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Electron-dense deposits following injection of Gold Sodium Thiomalate and Thiomalic acid.

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Electron-Dense Deposits Following Injection of
Gold Sodium Thiomalate and Thiomalic Acid
By WALTER
L. NORTON,DAVID
C. LEWISAND MORRISZWF
A
gold salt injected joints with the exception of V.B.,
from whom tissues were obtained from both knees.
Synovial biopsies were performed with a ParkerPearson needles in 4 patients. In one (V.B.), synovial tissue was obtained at surgical synovectomy.
Animal experiments. Young adult giant albino
rabbits were obtained from a local supplier. An
experimental synovitis was produced by the local
injection of 0.5 ml. of Phytohemagglutinin M
(Difco) (PHA) into the knee joint 48 hours prior
to the intra-articular injection of gold sodium
thiornalate (GST) or thiomalic acid (obtained
through the courtesy of Dr. Richard Smith, Merck,
Sharp and Dohme, West Point, Pennsylvania).
Thiomalic acid was dissolved in sterile phosphate
buffered saline, pH 7.4, prior to injection. Dosage
schedules are outlined in Table 2. Animals were
sacrificed 24 hours after the last test injection, and
synovial tissues processed for electron microscopy.
Specimens were coded, so that the form of therapy
was not known at the time of electron microscopic
examination.
Spectrographic analysis of the thiomalic acid
MATERIALS
AND METHODS
was performed by Dr. Morton Mason, Director of
Human synovial tissue and synovial exudate the Dallas City-County Criminal Investigation
cells. Synovial tissues and synovial exudate cells Laboratory. No significant contamination by heavy
were obtained from the knees of 4 patients with metals was found.
definite rheumatoid arthritis,? one patient with
Electron microscopy. All tissues were fixed in
juvenile rheumatoid arthritis, and one patient with veronal buffered osmium tetroxide, pH 7.4, depigmented villonodular synovitis. All had received hydrated in graded alcohol solutions and propyintra-articular injections of gold sodium thiornalate lene oxide, and embedded in Maraglas-Cardolite
(Myochrysine, Merck, Sharp and Dohme) and have mixture.9 Sections were supported on uncoated
been included in a previous report.5 The patient copper grids, stained with lead,lO uranyl acetate,ll
with juvenile rheumatoid arthritis received sys- or viewed unstained in an RCA EMU 3G electron
temic gold salt therapy following intra-articular microscope.
injection in one knee. Previous gold salt therapy
RESULTS
in these patients, and other relevant data, are given
Human
arthritis.
All 5 specimens of synin Table 1.
ovial
membrane
examined
were found to
All tissues and cells were obtained from the
of soluble gold salts to
animals results in the widespread distribution of gold throughout the body, the
highest concentrations being found in the
reticuloendothelial system, synovial lining
and idlammatory ~ i t e s . l -In
~ rheumatoid
arthritis, relatively large amounts of gold
have also been found to localize in the
synovial lining.5 Ashworth, Hess and Ziff
noted electron-dense deposits in animal
tissues following the administration of gold
thiomalate.6 The present report describes
the occurrence of characteristic electrondense material within the synovial lining
cells of patients with rheumatoid arthritis
treated with gold thiomalate. Animal experiments indicate that these changes are dependent on thiomalic acid.
DMINISTRATION
From the Department o f Internal Medicine,
Rheumatic Diseases Unit, University of Texas.
Southwestern Medical School at Dallas.
Supported by USPHS Project Grant #An{09989, USPHS Training Grant #AM-05154, and an
Arthritis Foundation Clinic Study Center Grant.
WALTERL. NORTON,M.D.: Associate Professor
of Medicine, University of Tennessee, Aleniphis.
436
DAVIDC. LEWIS, M.D.: Instructor in Medicine,
Harvard Medical School, Assistant in Medicine,
Beth Israel Hospital, Bosion, Massachusetts; formerly Trainee in Arthritis, University of Texas
Sorithwestern Medical School at Dallas. MORRIS
ZIFF, PH.D., h1.D.: Professor of Internal Medicine,
University of Texas, Southwestern Medical School
at Dallas.
