Note: Descriptions are shown in the official language in which they were submitted.
BAC~GROUND OF T~E INVENTION
Field of the Invention
This invention rela-tes to a process of dissociating a
culture of mycobacteria on liquicl media. More particularly, the
present invention relates to the production of phenotypes of
mycobacteria, at :Least two of which have been Eouncl to
possess interesting biological activities.
_escription of the Prior Art
As early as 1927, Petro-Ef (Microbic dissociation: the
tubercle bacillus Proc. Soc. Exp. Biol. Med., 2~: 632-63~) has
shown that several types of tubercle bacilli dissociated into 2
distinct variants when cultured on the solid gentian-violet-egg
medium: the so-called rough and smooth colonies. In subsequent
years, and more recently, the dissociation phenomenon of myco-
bacteria has been confirmed repeatedly. With the advent of
transparent solid media, the previous classification into rough
and smooth variants was further subdivided into Rough C, R, F,
etc, and Smooth S, D, T, etc, based on the microscopic charac-
teristics of colonies. Some of these colonial forms appear to
be specific for a given species of tubercle bacilli and of
atypical mycobacteria, they can thus be of value-in early iden-
tification. Other investigators working mainly with BCG sub-
strains, have described the spreading, intermediate and non-
spreading types of colonies.
Investigations have so far shown that thereis no
conclusive proof that the different colonial forms represent
true mutations of mycobacteria. However, many factors such as
the composition of solid media, cultural conditions, etc., can
influence colony morphology. Thus, it would appear that the
dissociation phenomenon represents changes in the phenotype of
mycobacteria.
Early investigations had shown that differences in
: ~ ' ~ ' ,' ~;
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the protein, glycogerl, carbohydrate, and lipid contents exist
between the rough and smooth variants of a few strains of mamma-
lian tubercle bacilli. However, the relationship between these
constituents and the colonial forms is still not well defined.
More recently, Fregnan et al. have r~por-ted a good correlation
bekween the mycoside content and the colony morphology of atypical
mycobacteria.
It is an object of the present invention to provide a
process whereby mycobacteria are dissociated i~D phenotypes.
It is another object of the present invention to
provide conditions under which mycobacteria are cultured to
produce phenotypes thereof.
SUMMARY OF THE INVE~TION
These and other objeets of the invention can be
attained by culturing myeobaeteria on a liquid medium capable of
promoting the growth of the myeobaeteria, the culturing being
carried out under partial anaerobic conditions and at a pH
between 4.5 and 8.5.
In accordance with a broad aspee~ of the invention,
there is provided a proeess which comprises culturing a myeobae-
` teria under partial anaerobie eonditions in eulture glass or
plastie bottles containing a liquid medium selected from the
group consisting of Sauton's medium, Long's medium9 Proskauer and
Beckts medium and Middlebrook's medium, said liquid medium par-
: s
tially filling said culture glass or plastic bottles, said
culture glass or plastie bottles being incubated on their flat
` side at 37.5C during 14 to 21 days, said glass or plastic bottles
being closed with a plastie Bakelite screweap, the pH of said
culture medium being maintained between 4.5 and 8.5, said cultur-
ing being carried out during about 14 to 21 days, thereby obtaining
surface bacterial pellicles eonsisting of a mixture of smooth9
filamentous and rough baeterial islets, respeetively eonstituting
~ -2-
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Phenotypes 1, 2 and 3 of said substrain of BCG, separa-tely sub-
culturing each of said Phenotypes 1, 2 and 3 in new supply of said
liquid medium and isolating each of said Phenotypes 1, 2 and 3
in pure state.
The mycobacteria which can be used in the process
accordin~ to the invention may include the following well known
s-trains of microorganisms: all substrains of Bacillus Calmette-
Guérin (BCG), M. tuberculosis (H37Rv), M. kansasii (P-8), a sco-
tochromogen (P-5) and M. intracellulare (P-2). Those strains of
microorganisms are all readily available to the public from well-
known depositories. For exemple, they can be found at the ATCC
and are also available to the public at the Institut de Microbiolo-
gie et dtHygiène de Montréal, of Montreal, Canada.
