cytes. Since these mitogens were also found to stimulate the production of a factor chemotactic for monocytes ( M N L CTX), they may contribute to the pathogenesis of periodontal disease.
Human Lymphoproliferative Reaction to Food Products
MATERIALS
Possible Role in Periodontal Inflammation
Extraction
J.
H . RADENTZ* BAKER
L . C . ALTMAN
J. J . OPPENHEIM
Procedure
z
M A N Y C O N T R I B U T I N G F A C T O R S have been implicated in the etiology of periodontal disease. The classic studies of Löe and his associates implicate bacterial components of plaque as the prime initiators of gingivitis. Glickman and S m u l o w and subsequently Ramfjord and A s h have presented studies indicating that occlusal trauma is a major contributing factor in the exacerbation of the disease. Ivanyi and Lehner and Horton et a l . have demonstrated that oral bacteria and plaque selectively stimulate the lymphocytes of patients with periodontal disease, suggesting that cellular immune responses may be related to the inflammatory process associated with periodontal destruction. Food has also long been sus pected of being implicated in the etiology of periodontal disease, but proof of this relationship has been lacking. Prichard has observed that food impaction was evident in 107 intrabony defects in 82 patients. Food impaction has also been implicated in the development of periodon tal abscesses and in some cases of periodontitis. 1
2 , 3
4
5
6 , 7
12
8
9
METHODS
Fifty grams of uncooked corn and 50 gm of corn which had been popped were placed separately in a Waring Blendor and homogenized without liquid for two minutes at 25°C. The resulting homogenate was mixed with 500 cc of cold isotonic (pH 7.2) phosphate-buffered saline (PBS) and homogenized for an additional Five minutes. The mixture was allowed to settle for 15 minutes and decanted. The liquid extraction procedure was repeated two additional times utilizing deionized H O in place of the PBS. The supernatants of the three homogenization procedures were combined, mixed, and centrifuged in a Servall model R-C-2 centrifuge using a G S A rotor at 550 x g for ten minutes, decanted, and placed in dialysis bags. One thousand cubic centimeters o f liquid were preevaporated from the dialysis bags, leaving 500 cc of concentrated extract within the bag. The extract was centrifuged at 500 x g for ten minutes and the liquid was decanted. Sterility was achieved by exposing the liquid extract to 4500 rad in a 250 KVCP Westinghouse Dual Quandrocondex x-ray unit and by the addition o f 100 g / m l o f gentamycin.f The solution was frozen at -20°C and stored for future use. As described previously 100 gm of various types of nuts were pulverized in a Blendor with 200 ml of PBS for eight minutes and centrifuged at 1600 x g for eight minutes. The middle aqueous layer, which contained most of the mitogenic activity, was recentrifuged and filtered successively through a Whatman no. 42 and 0.45 Millipore filters to obtain sterile extracts. The protein content of the two extracts was determined by a sensitive modified biuret procedure in which absorbance was determined at 300 m M . The carbohydrate content was determined by the Wingler H S 0 orcinol procedure. The representative extract of corn contained 2.5 mg of protein per ml and 0.7 mg of carbohydrate per ml, while the walnut contained 23 mg of protein per ml and 14 mg of carbohydrate per ml.
by W.
AND
10
The possibility that some foods might well contribute to periodontal disease by stimulating the cellular immune responses was therefore tested. In particular, the effects of corn and nut extracts on lymphocytes were studied because corn husks and possibly fragments of nuts have been observed within periodontal abscess cavities. Such abscesses, rather than being of bacterial origin, may well have been caused by foreign body or immunological reactions to these impacted foods. We, therefore, tested the in vitro lymphoproliferative response of peripheral human lymphocytes as a means of detecting possible cell-mediated immunity to components of corn and nuts. However, rather than detecting antigenic sensitization, we have found that these seeds contain nonspecific mitogenic activators of all adult and cord blood leuko
1 3
2
4
14
11
Leukocyte
Cultures
Eighty to 100 ml of peripheral human blood were obtained by venipuncture in heparinized ( > 2 0 units/ml) syringes from male and female volunteers. The red blood cells were sedimented for 1.5 hours at 37°C and the leukocyte-rich supernatant plasma was recovered. The white cells were suspended in R P M I 1640 tissue culture media containing 2 mM glutamine, 50 units of penicillin per ml, 50 jig of streptomycin per ml, and 20% autolo gous plasma at 2 x 1 0 cells per ml. The cell suspension
Laboratory of Microbiology and Immunology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20014 and Litton Bionetics, Kensington, Maryland. * Visiting Scientist from U.S. Army Institute of Dental Research, Walter Reed Army Medical Center, Washington, D.C. 20012.