ARTHnITIS A N D RHEU\fATIS\I,
VOL.
11, N O , 3
(JUNE
1968)
DEPOSITS FOLLOWING INJECrON OF GOLD SALTS
Fig. L-Synovial tissue from atient with rheumatoid arthritis. Mononuclear cells
contain many membrane boun ed structures, presumably phagosomes, filled with
very dense filamentous deposits. x 14,600.
B
Fig. B.--Synovial lining cell from patient with rheumatoid arthritis contains
characteristic dense deposits within membrane bounded structures. A lysosome at
three o’clock shows deposition of a small amount of dense material just beneath the
limiting membrane, which is ruptured. Dense material appears to extend beyond
the limiting membrane of the structure at six o’clock, This may represent an autophagosome. Note beaded structure of deposit. Fibrin-like material is present in the
extracellular space. x 58,000. The structure at 11 o’clock is shown enlarged in Fig. 3.
43?
43s
NORTON, WWIS, ZIFF
Fig. 3.-One of the structures from Fig. 2 is shown at a higher magnification.
There are both rod-like and round deposits. In several areas the rod-like particles
appear to be composed of small droplets arranged in a linear pattern. x 140,000.
Fig. 4.--Inclusion from a patient with rheumatoid arthritis containing dense
deposits on the left shows the localization of characteristic material within a membrane bounded structure. Similar deposits on the right appear to be free in the
cytoplasm. x 72,500.
439
DEPOSITS FOLLOWING INJECTION OF GOLD SALTS
Table 1.-Patients
Included in Study and Prior Gold Salt Therapy
Intra-akicular Gald Salt Therapy
Duration
of
therapy
Diagnosiso injections (rng.1 (wks.)
Total
Patient
Number of dose
Time since
previous
injection
Age
Sex
K.R.
59
M
RA
8
400
10
2 Days
J.L.
43
M
RA
7
325
10
7Days
I.H.
26
F
PVNS
10
475
9
11Days
R.D.
42
F
RA
7
325
10
28Days
V.B.?
14
F
Juvenile
RA
10
475
10
12Months
J.B.
56
M
RA
3
150
2
1Hour
Procedure
Synovial
biopsy,
Synovial
fluid
aspiration
Synovial
biopsy
Synovial
biopsy
Synovial
biopsy
Bilateral
surgical
synovectomy
Synovid
fluid
aspiration
*R.A. = Adult rheumatoid arthritis; PVNS = pigmented villonodular synovitis.
tAlso received systemic Myochrysine therapy for six months prior to synovectomy. Total
intramuscular dose was 550 mg. The last intramuscular injection was given three weeks
prior to surgery. Gold salt injections were given into the left knee, and synovectomy was
done in both knees.
contain characteristic intracellular electrondense deposits (Figs. 1-4) which appeared
to be composed of aggregates of fine filamentous material. At higher magnifications,
the filamentous strands comprising the deposits were usually noted to be somewhat
beaded, and were made up of small dense
granules measuring approximately 40 to 150
A in diameter (Figs. 3, 4). Although these
granules were usually found within membrane limited structures with the appearance of lysosomes and phagosomes, they
were also found free within the cytoplasm.
Similar material could not be identified in
extracellular sites. In the synovial tissues,
the deposits were found within synovial
lining cells and in mononuclear cells of the
underlying subsynovial tissues.
In the case of the 4 adult patients with
rheumatoid arthritis and one patient with
pigmented villonodular synovitis in whom
deposits were observed, GST had been injected intra-articularly prior to biopsy. It is
significant, however, that deposits were also
noted in both knee joints of V.B., a patient
with juvenile rheumatoid arthritis. This patient had received local injections in only
one knee, and a course of intramuscular injections prior to surgery (Table 1). The
amounts of material observed in both knees
were relatively smaller in amount than in
the others (Fig. 5 ) .
Less material was found in the cells of
the synovial membrane. In the former, deposits were seen most frequently within
mononuclear cells, even though polymorphonuclear cells predominated in the cell
population. Similar deposits have not been
previously noted in the synovial membranes
of 19 patients with rheumatoid arthritis12
and 5 patients with Reiter's syndrome13
who had not been treated with gold salt.
