close

Вход

Забыли?

вход по аккаунту

?

Susceptibility of cultures of rheumatoid tissues to rubella virus infection.

код для вставкиСкачать
Susceptibility of Cultures of Rheumatoid Tissues
to Rubella Virus Infection
Michael D. Parker, Donald E. McCollum and Grace P. Kerby
Previous reports by others of resistance of cultured rheumatoid synovial
cells to experimental virus infection prompted extension of such studies
to rheumatoid fibroblasts from other tissues. Strains of cells derived from
rheumatoid skin, synovium and a nodule and from nonrheumatoid skin
and synovium were inoculated in vitro with rubella virus (RV). All of the
tissues studied proved equally susceptible to RV infection.
Rheumatoid synovial cells grown as a monolayer in tissue culture differ from cells derived
similarly from patients with other types of arthritis. To summarize briefly, rheumatoid cells
show increased lysosomal activity (1), solubilize
cartilage matrix (2), have a decreased life
span (3), form hyaluronic acid at an accelerated
rate but of abnormal intrinsic viscosity (4, 5),
metabolize glucose more rapidly (5), are less
responsive to hydrocortisone suppression in
formation of hyaluronate (4), are less affected
by polypeptide “activator” ( 5 ) , and are reported to be completely resistant to infection
with RV (6) and relatively resistant to infection with Newcastle Disease Virus (NDV)
(7). The increased lysosomal activity, the
more rapid metabolism of glucose and the
From the Department of Medicine, Division of Rheumatic and Genetic Diseases, and the Department of
Surgery, Division of Orthopaedic Surgery, Duke Hospital,
Durham, North Carolina.
MICHAEL D. PARKER, MD: Fellow in Rheumatology,
Department of Medicine, Duke Hospital, Durham, North
Carolina; DONALD E. MCCOLLUM, MD: Professor of Orthopaedic Surgery, Duke Hospital, Durham, North Carolina; GRACE P. KERBY, MU: Professor of Medicine, Duke
Hospital, Durham, North Carolina.
Supported by United States Public Health Service Grant
AM 12441.
Reprint requests should be addressed to: Dr. Grace P.
Kerby.
Submittedfor publication Oct 25,1971; accepted Jan 12,
1972.
ability to differentiate A and B type synovial
cells are properties of both intact rheumatoid synovium (8-1 1) and cultured cells
(1, 3, 5). These observations demonstrate
that cells grown in vitro from rheumatoid
synovium retain properties of the intact
synovium from which they were derived. T h e
studies (6, 7) reporting resistance of cultured
rheumatoid synovial fibroblasts to experimental virus infection, therefore have increased speculation that a virus infection is
important in the pathogenesis of rheumatoid
arthritis (RA) (3, 5, 6, 12).
The present study was undertaken to determine if fibroblasts derived from rheumatoid
skin and rheumatoid nodules would also show
the resistance to RV infection reported by
Grayzel and Beck (6) for rheumatoid synovial
fibroblasts. For this purpose, rheumatoid skin
and nodule fibroblasts were inoculated with
RV. Strains of nonrheumatoid skin, rheumatoid
synovium and nonrheumatoid synovium served
as controls for comparison. Rubella virus was
assayed from the supernatant medium of virusinfected cultures.
Resultant data indicated that fibroblasts from
rheumatoid skin and a single rheumatoid nodule uniformly were susceptible to RV infection. Unexpectedly, rheumatoid synovial
fibroblasts also showed no resistance to RV
infection under the conditions employed.
Arthritis and Rheumatism, Vol. 15, No. 3 (May-June 1972)
275
PARKER E l AL
Table 1. Source of Surgical Tissue Cultures
~
Patients
______
_ _ _ _ _ _ _ ~
Age
Race
Sex
Clinical diagnosis
Tissue'
N
W
W
W
W
W
W
W
W
M
M
DHW
DW
CH
EM
GH
AR
JB
MK
BT
62
61
51
61
56
71
23
42
19
24
50
Sk
Sk, SY
Sk
Sk
Sk, SY
SY
SY
Sk, SY
SY
SY
Sk
CP
MS
PH
JM
ED
BH
IC
55
50
37
41
53
61
54
W
W
Osteoarthritis
Osteoarthritis
Osteoarthritis
Osteoarthritis
Osteoarthritis
Osteoarthritis
Osteoarthritis
Trauma
Trauma
Trauma
Aseptic necrosis
of femoral head
Rheumatoid arthritist
Rheumatoid arthritis
Rheumatoid arthritis
Rheumatoid arthritis
Rheumatoid arthritis
Rheumatoid arthritis
Rheumatoid arthritis
HL
cc
N
N
w
W
W
W
W
F
M
F
F
F
F
M
M
M
F
F
F
F
F
F
F
Sk
Sk. SY
Sk. SY
Sk
Sk, SY
Sk, SY. N
SY
"Sy = Synovium, Sk = Skin, N = Rheumatoid nodule
tRheumatoid patients satisfied the criteria of the American Rheumatism Association for the diagnosis of rheumatoid arthritis.