6
†Schering Corporation, Bloomfield, N.J. 562
Volume 46 Number 9
Human Lymphoproliferative
Response
563
was divided into 1-ml aliquots and cultured at 37°C for six days in 1 dram glass vials and an atmosphere of 5% C O in air as described previously. Stimulants were added in volumes of 0.1 ml and control cultures were not stimulated. The efficacy of the lymphocyte response was monitored by testing their responses to nonspecific mitogenic stimulation with 2 g of concanavalin A per m l ‡ and to the antigen streptolysin-0,§ 0.05 ml of 1:25 dilution per culture. Various dilutions of extract of corn or nuts were added as indicated. For the final six hours of incubation the cultures were exposed to 0.1 c of either C - or H-thymidine (sp act 36.6 ^ C i / m m o l ) . | | The cells were harvested using a modified "semiautomatic" multiple sample processor which retained the cells and the radioactivity on glass fiber filters. ¶The radioactivity was counted in a Packard model 133375 Tri-Carb liquid scintillation spectrometer. 6
z
1 4
3
Human cord blood leukocytes were cultured in the same manner as adult cells but modified to seven days of culture with a4½hour pulse of H-thymidine (sp act 6 Ci/mmol). 3
Assay for Mononuclear
Leukocyte
Chemotaxis
The supernatants o f leukocytes which had been exposed to various stimulants were analyzed for human monocyte chemotactic activity ( M N L C T X ) utilizing the system of Snyderman et a l . Human peripheral leukocytes were isolated, washed three times, and suspended in R P M I 1640 culture media with ½% human A B plasma at 2 x 1 0 cells per ml. The cells were then aliquoted into 2-ml volumes in 16 x 125 mm Falcon plastic tubes, half of which were exposed to 0.2 ml of a test stimulant while the other half were cultured with no added stimulants. Cell cultures were incubated at 37°C for 24 hours in an atmosphere of 95% air and 5% C 0 . Control cultures were then exposed to the same stimuli as the test cultures and all specimens were centrifuged at 200 x g for ten minutes. The sterile supernatants were then decanted and tested for M N L C T X factor as described previously. 1 5
FIGURE
1.
Comparison
1A
of C-TdR
incorporation
by
stimu-
lated and unstimulated adult leukocytes in a six-day culture in vitro. Each point represents the mean counts per minute of triplicate cultures.
6
2
15
RESULTS
Leukocyte cultures from seven adult patients demonstrated significant proliferation as measured by C thymidine ( C - T d R ) incorporation when exposed to extracts from corn or popped corn (Fig. 1). The extract of popped corn stimulated the leukocytes to a lesser degree than the corn extract and both stimulated less than concanavalin A or streptolysin-O, although the incorporation of C-thymidine was significantly higher than in the unstimulated cultures. Furthermore, leukocyte cultures from almost all of the more than 20 adult subjects tested during the course o f this study have shown significant reactions to these extracts. 1 4
14
14
The relationship of allergy to lymphocyte transformation by corn extract was also investigated. The reactivity of lymphocytes from two individuals who manifested significantly elevated histamine release** from their blood basophils in response to corn extract was therefore determined. There was no significant difference between the reactions of the two donors that were found to be allergic to corn and the majority of nonallergic subjects, suggesting that the corn extract might have a nonspecific mitogenic effect on lymphocytes. This possibility was tested with cord blood leukocytes which have the ability to respond uniformly to mitogens but not to antigens in the absence of sensitization. Figure 2 shows that 23 out of 24 cultures of cord blood leukocytes gave a positive response to corn. The mean response to corn is about the same as the mean response to concanavalin A, indicating that corn extract acts as a nonspecific mitogen (Fig. 2). Dose-response studies indicated that a 1:4 dilution of the original corn extract usually resulted in maximal stimulation but the degree of stimulation generally decreased with increasing dilution (Fig. 3). 11
Investigation of the kinetics of the leukocyte response to corn extract indicated low but significant increases in C-thymidine uptake by the third day in culture with 14
‡Miles Yeda, Ltd., Kankakee, 111. § Difco, Detroit, Mich. ||Schwarz/Mann, Orangeburg, N . Y . If Reeve Angel, Clifton, N . J .
** T h e histamine assay was kindly supplied by R. Siraganian and W. H o o k , Clinical Immunology Section, L M I , N I D R .
564
Radentz,
Baker, Altman,
J. Periodontol. September, 1975
Oppenheim
14
F I G U R E 4. Kinetics of C-TdR incorporation by stimulated and unstimulated leukocytes in vitro. Each point represents the mean counts per minute of triplicate cultures. 3
2. The in vitro H-TdR incorporation by stimulated and unstimulated newborn cord blood leukocytes. Each point represents the mean counts per minute of triplicate cultures from different donors. FIGURE
maximum blastogenesis occurring by the sixth day. This relatively slow rate of response of lymphocytes to corn extract resembles the kinetics of the response to suboptimal doses of the mitogen concanavalin A and the antigen streptolysin-0 (Fig. 4). This differs from the earlier peak response that is typically seen at three or four days in response to optimal doses of potent lectin mitogens. 14
Similar increases in C-thymidine incorporation were obtained with adult peripheral leukocytes or cord blood leukocytes (Fig. 5) that were stimulated with extracts of black walnuts, almonds or pecans; both the degree and rate of response to these nonspecific nut m i t o g e n s were similar to the results observed with corn. Since the production of lymphokines by activated lymphocytes is thought to be a more reliable correlate of cell-mediated immunity, we investigated the stimulating effect of the extracts of corn and black walnuts on leukocytes for a factor chemotactic for mononuclear leukocytes. The extracts of corn, popped corn, and black walnuts all possessed the ability to stimulate leukocytes to produce this lymphokine (Table 1). 12
DISCUSSION
There is considerable documentation in the literature that several types of corn products can cause various kinds of inflammatory reactions. Corn starch has been found to produce a sterile type of granulomatosis lesion when dusted into an abdominal surgical s i t e . It was also suspected of producing a fibrous type of giant cell lesion when inserted into a dental extraction site as part of the 18
14
3. The incorporation of C-TdR by adult leukocytes exposed to varying dilutions of corn extract in vitro. Each point represents the mean counts per minute of triplicate cultures. FIGURE
Volume 46 Number 9
Human Lymphoproliferative
Response
TABLE 1. Chemotactic Activity for Human Mononuclear in Supernatants of Lymphocyte Cultures Material tested 1. Control (no stimulation) 2. 3. 4. 5.
