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Mseleni disease serum is not harmful to cultured chondrocytes.

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BRIEF REPORT
MSELENI DISEASE SERUM IS NOT HARMFUL TO CULTURED
CHONDROCYTES
YU CHANGLONG, JOHN E. FINCHAM, GEORGE C. WRIGHT, JR.,
JOSHUA J. F. TALJAARD, and LEON SOKOLOFF
The origins of Mseleni disease, an acquired
polyarticular degenerative joint disease, are unknown.
We examined sera from 12 patients with the disease, 5
unaffected Mseleni residents, and 5 Durban residents.
The effects of sera from the 12 patients with Mseleni
disease on DNA or sulfated proteoglycan synthesis by
cultured rabbit or human infant articular chondrocytes
were no different from those of control sera. Cells
derived from 2 children contained no stainable DR
antigens; coculture had no impact on either of the above
measures of cell function.
Mseleni disease is an acquired polyarticular
degenerative joint disease that is endemic to northern
Zululand, Republic of South Africa. Almost nothing is
known of its etiology, but speculation has included
nutritional deficiencies, toxic compounds, and even
slow-acting virus infection (14). It has been proposed
From the Department of Pathology, State University of
New York at Stony Brook, and the National Research Institute for
Nutritional Diseases, the South African Medical Research Council,
Tygerberg, Republic of South Africa.
Supported by grant AM-17258-11 from the National Institutes of Health.
Yu Changlong, MD: Visiting Scholar, Sports Medicine
Institute, Beijing, People’s Republic of China; John E. Fincham,
BVSc: National Research Institute for Nutritional Diseases, South
African Medical Research Council, Tygerberg, Republic of South
Africa; George C. Wright, Jr., MS: Research Associate, Department
of Pathology, State University of New York at Stony Brook; Joshua
J. F. Taljaard, MD: Professor and Head, Chemical Pathology,
Tygerberg Hospital, Tygerberg, Republic of South Africa; Leon
Sokoioff, MD: Professor of Pathology, State University of New
York at Stony Brook.
Address reprint requests to Leon Sokoloff, MD, Department of Pathology, Health Sciences Center, State University of
New York at Stony Brook, Stony Brook, NY 11734.
Submitted for publication January 2, 1986; accepted in
revised form August 12, 1986.
Arthritis and Rheumatism, Vol. 30, No. 3 (March 1987)
elsewhere that chondrocytes of skeletal cartilages are
the selective target of the damaging agent; also presented elsewhere is the rationale for cell culture methods used to determine the nature of the causative
factor (1). The following study was undertaken to
probe whether a harmful agent in any of the 3 categories above could be detected in the serum of affected
individuals. None was found.
PATIENTS AND MEHODS
Sera. The study subjects were 12 adult patients
with Mseleni disease, 5 unaffected residents of the
village of Mseleni, and 4 black subjects from the city of
Durban (Table 1). None of the subjects were receiving
prescribed medication. Blood was drawn into trace
element-analysis grade Vacutainer tubes (6526; Becton Dickinson, Sunnyvale, CA). One aliquot of serum
was transferred to acid-cleaned, autoclaved borosilicate bottles and shipped air freight on dry ice to State
University of New York (SUNY) at Stony Brook.
Another aliquot was analyzed on a Technicon Autoanalyzer for its content of total protein, albumin,
calcium, inorganic orthophosphate, magnesium, yglutamyl transpeptidase, and alkaline phosphatase,
and for immunoreactive parathyroid hormone (Cterminal analysis; Immuno Nuclear, Stillwater, MN).
Sera were ultrafiltered through Millex HA sterilizing
filters, 0.45 Frn (Millipore, Bedford, MA), immediately
prior to use. A pool of serum was prepared from 3
adult laboratory personnel at SUNY as a standard for
the African specimens.
