A Cementum-Bound Antigen: Its Reaction With Serum Antibody and Localization, In Situ.*
periodontal pocket.11'12 This observation should not be surprising if one considers the wealth of bacterial end products (e.g., endotoxins) endogenous to plaque, which
in a very intimate association to the root surface. The present study employed a modified thin layer adsorption technique13 whereby a low pH extraction of cementum-bound antigen (CBA) from teeth exposed to a periodontal pocket was shown to react specifically with human serum antibody. In addition, an indirect immunofluorescence method was used to localize the presence of CBA in situ along the cementum surface. are
by Richard J. BravmanI Donald L. Everhart^ S. Sigmund Stahl
Materials
and
Methods
Collecting Blood Blood samples were collected IV at New York University Dental Center from 19 individuals ranging in age from 25 to 40 years. All of these patients suffered from periodontal disease ranging from gingivitis to advanced
The initiating agents in periodontal disease have been shown to originate from the bacterial accumulation in plaque.1"4 a direct relationship has been demonstrated2 between an increase in dental plaque and a concomitant increase in gingival inflammation. Antigens penetrating the gingival epithelium are capable of stimulating regional lymph nodes to produce immunocytes.5 Plasma cells which accumulate locally, may elaborate specific antibodies which form antigen-antibody complexes capable of activating the complement system. In the presence of continuing challenge by bacterial antigens, this process would be expected to become chronic and lead to tissue changes similar to those found in chronic periodontal disease. Endotoxins are potent inflammatory agents capable of inducing an acute inflammatory response when introduced locally into many animal species, including man.6 Aside from these direct pathological effects, it has been shown that the endotoxin of most gram negative bacteria are effective immunogens. Furthermore, these endotoxins can be removed from the cementai surface of periodontally involved teeth.6'7 Rizzo8 was able to demonstrate endotoxin adsorption into ulcerated gingival tissue but not into intact sulcular epithelium. More recent studies,9 however, demonstrate the ability of tritiated endotoxin to penetrate "clinically healthy" intact gingival crevicular epithelium. The critical function of cementum, that of anchoring periodontal ligament onto the root surface of a tooth, may be compromised by the presence of cementum-bound endotoxin.10 Only recently has the presence of an immunogen been demonstrated on the cementum of teeth exposed to a
Periodontitis.
Fourteen of these blood samples were taken from a random group of patients and designated as group A, while five samples were from patients in need of dental extractions specifically due to advanced Periodontitis. This group was designated as group B. The blood was left to clot, followed by centrifugation and removal of sera. All samples were preserved by the addition of 0.1% sodium azide and stored at 40°C.
Collecting Teeth Twenty-two individuals ranging in age from 25 to 40 years visited the Oral Surgery Clinic at New York University Dental Center in need of dental extractions. No significant medical history was noted in any patient. Extractions were indicated due to advanced Periodontitis (16 teeth), and bony impactions (six teeth). Eleven of the teeth from patients with advanced Periodontitis and three of the bony impacted teeth were tested in conjunction with the heterologous sera from group A. The remaining eight teeth were tested with group sera, where each of the five teeth exposed to periodontal pockets were matched with homologous patient sera. Three bony impacted teeth were tested against all five sera. For the purpose of this study, the teeth extracted for severe Periodontitis were considered the experimental group, while the impacted (bony) teeth were considered controls.
Immediately following surgery, the extracted teeth were placed in normal physiological saline, containing 0.1% sodium azide, and were stored at 4°C for no longer than 1 week. Teeth and sera from group A were used with the indium adsorption technique, whereas those from group were used for the detection of CBA in situ.
