphocyte _{proliferation} and development and its deficiency leads to SCID in humans

(Conlon

and Law,

2004)･

The isozyme ADA2 is the

_major

component

(73%)

of the activity oftotal ADA in the

serum _of healthy _persons

(Merrikhi, 2001).

ADA2 has been _suggested to be an

indicator _{of macrophage activation} or山mover

(Casal

et_al,

_2002)･

ADA2 is increased in many diseases, _particularly those associated with the immune system: for example rheumatoid arthritis, psoriasis and sarcoidosis. The plasma ADA2 isoform is also increased in most cancers. In animals according to a _study by Tanabe

(1993),

there

was no serum ADA2 _activityin cows _and rats, whereas other species such as dogs,

cats and plgS Showed only slight levels･ However, a _{recent study} in rats, by Conlon and Law

(2004),

ADA2 was _shown to be in greater quantities in macrophages than

monocytes and also that these cells released ADA2 into their surroundings following

1.3 GENERAL OBJECTIVES

i)

To _{evaluate the usefulness of ADA} as an _{enzyme of clinical slgnificance} in

canine and feline disease.

ii)

To inve･stlgate _{the relationship} between ADA _andthe immune _{sysf-em with} particular reference to lymphocytes in dogs and cats.

iii)

Study ADA _activity as a _prognostic factor for disease _progression illSOme

CHAPTER

2

PLASMA

ADENOSINE

DEAMINASE

ACTIVITY

IN

NORMAL

AND

DISEASED

DOGS

AND

CATS

2. 1 INTRODUCTION

ADA _{is widely} distributed in human _{tissues with the highest activity}found in the _{spleen and gastrointestinal} tract. _{It is considered} as an _auxiliary diagnostic tool and a _{reliable marker ofhuman山berculosis}

_(Orpbanidou

_et

al,

1996).

ADA activity is

increased in _{several other} diseases including hepatic disease

_(Goldberg,

_1965),

cutaneous leishmaniasis

(Erel

et al,

1998),

meningitis

(Babeti

et al,

2001),

leukemia

(Morisaki

et _ai,

_1985),

1ympboma

(Ganesbaguru

et _ai,

_1981),

_{nepbrotic syndrome}

(Misra

et ai,

1997),

brucellosis

(Cesur

et _ai,

2004),

hepatitis

(Vasudha

et _al

(2006),

pneumonia

(Nishikawa

et al

1988)

and sarcoidosis

(Taylor, 1986).

In addition ADA has been _{proven useful} in differentiating Causes _Of some diseases _such as _menlngltlS

(Baheti

et al,

2001), jaundice

(Goldberg,

1965),

peritonitis

(Leksrisakul

et al,

2001)

and hepatic disease

(Nisbikawa

et _al,

_1986).

In humans, the diagnostic _{value orADA}

activityin

various body fluids has also been analyzed, such as _{in the sputum of}

patients with pulmonary tuberculosis in which the enzyme was _{elevated compared} to

that from cancer _{and obstructive} lung diseases

(Dilmac

et _al,

2002).

Saracoglu et _al

(2005)

analyzed ADA in patients with oral and laⅣngeal cancer, in which ADA was

2.2. OBJECTIVE

To investigate P-ADA _{activity in normal and} in different _{canine and} feline diseases _{in order} to _{establish whether age has} an _{effect on} ADA _{activityand whether}

ADA _activity lS _{Ofany clinical value.}

2.3. MATERIALS AND METI10DS 2.31. ANIMALS

Blood was _collected from _normal dogs

_(n-

_{42, 20 males,} 22

_females)

_and cats

(n-16,

6 males and 10

females).

The age ranged from 4.6±2.9 yrs in dogs and 6.6± 4.6 years in cats. The dogs with lymphoma comprised of 7 males and 6 females with age 7.3±2.7 years. The age of dogs with hepatitis was 5.0±2.3 _{years of which} 6 were _{male and} 4 female. The dogs _with tumors were _{comprised of 6 males} 4 females

and the age ranged丘om 10.0±3.0 years. The dogs with demodicosis were _{2 males}

and 1 female with age of 5.Oj:6.0 years. The FIV positive cats were _{compnsed of4}

males and 4 females and the age ranged from 9.Oj=2.5 _years. The cats with CRF composed of 2 males and 3 females and the _{age ranged} from 7.Oj=3.0 _years. Heparinized blood was _collected largely &om the

jugular

vein.

2.32. MEASUREMENT OF PIADA ACTIVITY

Heparinized blood was _centrifuged at 1500 rpm for 15 minutes and the plasma analyzed fわr ADA activity. P-ADA activity was _{assayed using} a _{commercial ADA kit}

(Serotec,

AD⊥, Sapporo,

_Japan.)

_using an _autoanalyzer

(Accute,

Toshiba-40FR

Statistical _analyses were _{ca汀ied out uslng Student T} test _and Pearson's correlation. Probabilitywith values P<0.05 were _{considered statistically significant.}

Data are _summarized as _mean±SD.

2.4. REStJLTS

P-ADA _activityin healthy _normal dogs

_(n-42)

_was 3.44j=2.02

_(IU/L),

in dogs

less than _{5 years}

(n-25)

2･9±2･17

_(IU/L)

_{and those} over 5 years

(a-17)

4.4±2.4

(IU/L,

Fig･

_2-1)･

Dogs _older than 5 years had a _{signi丘cantly} higher P-ADA than the younger dogs

(P<0･05)･

There was no _correlation between _{age and} P-ADA in the

normal dogs･ P-ADA activitywas significantly elevated in lymphoma

(n-13),

hepatic disease

_(n-10),

_and demodicosis

_(n-3)

in Fig 2-2｡ There was no increase in P-ADA

activityin dogs with other tumors

(n- 10).