Although any liquid medium which can promote the
growth of mycobacteria can be used according to the invention,
the following media are preferred:
1.- Sauton's medium,
2.- Long1s medium;
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3.- Proskauer and Beck's medium,
4.- Middlebrook~s medium.
According to the invention, the culturing is preferably
carr:ied out at a temperature of about 37.5C cluring about 1
to 21 clay~.
The process according to the invention may be adapted
to obtain surface bacterial pellicles consisting of a mixture
of smooth, filamentous and rough bacterial islets, respectively
constituting Phenotype 1, 2 and 3 of the mycobacteria.
After the bacterial pellicles are obtained, they can
be separately sub~cultured into Phenotypes 1, 2 and 3 in the same
liquid medium followed by isolation of each of the Phenotypes
1, 2 and 3 in pure state.
It is thereafter possible to separately effect several
passages of each Phenotypes 1, 2 and 3 on the liquid medium.
Preferably, four such passages are used.
Finally each Phenotype can be isolated from the ~
mixture of Phenotypes 1, 2 and 3. ;. . .
BRIEF DESCRIPTIO~ ~F THE DRAWI~GS ~ ..
~ 20. The invention will be illustrated by means of the - ~;
- i following drawings, in which~
` FIGURE 1 is a view of the surface growth of BCG show-
: ing the mixture of smooth, filamentous and rough bacterial islets
FIGURES 2A, 2B and 2C are individual views of
morphological patterns of the surface growth of Phenotypes 1, 2
and 3 isolated from BCG;
FIGURE 3 shows bacterial growth curves of Phenotype 1 .
and Phenotype 3 isolated from BCG; :
FIGURE 4 shows the influence of pH of Sautonss medium
on the growth of Phenotypes 1 and 3 isolated from BCG;
~ FIGURES 5A, 5B and 5C show precipitationr~ctions
: between soluble extra ~Sof BCG, of its 2 phenotypes and BCG
_ 3 ~
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antiserum.
FIGURE 6 represents curves of the mean tumor volume
after treatment of ascites nodules with BCG, Phenotype 1, Pheno-
type 3 and Sauton's meclium.
FIGURE 7 i9 the same as FIGURE 6 except that it shows
~he results of a second experiment.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
MICROORGANISMS USED:
Species of mycobacteria included the Montreal substrain
of Bacillus Calmette-Guérin (BCG), M. tuberculosis (H37Rv), M.
kansasii (P-8), one scotochromogen (P-5) and M. intracellulare
(P-2) which as stated above are easily available. All these
strains were maintained on glycerin potato slants.
PREPARATION OF MICROORGANIS~ BEFORE USE:
Prior to their use the bacilli were cultured as sur-
face pellicle on Sauton's medium ~125ml) contained in 250 ml flat
bottom flasks. The seeded flasks were closed as usual with a
cotton plug and incubated at 37.5C.
INDUCTION OF PHENOTY_ES:
Small inocula withdrawn from the flat bottom flasks
were used to seed the surface of 50-ml of Sautonts medium
; (pH 7.2) contained in 200 ml culture glass bott~es (Cabinet Oval,
Dominion Glass, Montreal, Canada). The bottles were tightly
closed with a Bakelite screwcap and incubated on their flat side
at 37.5C during 14 to 21 days. The bottles were observed
every 2-3 days in order to note the morphological characteristics
of the developping surface pellicles durin~ the incubation period.
Instead of the uniformly rough pellicle which characterized after
. . .
the incubation period, the surface growth of mycobacteria in
` 30 flat bottom flasks, the surface growth in bottles consisted of ~ -
a mixture of smooth,filamentous and rough bacterial islets as
shown in FIGURE 1 of the drawings This phenomenon occurred
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spon-taneously sometimes after the first passage in these e~peri-
mental conditions but more frequently after the 2nd or 3rd pas-
sage. Moreover, it has been observed with all the mycobacterial
strains used,i.e. with BCG, M. tuberculosi~s H37Rv and 3 represen-
tative strains of a-typical mycobacterla.