A portion of this material was presented at the 174th meeting of the American Chemical Society, September, 1977. t This work was submitted for partial fulfillment of the M.S. degree at New York University, Graduate Division. Source of Support: Department of Microbiology Life. Savers Educational Fund. Present Address: Tufts University School of Dental Medicine Boston, MA 02111. For reprints contact Donald L. Everhart, New York University Dental Center, 421 First Ave., New York, NY. *
Indium Adsorption
Technique: Preparation Cementum was root planed from the surface of all teeth in this group, using a curet. Cementum from the 11
656
Volume 50 Number 12
Cementum-Bound Antigen
periodontally
involved teeth, was pooled as was the from the impacted teeth. The cementum, suspended in normal saline, was centrifuged for 10 minutes at 2500 rpm and the supernatant discarded. The cementum was then extracted, at room temperature, by the addition of 2 ml/curettaged tooth of glycline-HCl buffer, pH 2.514 and continually mixed for 20 minutes. This suspension was centrifuged for 10 minutes at 2500 rpm and the supernatant retrieved. The cementai extract was then dialyzed against several consecutive changes of distilled water followed by normal saline, and lyophilized until further use. Just prior to use, the lyophilate was resuspended in normal saline and dialyzed against the same. Normal saline was the sole diluent used. The use of phosphate or borate buffers in dilutions will prevent the adsorption of protein onto the indium surface.13 Indium coated slides were prepared by evaporating molten indium powder, 325 mesh-M5N,* onto the surface of alcohol-cleaned Fisher glass slides (75 X 25 mm). Evaporation was carried out at about 10~5 Torr in a Veeco "400 series" vacuum evaporatori until the slides appeared moderately brown in transmitted light.13'15
657
1
cementum
Detection
of CBA by Indium Method
A modified method of Giaever13 was used to determine
antibody in the sera samples. The method was as follows: A capillary tube (1.6 X 100 mm) was used to transfer a drop of CBA to the indium surface of the prepared glass slide. The slide was subsequently left to incubate for 3M> hours at 23 °C in
a
moist chamber, to allow time for
complete adsorption of CBA to the surface of the indium particles. Following incubation, the CBA drop was removed under vacuum, through a disposable glass pipette,
rinsed for 10 seconds with distilled water, and the indium surface dried immediately with compressed air. Observation of the slide through transmitted light at this time, allowed the visualization of a dark brown dot where the initial layer of antigen had been adsorbed. The dot was then overlayed with a drop of larger circumference using the human serum. Incubation in a moist chamber was continued for 30 minutes at 23 °C followed by the identical procedure for washing and drying as previously described. AU sera were tested in the same manner on a slide adsorbed with paired drops of CBA and extraction from bony impaction (EBI). A second control was the placing of rabbit sera over CBA and this gave a negative reaction.
of CBA in situ Longitudinal sections (0.17 mm) were prepared from all teeth with a ground sectioning machine and stored, in normal saline containing 0.1% sodium azide, at 4°C Detection
until used. *
Ventrón Corp., Danvers, MA. Instrument Inc., 400 Series, Plainview, NY.
t Veeco
Figure 1. Indium adsorption. CBA extract overlayed with human sera samples. CBA extract dots (2) in lower right hand corner of the slide were not overlayed with serum.
seven
Each of the sections of periodontally involved teeth and bony impactions were placed into a modified Leighton tissue culture tube which contained 1.0 ml of a 1:10 dilution in borate saline of homologous serum and incubated for 30 minutes at 37°C in a water bath. In addition, sections from two of the periodontally involved teeth were also incubated initially with heterologous serum in place of homologous serum. Each section was thoroughly rinsed with a continuous stream of borate saline followed by three consecutive transfers in 30 ml borate saline and a final rinse in distilled water. Each section was then incubated in an identical fashion with 1.0 ml of a 1:10 dilution with borate saline of rhodamine conjugated goat antihuman antibody! (with the combined specificities: IgG, IgA, IgM and Kappa and Lambda), and washed as previously described. Sections were immediately mounted with buffered glycerol mounting medium onto slides. Glass cover slips were placed over the glycerol coated section and finger nail polish was used as a sealant around the edges. From the conclusion of this mounting step, all slides not immediately being viewed were stored in the dark. Slides were
Cappel Laboratories, Cochranville,
PA
658
J. Periodontol.
Bravman, Everharí, Stahl
December, 1979
showed that a range of antibody titers existed to CBA. The results (Fig. 1) demonstrate a variety of responses to CBA. A positive antigen-antibody reaction is evident (Fig. 1 and Fig. 2) when the original antigen dot remains visible in the center of the overlayed film of serum. From Figure 2 it is clear that human antibody is specific for the glycine-HCl extraction of CBA and not to the identical extraction of cementum from bony impacted teeth. Localization
of CBA
in situ
Having established the fact- that human sera, and specifically that obtained from patients with periodontal disease, contains antibody which reacts specifically with a cementum-bound antigen, the next logical step was to Figure 2. Indium adsorption. Each slide demonstrates (from to right): CBA overlayed with human serum (positive rx.); EBI overlayed with human serum (negative rx.); a diagramatic representation of the previous results. Three different serum samples were utilized, one for each horizontal row.
left
mgure 3. Ground section of a periodontally diseased pocket.
tooth
previously exposed
localize its presence on the root surface of a sectioned tooth. Indirect immunofluorescence was consistently present on sections from periodontally involved teeth reacted with periodontal serum, but never detected on the cementai surface of bony impacted teeth. After a careful scan of the tooth contour, CBA was detected on the apical one-third of the root surface in each of the teeth extracted from patients with advanced periodontal disease. The clinical pocket in these patients extended to the apical one-third of the tooth. The presence of bacterial antigens therefore would be expected in the vicinity
to a
viewed using a Zeiss Universal Microscope with a darkfield condenser under oil immersion and an Osran HBO200 light source. In the light path were built in antithermal filters and a BG-12 exciter filter. Barrier filters 41 and 47 were used for very sharp wave cut offs. Photographs were taken with a 35 mm Nikon equipped with Kodak high speed extrachrome (daylight) film rated at ASA 160. The exposure time for optimal detection of rhodamine fluorescence was found to be 8 minutes.