MATERIALS AND METHODS
line of green monkey kidney cells by the method of Liebhaber (14) and stored in aliquots at -90" C for later use as
inoculum for the cultured tissues.
Tissue Cultures
Rheumatoid and nonrheumatoid synovium, skin and a
single rheumatoid nodule were cultured from surgical specimens in Dulbecco's Medium plus 15% heat-inactivated
fetal calf serum (growth medium). Incubations were carried
out at 37O C in a constantly-flowing moist atmosphere of air
and 10%COz The synovial lining membrane was carefully
dissected from underlying tissues and sliced into 1 to 2 sq
mm fragments for use in the cultures. Skin was cultured as
previously described (13). The rheumatoid nodule was
enucleated from the surrounding normal tissue and the
central portion was cultured as 1 to 2 sq mm fragments.
Growth medium was changed every 5 days. By 4 to 8
weeks, fibroblasts had grown out from the tissues to form a
confluent monolayer. Cell strains were harvested and subcultured by brief exposure to 250 mg% trypsin in calciumand magnesium-free bicarbonate buffered saline. Thirteen
strains of skin, 12 strains of synovium and 1 strain of
rheumatoid nodule fibroblasts were established (Table 1).
Cells were used in the experiments from the third to fifth
passage.
Preparationof Rubella Virus lnoculum
Rubella virus strain Mag V' was cultured in the Vero
276
Tissue Inoculationwith Rubella Virus
Skin Specimens. Duplicate 30 ml plastic disposable
flasks were inoculated with 3 x lo5 rheumatoid or
nonrheumatoid skin fibroblasts. The cells were grown to
confluence, washed with phosphate-buffered saline (PBS)
and inoculated with RV, 5 x lo6 plaque-forming particles
(pfp). Rubella virus-infected cultures were grown in virus
growth medium (VGM) containing minimal essential medium plus 5% heat-inactivated, kaolin-treated, agammaglobulin, newborn calf serum (14). After 24 hours, the
cell sheet was washed carefully with PBS and fresh VGM
was added. Virus growth medium was changed daily and on
days 5, 10 and 15 was stored at -90" C for later RV titration. Control noninoculated cells were handled in an
identical manner except that .1 ml VGM was substituted
for the RV inoculum. All cultures were placed on a turntable revolving at .2 rpm and inclined at 2" to effect constant
mixing.
* Kindly supplied by D r H. Liebhaber.
Arthritis and Rheumatism, Vol. 15, No. 3 (May-June 1972)
RHEUMATOID TISSUES AND RUBELLA VIRUS
R A SKIN
5-Day I n c u b a t i o n
10-Day I n c u b a t i o n
.
c
E
a
15-Day I n c u b a t i o n
-6
CP
PH
ED
BH
JW
60 mm plastic disSynovial S p c c i t ~ t ~Duplicate
~.
posable petri plates were planted with 2 x lo5 synovial
cells. Petri plates were incubated at 37" C overnight to allow cell attachment and then were inoculated with RV, l x
1O6 pfp. After 1 hour incubation the cell sheet was washed
carefully and incubated at 37' in growth medium. O n the
fifth and twelfth days of incubation the cell sheet was
washed three times with PBS and flooded with 3.0 ml of
Dulbecco's medium plus 2% heat-inactivated fetal calf
serum (15). After 48 hours incubation (ie, on the seventh
and fourteenth days of incubation), the medium was aspirated and stored at -90" for later titration. Control noninoculated cultures were handled in the same way except
that an equal volume of medium was used instead of the RV
inoculum.
