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Detection of cytoplasmic deoxyribonucleic acid and nucleoproteins with antinuclear serum
DISCUSSION Detection
of Cytoplasmic Acid
and
PRELIMINARY
Deoxyribonucleic
Nucleoproteins
Antinuclear
AISD
with
Serum
A recent st,udy (1) has demonstrated that antinuclear factors in the serum from patients with systemic lupus erythematosus or related diseases could be detected using cells grown in culture and the indirect immunofluorescent technique. It was found that the antinuclear factors were highly specific and reacted only with substances in the nucleus or at the site of the nuclear membrane. Some of the sera tested had the ability to react primarily with the chromosomes of cells in various stages of mitosis. Stollar and Levine (2) and others (S, 4) have demonstrated by complement fixation that antinuclear serum reacts with deoxyribonucleic acids (DNA) derived from various sources, including DNA extracted from bacteriophage (2). It, therefore, occurred to one of us (F. R.) that it might be possible to use such scra and the immunofluorescent method for the detect’ion of virus nucleoproteins or DNA in the cytoplasm of infected cells. A system already under study was employed to test this hypothesis. It had been previously clemonstrat’ed that a number of lymphoblastic lymphomas of Swiss mice could be transmitted with cells from a solid form of the tumor or with ascitic cells (5, G) . Electron microscope examination demonstrated masses of viruslike particles within the cytoplasmic matrix; these particles consisted of two concentric spherical incmbrancs with centers that were often less electron dense than the membrane. Relatively few of the cells of the tumor or cells in t,hc ascitic fluid appeared to harbor these viruslike st,ructures. The relationship of these particles to the etiology of the tumor remains obscure because attempts to transmit thcsc ncoplastic diseases with cell-free filtrates from preparat’ions of the tumors remain equivocal. Slides containing ascites cells and imprcssion smears from the solid tumors were prepared. The cells were air dried and reacted with antinuclear serum known to contain antibodies directed primarily against the
REPORTS
497
chromatin. After suitable washing, these cells were allowed to react with an antihuman gamma globulin horse pseudoglobulin that had been labeled with fluorescein isothiocyanate. The method used has been previously described (1). The specimens were then washed, mounted in Elvanol (7)) and viewed through the fluorescence microscope. Similar preparations were treated for 10 minutes with an ethanol (6 parts), glacial acetic acid (2 parts), formaldehyde (1 part) fixative and were stained with acridine orange as described by Armstrong (8). Reaction of antinuclear serum and the immunofluorescent reagent with the nuclei of the cells as well as with small spherical bodies in the cytoplasm was readily noted (Figs. 1 and 2), These cytoplasmic bodies varied in size, but could be demonstrated only in relatively few cells. It appeared that during the earlier transplantations of the tumors more cells contained these cytoplasmic inclusions, a finding consonant with conclusions drawn from the electron microscope studies. Staining with acridine orange yielded similar results; the redstaining cytoplasm of a few cells contained green spherical bodies identical in color to the nuclear material. The bodies were easier to visualize by the immunofluorescent technique, however, because of the total absence of other cytoplasmic fluorescence. The cytoplasmic inclusions observed with the antinuclear-immunofluorescent technique appear to correspond in number and size to the masses of particles seen by examination with the electron microscope. Continuous observations carried out over a period of one year with antinuclear sera and a variety of cultured cells from various sources had failed to reveal any reaction with cytoplasmic components (1) . This technique may, therefore, be useful for the detection of nuclear materials or viral nucleoproteins in the cytoplasm; such studies would presumably be limited to observations of DNA-cont,aining viruses since the majority of antinuclear sera do not appear to react with ribonucleic acids. Recent observations have revealed, however, that some human sera have substances that can
498
DISCUSSIO?J
FIG. 1. Immunofluorcscrnt lymphoma 5730 harvested 12 body that has reacted with visible because they did not
XSD
PRELIMIN.1RT
REPORTS
passage of pllotornicrograph of ascite> cells from the twelfth days aft,er transfer. The arrow points to a cptoplasmic inclusi on the antinuclear serum. Other cytoplasmic components are nlot fluoresce. Magnification: X 720.
