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Immune response to sheep erythrocytes with cytoxan and hybaroxia
LIFE SCIENCES Vol . 6, pp . Printed in Great Britain .
1009-1012,
1967,
Pergamon Press Ltd .
IMMUNE RESPONSE TO SHEEP ERYTHROCYTES WITH CYTOXAN AND HYBAROXIA* Benjamin V . Siegel Department of Pathology University of Oregon Medical School, Portland, Oregon (Received 9 February 1967) Previous work from this laboratory (1) has shown that a regimen of longterm hyperbaric oxygenation (hybaroxia, HPO) exerts a protective effect on mice infected with a leukemogenic virus .
Also, this HPO treatment has been
demonstrated to cause a transient decrease in size of thymuses and spleens of otherwise normal animals (2) .
These observations suggested that the effect
of HPO might be on proliferation of lymphoid tissues and it was speculated that hyperbaric oxygenation, alone, or in conjunction with other agents, would exert a depressing effect on the immune response . Experimental The HPO regimen employed has been described in detail (1) and, briefly, was as follows .
Five-week-old female Balb/c/jax mice were subjected to 97~
oxygen at 3 .4 atmospheres absolute pressure for 30 minute perfôds, twice daily, five days per week .
Temperature insi~ the chamber was maintained at 70°F+go
by means of a thermostatically controlled water jacket surrounding the chamber . The hyperbaric oxygen regimen was started on the day of immunization and was continued throughout the experiment .
No discomfort or convulsive behavior
was observed either during or following oxygen treatment .
Immunization con-
sisted of a single intraperitoneal injection of 0 .25 ml of a 33~ suspension of washed, packed sheep erythrocytes .
Antibody titers were determined on two-
fold serial dilutions of individual mouse sera by the direct agglutination of added sheep red cells .
Levels of mercaptoethanol-resistant antibody (3) were deter-
mined on serum samples allowed to react with 0 . 05 M 2-mercaptcethanol (ME)
* Supported by U. S. Atomic Energy Commission Contract RLO-1927-I2. 1009
1010
II~1lJNE RESPONSE
Vol . 6, No . 10
for 30 to 80 minutes prior to carrying out aerial dilutions .
Cytoxan (Mead-
Johaean Laboratory) was administered as a single iatraperitoneal i~jectioa of 40 mg/kg or 80 mg/kg body weight, four hours after immunization and several hours prior to the initial administration of hyperbaric oxygen . Results and Discussion Fig . 1 shows both the total and mercaptoethaaol-reeistani serum agglutinin response of control mice and mice treated with a single dose of 80 ' mg/kg body weight of cytoxan .
As noted is Fig . lA, maximum titers of total
antibody in control mice were reached by 4 days following immunization, and persisted for the remainder of the 26 day experimental period .
Antibody titers
is cytoxan treated mice were observed to be markedly depressed early after immunization . 13awever, these titers increased with time and by 28 days
A-TOTAL ANTIBODY
B-MERCAPTOETHANOLRESISTANT ANTIBODY
192 96
a
48
o-o Control mice
~-~ HPO treated mice A-~ 80 mg/kg cytoxan treated mice 6
~~ 80mq/kg cytoxan plus HPO treated mice
i ~~ ~
/ ~i
Days
FIG. 1 Berum agglutinin titers to sheep red blood cells in control mice and mice treated with a single dose of 80 mg/kg body weight of cYtaxaa . Each point represents the mean titer for six animals .
voi .
s,
xo .
10
IrII~fUNE RESPONSE
post-immunization were identical to those of the controls .
1011
By 7 to 9 days fol-
lowing immunization all antibody detected in control animals was ME-resistant, whereas antibody in the oytoxan treated mice did not become totally ME-resistant until days 14 to 19 (Fig .1B) . These observations suggest that although attainment of peak circulating titers for both total and ME-resistant antibody was retarded by cytoxan treatment, the 198 to 78 immunaglobulin sequence remained normal . In a comparable study employing a single injection of 40 mg/kg body weight of cYtoaran, total antibody reciprocal titers at 4 days averaged 12, in contrast to 192 for control animals .
No mercaptoetbanol resistance was evident at this time .
Total recovery to control levels of both types of a~ibody was reached by 7 days post-immunization .
These results would indicate that the rate of recovery to
peak control levels was inversely related to the dose of cytaxan employed .
A
reversible suppressive effect of cytoxan on the differentiaüon and/or division of immunopotential cells previously provided with antigenic i~ormsricn might account for this traneie~ retardattcn and subsequent recovery .
