]. Periodontal Res. 13: 37^5, 1978
Periodontal status of patients with abnormalities of the immune system p. B. ROBERTSON, T, E, WRIGHT, III, B. F. MACKLER, D , M , LENERTZ AND B, M, LEVY
The University of Connecticut School of Dental Medicine, Farmington, Connecticut and The University of Texas Dental Branch and Dental Science Institute, Houston, Texas Patients with immune system abnormalities were pair-matched to immunocompetent clinic patients by age and Plaque Index, Clinical examination included comprehensive health histories with summaries of antibiotic experience. Plaque Index (P1,I.), Gingival Index (G.L), Periodontal Disease Index (P,D,I,), caries experience (DMF-T) and full mouth radiographic surveys. Efficiency of matching was confirmed as tests of significance between sample means for age (11.3, 11,3) and PLI. (1.33, 1.34) showed no difference between the immunodeficient and clinic patient groups respectively. No clinical or radiographic evidence of attachment loss was noted in either group, A significant and consistent difference was observed in gingival inflammation and caries experience. The G.I. was lower (x = 1,05) in immunodeficient patients than that recorded in clinic patients (x = 1,46) in all pairs. Similar results were observed for caries experience, and 8 immunodeficient patients demonstrated DMF-T scores of zero. :
,'
(Accepted for publication May 12, 1977)
Introduction
There is considerable evidence to suggest that plaque-induced effector mechanisms play a major role in the pathogenesis of periodontal disease, A number of investigations have implicated both cellular and humoral pathways in periodontal tissue destruction (Ellison 1970, Genco, Evans & Ellison 1969, Oppenheim & Horton 1973). Correlations of varying magnitudes have been demonstrated between the clinical severity of periodontal disease and plaque/ oral bacterial antigen-induced peripheral
'
' '
blood lymphocyte blastogenesis (Ivanyi & Lehner 1971, Horton, Leskin & Oppenheim 1972, Mackler, et al. 1974), serum antibody levels to oral bacteria (Nisengard & Beutner 1970), crevicular fluid immunoglobulins (Brandzaeg 1973) and salivary IgA concentrations (Orstavik & Brandtzaeg 1975). One experimental approach to the interaction of periodontal disease and the immune system is the study of oral manifestations in patients with primary immunodeficiencies. Accordingly, a long-term study was designed to investigate the oral status of immunodeficient and immunocompetent
Supported by United States Public Health Service Contact DE-52454
38
R O B E R T S O N ,
W R I G H T ,
I I I , M A C K L E R ,
L E N E R T Z
A N D
L E V Y
Table I
Clinical profile of immunodeficient patients The diagnosis is that given by the clinical group and/or physician primarily responsibie for the patient's care, Dentai care experience is grouped into categories of no previous dental care (A), dental care only on an emergency basis (B), and regular dental treatment (C) Patient Code 01-02
3
01-04
4
01-08
J
•
5
01-03
7
01-07
7
01-09
,
01-05
•-
8
• • • ; ' '
9
01-10
12
01-06
14
01-01
43
02-01 02-05 02-04 02-03 02-02 03-04 03-01
•
•
'
8
12 15
24 8 17
17
03-03 04-02 04-01 04-03 04-04
Dental Care Experience
Primary Diagnosis
• :
5
5 8 13
Seiective IgA deficiency Seiective IgA deficiency Selective IgA deficiency Seiective igA deficiency Seiective IgA deficiency Selective IgA deficiency Selective IgA deficiency • Seiective IgA ' deficiency ••Seiective IgA deficiency .; Selective IgA deficiency Agammaglobuiinemia Agammaglobulinemia Agammaglobuiinemia Agammagicbulinemia l-lypogammaglobulinemia Neziof's Syndrome Generaiized Severe Dermatitis ImmunoSLjppressed Chronic Mucotaneous Gandidiasis Neutrophiiopenia Lazy Uupicitis Dysgammagiobulinemia Dysgammaglobulinemia
patients matched in age and oral hygiene, to assess both humoral and cellular immune parameters in both groups and to study clinical and immunological changes during the course of periodontal therapy. This report describes the first phase of the investigation and compares oral findings in immunodeficient patients with those of ageand Plaque Index-matched patients with normal immune systems.