The bacilli isolated from each of -these islets will
hereafter be referred to as Phenotypes 1, 2 and 3, respectively.
Other liquid media such as the Long's, the Proskauer
and Beck's and the Middlebrook's media gave similar results.
However, with Longls medium the relative proportion of smooth
islets was increased by comparison with the other 2 media for
corresponding subcultures of similar ages.
When the culture bottles containing the Sauton's
medium were closed with a cotton plug instead of a screw cap,
mycobacteria did not dissociate into phenotypes, flat bottom
flasks tightly closed with a rubber stopper inhibited almost
completely the bacterial growth. This should clearly establish
that the process according to the invention should stric~ly be
carried out under partial anaerobic conditions.
20 ISOLATION AND PURIFICATION OF THE PHENOTYPES: :
Selected inocula originating from the smooth, filamen-
tous and rough bacterial islets were then subcultured on Sauton~s
medium (pH7.2) contained in 3 series of culture bottles~ -In
these conditions, Phenotype 1 was readily isolated in a pure
state and it was maintained as such by sequential passages at 14
days intervals. In contrast, Phenotypes 2 and 3 were always
contaminated to some extent with Phenotype 1 even after several
passages under the same experimental conditionsO
, .
FIGURE 2 illustrates the morphological patterns of the
surface pellicles of the 3 phenotypes of sCG after purification
-- by sequential passages. Phenotype 1 derived from the smooth
bacterial islets shown in FIGURE 1 and gave a very thin and uni-
form pellicle that covered all the surface of the culture medium
: ' .. . . .
s
and a~ter 14 to 16 days of incubation climbed along the sides of
the glass bottle. Phenotype 2 may be derived from 2 sources,
it can be isola-ted directly from the filamentous bacterial
islets as shown in EIGURE 1 by selecting appropriate inoculum.
I~ has also frequently been observed -that Phenotype 1 loses its
morpholocJ:ic characteristics after 3 to 4 passages and converts
spon-taneously into Phenotype 2. The surface pellicle of Pheno-
type 2 was characterized by the presence of long and tortuous
bacterial filaments. In old cultures (21 days) these filaments
became broader and can cover all the surface of the medium. This
phenotype was not stable since after 5 to 6 passages it fre-
quently converted into Phenotype 3. The surface growth.. of -
Phenotype 3 was constituted by rough bacterial clumps of variable :
sizes which, even in old cultures, never covered all the surface .
of the liquid medium. Like Phenotype 2, it was not stable since
on a few occasions Phenotype 3 has converted into Phenotype 2. ~:
As seen in FIGURE 2, both Phenotypes 2 and 3 were contaminated
to some extend with Phenotype 1. .
. These 3 phenotypes were isolated in this manner from
all strains of mycobacteria listed above. Furthermore, all three
phenotypes possessed the staining properties of mycobacteria as
~ demonstrated by the Zielh-Neelsen technique.
-. It should be emphasized that after their isolation, ~.
the 3 phenotypes were cultured on Sauton's medium contained in
flat bottom flasks, in these conditions, all three phenotypes
` lose gradually their growing characteristics in such a way that :~
after 3 to 4 passages they had completely reverted to the original
` strain. Moreover, after completion of the reverting phase, the
.~ 3 phenotypes can be isolated again from each revertant.
B~CTERIAL GROWTH CURVES OF PHENOTYPES.l AND.
Two series of culture bottles were first inoculated
. respectively with Phenotypes 1 and 3. After various periods of
: - 6 ~
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incubation (up t~ 24 days) at 37.5C, groups of 3 bottles, chosen
at random, were removed from the incubator and their poolecl
content was filtered on a Buckner Eunnel. The bac.illary mass
was dried till constant weight in an oven at 110C. The y.ield
o:f. dry bacill.i was calculated as mg per 100 ml of Sauton's
medium.
