Proc. Nat. Acad. Sci. USA Vol. 72, No. 9, pp. 3656-3660, September 1975
Immunology
Enhancement of immunity against murine syngeneic tumors by a fraction extracted from non-pathogenic mycobacteria (immuno-adjuvant/murine leukemia/endotoxin sensitivity/virus resistance)
A. LAMENSANS*, L. CHEDID*, E. LEDERERt, J. P. ROSSELETt, R. H. GUSTAFSONt, H. J. SPENCERt, B. LUDWIGf, AND F. M. BERGERt *
Institut Pasteur, 75015 Paris, France; t Institut de Biochimie, Universit6 Paris-Sud, 91400 Orsay, France; and * Wallace Laboratories,
Cranbury, New Jersey 08512
Communicated by Jacques Monod, May 20, 1975
ABSTRACT The data reported here demonstrate that a preparation extracted from nonpathogenic mycobacteria such as Mycobacterium smegmatis and hereafter referred to as interphase material protected mice against Ehrlich ascitic carcinoma, L-1210 leukemia, and another syngeneic lymphoid leukemia. Furthermore, mice treated by this preparation were much less susceptible to endotoxins than when stimulated by BCG (bacillus Calmette-Guerin) or M. smegmatis cells. Moreover, guinea pigs treated by interphase material administered in Freund's incomplete adjuvant showed an increased immune response, yet their sensitivity to tuberculin was much weaker than that of controls sensitized with Freund's complete adjuvant. Finally, resistance to Columbia SK virus infection could be demonstrated when interphase material was administered to mice prior to virus challenge.
the press with cooling. The resulting homogenate was centrifuged for 90 min at 5000 X g, yielding a multiphase system consisting of a lower cell debris layer, a lower opaque aqueous layer and an upper hexane layer containing a sedimented gelatinous phase. The aqueous phase was withdrawn from the system by aspiration and discarded. The hexane phase containing the gelatinous sediment was decanted and centrifuged at 10,000 X g for 1 hr. The gelatinous material was separated from the hexane by decantation, suspended in distilled water, and the suspension was freed from residual hexane by heating to about 380 under reduced pressure. The aqueous suspension was freeze-dried to yield 29.0 g of a fluffy, light colored amorphous solid hereafter referred to as M. smegmatis interphase material (IPM). A similar procedure was used for obtaining interphase material from M. phlei and M. kansasii. M. smegmatis IPM is insoluble in neutral aqueous systems and only partly soluble in organic solvents. It is characterized by the following elemental analysis: C, 51-57%; H, 7.5-8.7%; N, 5.8-6.5%; P, 0.5-1.2% and by the following composition: neutral carbohydrates (arabinose, galactose, glucose), 21-23%; amino sugars, 0.8-1.8%; amino acids (as peptides or proteins), 24-28%; free lipids, 40-43%. These data as well as the presence of a,a'-diaminopimelic acid and approximately equimolar amounts of glucosamine and muramic acid indicate that IPM is essentially a cell wall constit-
It is now well established that mycobacteria have the ability to stimulate the immunity of the host against tumors (1-2). However, these organisms have also the capacity for rendering mice very susceptible to endotoxins (13) and for inducing delayed hypersensitivity to tuberculin. Our previous studies have shown that delipidated cells as well as purified cell walls of Mycobacterium smegmatis or M. kansasii had greater antitumor activity than killed cells of bacillus Calmette-Guerin (BCG), although they were devoid of several of the noxious effects of whole mycobacterial cells (14). In the following study various biological activities of a preparation extracted from nonpathogenic mycobacteria and hereafter referred to as interphase material (IPM) were evaluated. MATERIALS AND METHODS Cultivation of Cells. All cells were grown in Sauton's medium at 370. M. tuberculosis BCG strain Pasteur was grown for 14 days and M. smegmatis strain NBR 21732 ATCC for 9 days. Isolation of Interphase Material. Two hundred grams of freshly harvested M. smegmatis cells were suspended in 2 liters of saline solution, and the cells were dispersed by agitation in a Waring Blendor for about 10 min at room temperature. To the smooth suspension there was added 1.2 liters of hexane and 24 ml of polysorbate-80 while under mechanical agitation. The resulting emulsion was passed through a cooled continuous flow Manton-Gaulin pressure cell at 9000 pounds/inch2 (62 MPa) and with an effluent temperature not exceeding 300. The mixture was recycled twice through
uent. Based on electron microscopic examination and ultracentrifugal studies, IPM appears to be a heterogeneous material of varying particle sizes. It can be readily dispersed in water, saline solution, or in a vehicle consisting of 10% polyethylene glycol-400 in saline solution. In water containing 0.02% polysorbate-80, IPM forms a stable milky dispersion suitable for injection. Evaluation of Antitumor Activity. Leukemia L-1210. Materials to be tested were administered to groups of 10 B6DF1 mice in single doses of 100 ,ug per mouse by intraperitoneal (i.p.) injection of the test material in Hanks' BSS solution. Following implantation of L-1210 leukemia cells mortality response was recorded for each group and the data were plotted on probit coordinates versus time in days. Mean time to death (Y50) and standard deviation from the mean were determined graphically, and probability values were calculated by the Student t test. In this system, a delay in mean time to death of 2 days or more has been found to be highly significant (P = 0.001), and substances producing delays of this magnitude have been designated as being active.