Prior Antibiotic Therapy
I
A
A
• t Penicillin
G A A. G G
Penicillin
A
A C G
,e > B A B
A A
Amplcillin Erythromycin Amoxicillin Penicillin
Erythromycin Streptomycin
C B
,
Methods and Materials
Twenty-three patients with primary immunodeficiencies were identified (Table 1). Ten patients manifested selective IgA deficiency, 5 had agammaglobulinemia, 3 had cellular defects, 4 had leukocyte-related abnormalities and 1 had a severe combined immunodeficiency. Comprehensive medical and dental histories, which included sum-
I M M U N O D E F I C I E N C I E S
A N D
maries of present and past antibiotic experience, were obtained from each patient. The periodontal and general oral health status of the immunodeficient patients was evaluated using the Plaque Index (Silness & Loe 1964), Gingival Index (Loe & Silness 1963), Periodontal Disease Index (Ramfjord 1967) and DMF-T, A complete oral mucosal, periodontal and dental examination was performed. Full mouth periapical and bitewing radiographs were obtained on all but four patients, who received Panorex radiographic surveys. The mesial, facial, distal and lingual surfaces of all teeth were assessed in the Plaque, Gingival and Periodontal Disease Indices, The DMF-T was established using both the clinical and radiographic examination. As each primary immunodeficient patient was identified and characterized, clinic patients from the Dental Branch OutPatient Clinic were acquired and matched for age and Plaque Index Score, Most of the clinic patients were drawn froin a group participating in ongoing studies of gingivitis in children/adolescent conducted by the Department of Periodontics, University of Texas Dental Branch, Evaluation of matched clinic patients was performed exactly as previously described for immunodeficient patients, and 22 matched patient pairs were thus derived. Whole saliva (rubber band stimulated) and heparinized venous blood were obtained from both groups and submitted for clinical laboratory testing and immunological characterization. These studies are the subject of a report in preparation on the second phase of the investigation and serve here to indicate confirmation of the clinical diagnosis of immunodeficient patients and the immunocompetence of matched clinic patients. All patients with a primary diagnosis of selective IgA deficiency or agammaglobulinemia demonstrated low to undetectable levels of IgA in either serum or
P E R I O D O N T A L
D I S E A S E
39
saliva using Meloy radial immunodiffusion plates (Meloy Laboratories, Springfield, VA,) and/or Bio-Rad immunobead reagents (Bio-Rad Laboratory, Richmond, VA.). No compensation by IgM was observed (Mackler et al. unpublished data). The cellular deficient patients, whose diseases included a severe combined immunodeficiency disease (03-02), a Nezloff variant (03-04) and a generalized severe dermatitis (03-01), had negligible blastogenic responses to phytohemagglutinin (PHA), Patient (03-03), diagnosed as having mucocutaneous candidiasis, responded quite well to PHA but had delayed skin test anergy. The patients in the 04 category included 04-01, lazy lupicitis (Leukocyte) syndrome; 04-02, neutrophiiopenia; and 04-03 and 04-04, dysgammaglobulinemia. Of these patients, 04-01 had a phagocytosis index (latex uptake) of 0.1 % while 04-02 had very low levels of circulating phagocytes. The diagnosis of patients 04-03 and 04-04 was primarily based on clinical data since both had normal circulating lymphoid subpopulations and immunoglobulin levels.
Results
Profiles of immunodeficient patients are shown in Table I, Patients were divided into 4 general categories: selected IgA deficiency (01), agammaglobulinemia (02), cellular defects (03) and other immune abnormalities (04). Previous dental experience of the immunodeficient patients varied considerably. Nine patients reported no previous dental care (A), 4 patients obtained care only on an emergency basis (B) and 9 patients had received regular dental treatment with recalls every 6 to 12 months (C). No relationship was observed between dental care experience and any of the clinical indices. Eight immunodeficient patients reported extensive past antibiotic experience with
40
R O B E R T S O N ,
W R I G H T , f
- ,
I I I , M A C K L E R ,
L E N E R T Z
A N D
L E V Y
.