Alternatively,
antigenic information may persist in a form available to a target call pool which is renewed following destruction of host immunopotential target cells by cytaa~an . Previous observations from this laboratory (1, 2) have snggesbed that a nontoxic regimen of hyperbaric oxygenation could have a direct or potentiatlng efteot in depressing antibody levels .
The present results (Fig . 1) would indicate that
HPO treatment did not exert any marked effects an total or meroaptoethanolresietant antibody levels of either normal or
treated mice .
Ia an ancil-
lary experiment, administration of hyperbaric oxygen to mice receiving a dose of 40 mg/kg body weight of aytoxan was also without effect an antibody levels . Ia this connection, it should be noted that the twofold aerial dilution method employed here is useful only is detecting rather large changes in antibody levels, so that variations less than doubling or halving of antibody would remain undetected .
Other workers have described potentiating effects of hybaroxla .
For
example, Lottsfeldt et al (4) reported increased survival of leukemic mice treated with hyperbaric oxygen in conjunction with radiation and cytoxan, while Nathaasoa
1012
II~QHUNE RESPONSE
Vol . 6,
No .
et al (5) observed inhibition of tumor growth and an increased survival by a regimen of combined HPO with a cyclophosphamide congener .
The present lack
of effect of hyperbaric oxygen treatment on the mouse circulating antibody response using red cell antigens and cytoxan does not exclude the possibilities of demonstrable oxygen effects on the antibody response of animals immunized with different types of antigenic stimuli, or treated in conjunction with other immunosuppressive agents . Summary Concurrent administration of 97°,~ oxygen at 3 .4 atmospheres absolute pressure for 30 minute periods twice daily, five days per week, was without significant éffect in potentiating immunosuppression to sheep erythrocytes by single doses of 40 mg/kg and 80 mg/kg body weight of cytoxan .
Possible HPO
potentiatfng effects with other antigen-immunosuppressive systems are not excluded, and such studies are presently in progress . References 1.
B . LIBET and B . V . SIEGEL, Cancer Res ., 22, 737 (1982) .
2.
B . V . SIEGEL, unpublished observations .
3.
H . F . DEUTSCH and J . I . MORTON,
c
4.
F . I . LOTTSFELDT, S . SCHWARTZ Inst . , 36, 37 (1966) .
and W . KRIVIT, J. Nat . Cancer_
5.
L . NATHANSON, B . BROWN, C . MADDOCK and T . C . HALL, Cancer , 19, 1019 (1966) .
ce, 125, 600 (1956).
10
1009-1012,
1967,
Pergamon Press Ltd .
IMMUNE RESPONSE TO SHEEP ERYTHROCYTES WITH CYTOXAN AND HYBAROXIA* Benjamin V . Siegel Department of Pathology University of Oregon Medical School, Portland, Oregon (Received 9 February 1967) Previous work from this laboratory (1) has shown that a regimen of longterm hyperbaric oxygenation (hybaroxia, HPO) exerts a protective effect on mice infected with a leukemogenic virus .
Also, this HPO treatment has been
demonstrated to cause a transient decrease in size of thymuses and spleens of otherwise normal animals (2) .
These observations suggested that the effect
of HPO might be on proliferation of lymphoid tissues and it was speculated that hyperbaric oxygenation, alone, or in conjunction with other agents, would exert a depressing effect on the immune response . Experimental The HPO regimen employed has been described in detail (1) and, briefly, was as follows .
Five-week-old female Balb/c/jax mice were subjected to 97~
oxygen at 3 .4 atmospheres absolute pressure for 30 minute perfôds, twice daily, five days per week .
Temperature insi~ the chamber was maintained at 70°F+go
by means of a thermostatically controlled water jacket surrounding the chamber . The hyperbaric oxygen regimen was started on the day of immunization and was continued throughout the experiment .
No discomfort or convulsive behavior
was observed either during or following oxygen treatment .
Immunization con-
sisted of a single intraperitoneal injection of 0 .25 ml of a 33~ suspension of washed, packed sheep erythrocytes .
Antibody titers were determined on two-
fold serial dilutions of individual mouse sera by the direct agglutination of added sheep red cells .
Levels of mercaptoethanol-resistant antibody (3) were deter-
mined on serum samples allowed to react with 0 . 05 M 2-mercaptcethanol (ME)
* Supported by U. S. Atomic Energy Commission Contract RLO-1927-I2. 1009
1010
II~1lJNE RESPONSE
Vol . 6, No . 10
for 30 to 80 minutes prior to carrying out aerial dilutions .
Cytoxan (Mead-
Johaean Laboratory) was administered as a single iatraperitoneal i~jectioa of 40 mg/kg or 80 mg/kg body weight, four hours after immunization and several hours prior to the initial administration of hyperbaric oxygen . Results and Discussion Fig . 1 shows both the total and mercaptoethaaol-reeistani serum agglutinin response of control mice and mice treated with a single dose of 80 ' mg/kg body weight of cytoxan .