P-ADA _activity in healthy _nomal cats was _{48.6± 14.6}

_(IU/L).

_{Cats less than 5}

years

(n-8)

had P-ADA _activityof 32･4j=3.28

_(IU/L)

_and over 5 year olds

(n-8),

55･l j: i l･9

_(IU/L,

Fig･

_2-3).

Cats _older than _{5 years}

_(n-8)

had a _{significantly} higher

P-ADA _{activitythan the younger} ones

(P<0.01).

There was a _{positive correlation}

between _{age and} P-ADA _{activity in the nonnal} cats

(r

-0.48, P<0.05, Fig.

_2-4).

FIV positive cats ADA activity was as fわllows: 49･8± 16･6

(IU/L),

AC 35.6±4.97

(IU/L)

and ARC 64･1 ±9･1

(IU/L,

Figs･ 2-5 & Fig･

216)I

P-ADA activitywas significantly increased in cats in the ARC stage of FIV infection when compared with the AC

(P<0･005)

and control groups

(P<0.05).

P-ADA activity in cats _with CRf was 48.6±

0･36 IU/L

_(Fig･

_2-5)･

There was no _significant difference between _{controls and} cats

2.5. DISCUSSION

Our _{results also show} that in both dogs _and cats, _{animals younger} than 5 years had _{significantly} lower P-ADA _{activitythan older} ones. This is further highlighted by

the positive con-elation seen between _{age and} PIADA _activity ln _Cats. Our _results

concur with those of Vasudba et _al

₍₂₀₀₆₎

_{who also obseⅣed} higher levels in semm

ADA in older people compared to _younger ones･ _{In this study canine} P-ADA _activlty

was lower than _{that of the feline species. In addition, normal} P-ADA _activityin cats

has been _reported to be higher than other species including dogs, rabbits, cows, _plgS,

horses _and rats

(Tanabe, 1993).

It is difficult to explain the complexities that are

involved in the differences _{of P-ADA activitybetween} the different _species, however factors involved _{in plasma enzyme modulation may play} a key _{role. The plasma}

activityof an _enzyme depends on _several factors including _{the enzyme concentrations}

in different _{tissues, the intracellular} location _{of the enzyme,} rate of synthesis of the enzyme, severity of tissue and cellular damage, the molecular size of the enzyme and the rate _{of clearance of the enzyme} from _plasma. The 'normal'serum _{enzyme activity} probably reflects a balance between _{physiologlC Cell death and} degradation/activation

by _{the macropbage system} or, less commonly _excretion. It is therefわre possible that

any of the afore-mentioned factors may be responsible for differences seen in P-ADA

activityof dogs and cats.

In animals, total serum ADA _activity is reported to be _elevated in bovine leucosis

_(Chikuma,

1997 _and Yasuda et al,

1996),

liver diseases

(Abd

Ellab et al, 2004 _and Chikuma,

₁₉₉₇₎

_and tuberculosis

_(Silva

et al,

2006);

canine liver disease

(Aitug

and Agaoglu, 2000 and Tanabe,

1993)

and fTeline infectious peritonitis

(Tanabe,

In dogs P-ADA _activity was _{markedly elevated} in lymphoma, hepatic disease

and demodicosis. Our results are in agreement _{with previous s山dies} in human that

showed that serum was elevated in lymphoma

(Vezzoni

et _{al, 1984,}

_1985).

Canine demodicosis, a common _{skin disease of dogs} in which _{proliferation ofDemodex canis,}

is associated with the development of cutaneous lesions. Caswell et al

(1997)

have

recently demonstrated that this disease is characterized by lymphocytic folicullitis and peripheral blood increase of cytotoxic T lymphocytes. The increase of P-ADA activity

seen in demodicosis _{may therefore} be a _{renection of activation and mobilization of}

these cells. The current _{results concurred with} a _study on ADA in dogs _with induced liver _toxicity,by Altug _and Agaoglu,

_(2000),

_{that also revealed} high ADA _activity･ Abd Ellah et al

(2004)

reported that serum _{ADA activity lVaS increased in co常s vitb}

liver disease _{and suggested} a _possible link to the degree _{of hepatocellular} damage. Furthermore, in human _studies high serum ADA _activityhas been _{reported in chronic} bepatltlS, liver _{cirrhosis, chronic active} hepatitis _and bepatoma _{and the authors} suggest that serum ADA isozymes _may be a new _marker for liver disease

(Kobayashi

et _al,

1993).

Increased ADA activity also paralleled liver damage demonstrated histopathologically _{in the} acute group in this s山dy. Determination of P-ADA activity may be useful in the assessment of liver disease in dogs･ This may be ofparticular value in chronic hepatic disease where routine liver enzymes are _usually

unremarkable.

P-ADA _{activitywas significantly} higher in the ARC _group than in the AC _and control groups. There was no _significant difference between _{the ages of} cats in the AC

and those in the ARC stage of FIV infection･ These observations imply that P-ADA

activityis

up-regulated in advanced clinical disease･ These results were _consistent

between the infection _stages

_(Goto

et al,

1992).