Table II Clinical index scores for immunodeficient and immunocompetent patients Mean Clinical Index scores for patients manifesting seiective IgA deficiency or agammaglobulinemia and matched immunocompetent patients (Coiumn 1); patients manifesting cellular or other defects and matched immunocompetent patients (Coiumn 2); immunodeficient patients in all categories and matched immunocompetent patients (Column 3), mean ± S,E, of clinical indices and results of statistical analysis is shown in Column 3, IgA Deficiency And Agammaglobulinemia (1)
Age
Cellular and Other Defects (2)
All Categories (3)
ImmuhoDeficientPatients
ImmunoCcmpetent Patients
ImmunoDeficient Patients
Immunocompetent Patients
ImmunoDeficient Patients
Immunocompetent Patients
11,2
11,0
11,4
11,5
11,3
11,3
N,S,
1,25
1,29
1,40
1,38
1,33 ± 0,12
1,34 ± 0,11
N,S,
,85
1,40
1,21
1,49
1,05 ± 0,10
1,46 ± 0,10
p < 0,01
P,D,I,
1,04
1,23
1,25
1,60
1,13 ± 0,10
1,43 ± 0,11
p < 0,05
DMF-T
2,9
4,9
5,3
8,8
4,2 + 1,6
7,1 ± 1,4
p < 0,05
P1,l,
G,l,
those agents detailed in column five. Five of the eight patients were receiving antibiotics at the time of examination. The majority of immunodeficient patients in this population, however, reported a history of antibiotic experience comparable to that of the immunocompetent subjects, averaging a 10-days course every two years and almost universally associated in both groups with upper respiratory infections. Mean scores for age, PLI., G.L, P.D.I, and DMF-T in all patients are given in Table II. The clinical indices were highly interrelated, particularly measures of plaque accumulation and gingival inflammation. Product-moment correlation coefficients (r) between PLI, and G.I. in the immunodeficient and immunocompetent groups were -f 0,8361 and -1-0.8337 respectively. A similar positive relationship was observed between PLI, and P.D.I, with r values of -f 0.8427 and -1-0.8388 respectively. Attachment loss was not a finding in either
population. The G.I, and P.D.I, scores were therefore both measures of gingival inflammation, and the overall correlation between these indices was -f 0,8972. Efficacy of matching in immunodeficient and clinic populations based on age and plaque accumulation was confirmed and no statistical differences were apparent between the groups. A significant and consistent difference was observed in gingival inflammation and caries experience. The mean G.L scores of immunodeficient patients in all categories were less than those recorded in age and PLI.-matched immunocompetent patients. The lower levels of gingival inflammation manifested by immunodeficient patients was observed in all pairs constituting the study population. Mean DMF-T scores were also significantly lower in immunodeficient patients than the matched immunocompetent group. The caries findings were also consistent and reversal was observed in only three of the
IMMUNODEFICIENCIES
AND PERIODONTAL
Table III Mean clinical index scores of Immunodeficient patients with and without autibiotic experience Immunodeficient With Antibiotic Experience
Immunodeficient With No Antibiotic Experience
AGE
10,0
12,1
Pl,i,
1,3
1,4
G,l,
1,0
1,1
2,3
5,4
• '^
DMF-T
•
matched pairs. Eight immunodeficient patients demonstrated no clinically or radiographically detectable caries. In general, indices of gingival inflammation and caries were lower among patients with selective IgA deficiency or agammaglobulinemia (Column 1) than patients manifesting cellular or other immune defects (Column 2), Plaque accumulation was also consistently lower in the former patient groups. The effects of antibiotic administration were assessed by comparing clinical index scores of immunodeficient patients reporting extensive previous antibiotic experience to the remainder of the immunodeficient population as shown in Table III, No differences were observed in clinical indices between immunodeficient patients with and without extensive antibiotic experience in either plaque accumulation or gingival inflammation. While tiot statistically significant, caries experience of patients with a history of antibiotic therapy, which included four of eight patients with DMF-T scores of zero, was considerably lower than that of the remainder of the immunodeficient population. No correlation was found between caries experience and the nature of the immune abnormality. Oral radiographic examination of im-
DISEASE
41
munodeficient patients was unremarkable and was comparable with examination findings in the matched immunocompetent patients. Eruption sequetice, root morphology, integrity of the lamina dura, width of the periodontal ligament space and osseous architecture were comparable in radiographic surveys of both patient populations. Neither attachment loss, as measured by the P.D.I,, nor radiographic evidence of alveolar bone loss were observed in any immunodeficient patients. One of the immunocompetent patients demonstrated some attachment loss as a function of recession. Crevice measurements, in neither group exceeded 3.0 mm. The mean crevice depth in immunodeficient patients was 1.5 ±0.28 and in immunocompetent patients was 1.6 + 0,30, With one exception, no soft tissue abnormalities were observed on the tongue, lips, nasopharynx or oral mucosa of patients in either group. However, findings in patient number 03-03, who had clinical diagnosis of generalized chronic mucotaneous candidiasis, were so dissimilar to those in other immunodeficient patients that they deserve special comment. The gingiva of this patient was hyperplastic and demonstrated a severe erythema which extended from the gingival margin into alveolar mucosa. Several gingival areas exhibited hemorage upon air drying. The tongue was square shaped with no typical taper on its anterior aspect; it was deeply fissured and displayed a knife-edge lateral margin. All oral mucosal surfaces, particularly the tongue and lips, were covered by white firmly adherent masses which were shown by microbiological analysis to be primarily Candida atbicans. Similar involvement was noted on most skin surfaces, especially the scalp, ears, and nose. The candidiasis affecting the patient's lips, commissures, and perioral tissues severely limited opening the mouth and access to the teeth for adequate oral hygiene. The long
42
R O B E R T S O N ,
W R I G H T ,
I I I ,
M A C K L E R ,
it:
Fig. 1. Oral appearance of a severe combined immunodeficient patient. Severe combined immunodeficient patient exhibiting diffuse, dark, firmly adherent deposits, plaque, and no ciinical signs of inflammation. Photograph was taken through the piastic wall surrounding the patient.