Titration of Rubella Virus
Supernatant medium obtained from RV-inoculated tissue
cultures was titered for RV by the hemadsorption negative
plaque assay* of Marcus and Carver (16). Titers of RV
were expressed as pfp/ml. Appearance of plaques was prevented in controls by preincubation of the RV inoculum
with specific RV antiserum. A second series of RV titrations
was performed by the Vero cell cytopathic assay of
Liebhaber (17) using Vero cell cultures grown in optically
clear Falcon Plastic flasks. Flasks were observed directly for
development of RV-related cytopathic effect by using an
inverted phase microscope. T o determine whether any observed cytopathic effect was RV-related and not due to a
contaminant, the medium from the Vero cell flasks was assayed on day 10 for RV-specific hemagglutination by the
method of Stewart (18). Specificity of the hemagglutination
was clearly shown by use of convalescent human RV
* The Yale strain of GMK cells was kindly supplied by
D r P. 1. Marcus.
MS
Fig 1. Titers (in plaquefor ming particles/ml) of rube1la
virus (RV) assayed from supernatant medium of infected rheumatoid skin fibroblast cultures
5. 10 and 15 days following RV
inoculation. Initials identify
patients described in Table i.
antiserum (Microbiological Associates) and hyperimmune
rabbit RV (strain RA 27/3) antiserum (Flow Laboratories,
Inc). Such antiserum (in appropriate dilution and pretreated with kaolin and erythrocytes to remove nonantibody
inhibitors of agglutination and nonspecific agglutinins)
caused complete inhibition of the agglutination. Pre-rubella
virus immunization rabbit serum did not inhibit the agglutination. Furthermore, the hyperimmune antisera used did
not nonspecifically inhibit hemagglutination produced by
another virus, NDV.* At no time did supernatant medium
from noninoculated Vero cells cause spontaneous hemagglutination even after three weeks of incubation. By ten
days, supernatant medium from positive Vero cell cultures
always showed complete hemagglutination, whereas negative cultures showed no agglutination. Cultures not showing
complete hemagglutination by ten days did not subsequently become positive for RV-related hemagglutination or cytopathic effect over the next ten days. Positive hemagglutination appeared before marked cytopathic effect. The cytopathic effect was entirely like that described by Liebhaber (17) and was prevented by preincubation of the RV
with hyperimmune RV antiserum.
Acid Phosphatase
Intracellular lysosomal content of coverslip cultures of
synovium, skin and the rheumatoid nodule was estimated
by histochemical demonstration of acid phosphatase activity
by methods previously reported (I).
RESULTS
Growth of RV in Skin Fibroblasts
T h e RV titers of the supernatant fluid from
*American Type Culture Collection
Roakin, kindly supplied by D r T. R. Cate.
Arthritis and Rheumatism, Vol. 15, No. 3 (Yay-June 1972)
strain
N J-
277
PARKER ET AL
HC
AR
DHW
CC
OW
ET
experimentally infected rheumatoid and
nonrheumatoid skin fibroblast cultures are
shown in Figures 1 and 2. Rubella virus titers
of 1 x 104 to 1 x 106 pfp/ml were most frequently obtained, the range being 2.5 x lo3 to
2.3 x 106 pfp/ml. No overall difference was
apparent in RV titers of supernates from
rheumatoid and nonrheumatoid skin cultures,
although variation in titers was greater in
supernates from nonrheumatoid skin cultures.
At no time during the 15 days of incubation was
any cytopathic effect apparent in any of the
cultures. Two rheumatoid and two nonrheumatoid cell lines incubated for 21 days after RV
inoculation also failed to show any cytopathic
effect. When the titrations of supernates were
repeated using the Vero cell cytopathic assay for
RV, results were essentially the same.
Growth of RV in Synovial Fibroblasts
Titers of RV assayed from supernatant fluids
from infected synovial fibroblast cultures by the
RV-plaque assay method are shown in Figures
3 and 4. Titers were remarkably similar, all
being in the range of 1 x l o 3 to 1 x l o 4 pfp/
ml. There was no difference between RV titers
assayed from rheumatoid and nonrheumatoid
synovial fibroblasts, respectively. Figure 5 illustrates the appearance of hemadsorptionnegative plaques in an assay of supernatant
278
CH
Fig 2. Titers of rubella virus
(RV) assayed from supernatant
medium of infected non-rheumatoid skin fibroblast cultures
5, 10 and 15 days following RV
inoculation. Abbreviations as in
Fig 1.
fluid from RV-infected rheumatoid synovium.