Results
Antibody to Cementum-Bound Antigen A serological screening of 14 samples of sera collected from patients with varying degrees of periodontal disease
Figure 4. Darkfield micrograph 3 (Magnification, x 200).
of boxed in area from Figure
Volume 50 Number 12
Cementum-Bound Antigen 659 maintains with the surrounding periodontium. It is well established16 that the bonding of IgG or IgM to plaque antigens (or any antigen for that matter) forms immune complexes which can fix complement, resulting in the release of cytotoxin substances and other products responsible for the ultimate release of harmful lymphokines. The fact that no antigenic activity could be associated with the cementum from unexposed bony impacted teeth has great importance for at least two reasons. Primarily, it suggests that exposure of at least the crown is necessary for the presence of CBA. It also eliminates the possibility of induced antigenicity on the cementum as a result of any surface alterations incurred during extraction procedures.
1
Summary
Figure 5. Darkfield micrograph of boxed in area from Figure 3. Rhodamine fluorescence is evident along outer surface of cementum, indicating presence of antibody-antigen complex
(Magnification,
x
200).
of the sulcular base and perhaps some distance apical to the junctional attachment. A darkfield micrograph (Fig. 4) of this region clearly shows that it was the cementum in Figure 5, which was associated with the rhodamine fluorescence. Discussion Our results are the initial observations demonstrating that human antibody reacts with a cementum-bound antigen. The indium slide method demonstrated that CBA removed from cementum exposed to a periodontal pocket reacts with serum. As shown in Figure 1, the reaction varies in intensity suggesting that the concentration of antibody in the serum samples used was different. Since only 14 patients were tested, there was no attempt to quantitate titers or to establish a correlation between severity of periodontal disease and the presence, absence or degree of an antibody titer to CBA. We propose to follow this in further studies. The serum component was also identified in situ by reacting the serum with tooth sections and using indirect immunofluorescence to demonstrate the presence of antibody. The localization of antigen on the cementum has special meaning because of the intimate association it
The results which have been presented give clear evidence to the presence of an antigen(s) on the cementum of teeth exposed to a periodontal pocket, which is capable of reacting with human serum antibody. It was not possible to demonstrate human antibody reactivity against any components derived from the cementum of completely unexposed bony impacted teeth. Regardless of any speculations dealing with the character or origin of such antigens, their very presence would suggest they may have profound implications in the etiology and progression of the periodontal disease process. Acknowledgments
The authors wish to thank Dr. J. J. Hauser of Bell Laboratories and Dr. Steve Arnold, Department of Physics at New York University for the time and assistance offered in the preparation of indium slides. We also wish to thank the Department of Histology at the New York University Dental Center for the use of the sectioning machine.
References 1. Genco, R. J., Mashimo, P. ., Krygier, G., and Ellison, S. .: Antibody-mediated effecrs on the periodontium. J Periodontol 45: 330, 1974. 2. Loe, ., Theilade, E., and Jensen, S. B.: Experimental gingivitis in man. J Periodontol 36: 177, 1965. 3. Socransky, S. S.: Relationship of bacteria to the etiology of periodontal disease. J Dent Res 49: 203, 1970. 4. Zander, . ., Poison, A. M., and Heigl, L. C: Goals of periodontal therapy. J Peridontol 4 : 261, 1976. 5. Mergenhagen, S. E., Tempel, T. R., and Snyderman, R.: Immunologie reactions periodontal inflammation. J Dent Res 49: 256A, 1970. 6. Thomas, L.: The physiological disturbances produced by endotoxins. Ann Rev Physiol 16: 467, 1954. 7. Fine, D. H., Morris, M., Tabak, L., and Epstein, .: Extraction of endotoxin-like material from periodontally diseased cementum. J Dent Res 56: A45, 1977. 8. Rizzo, . .: Adsorption of bacterial endotoxin into rabbit gingival pocket tissue. Periodontics 6: 65, 1968. 9. Schwartz, J., Stinson, F. L., and Parker, R. B.: The passage of tritiated bacterial endotoxin across intact gingival crevicular epithelium. J Periodontol 43: 270, 1972. 10. Aleo, J. J., DeRenzis, F. ., Farber, P. ., and Varbon-
660
J. Periodontol. 1979
Bravman, Everhart, Stahl
December,
presence and biologic activity of cementumbound entotoxin. J Periodontol 45: 672, 1974. 11. Everhart, D. L., and Stahl, S. S.: A possible source of antigen(s) in periodontal disease. J Dent Res 55: B222, 1976. 12. Everhart, D. L., and Stahl, S. S.: The reaction of a component recovered from periodontally involved cementum with human serum. J Dent Res 56: 538, 1977. 13. Giaever, L: Visual detection of carcinoembryonic antigen on surfaces. J Immunol 116: 766, 1976. coer, A. P.: The
14. Campbell, D.H., Garvey, J. S., Cremer, . E., and Sussdorf, D. H.: Methods in Immunology, ed 2, 431. New York, W. A. Benjamin, Inc., 1963. 15. Giaever, I., and Laffin, R. J.: Visual detection of hepatitis antigen. Proc Nat Acad Sci 71: 4533, 1974. 16. Madder, B. F., Frostad, . B., Robertson, P. B., and
Levy, . M.: Immunoglobulin bearing lymphocytes and plasma cells in human periodontal disease. J Periodont Res 12: 37, 1977.