FIG. 2. Immunofluorescent photomicrograph of ascitrs cells from the fourth passage of lymphoma 5745 harvested 5 days after transfer. The arrow points to a cytoplasmic in&sic m body that has reacted with the antinuclear serum. Other cytoplasmic components are neot visible because they did not fluoresce. Magnification: X 1125.
DISCUSSION
AND
PRELIMINARY
combine with the nucleoli of cells in culture (I). Attempts to study the replication of viral RNA may be possible with the help of such reagents. We are presently investigating the replication of herpes simplex virus with the system described. It would also be of some interest to study the replication of vaccinia virus in this manner since the virus DNA has been localized in the cytoplasm of infected cells by other methods. ACKNOWLEDGMENT This work was supported in part by grants from t,he National Institute of Allergy and Infectious Diseases and the National Cancer Institute, United States Public Health Service. REFERENCES I. RAPP, F., J. Zmmunol. 88, in press (June 1962). 2. STOLLAR, D., and LEVINE, L., J. Immunol. 87,477-
484 (1961). H. R., DEICHER, H., and G., Proc. Sac. Ezptl. Biol. Med. 96,575-579 (1957). SELIWANN, M., Rev. Frank. e’tudes clin. et biol. 3, 558-584 (1958). DE HARVEN, E., and FRIEND, C., Symposium WI Tumor VirzLses, Nntl. Curbcer Inst. Morlograph No. 4, pp. 291312 (1960). FRIEND, C., DARCHUN, V., DE HARVEN, E., and HADDAD, J., Ciba Foundation Symposium on Tumor Vim~.scs of Murine Origin, pp. 193-213 (1962). RODRIGUEZ, J., and DEINHARDT, F., ~‘ilok)gg 12, 316317 (1960). ARMSTRONG, J. A., Ezptl. Cc~ll Research 11, 640643 (1956).
3. ROBBINS, KUNKEL, /t. 5.
6.
7. S.
W.
C., HOLMAN,
H.
FRED RAW CHARLOTTE
P. D. Wilson Research Foundation Hospital for Special Surgery Department of Microbiology and Immwnology Cornell University Medical College and Division of Experimental Pathology Sloan-Kettering Institute New York 21, New York Receked April 13, 1962
FRIEND
REPORTS
499
Virus-Polysaccharide II. Enhancement of Variants
interactions
of Plague Formation and of Poliovirus with Dextran
the Detection Sulfate
In a previous study (1) the effect of a sulfated agar polysaccharide on plaque formation by EMC virus was described. In limit.ed experiments with other natural and synthetic sulfated polysaccharides, it was shown that these anionic polymers inhibited the wild-type, small-plaque (rf) virus but had no effect on the large-plaque (r) mutant. In further experiments with various other polysaccharides, a highly sulfated sodium dextran sulfate (17% S)l was found to be strongly inhibitory for the largeplaque mutant as well as the r+ virus. When dextran sulfate was incorporated in the agar overlay at a concentration of 100 pg/ml the large plaques of the T mutant were converted into minute plaques identical with those of the r+ type. It was thus shown that whereas the r mutant produces large plaques under agar because of its resistance t,o inhibition by the agar polysaccharide, it is inhibited by dextran sulfate. In other experiments with a number of different viruses, it was found that dextran sulfate is a potent inhibitor for a number of viruses when tested by the plaque inhibition technique. Besides the smaller enteroviruses (ECHO and Coxsackie A9), plaque formation by a myxovirus (influenza B) and a large DNA virus (herpes) was found to be inhibited hy dextran sulfate. With type 1 poliovirus (Mahoney) it was unexpectedly observed that enhanced plaque formation occurred when dextran sulfate was added to the overlay medium at a concentration of 100 pg/ml. The diameter of the plaques with dextran sulfate in the agar was about twice that in plates without it. In the presence of dextran sulfate, enhanced virus multiplication was observed in liquid medium also, while a polycation, DEAE-dext.ran, suppressedboth cytopathic effects and virus growth. In this experiment, virus was adsorbed to monolayer cultures of HeLa cells at a low multiplicity of 0.001 in order to approximate the conditions that occur in multicyclic virus growth of plaque ’ Obtained
from
Pharmacia,
Uppsala,
Sweden.