Concanavalin A Corn Popped corn Black walnuts
565
Leucocytes
Chemotactic activity 8± 1* 83 77 23 26
± 11 ±8 ±4 ± 2
* Each value represents the mean and standard error of triplicate samples of monocytes which have migrated through 5.0 pores in a polycarbonate filter.
allergic response found only in sensitized individuals. Therefore, since these mitogenic extracts of corn and nuts are also capable of stimulating the production of mediators it is entirely conceivable that they may also be capable of inducing inflammatory reactions in vivo. Intradermal injections of mitogens, such as phytohemagglutinin, have been found to produce erythematous, indurated, cutaneous reactions similar to the delayed type of hypersensitivity reactions of sensitized individuals. The reaction peaked in 24 to 48 hours and histologically was characterized by a mononuclear cell infiltration of the area surrounding the cutaneous blood vessels and adnexa in the subcutaneous tissue and dermis. This reaction was accompanied by infiltration with polymorphonuclear leukocytes into the affected area. The periodontal abscess is histologically a similar type of lesion of undetermined etiology. The abscess has gener ally been attributed to obstruction of the clinical orifice of an intrabony defect, either by mechanical or physio logical means, with the concomittant accumulation of fluid and cells in the depths of the defect. The more superficial abscesses have been attributed to foreign bodies lodged in the sulcus (toothbrush bristles, e t c ) . A rather common finding during the treatment of these lesions is a corn or nut particle lodged in the abscess cavity. It is conceivable that the fragile epithelium of the crevice could be disrupted by the penetration of the hull and a cell-mediated hypersensitivity reaction initiated by the soluble mitogenic factor in the corn. This reaction might lead to massive tissue destruction with the release of lymphokines which would cause the inhibition of macrophage migration, the attraction of mononuclear cells, and the release of endogenous cytotoxic and cytolytic enzymes into the area of tissue destruction. The lesion resulting from this inflammatory response would be singularly more destructive and extensive than that resulting from a simple foreign body reaction. Food has long been suspected of exerting a significant effect on the development of periodontal disease, but its role has not been identified. Presumably, food particles provide nourishment for indigenous oral bacteria, whose potential for initiating periodontal disease has already been demonstrated. In addition, the detection of soluble factors within corn and nuts which are capable of 21
l4
5. The in vitro C-TdR incorporation by stimulated adult and newborn cord blood leukocytes. Each point repre sents the mean counts per minute of triplicate cultures from different donors. FIGURE
19
filler material in an antibiotic c o n e . Corn and its various derivatives have also been responsible for a wide variety of allergic responses. Intact corn starch particles can pass the intestinal barrier because of their high density and low particle size (6 to 15 ) through a process known as perabsorption. The particles have been found to be lodged in blood vessels, especially the small alveolar capillaries of the lung, and almost every organ of the b o d y . The ingress of the intact particles directly into the bloodstream offers a potential route for the development of sensitization to the material. Corn that is completely cooked (boiled) is relatively harmless as it cannot pass the intestinal barrier. Raw corn starch, on the other hand, is the major offender and is prevalent in short breads, cookies, pie crusts, popcorn, and tortillas. A large segment of our population may therefore be sensitized to the antigenic constituents of corn and should this material penetrate the epithelial barrier of the gingival crevice of a sensitized individual, a hyperim mune response would be expected to occur. Our findings indicate, however, that the in vitro blastogenesis of the adult and newborn leukocytes to corn as well as nut extracts are due to a ubiquitous nonspecific mitogenic stimulation rather than a classical 20
22
566
Radentz,
activating mediator
Baker, Altman,
lymphocytes in vitro
J. Periodontol. September, 1975
Oppenheim
to proliferate
and produce a
suggests that these materials may
penetrate into inflamed or traumatized gingival tissues and may also contribute directly to acute or chronic periodontal inflammation. SUMMARY
Extracts of corn and some nuts were found to ubiqui tously stimulate both adult and newborn cord blood lymphocytes to transform and produce a factor chemo tactic for monocytes. This indicates that corn and nuts contain a mitogen and are potential stimulators of the cellular immune response. The exposure of lymphocytes to
this
mitogen
in vivo
might
trigger a
destructive
inflammatory reaction in the surrounding tissues. These findings, therefore, suggest that foods such as corn and nuts may be responsible for some periodontal abscesses and may be contributing factors to the development of intrabony alveolar lesions and chronic periodontitis. REFERENCES
1. Löe, H. E., Theilade, E., and Jensen, S. B.: Experimen tal gingivitis in man. J Periodontol 3 6 : 177, 1967. 2. Glickman, I., and Smulow, J.: Alterations in the path way of gingival inflammation into the underlying tissue induced by excessive occlusal forces. J Periodontol 3 3 : 7, 1962. 3. Glickman, I., and Smulow, J.: The combined effects of inflammation and trauma from occlusion in periodontitis. Int Dent J 19: 393, 1969. 4. Ramfjord, S. P., and Ash, M. M.: Occlusion, p. 156. Philadelphia, W. B. Saunders Co., 1966. 5. Ivanyi, L., and Lehner, T.: The significance of serum factors in stimulation of lymphocytes from patients with periodontal disease by Veillonella alcalescens. Arch Allergy 4 1 : 620, 1971. 6. Horton, J. E., Leiken, S., and Oppenheim, J. J.: Human lymphoproliferative reaction to saliva and dental plaque de posits: An in vitro correlation with periodontal disease. J Periodontol 4 3 : 522, 1972. 7. Horton, J. E., Oppenheim, J. J., and Mergenhagen, S.