As noted is Fig . lA, maximum titers of total
antibody in control mice were reached by 4 days following immunization, and persisted for the remainder of the 26 day experimental period .
Antibody titers
is cytoxan treated mice were observed to be markedly depressed early after immunization . 13awever, these titers increased with time and by 28 days
A-TOTAL ANTIBODY
B-MERCAPTOETHANOLRESISTANT ANTIBODY
192 96
a
48
o-o Control mice
~-~ HPO treated mice A-~ 80 mg/kg cytoxan treated mice 6
~~ 80mq/kg cytoxan plus HPO treated mice
i ~~ ~
/ ~i
Days
FIG. 1 Berum agglutinin titers to sheep red blood cells in control mice and mice treated with a single dose of 80 mg/kg body weight of cYtaxaa . Each point represents the mean titer for six animals .
voi .
s,
xo .
10
IrII~fUNE RESPONSE
post-immunization were identical to those of the controls .
1011
By 7 to 9 days fol-
lowing immunization all antibody detected in control animals was ME-resistant, whereas antibody in the oytoxan treated mice did not become totally ME-resistant until days 14 to 19 (Fig .1B) . These observations suggest that although attainment of peak circulating titers for both total and ME-resistant antibody was retarded by cytoxan treatment, the 198 to 78 immunaglobulin sequence remained normal . In a comparable study employing a single injection of 40 mg/kg body weight of cYtoaran, total antibody reciprocal titers at 4 days averaged 12, in contrast to 192 for control animals .
No mercaptoetbanol resistance was evident at this time .
Total recovery to control levels of both types of a~ibody was reached by 7 days post-immunization .
These results would indicate that the rate of recovery to
peak control levels was inversely related to the dose of cytaxan employed .
A
reversible suppressive effect of cytoxan on the differentiaüon and/or division of immunopotential cells previously provided with antigenic i~ormsricn might account for this traneie~ retardattcn and subsequent recovery .
Alternatively,
antigenic information may persist in a form available to a target call pool which is renewed following destruction of host immunopotential target cells by cytaa~an . Previous observations from this laboratory (1, 2) have snggesbed that a nontoxic regimen of hyperbaric oxygenation could have a direct or potentiatlng efteot in depressing antibody levels .
The present results (Fig . 1) would indicate that
HPO treatment did not exert any marked effects an total or meroaptoethanolresietant antibody levels of either normal or
treated mice .
Ia an ancil-
lary experiment, administration of hyperbaric oxygen to mice receiving a dose of 40 mg/kg body weight of aytoxan was also without effect an antibody levels . Ia this connection, it should be noted that the twofold aerial dilution method employed here is useful only is detecting rather large changes in antibody levels, so that variations less than doubling or halving of antibody would remain undetected .
Other workers have described potentiating effects of hybaroxla .
For
example, Lottsfeldt et al (4) reported increased survival of leukemic mice treated with hyperbaric oxygen in conjunction with radiation and cytoxan, while Nathaasoa
1012
II~QHUNE RESPONSE
Vol . 6,
No .
et al (5) observed inhibition of tumor growth and an increased survival by a regimen of combined HPO with a cyclophosphamide congener .
The present lack
of effect of hyperbaric oxygen treatment on the mouse circulating antibody response using red cell antigens and cytoxan does not exclude the possibilities of demonstrable oxygen effects on the antibody response of animals immunized with different types of antigenic stimuli, or treated in conjunction with other immunosuppressive agents . Summary Concurrent administration of 97°,~ oxygen at 3 .4 atmospheres absolute pressure for 30 minute periods twice daily, five days per week, was without significant éffect in potentiating immunosuppression to sheep erythrocytes by single doses of 40 mg/kg and 80 mg/kg body weight of cytoxan .
Possible HPO
potentiatfng effects with other antigen-immunosuppressive systems are not excluded, and such studies are presently in progress . References 1.
B . LIBET and B . V . SIEGEL, Cancer Res ., 22, 737 (1982) .
2.
B . V . SIEGEL, unpublished observations .
3.
H . F . DEUTSCH and J . I . MORTON,
c
4.
F . I . LOTTSFELDT, S . SCHWARTZ Inst . , 36, 37 (1966) .
and W . KRIVIT, J. Nat . Cancer_
5.
L . NATHANSON, B . BROWN, C . MADDOCK and T . C . HALL, Cancer , 19, 1019 (1966) .
ce, 125, 600 (1956).
10