Abstracts Recording of the Retruded Position of the Mandible Patients With Mandibular Dysfunction
in
A Method for Evaluation of Initial Tissue Response to
Biomaterials
Helkimo, M., and Ingervall, B. Acta Odontol Scand 36: 167, No. 3, 1978.
Wennberg, ., Hasseigren, G., and Tronstad, L.
Ten patients (ages 19-55 years), all of whom had symptoms of pain and dysfunction of the masticatory system, were placed in the retruded position and this position recorded before treatment was initiated. Patients were treated with a maxillary bite splint and all occlusal interferences were eliminated. A second recording was performed within 6 months after the initial recording. The occurrence of objective and subjective symptoms was reduced in all patients by the second recording and six were almost symptom free. In a comparison between the passive terminal hinge, active terminal hinge and habitual closure, the passive terminal hinge recording showed the best consistency. It was stated that when the functional state of the masticatory system was improved the mandible could be positioned somewhat further distally than during the more acute stages of the symptoms. The results were said to indicate that simple instruction in relaxation of the jaw for a passive registration is sufficient to obtain a clinically acceptable retruded position of the mandible in most cases. Department of Stomatognathie Physiology, Faculty of Odontology, University of Umeâ, S901 87 Umea, Sweden. Dr. Gary Galovic
into the thigh muscles of anesthetized and muscle tissue surrounding the implant removed to expose the three pronged base of the teflon body, which, when removed, left three indentations in the connective tissue. Silicate cement, zinc phosphate cement, and a 4% phenol solution were tested by placing each in an indentation and leaving for 15 minutes, with a control of isotonic saline placed in one out of every six indentations. The block of muscle tissue surrounding the test area was dissected out, and incubated for histochemicals demonstration of succinate dehydrogenase and lactate dehydrogenase as markers of the Krebs cycle and glycolysis. Histochemical examination showed no evidence of dehydrogenase activity in indentations filled with test materials, but dehydrogenase activity was noted in control indentations. The results were believed to indicate that in conjunction with the implantation method described, changes in dehydrogenase activity can be utilized as a valid criterion of initial tissue response to materials. Department of Endodontics, School of Dentistry, Dr. Gary Galovic S-21421 Malmö, Sweden.
The Occurrence of TMJ-Disorders in an Elderly Population as Evaluated by Recording of Subjective and Objective
Factors Influencing Clinical Use of Antimicrobial Agents in Elderly Patients
Symptoms
Helòe, B., and Helòe, L. A. Acta Odontol Scand 36: 3, No. 1, 1978.
self-perceived temporomandibular joint (TMJ) pain and clinically registered findings to selected variables, 241 subjects were To relate
studied, 113 males and 128 females, ages 65 to 79. Standardized questioning was used to elicit subjective symptoms of pain and clicking,
well as general rheumatism. Participants were then given a standard clinical examination to determine objective presence of TMJ dysfunction. The results revealed that 8% had TMJ pain, 14% reported clicking or crepitation, while clinically 27% had these symptoms. Women were found more likely to report subjective symptoms not clinically verified. The findings also indicated that although diagnosed disorders increased with age, the reports of symptoms decreased. No evidence was found that would support the theory that occlusal disharmony or early loss of molars had led to joint changes or to subjective symptoms. Department of Oral Surgery and Oral Medicine, Geitmyrsveien 71, Oslo 4, Norway. Dr. Gary Galovic as
Acta Odontol Scand 36:
Teflon bodies
67, No. 2, 1978.