Culture methods. Tests were carried out on
articular chondrocytes derived from rabbit and human
infant sources. The rabbit cells were used to determine
w
5OIM
42lF
50lF
32lF
50lF
40lM
55lM
65lF
50lF
30lF
71lM
38lF
48 t 3.5
46lM
4UF
281F
46lM
41 t 4.27
74
69
73
71
72 2 1.11
*
76
76
77
78
ND
77 0.48
72
72
80
81
74
74
63
78
73
69
68
78
74 t 1.52
TP
41
36
40
20
34 t 4.87
43
39
42
40
ND
41 t 0.91
42
39
42
43
39
34
36
33
40
34
37
40
38 t 1.0
Alb
2.23
2.13
2.25
I .85
2.12 t 0.09
2. I5
2.30
2.33
2.30
ND
2.27 t 0.041
2.25
2.20
2.30
2.33
2. I8
2.00
2.03
2. I5
2.28
2.20
2. I0
2.40
2.2 2 0.035
Ca
0.77
0.78
0.61
0.68
0.71 t 0.04
1.07
1.20
0.97
I .36
1.15 t 0.084
49
43
52
I77
t 32.3
38
68
77
91
ND
69 2 11.2
63
59
53
67
78
76
61
32
I48
52
53
105
71 t 8.71
Akl Ph
0
2
0
80
21 2 19.8 80
_f
0
6
II
0
ND
4 2.7
82
0
16
0
0
73
0
0
0
28
5
0
17 ? 8.6
0.95
0.90
0.75
0.76
0.75
0.82
0.93
0.62
0.67
0.78
0.74
0.80
0.79 t 0.029
0.77
0.81
0.70
0.73
ND
0.75 t 0.024
CGT
ME
I .07
I .36
1.65
I .26
ND
1.34 t 0.121
1.49
1.52
I .52
0.81
1.29
1.71
1.16
I .07
I .39
0.84
1.10
0.87
1.23 t 0.027
Pi
Serum composition*
15.2
15.6
10.1
24.3
16.3 t 2.94
9.8
11.0
10.3
9.0
ND
10 2 0.42
101.4
164.9
173.0
48.6
124.5 t 27.2
125.5
96.0
170.5
192.8
95.1
136 2 19.7
205.8
190.0
205.0
98.8
61.2
86.4
40.5
89.8
87.3
158.0
143.2
141.1
125.6 ~t16.25
24.6
13.3
8.5
10.5
13.9
9.9
14.0
10.8
15. I
42.0
13.5
14.6
15.9 t 2.65
DNA
(5% control)
iPTH
'W4
65.4
58.3
56. I
56.2
59 t 2.19
37.2
34.9
26.7
30.2
76.5
41.1 t 9.03
20.9
26.2
24.4
52.3
88.0
109.7
81.1
71.4
42.4
50.3
67.9
63.7
58.2 t 7.91
(5% control)
Rabbit
Humant
39. I
35.9
31.1
36.5
35.6 t 1.67
36.2
36.2
33.5
43.9
ND
37.5 t 2.24
27.8
39.8
37.3
35.4
29.4
38.3
25.1
41.7
40.9
35.8
24.5
27.7
33.6 t 1.83
DNA
(dflask)
Chondrocyte cultures
* Serum composition measured as follows: TP = total protein, gdliter; Alb = albumin, gmlliter; Ca = calcium, mmolesfiiter; Pi = inorganic orthophosphate. mmoleslliter: Mg =
magnesium, mmoleslliter; CGT = yglutamyl transpeptidase, units/liter; Alk Ph = alkaline phosphatase, units/liter; iPTH = immunoreactive parathyroid hormone, pmoles/liter; ND =
not done.
t Human chondrocytes cultured with serum pooled from 3 unaffected American individuals and evaluated as a standard for the African specimens (see Patients and Methods for
details).
Mean t SEM
Durban controls
53lM
38lF
44lF
28lF
65lF
Mean t SEM 46 t 6.3
Mean 2 SEM
Unaffected
Mseleni
residents
Mseleni disease
Aeelsex
Serum composition and effect on cultured chondrocytes
Serum grow
Table 1.
350
-
BRIEF REPORTS
6
T
RABBIT
HUMAN
2 5
a
m
+
za
4
?
n
‘2
2
07
E* ,
n
n
C
Y
u)
2
50
40
\
m
& 30
-
2
20
I0
-
0
I0
I
2.5
5
Illl
2.5
10
SERUM (%)
1
5
40
Figure 1. Effect of human serum concentration on growth and
sulfated proteoglycan synthesis by rabbit and human infant articular
chondrocytes in monolayer culture. The scatter bar represents mean
_f
SEM (n = 5 ) .
the effect of the sera on both growth-promoting activity and on sulfated proteoglycan synthesis. The human
cells were studied for growth-promoting activity only.
The tests on the rabbit cells were run in 4 separate
experiments, using rabbit chondrocytes in their first
passage, 5 flasks in each group. The culture methods
used were identical to those detailed in other publications from obr laboraiory (5-7). The human cells were
autopsy specimens obtained from the knees of a
newborn and a 4-month-old infant. These cells were
initiated by explant culture. The chondrocytes were
combined to provide the requisite numbers. After
initial monolayer culture with human serum, they were
subcultured with 10% fetal bovine serum (FBS).