periods of essentially no oral hygiene were reflected in the high caries experience observed (DMF-T = 28), It should be noted that the G.I. of this patient (G.I. = 2.]) was essentially equal to the age-and plaquematched immunocompetent patient (G.I. = 2,3), and no evidence of alveolar bone resorption was observed. One immunodeficient patient not included in the matched population, a child with severe combined immunodeficiency, was delivered germ-free on September 21, 1971, by Caesarian section and has been maintained under gnotobiotic conditions since that date. Oral radiographic surveys revealed a normal eruption pattern and no evidence of dental caries. On clinical examination, diffuse, dark, firmly adherent deposits were noted oa the clinical crowns of all teeth as shown in Figure 1, Portions of these deposits were submitted to Dr. John Ennever for X-ray diffraction. The patterns obtained were typical of biologic apatite, a major crystalline component of dental calculus. Approximately 30 nonpathogen bacterial strains have been cultured from the patient, three of which were isolated from the dental deposits. These oral micro-
L E N E R T Z
A N D
L E V Y
organisms, which were consistent morphologically with Gram positive cocci, demon- strated intracellular calcification when in- ' cubated in buffered metastable calcium phosphate containing essential nutrients and a critical level of mineral salts. Since the calcified deposits plus abundant soft debris generally covered the clinical crowns of all erupted teeth, a PLI, of 3,0 was assigned. G.L, however, was 0, as there were no clinical signs of inflammation, redness or bleeding on probing. The latter observations, particularly with respect to color changes, were tempered by the difficulty in visualization associated with the plastic wall which surrounded the patient. ;
Discussion
This report deals with clinical survey findings obtained in the initial phase of an ongoing investigation of the oral status of patients with immune deficiencies. Results of immunological characterization studies and interrelationships of immunological parameters to clinical observations in both normal and immunodeficient patients are in preparation. Clinical results clearly suggest that, while immune system abnormalities may have major consequences with respect to other organ systems, young immunodeficient patients do not manifest more gingival inflammation, early periodontal destruction, or dental caries than immunocompetent patients of similar age and oral hygiene. Indeed, in this investigation, immunodeficient patients with primarily humoral defects demonstrated significantly lower levels of gingival inflammation and dental caries than matched patients with normal immune systems. The observations are in concert with findings by Robertson and Cooper (1974) which suggested that abnormalities of the IgA system did not necessarily lead in any direct way to destruction to the
I M M U N O D E F I C I E N C I E S
A N D
teeth or their supporting structures. The results with respect to gingival inflammation are consistent with findings by Lindstrom and Folke (1973) and Orstavik and Brandtzaeg (1975) who reported a significant positive correlation between periodontal inflammation and salivary IgA concentrations. In the present study, immunodeficient patients in groups 01-02 who demonstrated low to absent serum and salivary IgA had less gingival inflammation than that observed in patients in groups 03-04 with normal IgA levels. The Plaque Index, however, was also lower in groups 01-02 than 03-04 and interpretation of these results must be viewed in the context of the strong correlation between plaque accumulation and gingival inflammation found in both immunodeficient and immunocompetent patients. The inflammatory response of gingiva, however, to essentially identical plaque scores in immunodeficient and immunocompetent matched patient pairs showed a consistent difference in that the mean ratio of G.L: PLI. in the immunodeficient group was 0.80 and in the immunocompetent group was 1.2. These observations suggest that basic differences are present either in the microbiological constitution of plaque and/or in the gingival response to similar plaques. The age of the patient population precludes any conclusions related to loss of periodontal supporting structures. Although antibiotic therapy in the immunodeficient population did not appear to affect the severity of gingival inflammation, it was correlated with caries experience. It should be noted that, in general, the reported antibiotics are active in the gram positive spectrum. Comparable results in humans were observed in dental studies of longterm systemic penicillin therapy for rheumatic fever or chronic respiratory disease (Handelman, Mills & Hawes 1966, Littleton & White 1964). The caries ex-
P E R I O D O N T A L
D I S E A S E
43