Repeat titration for RV by the Vero Cell cytopathic assay method confirmed the ability of
rheumatoid synovium to support RV growth
in titer s e q u a l t o those observed with
nonrheumatoid synovium.
The titer of RV derived from a single strain
of rheumatoid nodule fibroblasts was 2.1 x lo3
on day 7 and 3.5 x lo3 on day 14 following
inoculation. These values were similar to those
found with rheumatoid and nonrheumatoid
synovial fibroblasts.
Acid Phosphatase (Lysosomal) Content
and Pattern
Synovial fibroblasts exhibited more intense
histochemical staining for acid phosphatase
than did skin fibroblasts. In all cell strains the
amount and distribution of acid phosphatase
varied greatly from one cell to another. Some
but not all rheumatoid synovial fibroblasts displayed a singular pattern of diffuse cytoplasmic
staining for acid phosphatase. This staining
pattern was not evident in nonrheumatoid
synovial fibroblasts or skin fibroblasts.
DISCUSSION
Interest in the question of whether virus
infection may be important in the pathogenesis
Arthritis and Rheumatism, Vol. 15, No. 3 (May-June 1972)
RHEUMATOID TISSUES AND RUBELLA VIRUS
I
R A SYNOVIUM
a
7-Day Incubation
&$@
14-Day Incubation
4
In
$
--
10'
0
0)
-2
lo2
(z
Fig 3. Titers of rubella virus
(RV) assayed from supernatant
medium of infected rheumatoid
synovial fibroblast cultures 7
and 14 days following RV inoculation. Abbreviations as in Fig 1.
10'
BH
PH
ED
MS
IC
of RA (3, 5, 6, 12) is sharpened by the fact that
two persistent characteristics of rheumatoid
synovium in vitro (accelerated synthesis of
hyaluronate and increased cellular acid phosphatase content) can be induced experimentally
in specific cell systems by exogenous virus
infection (19, 20). Although at present there is
no direct evidence that a virus is basic to the
pathogenesis of rheumatoid disease, the recent
study (6) demonstrating an absolute resistance
of rheumatoid synovium to experimental RV
infection suggested the possibility that a previously established virus infection might exist in
rheumatoid synovium.
Because of the potential importance of the
RV resistance study to an understanding of the
pathogenesis of RA, the present investigation
was undertaken to explore resistance of other
rheumatoid tissues, namely skin and a rheumatoid nodule, to RV infection. No such
resistance could be demonstrated. Fibroblasts
derived from rheumatoid skin and a rheumatoid
nodule supported growth of RV to levels of titer
equal to those found with fibroblasts derived
from nonrheumatoid skin.
T h e possible implications of this finding are
clouded by the subsequent finding that strains
of rheumatoid synovial fibroblasts used in this
study were invariably susceptible to RV infection, an observation contrary to that reported
NON-RA SYNOVIUM
7-Day Incubation
.
e
14-Day Incubation
E
.e 10'
e
g
u)
103
0
Fig 4. Titers of rubella virus
(RV) assayed from supernatant
medium of infected non-rheumatoid synovial fibroblast cultures 7 and 14 days following
RV inoculation. Abbreviations as
in Fig 1.
=
u
n lo2
10'
AR
Arthritis and Rheumatism, Vol. 15, No. 3 (May-June 1972)
EM
CC
CH
GH
JB
MK
279
PARKER ET AL
Fig 5. Rubella virus (RV) titration from a rheumatoid synovial fibroblast culture. Petri plate demonstrates hemadsorption-negative (dark) plaques
representing cell areas infected with RV. lnoculum
for this plaque assay was supernatant medium
from RV-infected rheumatoid synoviurn 14 days
following virus inoculation.
by Grayzel and Beck (6). Rubella virus was
detected in the supernatant fluids of rheumatoid
synovial fibroblasts in the same titers as those
from nonrheumatoid synovial fibroblasts. Use
of commercial RV hyperimmune antiserum
identified both the plaque-forming agent and
the Vero cell cytopathic agent in this study as
RV. By using two RV assay systems, dependent
upon three different properties of the virus
(production of specific hemagglutinin, development of cytopathic effect in Vero cells and
prevention of cellular hemadsorption associated
with NDV), the likelihood that a virus contaminant was being assayed seems remote.