of Cytoplasmic Acid
and
PRELIMINARY
Deoxyribonucleic
Nucleoproteins
Antinuclear
AISD
with
Serum
A recent st,udy (1) has demonstrated that antinuclear factors in the serum from patients with systemic lupus erythematosus or related diseases could be detected using cells grown in culture and the indirect immunofluorescent technique. It was found that the antinuclear factors were highly specific and reacted only with substances in the nucleus or at the site of the nuclear membrane. Some of the sera tested had the ability to react primarily with the chromosomes of cells in various stages of mitosis. Stollar and Levine (2) and others (S, 4) have demonstrated by complement fixation that antinuclear serum reacts with deoxyribonucleic acids (DNA) derived from various sources, including DNA extracted from bacteriophage (2). It, therefore, occurred to one of us (F. R.) that it might be possible to use such scra and the immunofluorescent method for the detect’ion of virus nucleoproteins or DNA in the cytoplasm of infected cells. A system already under study was employed to test this hypothesis. It had been previously clemonstrat’ed that a number of lymphoblastic lymphomas of Swiss mice could be transmitted with cells from a solid form of the tumor or with ascitic cells (5, G) . Electron microscope examination demonstrated masses of viruslike particles within the cytoplasmic matrix; these particles consisted of two concentric spherical incmbrancs with centers that were often less electron dense than the membrane. Relatively few of the cells of the tumor or cells in t,hc ascitic fluid appeared to harbor these viruslike st,ructures. The relationship of these particles to the etiology of the tumor remains obscure because attempts to transmit thcsc ncoplastic diseases with cell-free filtrates from preparat’ions of the tumors remain equivocal. Slides containing ascites cells and imprcssion smears from the solid tumors were prepared. The cells were air dried and reacted with antinuclear serum known to contain antibodies directed primarily against the
REPORTS
497
chromatin. After suitable washing, these cells were allowed to react with an antihuman gamma globulin horse pseudoglobulin that had been labeled with fluorescein isothiocyanate. The method used has been previously described (1). The specimens were then washed, mounted in Elvanol (7)) and viewed through the fluorescence microscope. Similar preparations were treated for 10 minutes with an ethanol (6 parts), glacial acetic acid (2 parts), formaldehyde (1 part) fixative and were stained with acridine orange as described by Armstrong (8). Reaction of antinuclear serum and the immunofluorescent reagent with the nuclei of the cells as well as with small spherical bodies in the cytoplasm was readily noted (Figs. 1 and 2), These cytoplasmic bodies varied in size, but could be demonstrated only in relatively few cells. It appeared that during the earlier transplantations of the tumors more cells contained these cytoplasmic inclusions, a finding consonant with conclusions drawn from the electron microscope studies. Staining with acridine orange yielded similar results; the redstaining cytoplasm of a few cells contained green spherical bodies identical in color to the nuclear material. The bodies were easier to visualize by the immunofluorescent technique, however, because of the total absence of other cytoplasmic fluorescence. The cytoplasmic inclusions observed with the antinuclear-immunofluorescent technique appear to correspond in number and size to the masses of particles seen by examination with the electron microscope. Continuous observations carried out over a period of one year with antinuclear sera and a variety of cultured cells from various sources had failed to reveal any reaction with cytoplasmic components (1) . This technique may, therefore, be useful for the detection of nuclear materials or viral nucleoproteins in the cytoplasm; such studies would presumably be limited to observations of DNA-cont,aining viruses since the majority of antinuclear sera do not appear to react with ribonucleic acids. Recent observations have revealed, however, that some human sera have substances that can
498
DISCUSSIO?J
FIG. 1. Immunofluorcscrnt lymphoma 5730 harvested 12 body that has reacted with visible because they did not
XSD
PRELIMIN.1RT
REPORTS
passage of pllotornicrograph of ascite> cells from the twelfth days aft,er transfer. The arrow points to a cptoplasmic inclusi on the antinuclear serum. Other cytoplasmic components are nlot fluoresce. Magnification: X 720.