perience of patients receiving penicillin was approximately two-thirds less than that in the untreated groups. This caries protection persisted for several years after cessation of therapy, Littleton and White (1964) also assessed the clinical status of periodontal tissues using the Periodontal Index and found no statistically significant differences between groups with and without histories of extensive penicillin therapy. Clinical observations on the severe combined immunodeficient patient appear to confirm earlier findings in germ-free animal models (Fitzgerald & McDaniel 1960) and suggest that calcified deposits in the absence of an organized plaque will not induce clinical gingival inflammation. The finding of some gingival inflammation in the immunodeficient patients would appear to question the concept that periodontal disease is mediated by host immune responses to dental plaque antigens. However, this early gingival inflammation (G,I, = 1,05) may have represented nonimmune host inflammatory responses to plaque. In the absence of specific host immunity, plaque can be inflammatory (Mackler 1974), chemotactic for polymorphonuclear leukocytes (Ward et al. 1968, Tempel et al. 1970, Lindhe & Hellden 1972), destroy fibroblasts (Baboolal et al. 1970, Mlinek et al. 1974), and its lipopolysaccharide components can activate the alternate complement pathway (Snyderman et al. 1968). In immunocompetent patients, the higher levels of gingival inflammation may have reflected the additional contribution of their functional immune responses to plaque during the early stages of the disease. Three major observations were made throughout the course of the investigation. Firstly, the presence and severity of early periodontal disease in both groups was directly related to the accumulation of microbial plaque. Indeed, in a patient main-
44
R O B E R T S O N ,
W R I G H T ,
III, M A C K L E R ,
L E N E R T Z
A N D
L E V Y
vitro correlation with periodontal disease. J. Periodontal Res. 43: 522-527, Ivanyi, L, & Lehner, T, 1971, Stimulation of lymphocyte transformation by bacterial antigens in patients with periodontal disease. Arch. Oral Biol. 16: 1117-1121, Lindhe, J, & Hellden, L, 1972, Neutrophil chemotactic activity elaborated by human dental plaque, /, Periodontal Res. 7: 297-303. Lindstrom, F, D. & Folke, L E, 1973. Salivary IgA in periodontal disease, Acta Odont. Scand. 31: 31-34. Littleton, N, W, & White, C, L, 1964, Dental findings from a preliminary study of children receiving extended antibiotic therapy, /, Am. Dent. Assoc. 68: 520-525, Loe, H, & Silness, 1. 1963, Periodontal disease in pregnancy, I. Prevalence and severity. Acta Odont. Scand. 21: 533-551, Mackler, B. F., Altman, L, C , Rosenstreich, D, L., Oppenheim, J, 1, & Mergenhagen, S, E. 1974, Blastogenesis and lymphokine synthesis by T and B lymphocytes from patients with periodontal disease. Infect, lmnnm. 10: 844-850, Mackler, B, E, 1975, Plaque dialyzate effects on human lymphocyte blastogenesis and inflammatory responses. Arch. Oral Biol. 20: 423^28. Mackler, B, E,, Waldrop, T. C , Schur, P., Robertson, P. B. & Levy, B, M. Submitted 1977. IgG subclasses in human periodontal References disease, /. Periodontal Res. Baboolal, R,, Mlinek, R, & Powell, R, N, 1970, Mlinek, A,, Buckem, A,, Hennig, S. & Begleitter. A, 1974. Cytotoxic effects of soluble A study of the effects of gingival plaque plaque extract on cells in vitro J. Periodontal extracts on cells cultured in vitro. J. PerioRes. 9: 342-348, dontal Res. 5: 248-254, Brandtzaeg, P, 1973, Immunology of inflam- Nisengard, R, 1, & Beutner, E, H. 1970, Immunologic studies of periodontal disease, V. matory periodontal lesions. Int. Dent. J. IgG type antibodies and skin test responses Lond. 23: 438-454, to actinomyces and mixed oral flora. J. Ellison, S, A, 1970, Oral bacteria and perioPeriodontol. 41: 149-152, dontal disease, J. Dent. Res. 49: 198-202, Fitzgerald, R, J, & McDaniel, E, G, 1960, Oppenheim, 1, J, & Horton, J, E, Comparative Dental calculus in the germ free rat. Arch. Immunology of the Oral Cavity. DHEW Oral Biol. 2: 239-244, Publication No. 73-438, U.S. Government Printing Office, Washington, D, C, Genco, R, J,, Evans, R. T. & Ellison, S, A. 1969, Dental research in microbiology with Orstavik, D, & Brandtzaeg, P. 1975, Secretion emphasis on periodontal disease, J. Am. of parotid IgA in relation to gingival inDent. Assoc. 78: 1016-1024. flammation and dental caries experience in man. Arch. Oral Biol. 20: 701-704. Handelman, S, L,, Mills, J. R. & Hawes, R, R, 1966. Caries incidence in subjects receiving Ramfjord, S,P, 1967. The periodontal disease long term antibiotic therapy, /, Oral Therap. index (PDI). J. Periodontol. 38: 602-610. and Pharmacol. 2: 338-345, Robertson, P. B, & Cooper, M. D. 1974. Oral Horton, J, E,, Leiken, S, & Oppenheim, 1, 1, manifestations of IgA deficiency. In The Im1972. Human lymphoproliferative reaction munoglobulin A System. Eds. 1, Mestecky of saliva and dental plaque deposits: An in and A, R. Lawton. Adv. Exp. Med. & Biot.,
tained in a controlled environment conducive to the absence of a mature oral microflora, no signs of disease were observed under the most adverse systemic conditions. Secondly, young immunodeficient patients manifested less gingival inflammation and caries than age and plaque matched immunocompetent patients. Extensive antibiotic experience appeared to be responsible for some reduction in caries scores but no appreciable effect was observed with respect to indices of gingival inflammation. In general, no oral developmental abnormalities or defects in supporting structure integrity were noted as a consequence of immune deficiency disease. Finally, the results supported the speculation that effector mechanisms of plaque-induced gingivitis are multifactorial in nature. While clinical scores for early periodontal disease were consistently greater in the immunocompetent group, none of the pair-matched immunodeficient patients were completely free of gingivitis.