FIGURE 3 shows the growth curves (pool data from 2
independant experiments) obtai.ned with Phenotype 1 and Phenotype
3 isolated from BCG and cultured on Sauton's medium at pH 7.2.
Although the lay phase was a little longer for Phenotype 1, the . .
rate of growth was about the same for both phenotypes. Moreover,
during all the incubation period the yield of Phenotype.3 was
larger than that of Phenotype 1.
A large batch of Sauton's medium was prepared and
divided into 10 lots. Each lot was adjusted to the desired pH .
(from 4.5 to 9.0 with an encreament of 0.5) with concentrated .
NH40H prior to its distribution into culture bottles. Each
series of bottles was firs. respectively seeded with inocula of
both phenotypes grown on Sauton's medium at pH 7.2 and, for the
; 20 next 3 passages, with the phenotypes growing at the appropriate
pH. The yield of dry bacilli (mean value of these 3 passages) ~ ...... .
in mg per 100 ml of medium at each specified pH was determined ~.
to evaluate the results.
The pH of culture filtrates following the incubation
period of 14 days in closed culture bottles drop from 7.2 to 6.7,
whereas in flat bottom flasks, it increased slighly to 7.3-7.4~
On the other hand, no difference in the drop of pH was observed
between Phenotype 1 and Phenotype 3 after the incubation period.
The influence of pH of Sauton's medium on the growth
of Phenotype 1 and Phenotype 3 of BCG is shown in FIGURE 4. ~s
seen, Phenotype 1 grew well on Sauton's medium at pH between 5.5 : ~
and 7.5, with a maximum growth at pH 7.00 On the contrary, 2 ~ ~-
_ 7 _
.
.
~540~5
peaks of bacterial grow-th were observed with Phenotype 3: one
at pll 5.5 and the other at pH 7Ø Moreover, Phenotype 3 grew
well at pH as low as 5.0 and as high as 8Ø The higher yield of
Phenotype 3 at p~I 7.0 confirmed the clata shown in FIGURE 3.
~ Eter an incubation period of 14 days, Phenotypes 1 and
3 obtained ~rom a pool of at least 10 culture bottles were
isola-ted by filtration, washed with distilled wat~r, weighted
ancl disintegrated in a ball mill as described by R rrurcotte in .
1969 in Can. J. Microbiol., 15: 35-41. The bacillary debris
were removed by centrifugation at 10,000 X g for half an hour and l,
the supernatant was further centrifuged at 144,000 X g for 3 h.
,;,
The clear supernatant was dialyzed exhaustively against distilled
water and lyophilized. The yield of mycobacterial components
was expressed as mg of lyophilized products per gram (moist
weight) of bacilli. ~ :
The protein and carbohydrate composition of Phenotypes
1 and 3 isolated from five mycobacteria are presented in Table 1. ~ .
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TABLE 1
PROTEIN AND CARBOHYDRATE CONTENTS OF P~NOTYPES 1
AND PHENOTYPES 3 ISOLATED FROM MYCOBACTERIA
STRAIN PHENOTYPE PROTEIN CARBOHYDR~TE
BCG 1 51.9 25,3b
3 44.7 28.3
_
H37Rv 1 54.5 17.4
3 54.1 14.6
M. kansasii 1 38.3 32.3
3 36.5 26.6
Scotochromogen 1 44.7 28.4
3 41.3 25.3
M. intra-
cellulare 1 42.9 26.0
3 46.1 21~7
.
a) Protein and carbohydrate concentrations were determined by
the micro-Kjelkahl technique an~ the thymolsulfuric
- acid reaction, respectively.
b) in percent of dry bacilli. Each data represents a mean value
obtained from at least 2 separate experiments.
~.B. All cultures were 14 day-old.
For Phenotypes 3, the chemical analyses were done on
ba~illi selected irom the rough bacterial islets.