Abbreviations: IPM, interphase material extracted from mycobacteria; BCG, bacillus Calmette-Guerin; MST, mean survival time; LDso, mean lethal dose; i.p., intraperitoneal; i.v., intravenous. 3656
Immunology:
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Lamensans et al.
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16
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Proc. Nat. Acad. Sci. USA 72 (1975)
10
12
14
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3657
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FIG. 1. Delay in leukemia mortality in mice after treatment with IPM from M. smegmatis and whole BCG cells. Mice received 100 gg of IPM and BCG cells, i.p., 8 days prior to L-1210 implantation, 500 cells per mouse, i.p.
FIG. 2. Delay in leukemia mortality in mice after treatment with IPM from M. phlei and whole BCG cells. Mice received 200,ug of IPM and BCG cells, i.p., 16 days prior to L-1210 implantation, 500 cells per mouse, i.p.
Syngeneic lymphoid leukemia and Ehrlich ascitic carcinoma. Test materials were injected intraperitoneally into (C57BI X AKR)F1 hybrid mice 8 days before inoculating by the same route 103 syngeneic leukemia cells or 14 days before inoculating 104 Ehrlich ascitic carcinoma cells. The lymphoid leukemia used and the details of the techniques have been previously described (14). Antiviral Activity. Substances to be tested were suspended by sonication in polyethylene glycol 400. Groups of 30 mice (10 g body weight, Charles River CD-i strain) were inoculated either intraperitoneally or intravenously with the desired dose (10-100 mg/kg) of BCG or IPM and held for 14 days. At this time (day 0), 20 mice per group were challenged intraperitoneally with 20 LD50 (mean lethal dose) units of Columbia SK virus and the remaining 10 animals were sacrificed for measurement of spleen weights, 14 days after infection of test materials. Deaths were recorded daily, and the experiment was terminated at day 12. Deaths occurring before day 3 were excluded from the calculations. Mean survival time (MST) of untreated controls ranged from 4 to 5 days, and increase in MST was expressed as percentage increase compared to untreated controls.
Hyperreactivity to Endotoxin. BCG and IPM were injected intravenously in a dose of 300,gg in 0.2 ml of vehicle, 14 days before challenging by the same route mice with various dosages of endotoxin prepared from Salmonella enteritidis (Danysz strain) by the phenol:water procedure (15). The LDo was calculated according to the method of Reed and Muench (16). Adjuvant Activity and Sensitivity to Tuberculin. Male Hartley guinea pigs of the Pasteur Institute strain weighing 350 g were injected in both hind footpads with 0.1 ml of a water-in-oil emulsion made of equal volumes of a saline solution of ovalbumin (50 mg/ml, egg albumin, five times crystallized, B grade, Calbiochem) and of either Freund's complete adjuvant or Freund's incomplete adjuvant (Difco). The mycobacterial fractions (2 mg/ml) were added to the antigen in the saline solution. All animals were skin-tested 18 days later with 100 IU or 300 IU of old tuberculin. The titers of circulating precipitins and agglutinins were also measured. RESULTS Leukemia L-1210 In each experiment, two control groups of 10 mice each received either diluent only or Pasteur strain BCG. Eight or 16
Table 1. Syngeneic lymphoid leukemia. Comparative effect of BCG and M. smegmatis IPM on survival time of mice
Table 2. Ehrlich ascitic carcinoma. Comparative effect of BCG and M. smegmatis IPM on survival time of mice
Survival at 60 days
Dose: (,ug) MST Treatment* Controls BCG M. smegmatis IPM
i.p. (days)
Survival at 60 days
Survi-
vors/ total
%
2/112 11/70 15/49 6/20 35/80 22/50
1.8 15.7 30.6 30 43.7 44
Pt Treatment*
30 100 10 30 100
22 34 35 29 40 40
Immunology
Enhancement of immunity against murine syngeneic tumors by a fraction extracted from non-pathogenic mycobacteria (immuno-adjuvant/murine leukemia/endotoxin sensitivity/virus resistance)
A. LAMENSANS*, L. CHEDID*, E. LEDERERt, J. P. ROSSELETt, R. H. GUSTAFSONt, H. J. SPENCERt, B. LUDWIGf, AND F. M. BERGERt *
Institut Pasteur, 75015 Paris, France; t Institut de Biochimie, Universit6 Paris-Sud, 91400 Orsay, France; and * Wallace Laboratories,
Cranbury, New Jersey 08512
Communicated by Jacques Monod, May 20, 1975