Inigo et al

(1992)

also showed that

serum ADA _{activityprogressively and significantly} increases in symptomatic

HIV-infected.

Tbe increase seen in hepatic disease, 1ymphoma _and demodicosis _suggests that

ADA _activitymay be of value in the diagnosis of these conditions･ Demodicosis and

the ARC _{stage of FⅠV} infection are both _{characterized} by _chronic in凸ammatione

Therefore PIADA _activitymay be high _{in other conditions associated with chronic} innammation. Monitoring _of P-ADA _{activityhas potential} in FIV infection as

increases _{of the enzyme} in cats in the AC _{group may suggest progression of the}

disease to the ARC phase. Our results also suggest the importance of taking age into

consideration when interpreting P-ADA activity results in dogs and cats. Based on

.⊥ 喜 5 岩■ヽ ごと 遥 4 くJ < <

等3

よ Total (n=42 ) <5yrs (Jt=25) >5yrs (n-17)

Fig･ 2-I ･ Graph showing the nomal canine PIADA activity.P-ADA of dogs above 5

years was _{significantly} higher thanthose under 5 years

(*P<0.05).

Total represents the whole population.

?

i)

妄

>･ 'B U < < E) <

i

Controls Lymphoma IIepatic TITmor≦ _Demodicosis

(n-42) (a-13) disease (J]= ₁0) _(n-3)

(n-10)

Fig･ 2-21 P-ADA _{activity in normal and} dogs _with disease. There were _significant

increases in P-ADA _activityin dogswith lymphoma, hepatic disease _and demodlCOSis when compared with the controls

(***P<0.005).

′-■＼ ､■ヽ ■■

5

=コ ヽ■一′ >ヽ .t_>･ I+I∼ U i < ∈l <

よ

60 50 40 30 20 10 T(Itat

(n-16)

<5 vrs■/

(n-8)

>5vrst′

(n-8)

Fig･ 213･ Graph _{sh_owing} P-ADA _activityin healthy feline _controls･ P-ADA _activityof

cats above 5 years was significantly higher than those _under 5 years

(**P<0･01)･

80 70 ′■■ヽ i ii己!l =) 巳. ∃芦 Itf > '= U i く E) <

よ

60 50 40 30 10 Age

_(years)

15 20

Fig. 2-4. Relationship between _{age and} PIADA _activityin feline _controls

_(r-0･48,

′ヽ

tj

50 i) =コ E5

倉40

>･ '古 くJ < ₃₀ < (≡

可

20 白■ Co _ntrols

(n-捕)

Frv

(m-S)

CRF

(m-5)

tj^

_i =コ ヽ■■ 土･ ●冒 +-II U < < l∋ <

i

60 50 40 30 20 C ontrols

(n-16)

AC

(n=4)

ARC

(n-4)

Fig･2-61Diagram _showing P-ADA _activityin Fry-positive cats. A _significant increase

(***P<0･005)

was seen in the ARC _{group compared} tothe AC andthe controls

(P<0.05).

P-ADA _activity was _markedly increased in cats in the ARC stage of FrV infection.

CHAPTER

3

INVESTIGATION

OF

CLINICAL

VALUE OF ADA

IN

CANINE

LYMPHOMA

3. 1 INTRODUCTION

Results _of the _{previous study showed} that P-ADA _activlty lS elevated in canine lymphoma, in this study we investlgated _whether P-ADA has a _role in

monitonng of dogs undergolng Chemotherapy.

Canine lymphoma is a _progressive fatal _{disease caused by the malignant}

clonal expansion of lymphoid cells. Lymphoma most commonly arises from organized lymphoid tissues including bone marrow, thymus, lymph _{nodes and spleen･} In addition to these primary and secondary lymphoid organs, common _extra-nodal

sites include the skin, eye, central nervous _{system, testis and} bone. Lympbo皿a is a

neoplasm that affects dogs of all ages and gender. The etiology of canine lymphoma is notknown. A _{genetic component is suspected} as there appears to be a

breed-predisposition to this neoplasm such as Boxer, Basset Hound, Rottweiler, Cocker

Spaniel, St. Bernard, Scottish Temier, Airedale Terrier, English Bulldog _and Golden Retriever

_(Lurie

et _al,

2004).

Clinical features include 4 basic _anatomic forms _of

presentation: multicentric which is characterized by generalized lymphadenopathy; splenlC, hepatic and bone ma汀OW involvement; _{mediastinal mainly} characterized by mediastinal lymphadenopathy; alimentary, characterized by gastrointestinal infiltration _{and extranodal which may affect any organ} or tissue･ The definitive diagnosis _of lympboma can be _{obtained easily} by _either a _cytologlCal or

histopathologlCal _{evaluation of the affected organ system･} Factors _used in evaluatlng affected dogs include clinical cancer _stage, serum _calcium levels, histologlC _{grade and}

immuno-phenotype I

3

｡2G OBJEJ CTrvT五

To investigate _Whether ADA _{is of any clinical} or _{prognostic value} in dogs

with lymphoma undergolng Chemotherapy.