I M M U N O D E F I C I E N C I E S
A N D
Plenum Publishing, New York 45: 497-503, Silness, J. & Loe, H, 1964. Periodontal disease in pregnancy, II, Correlation between oral hygiene and periodontal condition, Acta Odont. Scand. 22: 121-135, Snyderman, R,, Gewurz, H. & Mergenhagen, S. E, 1968, Interaction of the complement system with endotoxic lipopolysaccharide. Generation of a factor chemotactic for polymorphonuclear leukocytes, 7. Exp. Med. 128: 259-275, Address: D e p t . of P e r i o d o n t i c s
.
' . "
;
School of Dental Medicine University of Connecticut Health Center Farmington, Cl. 06032 U.S.A.
". V
P E R I O D O N T A L
D I S E A S E
45
Tempel, T, R., Snyderman, R,, lordan, H. V, & Mergenhagen, S, E, 1970, Eactors in saliva and oral bacteria, chemotactic for polymorphonuclear leukocytes: their possible role in gingival inflammation, / . Periodontol. 41: 71-80, Ward, P, A,, Lepow, I. H. & Newman, L, J. 1968, Bacterial factors chemotactic for polymorphonuclear leukocytes. Am. J. Path. 52: 725-736, • ' '• ' •' ,
'- '
;
•
, : : ;,
. . :•: - - > ^
.
,.•"».
. '
".
• ... ,_ .
...
• •
.
Periodontal status of patients with abnormalities of the immune system p. B. ROBERTSON, T, E, WRIGHT, III, B. F. MACKLER, D , M , LENERTZ AND B, M, LEVY
The University of Connecticut School of Dental Medicine, Farmington, Connecticut and The University of Texas Dental Branch and Dental Science Institute, Houston, Texas Patients with immune system abnormalities were pair-matched to immunocompetent clinic patients by age and Plaque Index, Clinical examination included comprehensive health histories with summaries of antibiotic experience. Plaque Index (P1,I.), Gingival Index (G.L), Periodontal Disease Index (P,D,I,), caries experience (DMF-T) and full mouth radiographic surveys. Efficiency of matching was confirmed as tests of significance between sample means for age (11.3, 11,3) and PLI. (1.33, 1.34) showed no difference between the immunodeficient and clinic patient groups respectively. No clinical or radiographic evidence of attachment loss was noted in either group, A significant and consistent difference was observed in gingival inflammation and caries experience. The G.I. was lower (x = 1,05) in immunodeficient patients than that recorded in clinic patients (x = 1,46) in all pairs. Similar results were observed for caries experience, and 8 immunodeficient patients demonstrated DMF-T scores of zero. :
,'
(Accepted for publication May 12, 1977)
Introduction
There is considerable evidence to suggest that plaque-induced effector mechanisms play a major role in the pathogenesis of periodontal disease, A number of investigations have implicated both cellular and humoral pathways in periodontal tissue destruction (Ellison 1970, Genco, Evans & Ellison 1969, Oppenheim & Horton 1973). Correlations of varying magnitudes have been demonstrated between the clinical severity of periodontal disease and plaque/ oral bacterial antigen-induced peripheral
'
' '
blood lymphocyte blastogenesis (Ivanyi & Lehner 1971, Horton, Leskin & Oppenheim 1972, Mackler, et al. 1974), serum antibody levels to oral bacteria (Nisengard & Beutner 1970), crevicular fluid immunoglobulins (Brandzaeg 1973) and salivary IgA concentrations (Orstavik & Brandtzaeg 1975). One experimental approach to the interaction of periodontal disease and the immune system is the study of oral manifestations in patients with primary immunodeficiencies. Accordingly, a long-term study was designed to investigate the oral status of immunodeficient and immunocompetent
Supported by United States Public Health Service Contact DE-52454
38
R O B E R T S O N ,
W R I G H T ,
I I I , M A C K L E R ,
L E N E R T Z
A N D
L E V Y
Table I
Clinical profile of immunodeficient patients The diagnosis is that given by the clinical group and/or physician primarily responsibie for the patient's care, Dentai care experience is grouped into categories of no previous dental care (A), dental care only on an emergency basis (B), and regular dental treatment (C) Patient Code 01-02
3
01-04
4
01-08
J
•
5
01-03
7
01-07
7
01-09
,
01-05
•-
8
• • • ; ' '
9
01-10
12
01-06
14
01-01
43
02-01 02-05 02-04 02-03 02-02 03-04 03-01
•
•
'
8
12 15
24 8 17
17
03-03 04-02 04-01 04-03 04-04
Dental Care Experience
Primary Diagnosis
• :
5
5 8 13