E.: A role for cell-mediated immunity in the pathogenesis of periodontal disease. J Periodontol 4 5 : 3 5 1 , 1974. 8. Prichard, J. F.: Advanced Periodontal Disease: Surgical and Prosthetic Management, p 271. Philadelphia, W. B. Saunders Co., 1966. 9. Truluck, M. H.: Acute periodontal abscess, diagnosis and treatment. J NC Dent Soc 4 4 : 15, 1960. 10. Glickman, I.: Clinical Periodontology, p 143. Philadel phia, W. B. Saunders Co., 1972. 11. O'Brian, T. J.: Diagnosis and treatment of periodontal abscess. J Wise Dent Soc 4 5 : 77, 1969. 12. Marquardt, J. L., Snyderman, R., and Oppenheim, J. J.: Depression of lymphocyte transformation and exacerbation of Behcet's syndrome by ingestion of English walnuts. Cell Immunol 9: 262, 1973. 13. Colowick, S. P., and Kaplan, N. O. (eds): Methods in Enzymology, Vol. 3, p. 696, Ne w York, Academic Press, 1957. 14. Tottschalk, A.: Glycoprotein: Their composition, struc ture and function, p 284. New York, Elsevier Publishing Co., 1972. 15. Snyderman, R., Altman, L. C , Hausman, M. S., and Mergenhagen, S. E.: Human mononuclear leukocyte chemotaxis: A quantitative assay for humoral and cellular chemotac tic factors. J Immunol 108: 857 (1972). 16. Altman, L. C , Snyderman, R., Oppenheim, J. J., and Mergenhagen, S. E.: A human mononuclear leukocyte chemo tactic factor: Characterization, specificity and kinetics of production by homologous leucocytes. J Immunol 110: 801, 1973. 17. Ling, N. R., and Husband, E. M.: Specific and nonspe cific stimulation of peripheral lymphocytes. Lancet 1: 363, 1964. 18. Pemberton, M., and Johnson, M.: Dangers of corn starch powder. Br Med J 3 : 235, 1973. 19. Miller, W. A.: Foreign body reaction to dental cone containing starch. Dent Pract 18: 428, 1968. 20. Lietz, A.: Laboratory research in food allergy. J Asthma Res 7: 127, 1970. 21. Blaese, R. M., Weiden, P., Oppenheim, J. J., and Waldmann, T. A.: Phytohemagglutinin as a skin test for the evaluation of cellular immune competence in man. J Lab Clin Med SI: 538, 1973. 22. Glickman, I.: Clinical Periodontology, p 249. Philadel phia, W. B. Saunders Co., 1972.
Abstracts SYNDESMOTIC LIMITING MOVEMENT OF THE PERIODONTAL LIGAMENT Heners, M .
INSTRUCTION IN ORAL HYGIENE FOR A GROUP OF DENTAL STUDENTS: ITS EFFECT ON THEIR PEERS
Int Dent J 24: 319, June, 1974.
Newcomb, G . M .
Contactless electronic pick-ups were fitted to the maxillary right central incisors of ten subjects to determine whether the periodontium has a specific function-dependent movement. By measuring simultane ously the horizontal and axial movements under specific forces in the sagittal plane, the tooth movement was recorded by a length measure ment in which the tooth crown was used as a mechanical reference point for an electronic displacement pick-up. It was demonstrated that the periodontium has a specific movement characteristic which guides the tooth in predetermined paths and can only be insignificantly changed by the direction of loading. In addition, the fine structure of the collagenous tissue was shown to be functionally orientated. Prosthetic Department, Klinik FürZahn-, Mund- und Kieferkrankheiten, Weimarerstrasse 8, D 2300, Kiel, Germany
J Public Health Dent 34: 113, Spring, 1974. After exposure of one group of 10 students to individual instruction in toothbrushing, plaque indexes (Loe and Silness) were made at one and two weeks for them and for a second group who had not had individual instruction. Definitely lowered indexes were found in the instructed group and in the group that was exposed to the first group, even though brushing instruction was very minimal. This behavioral change is brought about by modeling influences. A control group who had no contact with these positively reinforced modeling influences, demon strated no improvement in plaque index. This method of instruction is advocated as having possibilities for large group learning. Department of Periodontics, Royal Adelaide Hospital, North Terrace, Adelaide, South Australia, Australia
Human Lymphoproliferative Reaction to Food Products
MATERIALS
Possible Role in Periodontal Inflammation
Extraction
J.
