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Studies of arthritis and other lesions induced in rats by injection of mycobacterial adjuvant. IV. examination of tissues and fluids for infectious agents

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Studies of Arthritis and Other Lesions Induced in Rats by
Injection of Mycobacterial Adjuvant. IV. Examination
of Tissues and Fluids for Infectious Agents
By JOHN T. SHARP, BYRON H. WAKSMAN,
CARLM.
AND SARABELLE
MADOFF
Adjuvant induced arthritis is believed
to be due to an immunological process
associated with the development of delayed hypersensitivity to a component
of the injected mycobacterial adjuvant.
However, since pleuropneumonia-like
organisms (PPLO) may cause arthritis
in rats, it was necessary to determine
whether such microorganisms play any
role in adjuvant disease. This article reports attempts to cultivate bacteria and
PPLO from affected tissues and to transfer disease to normal recipients. No evidence for an infectious agent was obtained.
-ON
Es opinate que arthritis inducite per
adjuvantes es le resultato de un process0
immunologic associate con le disveloppamento de un tardive hypersensibilitate
pro un componente del injicite adjuvante
mycobacterial. Tamen, viste que organismos pleuropneumonioide es capace a
causar arthritis in rattos, il esseva necessari detenninar si tal microorganismos
ha un rolo in morbo de adjuvante. Le
presente articulo reporta effortios de
cultivar bacterios e organismos pleuropneumonioide ab afficite tissues e de
transferer le morbo a recipientes normal.
Nulle evidentia pro le presentia de un
agente infectiose esseva obtenite.
I
N 1956 PEARSON REPORTED THE PRODUCTION of what we shall call
adjuvant disease, namely inflammatory articular and periarticular lesions
in rats following the injection of mycobacterial adjuvants.’ In the original and
subsequent studies, evidence was presented that the pathogenesis of this process depends on some immunological mechanism. However, bacteriological
studies revealed the presence of pleuropneumonia-like organisms ( PPLO ) in
the lungs and at the injection sites of a small proportion of affected animak2
Since PPLO may cause arthritis in rats, it seemed desirable to conduct further
bacteriological studies on animals obtained from B different supplier and used
in a separate laboratory.8*4The results of these studies have failed to reveal
any evidence that adjuvant disease is due to an infectious agent. Simultaneously, additional evidence for an immunological pathogenesis of this adjuvantinduced rat arthritis was obtained.5 Details of the bacteriological studies are
reported in the present article.
MATERIALSAND METHODS
White male and female Sprague-Dawley rats, usually weighing 150 to 200 grams, were
used. One group of six animals was derived from a caesarean-delivered stock cared for
From the Departments of Medicine, Hamard Mcdical School, and Massachusetts General
Hospital; the Department of Bacteriology and Immunology, Harvard Medical school, and
the Neurological S d c e , Massachusetts General Hospital; and the Department of Medidne,
UniverslCy of California School of Merllcine, Los Angelcs, California.
This is publication No. X X X of the Robert W . Lovett Memorial Unit for Study of Crip
pling Disease, Hamard Medkal School.
This study was supported by grants from the Commonwealth Fund, the N d b n a l lnstitutea of Health (A-1250,A-1286,B-919,and E-1!257),and the Eli Lffly Company.
169
170
SHARP,
WAKSMAN, PEARSOK
AND MADOFF
under special conditions and alleged to be PPLO free.'6 Arthritis was induced by inoculation of approximately .05 ml. of adjuvant (namely heat-killed tubercle bacillit suspended
in Bayol F, at a concentration of 3 mg. per ml. ) . Adjuvant inoculations were made in the
foot pad of one hind limb. During certain experiments turpentine, diluted 1:5 in olive
oil, was inoculated into one of the other three limbs of some animals to determine
the response to a sub-inflammatory challenge. Inoculation of this concentration of turpentine in olive oil into control nonadjuvant inoculated animals produced minimal swelling
which did not persist beyond 24 hours, whereas animals with adjuvant disease had intense
inflammatory reactions. The details of the clinical, histological, and immunological observations on these animals are reported elsewhere.5
Cultures of joint and periarticular tissues were performed immediately after the animal
was sacrificed with ether. The limbs to be cultured were amputated and cleansed with alcohol. Joint fluid was cultured when present. Joint capsule and periarticular tissues from
each limb were dissected, minced, and swabbed on culture plates. The culture medium
used was made by adding 10 per cent ascitic fluid to Field's tryptic digest agar base. The
pH of the ascitic fluid was adjusted to 7.4 by adding .2M KH,PO,. The culture medium
was made in large lots and each lot was simultaneously used for routine work in a laboratory
studying many strains of PPLO. Blood cultures were performed by inocculating 2 to 5 ml.