Seiective IgA deficiency Seiective IgA deficiency Selective IgA deficiency Seiective igA deficiency Seiective IgA deficiency Selective IgA deficiency Selective IgA deficiency • Seiective IgA ' deficiency ••Seiective IgA deficiency .; Selective IgA deficiency Agammaglobuiinemia Agammaglobulinemia Agammaglobuiinemia Agammagicbulinemia l-lypogammaglobulinemia Neziof's Syndrome Generaiized Severe Dermatitis ImmunoSLjppressed Chronic Mucotaneous Gandidiasis Neutrophiiopenia Lazy Uupicitis Dysgammagiobulinemia Dysgammaglobulinemia
patients matched in age and oral hygiene, to assess both humoral and cellular immune parameters in both groups and to study clinical and immunological changes during the course of periodontal therapy. This report describes the first phase of the investigation and compares oral findings in immunodeficient patients with those of ageand Plaque Index-matched patients with normal immune systems.
Prior Antibiotic Therapy
I
A
A
• t Penicillin
G A A. G G
Penicillin
A
A C G
,e > B A B
A A
Amplcillin Erythromycin Amoxicillin Penicillin
Erythromycin Streptomycin
C B
,
Methods and Materials
Twenty-three patients with primary immunodeficiencies were identified (Table 1). Ten patients manifested selective IgA deficiency, 5 had agammaglobulinemia, 3 had cellular defects, 4 had leukocyte-related abnormalities and 1 had a severe combined immunodeficiency. Comprehensive medical and dental histories, which included sum-
I M M U N O D E F I C I E N C I E S
A N D
maries of present and past antibiotic experience, were obtained from each patient. The periodontal and general oral health status of the immunodeficient patients was evaluated using the Plaque Index (Silness & Loe 1964), Gingival Index (Loe & Silness 1963), Periodontal Disease Index (Ramfjord 1967) and DMF-T, A complete oral mucosal, periodontal and dental examination was performed. Full mouth periapical and bitewing radiographs were obtained on all but four patients, who received Panorex radiographic surveys. The mesial, facial, distal and lingual surfaces of all teeth were assessed in the Plaque, Gingival and Periodontal Disease Indices, The DMF-T was established using both the clinical and radiographic examination. As each primary immunodeficient patient was identified and characterized, clinic patients from the Dental Branch OutPatient Clinic were acquired and matched for age and Plaque Index Score, Most of the clinic patients were drawn froin a group participating in ongoing studies of gingivitis in children/adolescent conducted by the Department of Periodontics, University of Texas Dental Branch, Evaluation of matched clinic patients was performed exactly as previously described for immunodeficient patients, and 22 matched patient pairs were thus derived. Whole saliva (rubber band stimulated) and heparinized venous blood were obtained from both groups and submitted for clinical laboratory testing and immunological characterization. These studies are the subject of a report in preparation on the second phase of the investigation and serve here to indicate confirmation of the clinical diagnosis of immunodeficient patients and the immunocompetence of matched clinic patients. All patients with a primary diagnosis of selective IgA deficiency or agammaglobulinemia demonstrated low to undetectable levels of IgA in either serum or
P E R I O D O N T A L
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39
saliva using Meloy radial immunodiffusion plates (Meloy Laboratories, Springfield, VA,) and/or Bio-Rad immunobead reagents (Bio-Rad Laboratory, Richmond, VA.). No compensation by IgM was observed (Mackler et al. unpublished data). The cellular deficient patients, whose diseases included a severe combined immunodeficiency disease (03-02), a Nezloff variant (03-04) and a generalized severe dermatitis (03-01), had negligible blastogenic responses to phytohemagglutinin (PHA), Patient (03-03), diagnosed as having mucocutaneous candidiasis, responded quite well to PHA but had delayed skin test anergy. The patients in the 04 category included 04-01, lazy lupicitis (Leukocyte) syndrome; 04-02, neutrophiiopenia; and 04-03 and 04-04, dysgammaglobulinemia. Of these patients, 04-01 had a phagocytosis index (latex uptake) of 0.1 % while 04-02 had very low levels of circulating phagocytes. The diagnosis of patients 04-03 and 04-04 was primarily based on clinical data since both had normal circulating lymphoid subpopulations and immunoglobulin levels.