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Total nitrogen conten-t of entire bacilli was estimated
by the micro-Kjeldahl method and converted to proteins using
a conversion factor of 6.25. The protein content of hydrosoluble
extracts was ~letermined by the method of Lowry et al. Crystal~
:Line bovine albumin (~rmour and Co., Chicago, Ill.) was used as
standarcl.
Carbohydrates were determined by the -thymol-sulfuric
acid reaction as modiEied by Shetlar and Masters. D-glucose
was the standard. Before the determination of carbohydrates in
the entire mycobacterial cells, the bacilli were hydrolyzed in
2N HCl for 2 h at 100C as described previously.
As seen, with the excep-tion of M. intracullare,
Phenotypes 1 contained a little more nitrogenous components
than Phenotypes 3. Moreover, both M. kansasii phenotypes
possessed less nitrogen than those of other mycobacteria.
Higher carbohydrate contents were also found for Phenotypes 1,
with the exception of BCG. Moreover, t~e carbohydrate concentra-
tion in H37Rv phenotypes was the lowest, whereas it was approx-
imately the same for the other strains.
Phenotypes from a few strains of mycobacteria were
disintegrated mechanically and the yield of hydrosoluble extracts,
as well as their chemical composition, were determined.
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TABLE 2
YIE~D AND CHEMICAL COMPOSITION OF E~DROSOLUBLE COMPONENTS
EXTRACTED FROM PHENOTYPES 1 AND 3 OF A FEW STRAINS OF
MYCOBACTERIA
STRAINPHENOTYPE YIELD PROTEIN CARBOEIYDRATE
_ _ _ _ ,
BCG 1 69.9 55,2c 14 3c
3 52.8 56.3 17.9
H37Rv 1 64.7 47.5 13.5
3 39.1 61.8 17.1
10 M. kansa~ii 1 40.0 42.7 21.1
3 36.1 44.1 24.5
- .:
a) measured by the method of Lowry et al.
b) mg of lyophilized product per gram of moist bacilli
c) in percent of lyophilized product. ~;
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It is seen that larger quan-ti-ties of components can
be extracted from Phenotypes 1 than from Phenotypes 3~ However,
the protein and carbohydrate contents of extracts isolated from
Phenotypes 3 wer~ slighly higher than those of Pheno-types 1,
which is in contrast with the results obtained with the correspond-
ing entire bacilli (~able 1).
The antigenic composition of mycobacterial phenotypes
was determined by the immuno-diffusion technique using antisera
directed against the corresponding parental strains. For
example, antisera against the soluble extracts isolated from
the BCG strain and its 2 phenotypes, from M. tuberculosis ~I37Rv
and from M. kansasii were produced in rabbits according to an
immunization schedule described by R. Turcotte in 1969 in Can. J.
Microbiol, 15: 681-688. The micro immuno-diffusion technique
of Wadsworth (Int. Arch Allergy, 10: 355-358) as modified by
Crowle (J. Lab. Clin. Med., 52: 784-787) was used for comparing
the antigenic composition of phenotypes. According to the diffu-
sion patterns, each well of plastic templates was filled with
the various bacterial extracts (1% solution) and with undiluted
antisera. The plates were incubated at room temperature in a
moist atm~sphere for 4 days before final reading. After prolonged
washing of the plates with saline, they were stained with thiazine
red (0.1% in 1.0% acetic acid). ~ ;
FIGURE 5 shows the immuno-diffusion patterns obtained
with the parental BCG and its 2 phenotypes. As seen in FIGURE
5-A, at least 2 antigenic determinants were missing in
Phenotype 1 as compared to the parental BCG. Phenotype 3 seemed
to possess the same number of antigenic determinants as the
, ....... . .
parental BCG, though quantitative variations in the concentration
of these determinants existed between-these 2 preparations
~IGURE 5-B). Finally, the antigenic comparison of the 2 phenotypes
indicated that Phenotype 3 possessed at least 1 antigen which
- 12 _
.. . . .
S5
was absent from Phenotype 1 (FIGURE 5-C). Essentially the same
results were obt~inecl with the phenotypes from 2 other strains
(H37Rv and M. kansasli).