Animal experiments. All synovial tissues
which had been injected with either GST
or thiomalic acid (Table 2) were found to
contain dense deposits which appeared sim-
NORTON, LEWIS, ZIFF
Fig. S.-Small amounts of dense material within dense structures, presumably
lysosomes, of synovial lining cell of a patient with juvenile rheumatoid arthritis.
This tissue was obtained from a patient (V.B.) who had received 550 mg. of My+
chrysine by intramuscular therapy. The contralateral knee had been injected one
year previously (patient V.B.). x 29,800.
r
Fig. 6.43 ovial lining cell of rabbit joint injected with gold thiomalate contains
deposits simi ar to those seen in human tissues. x 27,400.
441
DEPOSITS FOLLOWING INJECroN OF GOLD SALTS
Fig. 7.--Synovial lining cell of rabbit joint injected with thiomalic acid. Dense
deposits appear similar to those in tissues treated with gold thiomalate, but appears
more filamentous, with less granular material. Tissues for thiomalic acid treated
animals usually lacked the fine granularity which was typical of gold thiomalate
treated tissues. x 24,300.
ilar to those seen in human synovial tissues
and synovial exudate cells. In these experiments, phytohemagglutinin was injected 2
or 3 days prior to injection of the other
agents in order to establish a synovial inflammatory reaction as previously described.14 The deposits in GST-treated rabbits appeared identical to those in human
tissues (Fig. 6). The deposits in thiomalic
acid-treated animals, although generally
similar in appearance and location (Fig. 7),
tended to be somewhat less granular. In
examining a number of animals with experimental arthritis induced by phytohemagglutinin as well as other substances,
similar deposits never have been noted:6
nor have they been found in synovial tissues from patients with rheumatoid arthritis
given local injections of colloidal gold,12 in
synovium of normal guinea p i g P or rabbit
synovial tissues injected with iron dextran."
DISCUSSION
A unique morphologic feature has been
identiEied in synovial tissues and synovial
exudate cells of patients treated with gold
sodium thiomalate. Characteristic electrondense deposits were concentrated heavily
in lysosomes and phagosomes, and were
found free in the cytoplasm in lesser
amounts. Therefore, it seems likely that the
material deposited may be formed intracellularly, following endocytosis and metabolism of gold salt, and concentrated within
autophagosomes. The studies of Persellin
and Z i f P also suggests that GST enters
peritoneal macrophage as an intact molecule. However, the possibility that extracellular material is avidly taken up and
concentrated within cells cannot be excluded by the present work.
Although most of the observations were
made in tissues which had received local
442
NORTON, LEWIS, ZIFF
Table 2.-Experimental
Rabbit
number
1
2
Procedures in Rabbit Knee Joints'
Day
3
1
PHAO Gold thiomalate,
13 mg.
PHA Thiomalic acid,
5.0mg.
3
PHA
-
4
PHA
-
4
5
Gold thiomalate,
13 mg.
Thiomalic acid,
5.0 mg.
Gold thiomalate,
15 mg.
Thiomalic acid,
6 mg.
Synovial Excision
*
Synovial Excision
-
6
Gold thiomalate, Synovial Excision
15 mg.
Thiomalic acid, Synovial Excision
6 mg.
"All rabbits received 0.5 ml. of PHA intra-articularly. Quantities given refer to amounts
injected into the knee joints.
injections of the materials under study, the
same changes were found in the synovial
tissue of a patient (V.B.) who had received
intra-articular therapy in the contralateral
knee a year previously, and subsequent
intramuscular GST therapy (Fig. 5 ) . Therefore, it is concluded that the changes with
GST, though related to local concentration,
were not solely dependent on the local injection of this agent, and occurred at the
therapeutic concentrations produced by systemic administration.
The nature of the reactions leading to
the disposition of this electron-dense material, and the nature of the material itself,
is unknown. However, it was clearly shown
that the filamentous deposits occurred following injection of thiomalic acid into experimental animals as well as following
administration of gold salt, although close
inspection indicated that the deposits after
thiomalic acid administration may not be
identical to the deposits which resulted
from gold salt administration.