FIG. 2. Immunofluorescent photomicrograph of ascitrs cells from the fourth passage of lymphoma 5745 harvested 5 days after transfer. The arrow points to a cytoplasmic in&sic m body that has reacted with the antinuclear serum. Other cytoplasmic components are neot visible because they did not fluoresce. Magnification: X 1125.
DISCUSSION
AND
PRELIMINARY
combine with the nucleoli of cells in culture (I). Attempts to study the replication of viral RNA may be possible with the help of such reagents. We are presently investigating the replication of herpes simplex virus with the system described. It would also be of some interest to study the replication of vaccinia virus in this manner since the virus DNA has been localized in the cytoplasm of infected cells by other methods. ACKNOWLEDGMENT This work was supported in part by grants from t,he National Institute of Allergy and Infectious Diseases and the National Cancer Institute, United States Public Health Service. REFERENCES I. RAPP, F., J. Zmmunol. 88, in press (June 1962). 2. STOLLAR, D., and LEVINE, L., J. Immunol. 87,477-
484 (1961). H. R., DEICHER, H., and G., Proc. Sac. Ezptl. Biol. Med. 96,575-579 (1957). SELIWANN, M., Rev. Frank. e’tudes clin. et biol. 3, 558-584 (1958). DE HARVEN, E., and FRIEND, C., Symposium WI Tumor VirzLses, Nntl. Curbcer Inst. Morlograph No. 4, pp. 291312 (1960). FRIEND, C., DARCHUN, V., DE HARVEN, E., and HADDAD, J., Ciba Foundation Symposium on Tumor Vim~.scs of Murine Origin, pp. 193-213 (1962). RODRIGUEZ, J., and DEINHARDT, F., ~‘ilok)gg 12, 316317 (1960). ARMSTRONG, J. A., Ezptl. Cc~ll Research 11, 640643 (1956).
3. ROBBINS, KUNKEL, /t. 5.
6.
7. S.
W.
C., HOLMAN,
H.
FRED RAW CHARLOTTE
P. D. Wilson Research Foundation Hospital for Special Surgery Department of Microbiology and Immwnology Cornell University Medical College and Division of Experimental Pathology Sloan-Kettering Institute New York 21, New York Receked April 13, 1962
FRIEND
REPORTS
499
Virus-Polysaccharide II. Enhancement of Variants
interactions
of Plague Formation and of Poliovirus with Dextran
the Detection Sulfate
In a previous study (1) the effect of a sulfated agar polysaccharide on plaque formation by EMC virus was described. In limit.ed experiments with other natural and synthetic sulfated polysaccharides, it was shown that these anionic polymers inhibited the wild-type, small-plaque (rf) virus but had no effect on the large-plaque (r) mutant. In further experiments with various other polysaccharides, a highly sulfated sodium dextran sulfate (17% S)l was found to be strongly inhibitory for the largeplaque mutant as well as the r+ virus. When dextran sulfate was incorporated in the agar overlay at a concentration of 100 pg/ml the large plaques of the T mutant were converted into minute plaques identical with those of the r+ type. It was thus shown that whereas the r mutant produces large plaques under agar because of its resistance t,o inhibition by the agar polysaccharide, it is inhibited by dextran sulfate. In other experiments with a number of different viruses, it was found that dextran sulfate is a potent inhibitor for a number of viruses when tested by the plaque inhibition technique. Besides the smaller enteroviruses (ECHO and Coxsackie A9), plaque formation by a myxovirus (influenza B) and a large DNA virus (herpes) was found to be inhibited hy dextran sulfate. With type 1 poliovirus (Mahoney) it was unexpectedly observed that enhanced plaque formation occurred when dextran sulfate was added to the overlay medium at a concentration of 100 pg/ml. The diameter of the plaques with dextran sulfate in the agar was about twice that in plates without it. In the presence of dextran sulfate, enhanced virus multiplication was observed in liquid medium also, while a polycation, DEAE-dext.ran, suppressedboth cytopathic effects and virus growth. In this experiment, virus was adsorbed to monolayer cultures of HeLa cells at a low multiplicity of 0.001 in order to approximate the conditions that occur in multicyclic virus growth of plaque ’ Obtained
from
Pharmacia,
Uppsala,
Sweden.