3.3. MATERIALS AND METHODS

Six dogs _with lymphoma _presented to Iwate UniversityVeterinary Teaching

Hospital were _{included in this s山dy. The dogs'slgnalments are shown in Table 3-1.}

A de丘nitive diagnosis was based on 丘ne-needle _asplration biopsy _cytologlCal

examination of enlarged superficial lymph nodes. Blood samples for ADA and other clinicopathologlCal tests were _collected at initial presentation _and on a _weekly basis,

in heparin _{and plaintubes.} Blood was _{also collected} in heparin from 42 healthy dogs

for ADA _analysis. Samples for ADA _analysis Were _Centrifuged at 1500 _rpm for 15

minutes and analyzed using an ADA _reagent kit

(Serotec,

ADIL, Sapporo,

_Japan)

in an _autoanalyzer

(Hitachi

7060,Tokyo,

Japan)

at 37oC.

Tbe Wisconsin _protocol

_(VCR,

CPA, Pred., DXR _and

_L-ASP)

was _employed over a 25-week course･ Disappearance _{of all measurable山mor} mass or lymph _node

Lymph _{node volume} cm3- length x _{width x} height x 3･14

6

Statistical analyses were _caⅡied out _uslng S山dent's T test _and Speaman's

Co汀elation. Results were reported as _mean± SD _and P<0･05 was _considered to be _of

statistical signi丘cance･

3.4｡ RESULTS

Normal ADA _{activityindogs} was 3.44j=2.02 IU/L. There was a significant difference in total P-ADA activitybetween the controls and the dogs with lymphoma

at first presentation

(P<0.005,

Fig.

3-1)･

A wide range of ADA activitywas seen in

the dogs with lympboma both at _{presentation and} in the course _{of the} treatment｡ There was no _significant difference in total PIADA _values due to sex or _age･ There was

however no _relationship found between lymphocytes, _red blood _{cell number} and

ADA _activityin dogs _with iymphoma.

Tbere was no _{con･elation} between _{clinical stage and} total P-ADA _activity

(r-0.28, P>0.05).

There was no _association between total P-ADA _and lymph _node size during the course _{of therapy,} however one case

_{(No･ 4)}

_{ofmulticentric} lymphoma

(Fig.

3-2)

showed a _{strong relationship} between the two _{parameters･} There was no

relationship found between P-ADA activityand chemotherapy1

0ftbe three dogs that died, two cases

(Nos.

4 &

5)

_showed marked elevation in total P-ADA activityupon relapse after having relatively stabilized, however the dog _{with mediastinal} lymphoma

_(case

No･

₃₎

did not show such a _pattern

(Fig･

3-3)･

3.5. DISCUSSION

In this s山dy total P-ADA activities were higher in the dogs

with lympboma than the over 5 years101d controls･ ADA is found in most tissues, its activityis

greatest in the lymphoid tissues, and more _specifically the T lymphocytes

(Adams

and Harkness,

_1976)･

These higher levels _{of P-ADA activitycould} be a _{reflection of the}

prolifTerative activlty Of the tumors _{and the stage of differentiation reached by both the}

nomal and the neoplastic lympboid cells.

Our _results did not show any significant difference in total P-ADA between dogs _{with mediastinal and those with multicentric} lymphoma at presentation･ According to a _s山dy done in human lymph _{node samples} by Ganeshaguru et al

(1981),

the highest levels _{of ADA} were found in T-cell tumors _{of lymphoblastic and}

di乱se _{undifferentiated types while} in the B-celltype tumors, _{the level of} ADA varied with the proportions of T-cells in the tumor. Therefore, tumor _{characteristics}

including _{phenotype may} be _responsible for _the differences seen between P-ADA

activities in the two groups.

According to Vezzoni et al

(1985),

a _relationship was fわund between ADA

activityof various

histotypes

of non-Hodgkin's lymphoma in humans, and their grade

ofmalignancy･ Our results did not show any association between the cancer _{stage and}

total plasma

_activityat

presentation.

Total PIADA _activity was seen to decrease drastically _{upon the}

commencement _of treatment but then was seen to fluctuate throughout the course _and

appeared to be very variable. There was _{also great variabilityin} the PIADA _activityat presentation･ A study in humans, by Ponce et _ai

_(2004),

_reported that significant differences seen between the lymphoma

_subtypes

are _{suggestive of inherent} biological

features _{and clinical behavior of these} tumors. These findings _may account for the

variabilityseen in total P-ADA activityprior and during the chemotherapeutic regime･

The dramatic _{elevation of total P-ADA activity upon relapse in patients}

(Case

Nos. 4 &

₅₎

_{and the strong relationship noted} between lymph _{node size and} P-ADA

activity(case

No.

4)

reiterates the potential use _{of this enzyme} in patient monitoring･

ADA _{activl_ty Of ceiis缶.om} human _neoplastic lymph _nodes is _related to the proliferative activityof lymphomas

(Ungerer

et _al,

1992)･

This probably in turn

affects the plasma and a _{similar assumptlOn could be made} in dogs･

ADA _may be as _{useful in the diagnosis of canine} lymphoma like it is in human

medicine. However the tumor

histologicaltype

may be a

_major

determining factor,

because ADA has been _reported as a _{marker of human} lymphoblastic lymphoma that showed unusually bigb enzyme levels in a _study by Vezzoni et al

(1984)･

Since there is no information in the literature on ADA _{activityin canine} lymphoma or its

response to cbemotherapeutic treatment, this preliminary s山dy provides valuable insight on _{the potential oftbis enzy皿e} in the diagnosis _and assessment _{of the canine}

patient with lymphoma･ Our study lS, however, limited by the lack _of tumor

characterization. Based on _{these results, further studies} were carried out to investlgate the role of lymphocytes and neutrophils in P-ADA activityin canine lymphoma･