(3 prong)
were
implanted
rabbits, and after 8 weeks the animals
were
Moellering, R. C. Geriatrics 33: 83, February, 1978. Infection may be more difficult to control in elderly patients due to disease. Many host factors can influence dosage and effectiveness, including those related to degenerative diseases, deterioration of physiologic processes and nonspecific mechanisms that enhance certain adverse drug effects. Examples used for degenerative diseases included the use of oral antibiotics which may slow down the uptake of digitalis. Some antibiotics potentiate hypoglycémie agents in diabetes. Numerous agents may by their toxicity show increased cardiovascular disease. Antimicrobials can interact with coumarin type anticoagulants. Age-related physiologic deterioration was illustrated by the effects observed in the excretion of antimicrobials and the increase in serum levels. Gastric achlorhydria is able to enhance the absorption of antibiotics. There is evidence to show that hypersensitivity reactions to antimicrobial agents are more common in older than younger people. Infectious Disease Unit, Massachusetts General Hospital, Boston, Mass 02114. Dr. Larry Johnson
degenerative
periodontal pocket.11'12 This observation should not be surprising if one considers the wealth of bacterial end products (e.g., endotoxins) endogenous to plaque, which
in a very intimate association to the root surface. The present study employed a modified thin layer adsorption technique13 whereby a low pH extraction of cementum-bound antigen (CBA) from teeth exposed to a periodontal pocket was shown to react specifically with human serum antibody. In addition, an indirect immunofluorescence method was used to localize the presence of CBA in situ along the cementum surface. are
by Richard J. BravmanI Donald L. Everhart^ S. Sigmund Stahl
Materials
and
Methods
Collecting Blood Blood samples were collected IV at New York University Dental Center from 19 individuals ranging in age from 25 to 40 years. All of these patients suffered from periodontal disease ranging from gingivitis to advanced
The initiating agents in periodontal disease have been shown to originate from the bacterial accumulation in plaque.1"4 a direct relationship has been demonstrated2 between an increase in dental plaque and a concomitant increase in gingival inflammation. Antigens penetrating the gingival epithelium are capable of stimulating regional lymph nodes to produce immunocytes.5 Plasma cells which accumulate locally, may elaborate specific antibodies which form antigen-antibody complexes capable of activating the complement system. In the presence of continuing challenge by bacterial antigens, this process would be expected to become chronic and lead to tissue changes similar to those found in chronic periodontal disease. Endotoxins are potent inflammatory agents capable of inducing an acute inflammatory response when introduced locally into many animal species, including man.6 Aside from these direct pathological effects, it has been shown that the endotoxin of most gram negative bacteria are effective immunogens. Furthermore, these endotoxins can be removed from the cementai surface of periodontally involved teeth.6'7 Rizzo8 was able to demonstrate endotoxin adsorption into ulcerated gingival tissue but not into intact sulcular epithelium. More recent studies,9 however, demonstrate the ability of tritiated endotoxin to penetrate "clinically healthy" intact gingival crevicular epithelium. The critical function of cementum, that of anchoring periodontal ligament onto the root surface of a tooth, may be compromised by the presence of cementum-bound endotoxin.10 Only recently has the presence of an immunogen been demonstrated on the cementum of teeth exposed to a
Periodontitis.
Fourteen of these blood samples were taken from a random group of patients and designated as group A, while five samples were from patients in need of dental extractions specifically due to advanced Periodontitis. This group was designated as group B. The blood was left to clot, followed by centrifugation and removal of sera. All samples were preserved by the addition of 0.1% sodium azide and stored at 40°C.
Collecting Teeth Twenty-two individuals ranging in age from 25 to 40 years visited the Oral Surgery Clinic at New York University Dental Center in need of dental extractions. No significant medical history was noted in any patient. Extractions were indicated due to advanced Periodontitis (16 teeth), and bony impactions (six teeth). Eleven of the teeth from patients with advanced Periodontitis and three of the bony impacted teeth were tested in conjunction with the heterologous sera from group A. The remaining eight teeth were tested with group sera, where each of the five teeth exposed to periodontal pockets were matched with homologous patient sera. Three bony impacted teeth were tested against all five sera. For the purpose of this study, the teeth extracted for severe Periodontitis were considered the experimental group, while the impacted (bony) teeth were considered controls.
Immediately following surgery, the extracted teeth were placed in normal physiological saline, containing 0.1% sodium azide, and were stored at 4°C for no longer than 1 week. Teeth and sera from group A were used with the indium adsorption technique, whereas those from group were used for the detection of CBA in situ.