It was possible that the conditions under
which the synovial fibroblasts were cultured
favored outgrowth of fibroblasts essentially
different from those studied by others. T o test
this possibility (that the fibroblasts cultured
here from rheumatoid synovium did not differ
fundamentally from those cultured here from
nonrheumatoid synovium) the cells were
280
stained histochemically for acid phosphatase.
Diffuse cytoplasmic staining for acid phosphatase was seen only in rheumatoid synovial
fibroblast cultures, a characteristic finding
previously reported (1). Despite this documentation of the basic similarity between the
rheumatoid synovial fibroblasts used in this
study and those grown by others, the cell
population may be heterogeneous with RV
susceptible cells masking the presence of RV
resistant cells.
T h e demonstration of RV resistance in
rheumatoid synovium may be dependent upon
use of a particular strain of RV. The RV strain
used in the present study was not the one used
by Grayzel and Beck (6) but was identified as
R V by serological methods, using hyperimmune antiserum against both RA 27/3 strain
of RV and naturally acquired RV. However,
certain strains of RV recovered from natural
cases of rubella and propagated in vitro have
subsequently displayed altered growth potential in a specific cell line as a result of adaptation during previous passages (21).
Patients from whom tissues were derived
were not studied serologically for evidence of
RV immunity, however, it has been shown that
fibroblasts derived from patients demonstrating
serological immunity to RV nevertheless support RV growth in vitro (22). Moreover, it has
been reported by Chandler, Robinson and Masi
that titers of RV antibody of patients with RA
do not differ significantly from those of
controls (12).
Grayzel and Beck reported both resistance to
RV in rheumatoid synovial cells and development of RV-related cytopathic effect in
nonrheumatoid cells (6). Rubella virus-infected
nonrheumatoid synovial cells in this study did
not develop cytopathic effect even after 21 days
incubation. Cytopathic effect was observed,
however, under special circumstances but was
not related to RV. Synovial fibroblast studies
were initially performed by the method used for
skin fibroblasts designed to maximize cellular
RV growth. Under these experimental condi-
Arthritis and Rheumatism, Vol. 15, No. 3 (Yay-June 1972)
RHEUMATOID TISSUES AND RUBELLA VIRUS
tions nonrheumatoid, but not rheumatoid,
synovial fibroblasts developed cytopathic effects
leading to cell death in 10 to 14 days. However,
control cultures of nonrheumatoid cell lines not
infected with RV also developed cytopathic
effects under these special experimental conditions. It was clearly shown that the cytopathic
effect was related to use of agammaglobulin,
kaolin-treated, newborn calf serum in the virus
growth medium. T h e difference between
rheumatoid and nonrheumatoid synovial cell
response to this agent was striking and
unexplained. It may be of importance.
In summary, the present study demonstrated
that RV could be grown in fibroblasts cultured
from all rheumatoid and nonrheumatoid tissues
studied, including skin, synovium and a rheumatoid nodule. This finding clearly does not
invalidate the earlier report bf Grayzel and
Beck (6) describing a state of RV resistance in
rheumatoid synovium, but it does suggest that
conditions under which such a state is demonstrable may be restricted.
ACKNOWLEDGMENT
The authors are indebted to Dr J. W. Beard and Dr A.
Langlois for their generous assistance and advice during tbe
establishment of the tissue culture laboratory used for these
studies.