Table 3-1. Profile _ofdogswith lymphoma at _{first presentation}

CASE BREED AGE SEX LYMPHOMA CANCER Initial

NO. _(yrs) STAGE ADA

(IU/L) 1 2 3 4 5 6 Corgi Golden Retriever

Mixed breed dog

Maltese Poodle Sbi tzu Yorkshire terrier 4 8 ll 12 7 9 F F M M M M Multicentric Mediastinal Mediastinal Multicentric Mu lticentric Multicentric 5 1 5 5 4 5

′ヽ ,A ii=ヨ こ) ■■l ㌔-′ ヨ! .t>･ 'B U i < (∋

i

B< 8 6 4 2 0 Controls Lymphoma

(n-42)

(n=6)

Fig. 311. P-ADAー' _activityof controls and dogs with lymphoma･ Dogs with lymphoma had higher P-ADA _{activity than controls}

(***P<0･005)･

This _{result showed} that

冒

至】 巳.

普

> :a U < <

葛

i

1 2 3 4 5 Pred + VCR + + + CPA ● DXR L-AS】) ● 6 0 4 3 ′-ヽ 【;7 日 り ヽ-′ 43 月 【アノ P二 pせ ○ 貞 J∃ A 百 ∃l J 6 7 Ⅵ7eek ●

Fig. 3-2. Strong _relationship between P-ADA _activityand lymph _{node size in} case No･ 4withmulticentric lymphoma･ The _{corresponding} course _{of chemotherapeutic}

treatment _undertaken isalso _shown. Pred. was _{administered per} os _{every other day･}

冒

i) 亡.

杏

T 'j: もl < <

守

_I ≡≡ --(.A邪S +('A5F J +CAS君5 き 6 ? $ 9 Week Pred ● VC見 ● ● ● CPA ● DX乱 しAS‡I ● ◆ ● ●

Fig. 313. PIADA _{activityofthe} 3 dogs that died dming course _{of therapy.} There was a

sudden increase in P-ADA activityatweek 7, following relapse of the disease a_prior to death in case Nos. 4and 5, respectively. +; Point _{of death.} + _{;normal course of}

therapy. +

,A shows points Where there

was a _{variation in therapy &om normal in}

CHAPTER

4

INVESTIGATION

OF

ADA

ACTIVITY

IN LYMPHOCYTES

AND

NEUTROPHILS

IN

NORMAL

AND

DISEASED

ANIMALS

4.1. INTRODUCTION

In _{view of} the fact that PIADA _activityhas some _potential in _canine

lymphoma, we further investigated its source in peripheral blood _{cells･ Numerous}

reports suggest that spontaneous山mors in humans are _recognlZed as _antlgenic by the

host. In _many Instances, however, the malignant tissue fails to _evoke an immune

response capable of destroying the neoplastic cells. It has been suggested in a _study

by Green _and °han

_(1973),

that increased levels orAdo in lympbocytes may result in

an inabilityof the _cells to divide. The level _of ADA _{activitymight} influence the

capability of the immune system to respond

(Uberti

et al,

1976).

FIV is _atypical lentivirus _{that replicates preferentially} in T _{cells and} is

stmc山rally similar to HIV

_(Bendinelli

et al,

1995)･

Tberefbre in both human beings

and cats, the disease is characterized by severe impalrment _Of T _{cell functions and}

cellular immune response due to infection _{of CD4'ce11s}

_(Ackley

et _{al, 1990,} Joshi et

al,

2004).

Gradual reduction in both the percentage and the absolute number of CD4'

T _cells is one _{of the} most _{striking lmmunOloglCal consequences of FIV} infection

resulting in the reduction of the CD4/CD8 ratio

(Hoffman-Fezer

et al,

1992)･

increased incidence _{of lymphoma.} Unlike HIV infection _{where the pnmary receptor} for HIV is CD4,

_Shimojima

et _al

(2004)

identified that the primary receptor that

promotes viral binding and renders CD4+ cells permissive to infection, as CD134 in

FIV. Despite _{the progressive} deterioration _{of T cell function,} the _abilityof B _cells to

recognize and respond to T-independent antigenic stimulus was not _affected

(Torten

et _ai,

_i991).

FIV _{aiso replicates} in macrophages and astrocytes. Primary infection of

cats by FIV is associated _with a _{protracted asymptomatic phase} of several months or

years that in some cats culminates in the development of immunosuppression･ Ishida and Tomoda

(1990)

have proposed classi丘cation of FIV stages as fわllows: Primary

infection, AC, PGL _{characterized} by _generalized lymphadenopathy, ARC characterized by weight loss, bacterial and viral infections, and FAIDS characterized

by severe _{secondary and opportunistic chronic} infections, tumors and wastlng･

_fiematol_og!c man_ifestations of FIV infection include anemia, 1ymphopenia, nentropenia and thrombocytopenia.

4.2. OBJECTIVE

To investigate ADA _{activity in blood peripheral cells in canine} lymphoma _and FIV. The diseases _chosen were based on the results丘om the _survey obtained in the previous chapter.

4.3｡ MATERIALS AND METHODS 4｡31. ANIMALS

Heparinized blood was _collected from ll healthy dogs

(5

_{males and} 6

females)

aged 5.7j=3.0 years･ Eleven dogs with lymphoma

(7

males and 4

females)

aged 7･3 ±2･3 years･ Lymphoma diagnosis was _made by血e-needle asplration biopsy, wbicb was then _stained and examined.