ABSTRACT The data reported here demonstrate that a preparation extracted from nonpathogenic mycobacteria such as Mycobacterium smegmatis and hereafter referred to as interphase material protected mice against Ehrlich ascitic carcinoma, L-1210 leukemia, and another syngeneic lymphoid leukemia. Furthermore, mice treated by this preparation were much less susceptible to endotoxins than when stimulated by BCG (bacillus Calmette-Guerin) or M. smegmatis cells. Moreover, guinea pigs treated by interphase material administered in Freund's incomplete adjuvant showed an increased immune response, yet their sensitivity to tuberculin was much weaker than that of controls sensitized with Freund's complete adjuvant. Finally, resistance to Columbia SK virus infection could be demonstrated when interphase material was administered to mice prior to virus challenge.
the press with cooling. The resulting homogenate was centrifuged for 90 min at 5000 X g, yielding a multiphase system consisting of a lower cell debris layer, a lower opaque aqueous layer and an upper hexane layer containing a sedimented gelatinous phase. The aqueous phase was withdrawn from the system by aspiration and discarded. The hexane phase containing the gelatinous sediment was decanted and centrifuged at 10,000 X g for 1 hr. The gelatinous material was separated from the hexane by decantation, suspended in distilled water, and the suspension was freed from residual hexane by heating to about 380 under reduced pressure. The aqueous suspension was freeze-dried to yield 29.0 g of a fluffy, light colored amorphous solid hereafter referred to as M. smegmatis interphase material (IPM). A similar procedure was used for obtaining interphase material from M. phlei and M. kansasii. M. smegmatis IPM is insoluble in neutral aqueous systems and only partly soluble in organic solvents. It is characterized by the following elemental analysis: C, 51-57%; H, 7.5-8.7%; N, 5.8-6.5%; P, 0.5-1.2% and by the following composition: neutral carbohydrates (arabinose, galactose, glucose), 21-23%; amino sugars, 0.8-1.8%; amino acids (as peptides or proteins), 24-28%; free lipids, 40-43%. These data as well as the presence of a,a'-diaminopimelic acid and approximately equimolar amounts of glucosamine and muramic acid indicate that IPM is essentially a cell wall constit-
It is now well established that mycobacteria have the ability to stimulate the immunity of the host against tumors (1-2). However, these organisms have also the capacity for rendering mice very susceptible to endotoxins (13) and for inducing delayed hypersensitivity to tuberculin. Our previous studies have shown that delipidated cells as well as purified cell walls of Mycobacterium smegmatis or M. kansasii had greater antitumor activity than killed cells of bacillus Calmette-Guerin (BCG), although they were devoid of several of the noxious effects of whole mycobacterial cells (14). In the following study various biological activities of a preparation extracted from nonpathogenic mycobacteria and hereafter referred to as interphase material (IPM) were evaluated. MATERIALS AND METHODS Cultivation of Cells. All cells were grown in Sauton's medium at 370. M. tuberculosis BCG strain Pasteur was grown for 14 days and M. smegmatis strain NBR 21732 ATCC for 9 days. Isolation of Interphase Material. Two hundred grams of freshly harvested M. smegmatis cells were suspended in 2 liters of saline solution, and the cells were dispersed by agitation in a Waring Blendor for about 10 min at room temperature. To the smooth suspension there was added 1.2 liters of hexane and 24 ml of polysorbate-80 while under mechanical agitation. The resulting emulsion was passed through a cooled continuous flow Manton-Gaulin pressure cell at 9000 pounds/inch2 (62 MPa) and with an effluent temperature not exceeding 300. The mixture was recycled twice through
uent. Based on electron microscopic examination and ultracentrifugal studies, IPM appears to be a heterogeneous material of varying particle sizes. It can be readily dispersed in water, saline solution, or in a vehicle consisting of 10% polyethylene glycol-400 in saline solution. In water containing 0.02% polysorbate-80, IPM forms a stable milky dispersion suitable for injection. Evaluation of Antitumor Activity. Leukemia L-1210. Materials to be tested were administered to groups of 10 B6DF1 mice in single doses of 100 ,ug per mouse by intraperitoneal (i.p.) injection of the test material in Hanks' BSS solution. Following implantation of L-1210 leukemia cells mortality response was recorded for each group and the data were plotted on probit coordinates versus time in days. Mean time to death (Y50) and standard deviation from the mean were determined graphically, and probability values were calculated by the Student t test. In this system, a delay in mean time to death of 2 days or more has been found to be highly significant (P = 0.001), and substances producing delays of this magnitude have been designated as being active.