H . RADENTZ* BAKER
L . C . ALTMAN
J. J . OPPENHEIM
Procedure
z
M A N Y C O N T R I B U T I N G F A C T O R S have been implicated in the etiology of periodontal disease. The classic studies of Löe and his associates implicate bacterial components of plaque as the prime initiators of gingivitis. Glickman and S m u l o w and subsequently Ramfjord and A s h have presented studies indicating that occlusal trauma is a major contributing factor in the exacerbation of the disease. Ivanyi and Lehner and Horton et a l . have demonstrated that oral bacteria and plaque selectively stimulate the lymphocytes of patients with periodontal disease, suggesting that cellular immune responses may be related to the inflammatory process associated with periodontal destruction. Food has also long been sus pected of being implicated in the etiology of periodontal disease, but proof of this relationship has been lacking. Prichard has observed that food impaction was evident in 107 intrabony defects in 82 patients. Food impaction has also been implicated in the development of periodon tal abscesses and in some cases of periodontitis. 1
2 , 3
4
5
6 , 7
12
8
9
METHODS
Fifty grams of uncooked corn and 50 gm of corn which had been popped were placed separately in a Waring Blendor and homogenized without liquid for two minutes at 25°C. The resulting homogenate was mixed with 500 cc of cold isotonic (pH 7.2) phosphate-buffered saline (PBS) and homogenized for an additional Five minutes. The mixture was allowed to settle for 15 minutes and decanted. The liquid extraction procedure was repeated two additional times utilizing deionized H O in place of the PBS. The supernatants of the three homogenization procedures were combined, mixed, and centrifuged in a Servall model R-C-2 centrifuge using a G S A rotor at 550 x g for ten minutes, decanted, and placed in dialysis bags. One thousand cubic centimeters o f liquid were preevaporated from the dialysis bags, leaving 500 cc of concentrated extract within the bag. The extract was centrifuged at 500 x g for ten minutes and the liquid was decanted. Sterility was achieved by exposing the liquid extract to 4500 rad in a 250 KVCP Westinghouse Dual Quandrocondex x-ray unit and by the addition o f 100 g / m l o f gentamycin.f The solution was frozen at -20°C and stored for future use. As described previously 100 gm of various types of nuts were pulverized in a Blendor with 200 ml of PBS for eight minutes and centrifuged at 1600 x g for eight minutes. The middle aqueous layer, which contained most of the mitogenic activity, was recentrifuged and filtered successively through a Whatman no. 42 and 0.45 Millipore filters to obtain sterile extracts. The protein content of the two extracts was determined by a sensitive modified biuret procedure in which absorbance was determined at 300 m M . The carbohydrate content was determined by the Wingler H S 0 orcinol procedure. The representative extract of corn contained 2.5 mg of protein per ml and 0.7 mg of carbohydrate per ml, while the walnut contained 23 mg of protein per ml and 14 mg of carbohydrate per ml.
by W.
AND
10
The possibility that some foods might well contribute to periodontal disease by stimulating the cellular immune responses was therefore tested. In particular, the effects of corn and nut extracts on lymphocytes were studied because corn husks and possibly fragments of nuts have been observed within periodontal abscess cavities. Such abscesses, rather than being of bacterial origin, may well have been caused by foreign body or immunological reactions to these impacted foods. We, therefore, tested the in vitro lymphoproliferative response of peripheral human lymphocytes as a means of detecting possible cell-mediated immunity to components of corn and nuts. However, rather than detecting antigenic sensitization, we have found that these seeds contain nonspecific mitogenic activators of all adult and cord blood leuko
1 3
2
4
14
11
Leukocyte
Cultures
Eighty to 100 ml of peripheral human blood were obtained by venipuncture in heparinized ( > 2 0 units/ml) syringes from male and female volunteers. The red blood cells were sedimented for 1.5 hours at 37°C and the leukocyte-rich supernatant plasma was recovered. The white cells were suspended in R P M I 1640 tissue culture media containing 2 mM glutamine, 50 units of penicillin per ml, 50 jig of streptomycin per ml, and 20% autolo gous plasma at 2 x 1 0 cells per ml. The cell suspension
Laboratory of Microbiology and Immunology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20014 and Litton Bionetics, Kensington, Maryland. * Visiting Scientist from U.S. Army Institute of Dental Research, Walter Reed Army Medical Center, Washington, D.C. 20012.