A portion of this material was presented at the 174th meeting of the American Chemical Society, September, 1977. t This work was submitted for partial fulfillment of the M.S. degree at New York University, Graduate Division. Source of Support: Department of Microbiology Life. Savers Educational Fund. Present Address: Tufts University School of Dental Medicine Boston, MA 02111. For reprints contact Donald L. Everhart, New York University Dental Center, 421 First Ave., New York, NY. *
Indium Adsorption
Technique: Preparation Cementum was root planed from the surface of all teeth in this group, using a curet. Cementum from the 11
656
Volume 50 Number 12
Cementum-Bound Antigen
periodontally
involved teeth, was pooled as was the from the impacted teeth. The cementum, suspended in normal saline, was centrifuged for 10 minutes at 2500 rpm and the supernatant discarded. The cementum was then extracted, at room temperature, by the addition of 2 ml/curettaged tooth of glycline-HCl buffer, pH 2.514 and continually mixed for 20 minutes. This suspension was centrifuged for 10 minutes at 2500 rpm and the supernatant retrieved. The cementai extract was then dialyzed against several consecutive changes of distilled water followed by normal saline, and lyophilized until further use. Just prior to use, the lyophilate was resuspended in normal saline and dialyzed against the same. Normal saline was the sole diluent used. The use of phosphate or borate buffers in dilutions will prevent the adsorption of protein onto the indium surface.13 Indium coated slides were prepared by evaporating molten indium powder, 325 mesh-M5N,* onto the surface of alcohol-cleaned Fisher glass slides (75 X 25 mm). Evaporation was carried out at about 10~5 Torr in a Veeco "400 series" vacuum evaporatori until the slides appeared moderately brown in transmitted light.13'15
657
1
cementum
Detection
of CBA by Indium Method
A modified method of Giaever13 was used to determine
antibody in the sera samples. The method was as follows: A capillary tube (1.6 X 100 mm) was used to transfer a drop of CBA to the indium surface of the prepared glass slide. The slide was subsequently left to incubate for 3M> hours at 23 °C in
a
moist chamber, to allow time for
complete adsorption of CBA to the surface of the indium particles. Following incubation, the CBA drop was removed under vacuum, through a disposable glass pipette,
rinsed for 10 seconds with distilled water, and the indium surface dried immediately with compressed air. Observation of the slide through transmitted light at this time, allowed the visualization of a dark brown dot where the initial layer of antigen had been adsorbed. The dot was then overlayed with a drop of larger circumference using the human serum. Incubation in a moist chamber was continued for 30 minutes at 23 °C followed by the identical procedure for washing and drying as previously described. AU sera were tested in the same manner on a slide adsorbed with paired drops of CBA and extraction from bony impaction (EBI). A second control was the placing of rabbit sera over CBA and this gave a negative reaction.
of CBA in situ Longitudinal sections (0.17 mm) were prepared from all teeth with a ground sectioning machine and stored, in normal saline containing 0.1% sodium azide, at 4°C Detection
until used. *
Ventrón Corp., Danvers, MA. Instrument Inc., 400 Series, Plainview, NY.
t Veeco
Figure 1. Indium adsorption. CBA extract overlayed with human sera samples. CBA extract dots (2) in lower right hand corner of the slide were not overlayed with serum.
seven
Each of the sections of periodontally involved teeth and bony impactions were placed into a modified Leighton tissue culture tube which contained 1.0 ml of a 1:10 dilution in borate saline of homologous serum and incubated for 30 minutes at 37°C in a water bath. In addition, sections from two of the periodontally involved teeth were also incubated initially with heterologous serum in place of homologous serum. Each section was thoroughly rinsed with a continuous stream of borate saline followed by three consecutive transfers in 30 ml borate saline and a final rinse in distilled water. Each section was then incubated in an identical fashion with 1.0 ml of a 1:10 dilution with borate saline of rhodamine conjugated goat antihuman antibody! (with the combined specificities: IgG, IgA, IgM and Kappa and Lambda), and washed as previously described. Sections were immediately mounted with buffered glycerol mounting medium onto slides. Glass cover slips were placed over the glycerol coated section and finger nail polish was used as a sealant around the edges. From the conclusion of this mounting step, all slides not immediately being viewed were stored in the dark. Slides were
Cappel Laboratories, Cochranville,
PA
658
J. Periodontol.
Bravman, Everharí, Stahl
December, 1979
showed that a range of antibody titers existed to CBA. The results (Fig. 1) demonstrate a variety of responses to CBA. A positive antigen-antibody reaction is evident (Fig. 1 and Fig. 2) when the original antigen dot remains visible in the center of the overlayed film of serum. From Figure 2 it is clear that human antibody is specific for the glycine-HCl extraction of CBA and not to the identical extraction of cementum from bony impacted teeth. Localization
of CBA
in situ
Having established the fact- that human sera, and specifically that obtained from patients with periodontal disease, contains antibody which reacts specifically with a cementum-bound antigen, the next logical step was to Figure 2. Indium adsorption. Each slide demonstrates (from to right): CBA overlayed with human serum (positive rx.); EBI overlayed with human serum (negative rx.); a diagramatic representation of the previous results. Three different serum samples were utilized, one for each horizontal row.
left
mgure 3. Ground section of a periodontally diseased pocket.
tooth
previously exposed
localize its presence on the root surface of a sectioned tooth. Indirect immunofluorescence was consistently present on sections from periodontally involved teeth reacted with periodontal serum, but never detected on the cementai surface of bony impacted teeth. After a careful scan of the tooth contour, CBA was detected on the apical one-third of the root surface in each of the teeth extracted from patients with advanced periodontal disease. The clinical pocket in these patients extended to the apical one-third of the tooth. The presence of bacterial antigens therefore would be expected in the vicinity
to a
viewed using a Zeiss Universal Microscope with a darkfield condenser under oil immersion and an Osran HBO200 light source. In the light path were built in antithermal filters and a BG-12 exciter filter. Barrier filters 41 and 47 were used for very sharp wave cut offs. Photographs were taken with a 35 mm Nikon equipped with Kodak high speed extrachrome (daylight) film rated at ASA 160. The exposure time for optimal detection of rhodamine fluorescence was found to be 8 minutes.
