398
BIOCHEMICAL SOCIETY TRANSACTIONS
The use of Balb/c mice for the elimination of mycoplasma from infected hybridomas
KENNETH CARROLL and RICHARD O'KENNEDY
School of Biological Sciences, National Institute for Higher
Education, Glasnevin, Dublin 9, Republic of Ireland
Table 1. Growth periods fiw dijferent mycq.hsmu infected hyhridomas grown u s ascitic.turnours in Halhlc mice
Mycoplasma infections constitute a serious problem in all
routine culture of animal cells. In the production of monoclonal antibodies, their presence can lead to any of the
following problems: ( i ) failure to generate hybridomas; (ii)
poor performance of existing hybridomas and myelomas in
culture; (iii) the loss of antibody production and the consequent loss of valuable and sometimes irreplacable clones.
The need for routine screening of cells for mycoplasma
cannot, therefore, be overemphasized. While the detection of
mycoplasma is not very difficult, their elimination is. The use
of chemicals and antibiotics yields variable results and is
further compounded by the rapid development of resistant
strains of mycoplasma. The passage of infected cells in nude
mice has been reported to be highly effective in eliminating
mycoplasma (Van Diggelen et al., 1977) and can be applied
to most cell types. Here, in the absence of a nude mouse
colony, Balb/c mice were used to eliminate mycoplasmas
from syngeneic hybridomas. Success was achieved in four of
the five cases examined.
The hybridomas used were generated by the fusion of
Sp2/0 myelomas with immune spleen cells from Balb/c mice
immunized with Landschutz ascites tdmour cells. Mycoplasma were subsequently detected in these cells by the
fluorescent Hoechst 33258 test of Chen (1977) as modified
by M. Dooley and M. Clynes, NlHED (personal communication). Infected cells were harvested and grown as ascitic
tumours in Balb/c mice which had been pristane primed (0.5
ml of pristane given i.p. 10 days before inoculation with
cells). Between lxlOJ and lxlOh cells were given to each
mouse. The cells were harvested after 14-32 days, depending on the original inoculum size (see Table l), and were
H yhridoma
lnoculum size
7C1
8B I
9A
I OE3
1 ow
Number of days t o
harvesting ascites
1 x I or>
I x 10"
I x 10'
I x 10'
I x 10"
I9
20
27
32
17
either passaged into other mice or recovered aseptically into
culture where they could be re-examined for mycoplasma. In
general, however, cells were passaged three to four times
before being recovered into culture so as to try to ensure that
they would be completely free of mycoplasma and not pose a
threat to other uninfected cell cultures.
Of the five cells treated in this manner, four were successfully cleared of mycoplasma after three passages in Balb/c
mice. The fifth culture was passaged again and it remains to
be seen if it can be cleared of mycoplasma. This procedure
offers a simple and effective means of eliminating mycoplasma from valuable cultures. It offers a further important
advantage in that the cells are removed from the laboratory
where their maintenance would otherwise pose a serious
threat to other uninfected cultures.
This work was funded by St Luke's Cancer Rewarch Fund.
Chen, T. R. (1977) Exp. Cell Res. 104,255
Van Diggelen, 0. P., Seung-il Shin & Phillips, D. N. ( I 077) C h n c w
Res. 31,2680
Keceived 28 August I087
Inhibition of prostaglandin synthase by some low molecular mass fatty acids
J. PHILIP RYAN* and GORDON DAVlSt
*Department of Veterinary Physiology und Biochemistry, The
Veterinary College of Ireland, University College, Dublin 4
and t Department of Pharmacology, Trinity College, School
of Pharmacy, Dublin 4, Republic of Ireland
Prostaglandins have profound effects on the contraction of
smooth muscle. A number of long-chain unsaturated fatty
acids are known to inhibit prostaglandin biosynthesis. These
include analogues of arachidonic acid itself (Bowman &
Rand, 1980) and other unsaturated acids such as linolenic,
linoleic and oleic acid (Anon, 1969). Furthermore, studies on
rats have shown that stimulating the diabetic environment
modifies the synthesis of various prostaglandins and also that
cyclo-oxygenase inhibitors enhance the resulting ketosis
(Axelrod et al., 1981; Axelrod & Levine, 1982; Axelrod &
Cornelius, 1984). These observations led to the idea that
perhaps certain acids that elevate ketone body levels, or even
one of the ketones itself, may be capable of inhibiting
prostaglandin synthesis. In this paper a series of low molecular mass fatty acids and each of the ketone bodies were studied to test this hypothesis.