Heparinized blood was _{also collected} from 13 healthy controls and ll FIV positive cats･ The _control group was _{made up of 6 males and} 7 females _and the age

ranged丘om 6･7±4･5 years･ The FIV positive cats Ⅵ′ere _{comprised of 5 males and} 6

females _{and the age ranged} &om 9･Oj=2･5 _years･ Of the _positive cats, 7 were in the

AC _{stage of infection while} 4 were in the ARC stage･ All the cats included in this

work were _seronegative for Feline leukemia virus antigen and the FIV-positive group

was _seropositive fわr FIV _antibody

(IDEXX

Laboratories, Portland,

Maine)･

4.32. CELL SEPARATION

pBMC _and PMNC were _separated using the double densityHistopaque@

(sigma-Aldrich,

St. Louis, MO,

_USA)

_{separation according} to _{the method} described by Strasser et al

(1998).

Hl.077 was _carefully layered onto 4 ml Hl･1 19 and stored at

4oC _until use･ The _columns were kept on _{ice in separate conical} 12 _{ml centrifugal}

polypropylene山bes･ 4 ml ofblood was layered on the low gradient solution uslng a

21G hypodermic _needle on a _synnge･ The tubes were _centrifuged at 350 x _{g for 30}

min at room temperature _with a _swing-Out rotor _and the _process terminatedwithout applying brakes.

was layered over _{the Hl･119･ Six milliliters of whole} blood _was layered over _the

Hl.077 _{and. centrifuge} at700 x _{g for 20 minuteswith no} brake _{atroom temperature.}

After _{centrifugation} the top Inter-Phase layer consisting Of PBMC and the second layer of PMNC were _{collected and} transferred to _{separate 50 ml centrifuge}

山bes fわr washing. Washing was done _with D-PBS

(Sigma-Aldrich,

St. Louis, MO,

TuTSA)

and centrifuged at 200 x _{g for} lOmirluteS. Red bl100d ceiis were iysed _using

ammonium chloride buffer solution and the cells washed twice. The PBMCs were

cultured for 1 hour in RPMI-1640 media containing 10% FBS

(Sigma-Aldrich,

St. Louis, MO,

_USA)

at 37oC _and 5% CO2 in a humidified incubator to remove

monocytes. Lymphocytes were _then harvested for ADA _analysis.

4.33. MEASUREMENT OF LYMPHOCYTE _and NEUTROPHIL ADA ACTIVITY

The _cells were _{counted uslng} a hemocytometer _and lysed by _ultrasound. The

suspension was then _centrifuged and the cell lysate-supernatant was _analyzed for

ADA _{activitycontent.} P-ADA _{activitywas measured} as _{earlier described} in chapter 2･

Statistical _analyses were _carried out uslng Student's T test and Pearson's

correlation. Results were _presented as _{mean±SD and} P<0.05 was _considered to _{be of}

statistical signi丘cance.

4.4. RESULTS

4.41. CANINE LYMPHOCYTE/NEUTROPHIL ADA ACTVITY

control _{groupしADA activity} was 2.6± 1.6

(Ⅳ/106

_cells)

_and that of dogs

ADA _{activity of the dogs with} lymphoma _{when compared with the control group}

(P<0.05,

Fig.

4-1).

N-ADA _activity was _1.2±0.52

_{(IU/106 cells)}

_{in the controls}

and 0.4±0.23

(IU/106 cells),

in the dogs _with lymphoma･ Healthy _controls had higher N-ADA

activitythan dogs with lymphoma

(P<0･0001,

Fig.

4-2).

L-ADA was _{significantly}

higher _than N-ADA in both _groups

_(P<0･05).

There was, however, no _significant

correlation between L-ADA and P-ADA activities in neither group･

4.42｡ FELINE LYMPHOCYTE/NEUTROPHIL ADA ACTIVITY

Tbe _{control cats'L-ADA activity} was 0.78±0.67

(IU/106

_cells)

_{and of}

FIV-positive cats was 1,4±2.06

(Ⅳ/106 cells)

_and is shown in Fig. 4-3. There was no

significant difference between FIV positive and FIV negative L-ADA activity.The

AC _{groupしADA activity} was 0.68±0.28

_{(IU/106 cells)}

_and ARC _group was 3.55±

3･096

_{(IU/106 cells).}

There was a _significant diffei･enCe betweerl _{the ARC group and}

the AC _{and control groups}

_(P<0.05,

Fig.

_4-3).

N-ADA _{activity in the nomal} cats was 0.19±0.22

(IU/106 cells)

_and 0.22±

o･36

_{(〟/106 cells)}

in the FIV _positive cats･ In AC _and ARC _groups, N-ADA was 0.09

j=0･15

_(IU/106

cells)

and O･98-+0･54

(IU/106 cells),

respecfively･ N-ADA

_activity

was _{slgnificantly} higher in cats in the ARC _{stage of FIV} infection than the _other groups

(P<0･05,

Fig･

4-4).

L-ADA _{activitywas significantly} higher _than N-ADA in all groups

(P<0･05)･

There was a _{negative co汀elation} between P-ADA _{activity andし}

ADA _{activityin the control group}

(r--0.55, P<0.05, Fig.

4-5),

but there was none found in the FIV _positive cats･ Similarly there was no _relationship fTound between

P-4.5. DISCUSSION

This _{study reported for the first time, L-ADA activity,}in both _{the canine and} feline species･ In case _{of the dogs,} L-ADA was _{slgnificantly elevated} in the dogs _with

lympboma _{compared with the healthy controls}

_(P<0.05).