To see if the phenotypes posse~ distinctive antigens not
present in the parental strains, antisera directed agains the 2
BCG phenotypes were prepared and absorbed wi-th hydrosoluble
ex~rac~s of the parental BCG prior to their s-tudy by the immuno-
diffusion -techni~ue against their homologous antigens. The
absorption of both types of antisera with extracts of parental BCG
led to the disappearance of all the precipitin bands. These
results suggest that new antigens were absent in the phenotypes.
However, absorption of Phenotype 3 antisera with extracts of
Phenotype l prior to their reaction against Phenotype 3 antigens
led to the disappearance of all but one precipitin bands, thus
confirming the results presented in FIGURE 5-C.
~ suspension (about 1.0%) of Phenotypes l and 3
obtained from BC& was homogenized to disrupt bacillary clumps. -
The negative staining was performed according to the technique
of Brenner and Horne (Biochem. Biophys. ~cta., 34: 103-110).
The grids were examined under an electron microscope (Philips
300) and measurements of bacillary dimensions were done directly
on the TV monitor.
On electron microscopic examination, no difference in
cell wall structure, size and the number of granules inside the
cytoplasm were observed between the parental BCG and its 2
' :
phenotypes. However, the bacillary length of Phenokype l was
shorter than that of Phenotype 3, 1.37~ as compared to 1.45~
respectively (mean value obtained from 300 measurements). This
difference was highly significant (P<0.001) when analyzed by the
Student "t" test.
The culture of several species of mycobacteria on khe
surface of liquid media under partlal anaerobic conditions led
:;
!
to the dissociation of the original strain into 3 phenotypes
according to the morpholo~ical pattern of their surface growth.
Presently, it is believed that dissociation of myco-
bacteria into phenotypes was due mostly to -the fac-t that they
were growing in semi-an~erobic conditions. Obviously, such
conditions were deleterious to the complete development of
mycobacteria since the yield of bacilli (in m~/100 ml of medium)
in culture bo-ttles was about 4 times less than that ob-tained in
flat bottom flasks. ~Iowever, even though tubercle bacillus is
an obligate aerobe , it can grow in the experimental condi-
tions used in this study.
As will be shown below it has been found that Phenotype
- 1 enhances whereas Phenotype 3 inhibited the early development
of subcutaneous transplants of Ehrlich carcinoma in Swiss mice.
Other biological properties of these phenotypes will be published
in the near future.
`The biological activities of Phenotypes 1 and 3 wiIl
be described in the following tests.
Randombred male Swiss mice, 18-22 g, were fed standard
commercial diet and water.
Ehrlich carcinoma obtained origlnally from Dr. Roger
Daoust of the Montreal Cancer Institute were maintained routinely,
by intraperitoneal passages in Swiss mice at weekly intervals.
For the present experiments, ascites cells were harvested in
minimal essential medium (MEM) 199, washed 2 or 3 times in that
medium, and adjusted to the desired concentration after cellular
counting. Mice were inoculated sc in the lower left ventral
quadrant with 1 x 107 cells in a volume of 0.1 ml and distributed,
at random, in groups of 8. This site was chosen,instead of the
back of the animal, to evaluate the effects of BCG on the
extension of the tumor into the peritoneal cavity.
The parental BCG, and 2 phenotypes (1 and 3) derived
from it, were used. All 3 microorganisms were 14 days old when
- 14 -
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1~5~
harvested. Suspensions of bacilli, containing 10 mg (mois-t
weight)/ml Sauton medium 1:~, were prepared and kept frozen at
-50C until used. The number of viable U/ml, determined by
plate counting on solid Dubos medium, were :l3 X 107, 20 X 107,
ancl 26 X 10 Eo~ the parental BCG, phenotype 1, and pheno-type 3,
respect:ively.
Seven days af-ter the sc transplantation oE the ascites
cells, 0.1 ml of each bacillary suspension was injected directly
into the tumor nodule. Control cancerous mice were inoculated
with 0.1 ml Sauton medium diluted 1:~.