Adult male rats weighing approximately 250 g were used
to obtain rat spleens and stomach strips. Rat spleens were
washed in ice-cold Krebs solution, blotted dry and weighed.
The spleens were cut into small pieces and homogenized
manually in several volumes of ice-cold phosphate buffer (50
mM-Na,HPO,/SO mM-NaH,PO,; pH 7.4). The homogenate
was then centrifiged in a bench centrifuge at 1000 g for 1 0
min. The supernatant solution was further centrifuged at 4°C
in a Sorval Rotor at 10 000 g for 10 min to give a final supernatant solution which was centrifuged at 50 000 g for 60 min.
Using this procedure each gram of original spleen gave a
25-30 mg crude microsomal pellet which was used as the
source of prostaglandin synthase ( E C 1.14.99.1) activity.
The pellet was resuspended in phosphate buffer to which
hydroquinone ( 5 ,ug/ml) and reduced glutathione (10 pg/
ml) had been added. Two millilitre aliquots of a 1 :20 (w/v)
resuspension of the pellet were used in each assay.
Test inhibitors were made up as 500 mM solutions,
adjusted to pH7.4 with NaOH. Aliquots in the range of
0-800 pmol (0- 1.61111) were then added to polypropylene
vials and the total volume in each vial made up to 3.8 ml with
phosphate buffer. The vials were pre-heated in a water bath
at 37°C and at time zero 2.0 p g (0.2 ml) of arachidonic acid
was added. The vials were shaken vigorously at 120 cycles/
min for 30 min. Appropriate blanks and controls were
carried through with each batch of assays. After incubation
the reactions were stopped by heating the samples for 30 s in
boiling water. The total protein in these samples was found
to lie in the range 1-2 mg/ml by the method of Lowry et al.
(1951).
1988
624th MEETING. DUBLIN
399
Table 1. Test inhibitor vrrlues
Values in parentheses represent the number of observations. IDz5:25% inhibition dose;
ID5,,:50% inhibition dose. See text for details.
Inhibition of prostaglandin synthase
Test inhibitors
Ketone bodies
Acetone
Acetoacetic acid
B-Hydroxybutyric acid
Short-chain fatty acids
Acrylic acid
Pyruvic acid
ui.-Lactic acid
Acetic acid
Formic acid
Propionic acid
Butyric acid
Crotonic acid
Long-chain fatty acidst
Oleic acid
Linoleic acid
Linolenic acid
Reference inhibitors
Acetylsalicyclic acid
lndomethacin
ID25
ID,,,
(mM)
(mi)
>200(9)
> 200 (9)
- 200 (9)
Coefficient of
determination
-
-
-
-
-
-
> 200 (6)
> 200 (6)
> 200 (6)
> 200 (6)
> 200 (6)
Amount to abolish
reticuloruminal
motility in sheep
mmol (mean s.I).)*
*
-
63
56
50
0.997( 1 3)
0.987( 1 9)
0.992(25)
-
-2
-2
-
-I
-
0.064
0.120
0.004
0.009
-
1390 f 950 (30)
930 640 (49)
920 780 (10)
870 f 670 ( 1 2)
490 k 310 (21 )
160k110(7)
**
-
0.996 (24)
0.946(20)
*Derived from data by Leek et ul. ( 1 9 7 8 ~ )Upton
;
et ul. ( 1 977)and Ryan ( 1 98 1 u,h, I 982).
tEstimated from data o n sheep seminal vesicle and rat stomach (Anon, 1969).
Table 2. Inhihitiori ofprostaglandin synthase
Data derived from the actual values at 200 mhi, except where
indicated. See text for details.