of heart's blood into 100 cc. tryptic digest broth flasks containing 10 per cent horse serum.
After 5 to 7 days incubation, each blood culture flask was subcultured on ascitic fluid agar
plates. Agar plates inoculated with joint fluid or tissue, or subcultured from the blood culhire flasks were examined using the in situ staining technique of Dienes.7 All cultures were
sealed during incubation to prevent moisture loss and most cultures were made anaerobic
by the Fortner technique.*
In one set of experiments, attempts were made to transfer the disease by inoculating
blood and tissues of adjuvant-inoculated animals into normal recipients. Limbs with moderate or severe inflammation were chosen as donor tissues. The joint fluid and periarticular
soft tissues were removed, minced with sterile scissors, and ground in a Potter-Elvehjem
mill with sterile saline. One group of animals received the saline suspension. Another group
of animals received the same material emulsified in an equal volume of incomplete adjuvant
consisting of sterile Bayol F and Arlacel A (85:15).
Other groups of animals maintained in an entirely separate animal farm were inoculated
by various routes with the sediment from large volumes of broth cultures of the Preston
strain of PPLO, originally isolated from rodent artluitis.4 Cdturecl made from the inflamed
articular and periarticular tissues of these animals were carried out in a fashion identical
to that outlined above.
RESULTS
In the earlier studies, 51 animals with adjuvant disease were cultured. Seven
of 38 adjuvant injection sites yielded PPLO as did 2 of 36 lungs. One hundred
and twelve joint cultures and 31 cultures of various organs were negative.2
The additional studies detailed here have failed to provide evidence for an
infectious etiology of adjuvant disease. Ninety-five limbs from 44 rats with
adjuvant-induced arthritis were studied bacteriologically (table 1). Five cultures were overgrown with a spreading bacterium and cannot be considered
further. Pleuropneumonia-like organisms were isolated from none of the re-
'All animals were obtained from the Charles River Breeding Laboratories, Brookline,
Massachusetts.
tJamaica 22 strain, obtained through the courtesy of Dr. Jules Freund.
#The strain used in these experiments was kindly provided by Dr. William C. Kuzell
since the strain originally obtained from Dr. Preston and maintained in Dr. Dienes' laboratory
by repeated subculture was no longer virulent.
171
MYCROBACTERIAL ADJUVANT-INDUCED ARTHRITIS
Table 1.-Bacteriology of Adjuvant Znduced Arthritis
95
paw8 from 44 rat8 cultured
90 paws negative for PPM
75 paw8 negative for bacteria
5
paw8 unknown
-- culturer overgrown with spreading organism
(Proteus)
1 paw culture grew out a gram negative rod similar to Streptobacillur
moniliformls from adjwant injection site 35 day8 after inoculation
14 paw culturer contaminated with gram positive rods similar to B. mycoides.
There paws injected with one lot of improperly sterilized adjuvant
containing this organism.
maining 90 limbs. In one instance, a pleomorphic, gram-negative rod was recovered from an adjuvant injection site. This organism produced L forms spontaneously and required enriched media for growth, thus resembling Streptobacillus moniliformis. However, appropriate serum was not available for serological identification. In addition to this bacillus, a gram-positive rod similar to
Bacillus mycoides contaminated one lot of adjuvant and was recovered from
all of 14 limbs injected with this lot of material.
In all rats of the present study, the initial arthritic lesions were observed to
develop in uninoculated limbs between 10 and 18 days after inoculation of adjuvant, and fresh lesions continued to appear up to approximately 4 weeks.