Abbreviations: IPM, interphase material extracted from mycobacteria; BCG, bacillus Calmette-Guerin; MST, mean survival time; LDso, mean lethal dose; i.p., intraperitoneal; i.v., intravenous. 3656
Immunology:
10
Lamensans et al.
18
16
14
12
Proc. Nat. Acad. Sci. USA 72 (1975)
10
12
14
16
3657
18
FIG. 1. Delay in leukemia mortality in mice after treatment with IPM from M. smegmatis and whole BCG cells. Mice received 100 gg of IPM and BCG cells, i.p., 8 days prior to L-1210 implantation, 500 cells per mouse, i.p.
FIG. 2. Delay in leukemia mortality in mice after treatment with IPM from M. phlei and whole BCG cells. Mice received 200,ug of IPM and BCG cells, i.p., 16 days prior to L-1210 implantation, 500 cells per mouse, i.p.
Syngeneic lymphoid leukemia and Ehrlich ascitic carcinoma. Test materials were injected intraperitoneally into (C57BI X AKR)F1 hybrid mice 8 days before inoculating by the same route 103 syngeneic leukemia cells or 14 days before inoculating 104 Ehrlich ascitic carcinoma cells. The lymphoid leukemia used and the details of the techniques have been previously described (14). Antiviral Activity. Substances to be tested were suspended by sonication in polyethylene glycol 400. Groups of 30 mice (10 g body weight, Charles River CD-i strain) were inoculated either intraperitoneally or intravenously with the desired dose (10-100 mg/kg) of BCG or IPM and held for 14 days. At this time (day 0), 20 mice per group were challenged intraperitoneally with 20 LD50 (mean lethal dose) units of Columbia SK virus and the remaining 10 animals were sacrificed for measurement of spleen weights, 14 days after infection of test materials. Deaths were recorded daily, and the experiment was terminated at day 12. Deaths occurring before day 3 were excluded from the calculations. Mean survival time (MST) of untreated controls ranged from 4 to 5 days, and increase in MST was expressed as percentage increase compared to untreated controls.
Hyperreactivity to Endotoxin. BCG and IPM were injected intravenously in a dose of 300,gg in 0.2 ml of vehicle, 14 days before challenging by the same route mice with various dosages of endotoxin prepared from Salmonella enteritidis (Danysz strain) by the phenol:water procedure (15). The LDo was calculated according to the method of Reed and Muench (16). Adjuvant Activity and Sensitivity to Tuberculin. Male Hartley guinea pigs of the Pasteur Institute strain weighing 350 g were injected in both hind footpads with 0.1 ml of a water-in-oil emulsion made of equal volumes of a saline solution of ovalbumin (50 mg/ml, egg albumin, five times crystallized, B grade, Calbiochem) and of either Freund's complete adjuvant or Freund's incomplete adjuvant (Difco). The mycobacterial fractions (2 mg/ml) were added to the antigen in the saline solution. All animals were skin-tested 18 days later with 100 IU or 300 IU of old tuberculin. The titers of circulating precipitins and agglutinins were also measured. RESULTS Leukemia L-1210 In each experiment, two control groups of 10 mice each received either diluent only or Pasteur strain BCG. Eight or 16
Table 1. Syngeneic lymphoid leukemia. Comparative effect of BCG and M. smegmatis IPM on survival time of mice
Table 2. Ehrlich ascitic carcinoma. Comparative effect of BCG and M. smegmatis IPM on survival time of mice
Survival at 60 days
Dose: (,ug) MST Treatment* Controls BCG M. smegmatis IPM
i.p. (days)
Survival at 60 days
Survi-
vors/ total
%
2/112 11/70 15/49 6/20 35/80 22/50
1.8 15.7 30.6 30 43.7 44
Pt Treatment*
30 100 10 30 100
22 34 35 29 40 40