Preliminary experiments were carried out with
4 different concentrations of the American control
serum to determine the minimum volume required for
both species of chondrocyte, and to conserve the
limited amount of African samples which were available (Figure 1). The tests were carried out in Dulbecco-Vogt modified Eagle’s medium (DMEM), 450
mg of glucose/dl. The inoculum size was 3 x lo5
chondrocytes/T25 flask. The cells were first cultured
for 24 hours in DMEM supplemented with 10% FBS.
The medium was then replaced by DMEM plus 5%
test sera. Rabbit chondrocyte cultures were fed a
second time at 48 hours and labeled with radiosulfate
(3 pCi/mmole, 1 pCi/ml) for 20 hours prior to harvest
(total exposure to the test serum 96 hours). The
content of DNA in each flask was measured by the
diphenylamine method and radiosulfate incorporation
into macromolecules in the medium and trypsin fractions as previously described (5-7). Radioactivity is
reported as disintegrations per minute/pg of DNA. To
ensure distribution of the various serum test groups in
the 4 experiments, the sera were assigned by the
research associate, and then all tests were carried out
“blind” by the senior author. The American serum
was run in each experiment to standardize the data.
Statistical analysis. Because the tests on rabbit
chondrocytes were carried out in 4 separate experiments, the DNA and radiosulfate data were normalized to 100%. This was achieved by dividing the actual
values observed for the various African sera by that of
the corresponding American control. The human
chondrocytes were tested in a single experiment, and
the data were reported in absolute values. Pearson
correlations, univariate and multivariate analysis of
variance tests, and stepwise regression tests were
analyzed for cell culture, serum constituents, age, and
sex of the various groups (8).
RESULTS
Effect of serum concentration. For both the
rabbit and the human infant chondrocytes, 5% human
serum was approximately as effective as 10% FBS in
supporting growth (Figure 1). As the concentration of
human serum increased from 1.5%10%, in the rabbit
chondrocyte cultures, radiosulfate uptake declined,
and was never as great as the uptake with 10% FBS.
Radiosulfate incorporation by the human cells was
very low for both types of serum and at all concentrations.
African test sera. There were no significant
differences between the 2 control groups, nor between
the subjects with Mseleni disease and the control
subjects, with respect to the cell culture data or the
serum composition (Table 1). The DNA content of the
flasks and radiosulfate incorporation by rabbit
chondrocytes were negatively correlated (r = -0.684,
P < 0.001). Values of DNA for rabbit and human
chondrocytes did not correlate with each other.
351
BRIEF REPORTS
Table 2. Effect of coculture of human chondrocytes on response to serum: 2 further studies using
human chondrocytes separately and in combination*
Radiosulfate incorporation
Culture
Serum
H 104
H106
H104
+ H106
FBS
HS
FBS
HS
FBS
HS
DNA
(widflask)
43.3
49.1
37.9
47.4
42.6
48.6
t 1.19
t 1.09
t 1.69
t 0.95
t 1.25
2
0.57
10-3
dpm/pg DNA
%
pericellular
1.1 t 0.06
1.1 f 0.09
1.3 t 0.13
1.0 2 0.03
1.3 t 0.10
1.1 t 0.07
13.8 2 0.85
13.4 f 1.14
15.2 t 0.76
12.7 2 0.36
15.3 t 0.97
13.2 t 0.48
* Values given are mean t SEM (n = 5). FBS = 10% fetal bovine serum; HS = 10% human serum,
obtained from 3 unaffected American subjects (see Patients and Methods for details).
Radiosulfate incorporation by rabbit chondrocytes
correlated positively with serum calcium levels (P =
0.02). Sera that had elevated y-glutamyl transpeptidase levels contained significantly less calcium, inorganic orthophosphate, and albumin than did those without increased yglutamyl transpeptidase (P< 0.05).
Further studies of human chondrocytes. A question was raised about possible anomalous behavior of
the human chondrocytes, because the cells had been
cocultured from 2 different donors, and particularly
because class I1 antigens are expressed by activated
chondrocytes (9). For this reason, 2 further studies
were carried out with chondrocytes from 2 human
donors, ages 6 and 10 years. It was not possible to
employ the original strains because 1 had senesced in
vitro.
Growth and radiosulfate incorporation were
investigated, as described above, in the 2 chondrocyte
cultures, separately and in combination, with 10%
FBS or human serum. The data disclosed no interaction of the cocultures distinct from that of the individuals separately (Table 2).