REFERENCES
1. Goldfischer S, Smith C, Hamerman D: Altered
acid hydrolase activities in rheumatoid synovial
cells in culture. Am J Pathol52:569-578, 1968
2. Hamerman D, Janis R, Smith C: Cartilage matrix depletion by rheumatoid synovial cells in
tissue culture. J Exp Med 126:1005-1012,
1967
3. Smith C, Hamerman D: Significance of persistent differences between normal and rheumatoid
synovial membrane cells in culture. Arthritis
Rheum 12:639-645,1969
4. Castor CW, Dorstewitz EL: Abnormalities of
connective tissue cells cultured from patients
with rheumatoid arthritis. I. Relative unresponsiveness of rheumatoid synovial cells to hydrocortisone. J Lab Clin Med 68:300-313,
1966
5. Castor CW, Smith SF, Ritchie JC, et al: Connective tissue activation. 11. Abnormalities of
cultured rheumatoid synovial cells. Arthritis
Rheum 14:55-65,1971
6. Grayzel AI, Beck C: Rubella infection of synovial cells and the resistance of cells derived from
patients with rheumatoid arthritis. J Exp Med
126:367-373,1970
7. Smith C: Resistance of cultured synovial cells to
infection with exogenous virus. In Proceedings
of the Conference on Atypical Virus Infections-Possible
Relevance to Animal Models
and Rheumatic Disease. Edited by C L Christian, PE Phillips, RC Williams. New York, the
Arthritis Foundation, 1971, pp 26-30
8. Luscombe M : Acid phosphatase and catheptic
activity in rheumatoid synovial tissue. Nature
197:1010,1963
9. Roberts JE, McLees BD, Kerby GP: Pathways
of glucose metabolism in rheumatoid and nonrheumatoid synovial membrane. J Lab Clin
Med70:503-511,1967
10. Dingle J T M , Thomas DPP: In vitro studies on
human synovial membrane. A metabolic comparison of normal and rheumatdid tissue. Br J
Exp Pathol37:318-323,1956
11. Barland P, Novikoff AB, Hamerman D: Electron microscopy of the human synovial membrane. J Cell Biol 14:207-214,1962
12. Chandler RW, Robinson H, Masi AT: Serological investigations for evidence of an infectious
aetiology of rheumatoid arthritis. Ann Rheum
Dis 30:274-278,1971
13. Kelley WN, Wyngaarden JB: Effects of allopurinol and oxypurinol on purine synthesis in
cultured human cells. J Clin Invest 49:602609,1970
4. Liebhaber H, Pajot T , Riordan JT: Growth of
high titered rubella virus in roller bottle cultures
of V e r o cells. P r o c S a c E x p Biol M e d
130:12-14,1969
5. Parkman PD, Hopps HE, Meyer H M : Rubella
virus isolation, characterization, and laboratory
diagnosis. Am J Dis Child 118:68-77,1969
6. Marcus P I , Carver D H : Hemadsorptionnegative plaque test: New assay for rubella virus
revealing a unique interference. Science
149:983-986,1965
17. Liebhaber H, Riordan JT, Horstman DM:
Arthritic and Rheumatism, Vol. 15, No. 3 (May-June 1972)
281
PARKER ET AL
Replication of rubella virus in a continuous line
of African green monkey kidney cells (Vero).
Proc SOC Exp Biol Med 125:636-643,1967
18. Stewart GL, Parkman PD, Hopps HE, et al:
Rubella-virus hemagglutination-inhibition test.
N Engl J Med 276:554-557, 1967
19. Allison AC, Mallucci L: Histochemical studies
of lysosomes and lysosomal enzymes in virusinfected cell cultures. J Exp Med 121:477-478,
1965
20. Ishimoto N, Temin H M , Strominger J L :
Studies of carcinogenesis by avian sarcoma viruses. 11. Virus induced increase in hyaluronic
acid synthetase in chicken fibroblasts. J Biol
Chem 241 :2052-2057, 1966
21. Vaheri A, Sedwick WD, Plotkin SA: Growth of
rubella virus in BHK21 cells. I. Production, assay, and adaptation of virus. Proc SOCExp Biol
Med 125:1086-1092,1967
22. Heggie AD: Pathogenesis of the rubella
exanthem. Isolation of rubella virus from the
skin. N Engl J Med 285:664-667,1971
CAROL-NACHMAN-PRIZE FOR RHEUMATOLOGY
The purpose of this prize is to serve t h e advancement of clinical, therapeutic and experimental
research in the field of rheumatology. It will be awarded for outstanding research papers. For
the first awarding of the prize for 1972, information may be obtained from the SECRETARY OF
THE
BOARD
OF TRUSTEES, DR.
BADEN, GERMANY.
282
K.
MIEHLKE,
HEAD
PHYSICIAN,
RHEUMATICS
CLINIC,
WIES-
Deadline for submission of papers is on or before July 1, 1972.
Arthritis and Rheumatism, Vol. 15, No. 3 (May-June 1972)
Документ
Категория
Без категории
Просмотров
0
Размер файла
533 Кб
Теги
virus, culture, infectious, tissue, rubella, susceptibility, rheumatoid
1/--страниц
Пожаловаться на содержимое документа