Table 2 relates the time when the animals were sacrificed and cultures were
made, to the inoculation with adjuvant on the one hand and the type of local
treatment given on the other hand. While some animals were examined during
the latent period, the majority were studied during the stage of active inflammatory disease, and a few during recovery. Somewhat more than half of the
extremities cultured were intensely inflamed at the time the animals were
killed. A few animals were studied in the absence of any objective inflammatory disease. The details are shown in table 3.
Blood cultures taken from 14 rats 8 to 21 days after adjuvant inoculation
were negative for bacteria and PPLO. Cultures from the anterior chamber of
3 eyes, taken 22 days after inoculation with adjuvant, failed to show growth of
PPLO. Two of these were involved with iridocyclitis and one was unaffected.
A few colonies of gram-positive cocci were recovered from the anterior chamber of the healthy eye, the conjunctiva of one eye with iridocyclitis, and one
uninvolved eye. Gram-negative rods were isolated in small numbers from the
anterior chamber of one eye with iridocyclitis. The presence of these few
bacteria in the anterior chamber fluid is believed to represent contamination
from the conjunctival flora. A few attempts to culture bzlanitis lesions were unsuccessful because of overgrowth with a spreading micro-organism.
Attempts to transfer the disease with blood and inflamed tissues were negative. Table 4 outlines these experiments. Three animals were sacrificed 10 days
172
SHARP, WAKSMAN, PEARSON AND MADOFF
Table 2.-Cultures
of Paws from 44 Rats with Adjuvant Induced Arthritis
Time After Adjuvant in Days
8
10
12
4
4
4
13
17
18
22
19
21
25
28
36
1
1
4
7
1
3
4
50
Totals
Limbs Cultured
Not Inflamed
2
4
2
M
1nfl-d.
loci1 injection
Inflammd, turpentine
in jrcted
2
2
3
T0t.l
Limb.
No. of Mat.
Studied
14
2
19
21
2
2
Inflrcd. tuberculin
injected
Inflamed. idjwant
injected
1
1
2
3
3
5
2
3
9
9
15
6
3
3
3
5
2
10
3
5
1
4
3
4
5
1
3
4
10
4
17
1
1
4
1
2
6
2
39
4 - 9 5
44
2
after inoculation, when they showed no arthritis. Three other animals were
sacrificed at 17 days and two more at 28 days following adjuvant inoculation.
Each of these 5 had a moderate degree of arthritis. Blood was taken from each
animal and pooled in groups according to the day after inoculation of adjuvant.
Pooled joint capsule and periarticular tissues were prepared from the 17 and
28 day donors, suspended in saline or incomplete adjuvant and injected into
normal recipients. The pooled donor blood was given intravenously, into the
foot pad, or by a combination of subcutaneous and intraperitoneal injections.
Ten recipients of blood showed no disease although slight local swelling was
present for 3 or 4 days after the foot pad inoculations. The suspensions of
pooled joint and periarticular tissues were injected either into the foot pad or
intraperitoneally. Again no disease was produced in 3 animals although some
local swelling was observed following the foot pad inoculations.
All 6 of the animals derived from a caesarean-delivered stock developed moderate or severe adjuvant disease. Four of 22 animals inoculated with the Preston strain of PPLO developed acute polyarthritis. Limbs from these animals
yielded PPLO on culture in all instances.
Table 3.-Cultures of Paws from 44 Rats with Adjucant Znduced Arthritis
D e g r e e of I n f l a m m a t i o n i n Paw at T i m e of C u l t u r e
NO LOCAL INJECTION
TURPENTINE INJECTED
0
+
te
CH
Ccce
14
2
4
3
4
6
MAX
TOTALS
5
5
33
7
4
21
2
2
17
33
TUBERCULIN INJECTED
ADJUVANT INJECTED
TOTAL
4
8
4
6
95
"and t designates same animals.