Class I1 antigens were immunocytochemically
sought, using a mouse monoclonal antibody to human
HLA-DR (Becton Dickinson). The cells were grown
on glass coverslips, air-dried, and fixed in acetone.
Table 3. Effect of Mseleni disease serum on human chondrocytes
from a single donor*
Mseleni disease (6)
Mseleni control (4)
Durban control (4)
* Values given are mean
25.7 2 2.3
29.6 t 1.9
31.8 t 3.9
t SEM (n = 3).
336 2 26.5
268 f 25.4
312 f 27.4
Staining was carried out with biotinylated horseradish
peroxidase and avidin and run with positive and negative controls. Five hundred cells were counted. No
staining was found on the surface of the surviving
infant chondrocyte strain from the original study or on
those of the supplementary cultures. Pretreatment of
the coverslips with chondroitinase AC (Sigma, St.
Louis, MO) did not unmask stainable antigen.
In addition, sufficient serum remained to carry
out a smaller number of tests on chondrocytes from a
3-month-old infant. No significant differences in cell
proliferation or radiosulfate incorporation were found
among the 3 African groups (Table 3).
DISCUSSION
The effect of serum on growth and expression
of articular chondrocytes is powerful and is subject to
many variables, including specificities of serum and
chondrocyte according to species (9,batch, and heatinactivation (6). The content of platelet-derived
growth factor released during the coagulation and
processing of blood is a major source of the action of
the serum, but so too are other peptide and hormonal
components (6). Precaution was taken during the
collection of serum to minimize contamination with
endotoxin that also can affect the response of
chondrocytes.
Adult human articular chondrocytes ordinarily
do poorly with FBS, but prosper with human serum.
The results of this study demonstrate that infant
human chondrocytes differ from their adult counterparts in this regard: they proliferate readily with FBS.
Neither fetal bovine serum nor human serum results in
sulfated proteoglycan synthesis comparable to that
seen with rabbit cells. With a single exception (lo), no
352
BRIEF REPORTS
previous attempt to compare the action of human
serum samples from arthritic patients with the in vitro
behavior of cultured chondrocytes has been reported.
The present study was predicated on the possibility that Mseleni disease serum might be deficient in
factors necessary for normal growth of chondrocytes,
or perhaps contains a selective chondrotoxin, chondrocytotoxic antibody, or even a transmissible infectious agent. No support was found for any of these
possibilities; however, we recognize that such factors
may have been present at some earlier age and may no
longer be operative. The limitations of our failure to
demonstrate a chondrotropic virus needs no comment.
Acknowledgments. We are indebted to Dr. David
Mann for clinical evaluation of the Mseleni disease patients
and to Dr. C. H. J. Schutte for collection of blood in the
field. Dr. David Wooten graciously carried out the statistical
analyses and Dr. Jules Elias performed the immunocytochemical staining.
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2. Mselenijoint disease (editorial).Lancet K483-484, 1985
3. Fincham JE, Hough FS, Taljaard JJF, Capatos D:
Mseleni joint disease: an animal model? S Afr Med J
6751-57, 1985
4. Nurse GT: Slow virus in Mseleni disease? (letter).
Lancet II:784, 1985
5. Choi YC, Morris GM, Lee FS, Sokoloff L: The effect of
serum on monolayer cell culture of mammalian articular
chondrocytes. Connect Tissue Res 7: 105-1 12, 1980
6. Prins APA, Lipman JM, McDevitt CA, Sokoloff L:
Effect of purified growth factors on rabbit articular
chondrocytes in monolayer culture. 11. Sulfated proteoglycan synthesis. Arthritis Rheum 25: 1228-1238, 1982
7. Wei X, Wright GC Jr, Sokoloff L: The effect of sodium
selenite on chondrocytes in monolayer culture. Arthritis
Rheum 29:660464, 1986
8. Sokal RR, Rohlfe FJ: Biometry: The Principles and
Practice of Statistics in Biological Research. Second
edition. San Francisco, WH Freeman, 1981
9. Burmester GR, Menche D, Merryman P, Klein M,
Winchester R: Application of monoclonal antibodies to
the characterizationof cells eluted from human articular
cartilage: expression of Ia antigens in certain diseases
and identification of an 85-kD cell surface molecule
accumulated in the pericellular matrix. Arthritis Rheum
26: 1187-1 195, 1983
10. Schwartz ER, Kirkpatrick PR, Thompson RC: Sulfate
metabolism in human chondrocyte cultures. J Clin Invest 54: 1056-1063, 1974
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