Results
Antibody to Cementum-Bound Antigen A serological screening of 14 samples of sera collected from patients with varying degrees of periodontal disease
Figure 4. Darkfield micrograph 3 (Magnification, x 200).
of boxed in area from Figure
Volume 50 Number 12
Cementum-Bound Antigen 659 maintains with the surrounding periodontium. It is well established16 that the bonding of IgG or IgM to plaque antigens (or any antigen for that matter) forms immune complexes which can fix complement, resulting in the release of cytotoxin substances and other products responsible for the ultimate release of harmful lymphokines. The fact that no antigenic activity could be associated with the cementum from unexposed bony impacted teeth has great importance for at least two reasons. Primarily, it suggests that exposure of at least the crown is necessary for the presence of CBA. It also eliminates the possibility of induced antigenicity on the cementum as a result of any surface alterations incurred during extraction procedures.
1
Summary
Figure 5. Darkfield micrograph of boxed in area from Figure 3. Rhodamine fluorescence is evident along outer surface of cementum, indicating presence of antibody-antigen complex
(Magnification,
x
200).
of the sulcular base and perhaps some distance apical to the junctional attachment. A darkfield micrograph (Fig. 4) of this region clearly shows that it was the cementum in Figure 5, which was associated with the rhodamine fluorescence. Discussion Our results are the initial observations demonstrating that human antibody reacts with a cementum-bound antigen. The indium slide method demonstrated that CBA removed from cementum exposed to a periodontal pocket reacts with serum. As shown in Figure 1, the reaction varies in intensity suggesting that the concentration of antibody in the serum samples used was different. Since only 14 patients were tested, there was no attempt to quantitate titers or to establish a correlation between severity of periodontal disease and the presence, absence or degree of an antibody titer to CBA. We propose to follow this in further studies. The serum component was also identified in situ by reacting the serum with tooth sections and using indirect immunofluorescence to demonstrate the presence of antibody. The localization of antigen on the cementum has special meaning because of the intimate association it
The results which have been presented give clear evidence to the presence of an antigen(s) on the cementum of teeth exposed to a periodontal pocket, which is capable of reacting with human serum antibody. It was not possible to demonstrate human antibody reactivity against any components derived from the cementum of completely unexposed bony impacted teeth. Regardless of any speculations dealing with the character or origin of such antigens, their very presence would suggest they may have profound implications in the etiology and progression of the periodontal disease process. Acknowledgments
The authors wish to thank Dr. J. J. Hauser of Bell Laboratories and Dr. Steve Arnold, Department of Physics at New York University for the time and assistance offered in the preparation of indium slides. We also wish to thank the Department of Histology at the New York University Dental Center for the use of the sectioning machine.
References 1. Genco, R. J., Mashimo, P. ., Krygier, G., and Ellison, S. .: Antibody-mediated effecrs on the periodontium. J Periodontol 45: 330, 1974. 2. Loe, ., Theilade, E., and Jensen, S. B.: Experimental gingivitis in man. J Periodontol 36: 177, 1965. 3. Socransky, S. S.: Relationship of bacteria to the etiology of periodontal disease. J Dent Res 49: 203, 1970. 4. Zander, . ., Poison, A. M., and Heigl, L. C: Goals of periodontal therapy. J Peridontol 4 : 261, 1976. 5. Mergenhagen, S. E., Tempel, T. R., and Snyderman, R.: Immunologie reactions periodontal inflammation. J Dent Res 49: 256A, 1970. 6. Thomas, L.: The physiological disturbances produced by endotoxins. Ann Rev Physiol 16: 467, 1954. 7. Fine, D. H., Morris, M., Tabak, L., and Epstein, .: Extraction of endotoxin-like material from periodontally diseased cementum. J Dent Res 56: A45, 1977. 8. Rizzo, . .: Adsorption of bacterial endotoxin into rabbit gingival pocket tissue. Periodontics 6: 65, 1968. 9. Schwartz, J., Stinson, F. L., and Parker, R. B.: The passage of tritiated bacterial endotoxin across intact gingival crevicular epithelium. J Periodontol 43: 270, 1972. 10. Aleo, J. J., DeRenzis, F. ., Farber, P. ., and Varbon-
660
J. Periodontol. 1979
Bravman, Everhart, Stahl
December,
presence and biologic activity of cementumbound entotoxin. J Periodontol 45: 672, 1974. 11. Everhart, D. L., and Stahl, S. S.: A possible source of antigen(s) in periodontal disease. J Dent Res 55: B222, 1976. 12. Everhart, D. L., and Stahl, S. S.: The reaction of a component recovered from periodontally involved cementum with human serum. J Dent Res 56: 538, 1977. 13. Giaever, L: Visual detection of carcinoembryonic antigen on surfaces. J Immunol 116: 766, 1976. coer, A. P.: The
14. Campbell, D.H., Garvey, J. S., Cremer, . E., and Sussdorf, D. H.: Methods in Immunology, ed 2, 431. New York, W. A. Benjamin, Inc., 1963. 15. Giaever, I., and Laffin, R. J.: Visual detection of hepatitis antigen. Proc Nat Acad Sci 71: 4533, 1974. 16. Madder, B. F., Frostad, . B., Robertson, P. B., and
Levy, . M.: Immunoglobulin bearing lymphocytes and plasma cells in human periodontal disease. J Periodont Res 12: 37, 1977.