The density of the deposits suggested
that a heavy metal might be involved in
their formation. Spectrographic analysis of
the thiomalic acid ruled out the possibility
that exogenously administered heavy metal
played a part in this reaction. The participation of endogenous heavy metals such as
iron cannot be ruled out, even though iron
dextran administration does not result in
similar deposits.''
At the present time it is not possible to
relate any of the therapeutic or toxic properties of gold salt to the morphologic phenomenon observed, but it is noteworthy
that therapeutic concentrations of a sulfhydryl compound, either free or combined
with gold, are capable of producing specific
morphologic alterations in the cells of the
synovial membrane.
SUMMARY
Characteristic electron-dense deposits have been found in cells of the synovial membrane in 3 patients with rheumatoid arthritis, one with juvenile rheumatoid arthritis,
and one with pigmented villonodular synovitis. In 2 patients, deposits were also observed in mononuclear cells of the synovial fluid. The deposits were comprised of dense
filamentous material which were largely concentrated within phagosomes of mononuclear cells of the synovial lining and synovial exudate. Similar changes could be produced in experimental animals treated with gold thiomalate and with thiomalic acid
alone. Although the morphologic changes were independent of exogenously administered heavy metals, a reaction between the sulfhydryl compound and endogenous heavy
metals cannot be ruled out.
443
DEPOSITS FOLLOWING IN JECTON OF GOLD SALTS
SUMMARIOIN INTERLINGUA
Tissu synovial de patientes tractate con thiomalato de aura per via intrasynovial o
constitutional contine, il esseva trovate, distincte depositos intracellular que es electrono-opac. Viste que simile alterationes poteva esser producite in arthritis experimental in conilios per le tractamento con thiomalato de auro o con acido thiomalic, le
conclusion esseva formulate q u e le precipitatos opac non requireva auro exogene pro
lor production. Es suggestionate que acido thiomalic o su productos metabolic es capace a fonnar depositos opac con metallos endogene, possibilemente ferro.
REFERENCES
1. Jeffrey, J. R., Freundlich, H., and Bailey, D.
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Rheum. Dis. 17:52,1958.
2. Block, W. D., Buchanan, 0. H., and Freyberg,
R. H.: Metabolism, toxicity and manner of action
of gold compounds used in the treatment of arthritis. J. Pharm. Exp. Ther. 82:391, 1944.
3. Bertrand, J. J., Wain, H., and Tobias, C. A.:
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to arthritis. J. Lab. Clin. Med. 33:1133, 1948.
4. LaTonna, E. A., Brecher, G., Cronkite, E. P,
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(19SAu) in skeletal tissues and synovia of mice.
Arthritis Rheum. 6:1, 1963.
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administration of gold salts. Arthritis Rheum. 9:
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6. Hess, E., Ashworth, C. T., and Ziff, M.: Unpublished experiments.
7. Ropes, M. W., Bennett, G. A., Cobb, S., Jacox,
R., and Jessar, R. A.: Proposed diagnostic criteria
for rheumatoid arthritis. J. Chronic Dis. 5:630,
1957.
8. Parker, R. H., and Pearson, C. M.: A simplified synovial biopsy needle. Arthritis Rheum. 6:
172,1963.
9. Freeman, J., and Spurlock, B.: A new epoxy
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10. Millonig, G.: A modified procedure for lead
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11. Watson, M. L.: Staining of tissue sections
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13. Norton, W. L., Lewis, D. C., and Ziff, hl.:
Light and electron microscopic observations on
the synovitis of Reiter’s disease. Arthritis Rheum.
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14. Lewis, D. C., Jasin, 13. E., and Ziff, M.:
Cellular responses in the synovial membrane: arthritis produced by endotoxin and phytohemagglutinin. Arthritis Rheum. 92363, 1966.
15. Lewis, D. C., Jasin, H. E., and Ziff, M.:
Cellular responses in the synovial membrane: arthritis produced by endotoxin and by phytohemagglutinin. Arthritis Rheum. 9:863, 1966.
16. Wyllie, J. C., More, R. H., and Hanst, M. D.:
The fine structure of normal guinea pig synovium.
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17. Ball, J., Chapman, J. A., and Muirden, K. D.:
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