The _{result concurred with} that of Meier et _ai

(i976)

_{who reported} high L-ADA

_{activityvaiues}

in human _patients with iymphoma･ Muller et _al

(1982),

on _{the other} hand, _{reported reduced} levels _of

ADA _activity in Hodgkin lympboma. Carter et al

(1986)

have shown that there are

strong similarities of morphology and behavior between human non-Hodgkin's lympbomas _{and canine} lympbomas. Uberti et _al

₍₁₉₇₆₎

have _{suggested that variations} in purine or _{pynmidine concentrations within} the lympbocytes may result in reduced immune _response directed _{agalnSt山mor antlgenS and neoplastic cells. In addition,} previous studies in human have suggested that L-ADA activity may Offer insight into molecular aspects of the immune mechanism and hosト山mor interactions

(Su丘･in

et al, 1977 _and

_1978)･

The increase inしADA _{activity in the dogs with} lympboma _may be largely _a肘ibuted to山mor _{cells present in the peripheral} blood at_{presentation.}

The _{results showed} a _significant difference in L-ADA _{activitybetween} ARC

group and the other groups

(P<0.05).

Our _results concur _with those _{of Christensen} et

al

(1988)

who fわund that L-ADA activity was increased in HIV _{patients and} that this increase was _{only slgnificant} in the AIDS _and ARC _patients. Previous _studies have

repo朽ed that mitogen-stimulated lymphocytes _produce increased ADA

_(Hovi

et al,

1976)･

Therefore, the results seen in cats in the ARC _{stage of infection may} be a

reflection of the on-golng Immune _{aCtivation and virus multiplication} that has been reported in this phase of the disease.

IncreasedしADA _activity has been _reported in leprosy

_(Sebgal

et _al,

1992),

typhoid fever

(Galanti

et _al,

1981),

_and HIV infection

(Christensen

et _al,

1988)I

Decreased L-ADA _activityhas been fわund in diseases _causing an impairment _{of the}

immune _{response, such} as _acute lymphocytic leukemia in children

(Zimmer

et _al,

1975),

tumor _patients

_(Uberti

et _al,

_1976),

_{glomerulonephritis}

_(Klinger

et _al,

_1983),

chronic active liver diseases

(Nardieiio

et _ai,

₁₉₈₃₎

_{and renal adenocarcinoma}

(Sufrirl

et _al,

_1978).

The _{results show} that canine and feline neutrophils also produce ADA･ However, in both cases L-ADA _activity Was _{Significantly} higher _{than that of}

neutrophils. This finding concurs _with the findings _of other authors who have suggested that lymphocytes are an important _{component of ADA activlty･}Erel et _al

(1998)

made similar obseⅣations where L-ADA activity was _{signi丘cantly} higher than N-ADA _activity in both _{controls and patients with} leisbmaniasis. In dogs _Ⅵ′itb lymphoma, N-ADA _activitywas seen to _{significantly} decrease _compared to the

controls. S山dies in bumans with lymphoma, have shown some degree _{of neutrophil}

dysfunction _exists including _{reduced chemotaxis and adherence} to _{nylon wool} in

some _{patients which} improved following _{commencing therapy}

(Fliedner

et al, 1979 and McCoⅢnack et _al,

1978).

In addition, one _s山dy showed a _marked increase in cell

count, _enzyme release, phagocytosis and killing, following stimulation

(Fossat

et al,

1994).

These findings _suggest that, like in the case in humans, _neutrophils in canine

lymphoma _{may also exist in} a depressed state resulting ln a _reduction in enzyme

synthesis including ADA･ On the other hand, cats in the ARC stage of FIV infection had _{significantly elevated} N-ADA _{activity1evels.} In HIV infection, _patients had activated neutrophils that _showed increased _apoptosis, decreased _{viability and}

(pitrak,

1999 _and Kubes et al,

2003)･

In cats therefore the increased N-ADA _activity may be due activation status _{of the cells} in FIV infection _resulting ln increased

en2γme release･

Despite bigb _nomal P-ADA _activity in cats, L-ADA activity was _relatively

low, _contrary to _the case in dogs. Tanabe

(1993)

_{also obseⅣed} that tissue ADA

activlty Was much l10Wer thanP-ADA activity･In dogswith lymphoma, elevated L-ADA _{activity may} largely be a _{renectiorl Of} tumor _{cell presence} in the peripheral

blood･ _{In this regard} LIADA _{activity may} be useful in distinguishing lymphoma from

mere lymphocytosis due to other causesI In cats L-ADA _activitymay be an indicator

of the immune system status and phase of FIV infection･ Further experiments were

carried out to investlgate _{the role of T} or B-1ympnocytes in the selected canine and ll

1S ′ヽ

芸16

V

言14

▼■

5

12 ■■

こ

川 :E: .>_◆■ f; U < 6

i4

1

2 0 Controls

(I)-1I)

Lymphoma

(m≡ll)

Fig･ 411 I Sig山ficant increase in L-ADA activityin dogswith lymphoma compared to

the healthy _controls

_(*P<0･05)A

Dogswith lymphoma have higher LIADA _activity than healthy _controls.