Tesc inhibitor
Ketone bodies
Acetone
Acetoacetic acid
B-Hydroxybutyric acid
Short-chain fatty acids
Acrylic acid
Pyruvic acid
tx-Lactic acid
Acetic acid
Formic acid
Propionic acid
Butyric acid
Crotonic acid
Percentage inhibition
(at a dose of 200 mM)
Negl igible
12
25
20
Negligible
8
6
Negligible
60*
65t
74*
*Data derived by slight extrapolation of the linear regression
range o f the log dose-inhibition curves.
tThe same value was obtained from the regression curve.
T h e bioassay for prostaglandin E, (PGE,) activity was
carried out o n all samples using essentially the same method
as that developed in Vane’s laboratory (Piper & Vane, 1969;
Eckensfels & Vane, 1972). T h e inhibitors were tested at
several dose levels in the range 25-200 mM (final concentrations). T h e log dose-inhibition curves were linear as follows:
crotonate (25- 1 5 0 mM); butyrate (50-200 mM) and propionate (50-150 mM). T h e reference inhibitors were linear in
)
the following ranges: acetylsalicylate ( 12.5- 125 p ~ and
.
other acids and ketones
indomethacin (2.5- 15.0 p ~ )Some
gave slight inhibition at 1 0 0 o r 2 0 0 mM. Tables 1 and 2 summarize the results obtained. Except for the weaker inhibitors,
regression lines were fitted in the linear range and the inhibition parameters were calculated from these.
Vol. 16
T h e relative potency of a number of acids tested in these
experiments follows the same order as previously found in
markedly different experiments o n the abolition of reticuloruminal motility in sheep (Critchlow et al., 1980; Leek et uf.,
1978b; Upton et al., 1976, and references cited in Table 1).
Although the latter results may be explained in terms of
excitation of acid-sensitive receptors in the rumen, the
mechanism remains unclear. A n attempt to explain the
effects in terms of the ability of acids to elevate ketone bodies
and in particular crotonic acid has also been made (Ryan,
1981b ) .If there is some coincident relationship between the
two systems studied it may be concluded that it is not the
ketone bodies p e r se, but the acids themselves that elicit the
effects.
Anon ( 1969)Nutr. Rev. 27,3 18-320
Axelrod, L.& Cornelius, P. ( I 984) I’rostuglundins28,333-353
Axelrod. L.& Levine, L. ( 1982)Diubetes 3 1,994-I00 I
Axelrod, L., Lloyd-Jones, P., Ferris, A. M. & Martin, D. B. (1981)
Prostaglundins6,147-1 52
Bowman, W. C . & Rand, M. J . (1980) Textbook 01I’hormtrc,o/ogy
2nd edn., pp. 12-34,Blackwell Scientific Publications, London
and Melbourne
Critchlow, E. C., Leek, B. F., Upton, P. K. & Ryan, J. P. ( 1980)Fed.
I’roc.Fed. Am. SOC. Exp. Hiol. 39,890
Eckensfels, A.& Vane, J. R. (1972)Hr.J. I’hurmucol.45,451-462
Leek, B. F., Ryan, J. P. & Upton, P. K. (1978~)
J. I’hysiol.
(London)284,158- 1591’
Leek, B. F., Ryan, J. P. & Upton, P. ti. (l978b)Ir. J. Med. Sci. 147,
334
Lowry, 0.H.,Rosebrough, N. J., Farr, A. L. & Randall, R. J. ( 195 I )
J . Biol. Chem.193,265-275
Piper, P. J. & Vane, J. R. ( I 969)Nu/irre(London) 223,29-35
Ryan, J. P. ( 198 1 u ) I K C S Med. Sci. 9,16 1 - 162
Ryan, J. P. ( 1 98 1b ) Biochem.Soc. Trans. 9,334-335
Ryan, J. P.( 1 982)Int. Gout Sheep Kes. 2,49-57
Upton, P. K.,Ryan, J. P. & Leek, B. F. (1976)Ir. J. Med. Sci. 145,
307-308
Upton, P. K., Ryan, J. P. & Leek, B. F. ( 1 977)/’roc..Nl4/r, Soc~.36,9A
Received 23 September I987