2070 in incemplete adjuvant
40% in saline
5 % in incomplete adjuvant
Minced Periarticular Tissue
10% in saline
Pooled Blood
Material
2t
28
17
28
2t
3O
Moderate Arthritis
17
3*
Moderate Arthritis
Moderate Arthritis
Moderate Arthritis
None
Disease in Donors
10
Days after
Adjuvant
3
Donors
-
2.0 ml. IP
.2 ml. in foot pad
.5 ml. IP
.3 ml. in foot pad
1.2 ml. IP
0.4 ml. in foot pad
1.0 ml. Lp
0.4 ml. in foot pad
.2, .2 ml. in foot pad
1.0 ml. IV
.5 ml. SC and 1.5 ml. I€'
1.0, 2.0 ml. IV
.5 ml., .5 ml. in foot pad
.4 ml. in foot pad
1.0 ml. N
Dose and Route
Table I.--Passive Transfer Experiments
1
1
1
1
1
1
1
1
2
1
1
2
2
1
1
eipiente
No. Re-
No effect
No effect
Slight swelling, 1 week
No effect
Slight swelling 3-4 days
No effect
Slight swelling, 1 week
No effect
Slight swelling 3-4 days
No effect
No effect
No effect
Slight swelling 3-4 days
Slight swelling 3-4 days
No effect
Re8uIt.n
k.
5
5c
b
r
174
SHARP, WAKSMAN, PEARSON AND MADOFF
DISCUSSION
The studies reported here have yielded no evidence for an infectious etiology
of this adjuvant-induced disease of rats. A potentially pathogenic bacterium,
Streptobacillus moniliformis, was isolated from the adjuvant inoculation site of
a single animal. Since the media and techniques employed were appropriate
to permit recovery of this slightly fastidious organism, its absence from 89 other
limbs is believed to be adequate evidence to exclude it as the etiological agent.
No single culture in this study yielded pleuropneumonia-like organisms. That
the media and techniques were appropriate for recovering PPLO was demonstrated in a control experiment on animals with PPLO-induced polyarthritis.
The unsuccessful attempts to transfer disease using involved tissues constitutes
further evidence that this process is not infectious. Several studies have indicated that rat polyarthritis due to PPLO is readily transmitted with infected
t i s s u e ~ , 3 ~and
~ ~ ~one
J ” might anticipate that the process might be transferrable
if due to a virus. Finally, adjuvant disease is uninfluenced in its development
by treatment of the rats with antibiotics which are effective in preventing polyarthritis induced by known strains of PPL0.11*12J3Negative bacteriological results and negative attempts to transfer the disease, of course, do not exclude
completely an infectious etiology since it remains possible that the disease
under study may be caused by a strain of organism more fastidious than can be
grown on the media used. However, in the present instance there is strong
positive evidence that an immunological process is responsible for production
of adjuvant disease. This evidence is presented in detail elsewhere and is only
summarized here.j First, the use of adjuvant is a powerful immunizing technigue. Then, there is a regular latent period of 10 to 18 days between the injection of adjuvant and the appearance of the initial lesions of the illness. This
latent period is comparable to the time required for the appearance of a primary
immunological response. When convalescent animals or those that have recovered from an initial attack of adjuvant disease are given a second inoculation of adjuvant, the flare-up occurs with a shortened latent period of 4 to 7
days. The decreased latent period on re-exposure to an antigen is characteristic
of a secondary type of immunological response. Whole body irradiation and
cortico-steroid therapy, which abolish many immunological responses, suppress
the development of adjuvant disease in adult rats. The very yoiing rat, which
is immunologically unresponsive to many antigens, i s refractory to adjuvant
disease before the age of 3 or 4 weeks and does not become fully susceptible
until somewhat later. In addition, animals treated with tubercle bacilli at birth
are made “tolerant” to this antigen in a large proportion of instances and are
not susceptible to the development of adjuvant disease as adults. When the
bacteriological studies are considered in conjunction with the immunological
evidence, it appears reasonable to conclude that this experimental disease is
due to a systemic immunological response to an unidentified antigen. Evidence
to suggest that the disease results from a delayed hypersensitive response to a
component of the injected adjuvant is presented elsewhere.