6
†Schering Corporation, Bloomfield, N.J. 562
Volume 46 Number 9
Human Lymphoproliferative
Response
563
was divided into 1-ml aliquots and cultured at 37°C for six days in 1 dram glass vials and an atmosphere of 5% C O in air as described previously. Stimulants were added in volumes of 0.1 ml and control cultures were not stimulated. The efficacy of the lymphocyte response was monitored by testing their responses to nonspecific mitogenic stimulation with 2 g of concanavalin A per m l ‡ and to the antigen streptolysin-0,§ 0.05 ml of 1:25 dilution per culture. Various dilutions of extract of corn or nuts were added as indicated. For the final six hours of incubation the cultures were exposed to 0.1 c of either C - or H-thymidine (sp act 36.6 ^ C i / m m o l ) . | | The cells were harvested using a modified "semiautomatic" multiple sample processor which retained the cells and the radioactivity on glass fiber filters. ¶The radioactivity was counted in a Packard model 133375 Tri-Carb liquid scintillation spectrometer. 6
z
1 4
3
Human cord blood leukocytes were cultured in the same manner as adult cells but modified to seven days of culture with a4½hour pulse of H-thymidine (sp act 6 Ci/mmol). 3
Assay for Mononuclear
Leukocyte
Chemotaxis
The supernatants o f leukocytes which had been exposed to various stimulants were analyzed for human monocyte chemotactic activity ( M N L C T X ) utilizing the system of Snyderman et a l . Human peripheral leukocytes were isolated, washed three times, and suspended in R P M I 1640 culture media with ½% human A B plasma at 2 x 1 0 cells per ml. The cells were then aliquoted into 2-ml volumes in 16 x 125 mm Falcon plastic tubes, half of which were exposed to 0.2 ml of a test stimulant while the other half were cultured with no added stimulants. Cell cultures were incubated at 37°C for 24 hours in an atmosphere of 95% air and 5% C 0 . Control cultures were then exposed to the same stimuli as the test cultures and all specimens were centrifuged at 200 x g for ten minutes. The sterile supernatants were then decanted and tested for M N L C T X factor as described previously. 1 5
FIGURE
1.
Comparison
1A
of C-TdR
incorporation
by
stimu-
lated and unstimulated adult leukocytes in a six-day culture in vitro. Each point represents the mean counts per minute of triplicate cultures.
6
2
15
RESULTS
Leukocyte cultures from seven adult patients demonstrated significant proliferation as measured by C thymidine ( C - T d R ) incorporation when exposed to extracts from corn or popped corn (Fig. 1). The extract of popped corn stimulated the leukocytes to a lesser degree than the corn extract and both stimulated less than concanavalin A or streptolysin-O, although the incorporation of C-thymidine was significantly higher than in the unstimulated cultures. Furthermore, leukocyte cultures from almost all of the more than 20 adult subjects tested during the course o f this study have shown significant reactions to these extracts. 1 4
14
14
The relationship of allergy to lymphocyte transformation by corn extract was also investigated. The reactivity of lymphocytes from two individuals who manifested significantly elevated histamine release** from their blood basophils in response to corn extract was therefore determined. There was no significant difference between the reactions of the two donors that were found to be allergic to corn and the majority of nonallergic subjects, suggesting that the corn extract might have a nonspecific mitogenic effect on lymphocytes. This possibility was tested with cord blood leukocytes which have the ability to respond uniformly to mitogens but not to antigens in the absence of sensitization. Figure 2 shows that 23 out of 24 cultures of cord blood leukocytes gave a positive response to corn. The mean response to corn is about the same as the mean response to concanavalin A, indicating that corn extract acts as a nonspecific mitogen (Fig. 2). Dose-response studies indicated that a 1:4 dilution of the original corn extract usually resulted in maximal stimulation but the degree of stimulation generally decreased with increasing dilution (Fig. 3). 11
Investigation of the kinetics of the leukocyte response to corn extract indicated low but significant increases in C-thymidine uptake by the third day in culture with 14
‡Miles Yeda, Ltd., Kankakee, 111. § Difco, Detroit, Mich. ||Schwarz/Mann, Orangeburg, N . Y . If Reeve Angel, Clifton, N . J .
** T h e histamine assay was kindly supplied by R. Siraganian and W. H o o k , Clinical Immunology Section, L M I , N I D R .
564
Radentz,
Baker, Altman,
J. Periodontol. September, 1975
Oppenheim
14
F I G U R E 4. Kinetics of C-TdR incorporation by stimulated and unstimulated leukocytes in vitro. Each point represents the mean counts per minute of triplicate cultures. 3
2. The in vitro H-TdR incorporation by stimulated and unstimulated newborn cord blood leukocytes. Each point represents the mean counts per minute of triplicate cultures from different donors. FIGURE
maximum blastogenesis occurring by the sixth day. This relatively slow rate of response of lymphocytes to corn extract resembles the kinetics of the response to suboptimal doses of the mitogen concanavalin A and the antigen streptolysin-0 (Fig. 4). This differs from the earlier peak response that is typically seen at three or four days in response to optimal doses of potent lectin mitogens. 14
Similar increases in C-thymidine incorporation were obtained with adult peripheral leukocytes or cord blood leukocytes (Fig. 5) that were stimulated with extracts of black walnuts, almonds or pecans; both the degree and rate of response to these nonspecific nut m i t o g e n s were similar to the results observed with corn. Since the production of lymphokines by activated lymphocytes is thought to be a more reliable correlate of cell-mediated immunity, we investigated the stimulating effect of the extracts of corn and black walnuts on leukocytes for a factor chemotactic for mononuclear leukocytes. The extracts of corn, popped corn, and black walnuts all possessed the ability to stimulate leukocytes to produce this lymphokine (Table 1). 12
DISCUSSION
There is considerable documentation in the literature that several types of corn products can cause various kinds of inflammatory reactions. Corn starch has been found to produce a sterile type of granulomatosis lesion when dusted into an abdominal surgical s i t e . It was also suspected of producing a fibrous type of giant cell lesion when inserted into a dental extraction site as part of the 18
14
3. The incorporation of C-TdR by adult leukocytes exposed to varying dilutions of corn extract in vitro. Each point represents the mean counts per minute of triplicate cultures. FIGURE
Volume 46 Number 9
Human Lymphoproliferative
Response
TABLE 1. Chemotactic Activity for Human Mononuclear in Supernatants of Lymphocyte Cultures Material tested 1. Control (no stimulation) 2. 3. 4. 5.