Abstracts Recording of the Retruded Position of the Mandible Patients With Mandibular Dysfunction
in
A Method for Evaluation of Initial Tissue Response to
Biomaterials
Helkimo, M., and Ingervall, B. Acta Odontol Scand 36: 167, No. 3, 1978.
Wennberg, ., Hasseigren, G., and Tronstad, L.
Ten patients (ages 19-55 years), all of whom had symptoms of pain and dysfunction of the masticatory system, were placed in the retruded position and this position recorded before treatment was initiated. Patients were treated with a maxillary bite splint and all occlusal interferences were eliminated. A second recording was performed within 6 months after the initial recording. The occurrence of objective and subjective symptoms was reduced in all patients by the second recording and six were almost symptom free. In a comparison between the passive terminal hinge, active terminal hinge and habitual closure, the passive terminal hinge recording showed the best consistency. It was stated that when the functional state of the masticatory system was improved the mandible could be positioned somewhat further distally than during the more acute stages of the symptoms. The results were said to indicate that simple instruction in relaxation of the jaw for a passive registration is sufficient to obtain a clinically acceptable retruded position of the mandible in most cases. Department of Stomatognathie Physiology, Faculty of Odontology, University of Umeâ, S901 87 Umea, Sweden. Dr. Gary Galovic
into the thigh muscles of anesthetized and muscle tissue surrounding the implant removed to expose the three pronged base of the teflon body, which, when removed, left three indentations in the connective tissue. Silicate cement, zinc phosphate cement, and a 4% phenol solution were tested by placing each in an indentation and leaving for 15 minutes, with a control of isotonic saline placed in one out of every six indentations. The block of muscle tissue surrounding the test area was dissected out, and incubated for histochemicals demonstration of succinate dehydrogenase and lactate dehydrogenase as markers of the Krebs cycle and glycolysis. Histochemical examination showed no evidence of dehydrogenase activity in indentations filled with test materials, but dehydrogenase activity was noted in control indentations. The results were believed to indicate that in conjunction with the implantation method described, changes in dehydrogenase activity can be utilized as a valid criterion of initial tissue response to materials. Department of Endodontics, School of Dentistry, Dr. Gary Galovic S-21421 Malmö, Sweden.
The Occurrence of TMJ-Disorders in an Elderly Population as Evaluated by Recording of Subjective and Objective
Factors Influencing Clinical Use of Antimicrobial Agents in Elderly Patients
Symptoms
Helòe, B., and Helòe, L. A. Acta Odontol Scand 36: 3, No. 1, 1978.
self-perceived temporomandibular joint (TMJ) pain and clinically registered findings to selected variables, 241 subjects were To relate
studied, 113 males and 128 females, ages 65 to 79. Standardized questioning was used to elicit subjective symptoms of pain and clicking,
well as general rheumatism. Participants were then given a standard clinical examination to determine objective presence of TMJ dysfunction. The results revealed that 8% had TMJ pain, 14% reported clicking or crepitation, while clinically 27% had these symptoms. Women were found more likely to report subjective symptoms not clinically verified. The findings also indicated that although diagnosed disorders increased with age, the reports of symptoms decreased. No evidence was found that would support the theory that occlusal disharmony or early loss of molars had led to joint changes or to subjective symptoms. Department of Oral Surgery and Oral Medicine, Geitmyrsveien 71, Oslo 4, Norway. Dr. Gary Galovic as
Acta Odontol Scand 36:
Teflon bodies
67, No. 2, 1978.
(3 prong)
were
implanted
rabbits, and after 8 weeks the animals
were
Moellering, R. C. Geriatrics 33: 83, February, 1978. Infection may be more difficult to control in elderly patients due to disease. Many host factors can influence dosage and effectiveness, including those related to degenerative diseases, deterioration of physiologic processes and nonspecific mechanisms that enhance certain adverse drug effects. Examples used for degenerative diseases included the use of oral antibiotics which may slow down the uptake of digitalis. Some antibiotics potentiate hypoglycémie agents in diabetes. Numerous agents may by their toxicity show increased cardiovascular disease. Antimicrobials can interact with coumarin type anticoagulants. Age-related physiologic deterioration was illustrated by the effects observed in the excretion of antimicrobials and the increase in serum levels. Gastric achlorhydria is able to enhance the absorption of antibiotics. There is evidence to show that hypersensitivity reactions to antimicrobial agents are more common in older than younger people. Infectious Disease Unit, Massachusetts General Hospital, Boston, Mass 02114. Dr. Larry Johnson
degenerative