SUMMARY
Ninety-five limbs of 44 rats with adjuvant-induced arthritis were cultured
for bacteria and PPLO. Blood cultures were made from 14 animals. The aque-
MYCROBACTERIAL ADJUVANT-INDUCED ARTHRITIS
175
ous humor of 2 animals with iridocyclitis were also cultured. Attempts were
made to transfer the disease by injecting normal recipients with affected tissues or blood. No evidence for an infectious origin of this disease was obtained. Evidence suggesting that the process is due to an immunological mechanism was summarized.
REFERENCES
1. Pearson, C. M.: Development of arthritis, periarthritis and periostitis in rats
given adjuvants. Proc. Soc. Exp. Biol.
and Med. 91:95-101, 1956.
2. -, and Wood, F. D.: Studies of polyarthritis and other lesions induced in
rats by injection of mycobacterial adjuvant. I. General clinical and pathological characteristics and some modifying factors. Arth. & Rheumat. 2:
440, 1959.
3. Collier, W. A.: Infectious polyarthritis
of rats. J. Path. and Bact. 48:579589, 1939.
4. Findlay, G.M., MacKenzie, R. D.. MacCallum, F. O., and Klieneberger, E.:
The aetiology of polyarthritis in the
rat. Lancet 2:7-10, 1939.
5. Waksman, B. H., Pearson, C. M., and
Sharp, J. T.: Studies of arthritis and
other lesions induced in rats by injection of mycobacterial adjuvant. 11.
Evidence that the disease is a disseminated immunologic response to
exogenous antigen. J. Immunology. In
press.
6. Foster, H. L.: Large scale production
of rats free of commonly occurring
pathogens and parasites, Proc. of the
Animal Care Panel 8:92, 1958.
7. Dienes, L.: L organism of kliemberger
and streptobacillus moniliformis. J.
Inf. Dis. 8532442, 1939.
8. Fortner, J.: Ein einfaches plattenverfahren zur zuchtung strenger anaerobier ( anaerobe Baxillen, filtrierbare
anaerobe bakterien, spirochaeta pallida), Centralbl. f. Bakteriol. ( Abt.
I ) 108:155, 1928.
9. Woglom, W. H.,and Warren, J.: A
pyogenic, filtrable agent in the albino rat. J. Exp. Med. 68:513, 1938.
10. Klieneberger, E.: Studies on pleuropneumonia-like organisms. The L4
organism as the cause of Woglom’s
“Pyogenic virus”. J. Hyg. 39:260,
1939.
11. Pearson, C. M.: Development of Arthritis in the Rat Following Injection
With Adjuvant. In: Shaffer, J. H.,
LoGrippo, G. A., and Chase, M. W.:
International Symposium on the
Mechanisms of Hypersensitivity. Boston: Little, Brown and Co., 1959, p.
647.
12. Kuzell, W.C., and Mankle, E. A.: Cortisone acetate and terramycin in polyarthritis of rats, Proc. SOC.Ex. Biol.
and Med. 74~677,1950.
13. --, Gardner, G. M., Fairley, D. M., and
Tripi, H. B.: Chemotherapeutic Trials
in Experimental Polyarthritis of Rats,
In: Rheumatic Diseases, based on the
Proc. Seventh International Congress
on Rheumatic Diseases. Phila.: W
B. Saunders CO., 1952, pp. 409.
John T . Sharp, M.D., Formerly Instructor in Medicine, Harvard Medical School, and Assistant Physician, Massachusetts
General Hospital, Boston, Mass.; currently Associate Physician,
Division of Arthritis, Henry Ford Hospital, Detroit, Mick.
Byron H . Waksmun, M.D., Assistant Professor of Bacteriology
and Immimology, Harvard Medical School, and Associate
Bacteriologist, Mussachwetts General Hospital, Boston, Muss.,
National Neurological Resenrch Foundation Scientist.
Carl M . Pearson, M.D., Associate Professor of Medicine, University of California School of Medicine, Los Angeks, Cal.
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adjuvant, induced, tissue, fluid, examination, injections, lesions, mycobacterium, agenti, arthritis, infectious, rats, studies
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