Concanavalin A Corn Popped corn Black walnuts
565
Leucocytes
Chemotactic activity 8± 1* 83 77 23 26
± 11 ±8 ±4 ± 2
* Each value represents the mean and standard error of triplicate samples of monocytes which have migrated through 5.0 pores in a polycarbonate filter.
allergic response found only in sensitized individuals. Therefore, since these mitogenic extracts of corn and nuts are also capable of stimulating the production of mediators it is entirely conceivable that they may also be capable of inducing inflammatory reactions in vivo. Intradermal injections of mitogens, such as phytohemagglutinin, have been found to produce erythematous, indurated, cutaneous reactions similar to the delayed type of hypersensitivity reactions of sensitized individuals. The reaction peaked in 24 to 48 hours and histologically was characterized by a mononuclear cell infiltration of the area surrounding the cutaneous blood vessels and adnexa in the subcutaneous tissue and dermis. This reaction was accompanied by infiltration with polymorphonuclear leukocytes into the affected area. The periodontal abscess is histologically a similar type of lesion of undetermined etiology. The abscess has gener ally been attributed to obstruction of the clinical orifice of an intrabony defect, either by mechanical or physio logical means, with the concomittant accumulation of fluid and cells in the depths of the defect. The more superficial abscesses have been attributed to foreign bodies lodged in the sulcus (toothbrush bristles, e t c ) . A rather common finding during the treatment of these lesions is a corn or nut particle lodged in the abscess cavity. It is conceivable that the fragile epithelium of the crevice could be disrupted by the penetration of the hull and a cell-mediated hypersensitivity reaction initiated by the soluble mitogenic factor in the corn. This reaction might lead to massive tissue destruction with the release of lymphokines which would cause the inhibition of macrophage migration, the attraction of mononuclear cells, and the release of endogenous cytotoxic and cytolytic enzymes into the area of tissue destruction. The lesion resulting from this inflammatory response would be singularly more destructive and extensive than that resulting from a simple foreign body reaction. Food has long been suspected of exerting a significant effect on the development of periodontal disease, but its role has not been identified. Presumably, food particles provide nourishment for indigenous oral bacteria, whose potential for initiating periodontal disease has already been demonstrated. In addition, the detection of soluble factors within corn and nuts which are capable of 21
l4
5. The in vitro C-TdR incorporation by stimulated adult and newborn cord blood leukocytes. Each point repre sents the mean counts per minute of triplicate cultures from different donors. FIGURE
19
filler material in an antibiotic c o n e . Corn and its various derivatives have also been responsible for a wide variety of allergic responses. Intact corn starch particles can pass the intestinal barrier because of their high density and low particle size (6 to 15 ) through a process known as perabsorption. The particles have been found to be lodged in blood vessels, especially the small alveolar capillaries of the lung, and almost every organ of the b o d y . The ingress of the intact particles directly into the bloodstream offers a potential route for the development of sensitization to the material. Corn that is completely cooked (boiled) is relatively harmless as it cannot pass the intestinal barrier. Raw corn starch, on the other hand, is the major offender and is prevalent in short breads, cookies, pie crusts, popcorn, and tortillas. A large segment of our population may therefore be sensitized to the antigenic constituents of corn and should this material penetrate the epithelial barrier of the gingival crevice of a sensitized individual, a hyperim mune response would be expected to occur. Our findings indicate, however, that the in vitro blastogenesis of the adult and newborn leukocytes to corn as well as nut extracts are due to a ubiquitous nonspecific mitogenic stimulation rather than a classical 20
22
566
Radentz,
activating mediator
Baker, Altman,
lymphocytes in vitro
J. Periodontol. September, 1975
Oppenheim
to proliferate
and produce a
suggests that these materials may
penetrate into inflamed or traumatized gingival tissues and may also contribute directly to acute or chronic periodontal inflammation. SUMMARY
Extracts of corn and some nuts were found to ubiqui tously stimulate both adult and newborn cord blood lymphocytes to transform and produce a factor chemo tactic for monocytes. This indicates that corn and nuts contain a mitogen and are potential stimulators of the cellular immune response. The exposure of lymphocytes to
this
mitogen
in vivo
might
trigger a
destructive
inflammatory reaction in the surrounding tissues. These findings, therefore, suggest that foods such as corn and nuts may be responsible for some periodontal abscesses and may be contributing factors to the development of intrabony alveolar lesions and chronic periodontitis. REFERENCES
1. Löe, H. E., Theilade, E., and Jensen, S. B.: Experimen tal gingivitis in man. J Periodontol 3 6 : 177, 1967. 2. Glickman, I., and Smulow, J.: Alterations in the path way of gingival inflammation into the underlying tissue induced by excessive occlusal forces. J Periodontol 3 3 : 7, 1962. 3. Glickman, I., and Smulow, J.: The combined effects of inflammation and trauma from occlusion in periodontitis. Int Dent J 19: 393, 1969. 4. Ramfjord, S. P., and Ash, M. M.: Occlusion, p. 156. Philadelphia, W. B. Saunders Co., 1966. 5. Ivanyi, L., and Lehner, T.: The significance of serum factors in stimulation of lymphocytes from patients with periodontal disease by Veillonella alcalescens. Arch Allergy 4 1 : 620, 1971. 6. Horton, J. E., Leiken, S., and Oppenheim, J. J.: Human lymphoproliferative reaction to saliva and dental plaque de posits: An in vitro correlation with periodontal disease. J Periodontol 4 3 : 522, 1972. 7. Horton, J. E., Oppenheim, J. J., and Mergenhagen, S.