′ヽ 7J 4) U ∈> ■■

5

l■ll ヽ■′

倉

> :J5 くJ < < 負 < 1 2: 1.8 1.6 1.4 1.2 I 0.8 0.6 0.4 0.2 0 Controls

(n=11)

Lympho ma

(n=11)

Fig･ 4-2. N-ADA _{activities in healthy controls and} dogs _with lymphoma, Dogswith lymphoma had a _{significantly} lower N-ADAthanthe _{control group}

(****P<0.0001).

ゴ y. FF] E∃

邑

王､ ●芦 ミロ tJ < < 良 < i

CoJltT'OIs FIV+ AC ARC

(n=1 3) (n-11₎ (n=7) (n≡ヰ)

Fig1 4-3･ L-ADA _{activity of controls,} FⅣ+, AC _and ARC _{stages of FIV} infection･ The LIADA _{of the ARC group} was _{significantly elevated compared} to _{the other} two

groups

(*P<0･05).

Increase in LIADA occurs in cats inthe ARC _{stage of} FIV

J.t[ 富1･4 4) 1'e ).三 ;⊃

盲1

-≡

慧o･8

<

!

o･6 2: 0.4 Controls FTV十 AC ARC (n-;13) (b-11) (I)ヒ7) (n-4)

Fig･ 4-4･ N-ADA _{activities of controls,} FⅣ-positive, AC _and ARC _{stages of FIV} infection･ N-ADA _{activityinthe} ARC _group was _{significantly higherthanthe}

other groups

(*P<0.05).

′■■＼ ∽ l■■l lllll■■ q) U ＼丘 ∈〉 I■･･{ i? i) ■■ ヽ■′ iヽ .t> 1■■ U < < `∋ < l J 0 0 20 40 60 80 100

P-ADA Activity

_(IU/L)

Fig･ 4-5･ Negative _correlation between P-ADA _{activity and} L-ADA _activity ln _nOmal cats

(rニー0.55,P<0.05).

CHAPTER

5

INVESTIGATION

OF

ADENOSINE

DEAMINASE

IN T AND B

LYMPHOCYTES

5.1. INTRODUCTION

In the previous chapterしADA activity was _shown to be the

_major

component

of ADA activity, we further investigated _{the relationship} between T _and

B-1ymphocytes _and ADA _activity.

ADA is an _{enzyme capable of catalyzlng the cataboiism of purine} bases _and

whose prlnCIPal biologic activity is detected in T lymphocytes･ The role of this enzyme in cellular immune function was highlighted followlng the discovery _of reduced levels in patients with SCID. In humans, ADA activityis 5120-fold higher in

T lymphocytes than B lymphocytes

_(Sullivan

_and Osbourne,

_1977)･

In humans, ADA has been _considered a _{marker of cell-mediated} immunity

(Baganha

et al,

1990).

Low T-ADA _activity has _also been _reported in _{patients with multiple sclerosis}

(Vivekanandhan

et al,

2005).

A negative correlation was _observed between serum

ADA _and T lymphocytes _percentage in human _{patients wi也nephrotic syndrome}

(Misra

et al,

1997).

ADA activityhas been identified as a _{marker of T cell activation}

(Rose

et _al,

_2001).

Fur也ennore, _{several reports} have _observed that ADA interacts with CD 26 on _{the surface of T cells surface resulting} ln a _{COIStimulatory} effect in the activation or T cells

(Blazquez

et _{al, 1992,} Morimoto _and Schlossman, 1998, Cordero et _{al, 2001 and} Pacheco et _al,

_2005).

Most _{of the} lymphomas described _{in canines onglnate} from B lymphomas

(Teske, 1994).

These lymphomas react much better to _chemotherapy than do T _cell lymphomas, _{which make up} 10-38% _{of the} cases _and have a _much worse _prognosis

(Hahn

et _al,

1992).

It is generally accepted that FIV may be useful as a _model for AIDS･ Several

authors have reported that serum ADA _{activity is elevated} in HIV infection

(Gakis

et

al, 1989, Goto et al, i992 and Inigo et al,

1992).

Furthermore, a _relationship has been

described _{in which} serum ADA _{correlated with retroviral} infection in HIV infection

(Mastrioanni

et _al,

1987).

ADA is also reported to be a _{useful marker of progression}

EXPERIMENT A: INVESTIGATION OF T AND B LYMPHOCYTE ADA ACTIVITY IN DOGS AND CATS

5.2. OBJECTIVE

The _{present study} was _undertaken to investigate the role of T and B lymphocyte in ADA _activityin dogs _and cats.

5.3｡ MATERIALS AND METHODS 5.31. ANIMALS

Heparinized blood was _collected from 10 healthy dogs _{of various} breeds, _and

8 dogs _with lymphoma. The _{control group composed of 6 males,} 4 fTemales,with an

average age of 3,7± 1.8 years. The group of dogs with lymphoma composed 5 males,

3 females _and an _{average age of 7.5j=2.1 years.} None _{of the} dogs were _receiving

immunosuppressive _medication atthe time of sampling.

Heparinized blood was _collected from 10 healthy FIV-negative _and 8

FIV-infected cats. All the cats included in this work were _seronegative for Feline leukemia

virus antigen and the FIV-positive group was _seropositive fわr FIV _antibody

(IDEXX

Laboratories, Portland,

_Maine).

The cats were _{of mixed-breed with} the FIV-negative

group composed of 6 males, 4 females, with an _{average age of4･7±4･2 years･} The

FIV _{positive group composed 5 males,} 3 females _and an _average age of 7･5±2･1