Developing tumors were measured every 2-3 days with a
caliper, and the volume of the tumor mass was calculate~ accord-
ing to the formula used by Attia and Weiss (Cancer Res. 26: 1787-
1~300, 1966).
The P values were calculated by the Student's t test.
The effects of the 3 preparations of BCG on the early
development of sc transplants, as observed in 2 independant
experiments shown in FIGURES 6 and 7. During this period of
observation, the parental BCG had no effect on tumor growth, when
` 20 compared to the control group inoculated wlth Sauton medium In
~; contrast, phenotype 3 had a marked inhibitory effect on tumor
growth. Surprisingly, this inhibitory effect was detectable
within the first days of treatment and became more significant
at the end of the observation period (P<0.05 and P=0.1, as compared
to controls and parental BCG).
In contrast with phenotype 3 and parental BCG,phenotype
1 enhanced tumor growth and is thus a very usefull product in
research. I'his stimulating effect, which seemed to occur early
after treatment, was more evident thereafter. However, pheno-
" type 1 differed slightly from controls (P=0.1) and not from the
parental BCG.
In both experiments, the difference between the effectsof phenotypes 1 and 3 on tumor growth, at 17 and 20 days, was
- .: : - , " . : .; - , . , :, :~:
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highly significant (P<0.001).
To eliminate the possibility that the inhibiting and
stimulating effects could be due to differences in the size of
the local inflammatory reac-tions induced by the various BCG
preparations, groups of normal mice were inoculated sc with
similar doses Oe bacilli as those used in cancerous animals. As
expected, small inflammatory reactions were detected a-t the
inoculation sites. However, the si~e of these reactions and
their evolution were the same fcr all 3 BCG preparations.
After the 20th day, the mice were observed daily for
death, tumor regression, and extension of the tumor into the
peritoneal cavity. The mean survival time of the control animals
was 38.~ +1.22 days, whereas that of animals treated with
parental BCG, phenotype 3, and phenotype 1 was 42 ~ + 4.05,
43.1 ~ 4.53, and 34.8 ~ 2.71 days, respectively (pooled results
of the 2 expts). Statistical analyses of these date revealed
~ that phenotype 3 differed significantly from phenotype 1 (P<0.05)
;` and controls (P<0.05). Moreover, in both experiments, the group
of mice treated with phenotype 1 had the lowest incidence of
tumor regression, with most frequent extension of the tumor into
the peritoneal cavity. Finally, the effects of phenotype 3
were not significantly different from those of the original BCG.
~ To see whether similar inhibiting and stimulating
- effects could be observed when the ascites cells were implanted
intraperitoneally (ip), groups of 10 Swiss mice were inoculated
first with a mixture containing 107 tumor cells and 106 bacilli
(parental BCG, phenotype 1, or phenotype 3) and then treated,
7 days later, with the same dose of the appropriate micro- ;
organisms~ The results are shown in table 3. In ip transplanted
; tu~ors, phenotype 1 as well as phenotype 3 and the parental BCG
inhibited the tumbr development when compared to controls.
However, phenotype 3 appeared to be the most potent inhibitory
- 16 -
: . . ,
4~355
preparation (phenotype 3 vs. phenotype l:P=0.1).
TABLE 3
~FFECTS OF 3 PREPARATIONS OF BCG ON IP TRANSPLANTS OF ASCITES
CELLS IN SWISS MICE
Mlce Number of Mean
treated mice wlth survival p
with * tumoral time
regression (days-~SE) values
:
Parental BCG 0/10 24.7-2.91 <0.01
Phenotype 1 0/10 23.0+2.46 <0.02
Phenotype 3 1/10 29.3+3.64 <0.01 `:
Sauton medium
(control) 0/10 15.7+0.87 :
.
* At day 0, injection of mixtures of ascites cells and
bacilli, at day 7, ip injection of bacilli only.
When compared to ~he control group.
: ~ 17 -
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