E.: A role for cell-mediated immunity in the pathogenesis of periodontal disease. J Periodontol 4 5 : 3 5 1 , 1974. 8. Prichard, J. F.: Advanced Periodontal Disease: Surgical and Prosthetic Management, p 271. Philadelphia, W. B. Saunders Co., 1966. 9. Truluck, M. H.: Acute periodontal abscess, diagnosis and treatment. J NC Dent Soc 4 4 : 15, 1960. 10. Glickman, I.: Clinical Periodontology, p 143. Philadel phia, W. B. Saunders Co., 1972. 11. O'Brian, T. J.: Diagnosis and treatment of periodontal abscess. J Wise Dent Soc 4 5 : 77, 1969. 12. Marquardt, J. L., Snyderman, R., and Oppenheim, J. J.: Depression of lymphocyte transformation and exacerbation of Behcet's syndrome by ingestion of English walnuts. Cell Immunol 9: 262, 1973. 13. Colowick, S. P., and Kaplan, N. O. (eds): Methods in Enzymology, Vol. 3, p. 696, Ne w York, Academic Press, 1957. 14. Tottschalk, A.: Glycoprotein: Their composition, struc ture and function, p 284. New York, Elsevier Publishing Co., 1972. 15. Snyderman, R., Altman, L. C , Hausman, M. S., and Mergenhagen, S. E.: Human mononuclear leukocyte chemotaxis: A quantitative assay for humoral and cellular chemotac tic factors. J Immunol 108: 857 (1972). 16. Altman, L. C , Snyderman, R., Oppenheim, J. J., and Mergenhagen, S. E.: A human mononuclear leukocyte chemo tactic factor: Characterization, specificity and kinetics of production by homologous leucocytes. J Immunol 110: 801, 1973. 17. Ling, N. R., and Husband, E. M.: Specific and nonspe cific stimulation of peripheral lymphocytes. Lancet 1: 363, 1964. 18. Pemberton, M., and Johnson, M.: Dangers of corn starch powder. Br Med J 3 : 235, 1973. 19. Miller, W. A.: Foreign body reaction to dental cone containing starch. Dent Pract 18: 428, 1968. 20. Lietz, A.: Laboratory research in food allergy. J Asthma Res 7: 127, 1970. 21. Blaese, R. M., Weiden, P., Oppenheim, J. J., and Waldmann, T. A.: Phytohemagglutinin as a skin test for the evaluation of cellular immune competence in man. J Lab Clin Med SI: 538, 1973. 22. Glickman, I.: Clinical Periodontology, p 249. Philadel phia, W. B. Saunders Co., 1972.
Abstracts SYNDESMOTIC LIMITING MOVEMENT OF THE PERIODONTAL LIGAMENT Heners, M .
INSTRUCTION IN ORAL HYGIENE FOR A GROUP OF DENTAL STUDENTS: ITS EFFECT ON THEIR PEERS
Int Dent J 24: 319, June, 1974.
Newcomb, G . M .
Contactless electronic pick-ups were fitted to the maxillary right central incisors of ten subjects to determine whether the periodontium has a specific function-dependent movement. By measuring simultane ously the horizontal and axial movements under specific forces in the sagittal plane, the tooth movement was recorded by a length measure ment in which the tooth crown was used as a mechanical reference point for an electronic displacement pick-up. It was demonstrated that the periodontium has a specific movement characteristic which guides the tooth in predetermined paths and can only be insignificantly changed by the direction of loading. In addition, the fine structure of the collagenous tissue was shown to be functionally orientated. Prosthetic Department, Klinik FürZahn-, Mund- und Kieferkrankheiten, Weimarerstrasse 8, D 2300, Kiel, Germany
J Public Health Dent 34: 113, Spring, 1974. After exposure of one group of 10 students to individual instruction in toothbrushing, plaque indexes (Loe and Silness) were made at one and two weeks for them and for a second group who had not had individual instruction. Definitely lowered indexes were found in the instructed group and in the group that was exposed to the first group, even though brushing instruction was very minimal. This behavioral change is brought about by modeling influences. A control group who had no contact with these positively reinforced modeling influences, demon strated no improvement in plaque index. This method of instruction is advocated as having possibilities for large group learning. Department of Periodontics, Royal Adelaide Hospital, North Terrace, Adelaide, South Australia, Australia