Results
Profiles of immunodeficient patients are shown in Table I, Patients were divided into 4 general categories: selected IgA deficiency (01), agammaglobulinemia (02), cellular defects (03) and other immune abnormalities (04). Previous dental experience of the immunodeficient patients varied considerably. Nine patients reported no previous dental care (A), 4 patients obtained care only on an emergency basis (B) and 9 patients had received regular dental treatment with recalls every 6 to 12 months (C). No relationship was observed between dental care experience and any of the clinical indices. Eight immunodeficient patients reported extensive past antibiotic experience with
40
R O B E R T S O N ,
W R I G H T , f
- ,
I I I , M A C K L E R ,
L E N E R T Z
A N D
L E V Y
.
Table II Clinical index scores for immunodeficient and immunocompetent patients Mean Clinical Index scores for patients manifesting seiective IgA deficiency or agammaglobulinemia and matched immunocompetent patients (Coiumn 1); patients manifesting cellular or other defects and matched immunocompetent patients (Coiumn 2); immunodeficient patients in all categories and matched immunocompetent patients (Column 3), mean ± S,E, of clinical indices and results of statistical analysis is shown in Column 3, IgA Deficiency And Agammaglobulinemia (1)
Age
Cellular and Other Defects (2)
All Categories (3)
ImmuhoDeficientPatients
ImmunoCcmpetent Patients
ImmunoDeficient Patients
Immunocompetent Patients
ImmunoDeficient Patients
Immunocompetent Patients
11,2
11,0
11,4
11,5
11,3
11,3
N,S,
1,25
1,29
1,40
1,38
1,33 ± 0,12
1,34 ± 0,11
N,S,
,85
1,40
1,21
1,49
1,05 ± 0,10
1,46 ± 0,10
p < 0,01
P,D,I,
1,04
1,23
1,25
1,60
1,13 ± 0,10
1,43 ± 0,11
p < 0,05
DMF-T
2,9
4,9
5,3
8,8
4,2 + 1,6
7,1 ± 1,4
p < 0,05
P1,l,
G,l,
those agents detailed in column five. Five of the eight patients were receiving antibiotics at the time of examination. The majority of immunodeficient patients in this population, however, reported a history of antibiotic experience comparable to that of the immunocompetent subjects, averaging a 10-days course every two years and almost universally associated in both groups with upper respiratory infections. Mean scores for age, PLI., G.L, P.D.I, and DMF-T in all patients are given in Table II. The clinical indices were highly interrelated, particularly measures of plaque accumulation and gingival inflammation. Product-moment correlation coefficients (r) between PLI, and G.I. in the immunodeficient and immunocompetent groups were -f 0,8361 and -1-0.8337 respectively. A similar positive relationship was observed between PLI, and P.D.I, with r values of -f 0.8427 and -1-0.8388 respectively. Attachment loss was not a finding in either
population. The G.I, and P.D.I, scores were therefore both measures of gingival inflammation, and the overall correlation between these indices was -f 0,8972. Efficacy of matching in immunodeficient and clinic populations based on age and plaque accumulation was confirmed and no statistical differences were apparent between the groups. A significant and consistent difference was observed in gingival inflammation and caries experience. The mean G.L scores of immunodeficient patients in all categories were less than those recorded in age and PLI.-matched immunocompetent patients. The lower levels of gingival inflammation manifested by immunodeficient patients was observed in all pairs constituting the study population. Mean DMF-T scores were also significantly lower in immunodeficient patients than the matched immunocompetent group. The caries findings were also consistent and reversal was observed in only three of the
IMMUNODEFICIENCIES
AND PERIODONTAL
Table III Mean clinical index scores of Immunodeficient patients with and without autibiotic experience Immunodeficient With Antibiotic Experience
Immunodeficient With No Antibiotic Experience
AGE
10,0
12,1
Pl,i,
1,3
1,4
G,l,
1,0
1,1
2,3
5,4
• '^
DMF-T
•
matched pairs. Eight immunodeficient patients demonstrated no clinically or radiographically detectable caries. In general, indices of gingival inflammation and caries were lower among patients with selective IgA deficiency or agammaglobulinemia (Column 1) than patients manifesting cellular or other immune defects (Column 2), Plaque accumulation was also consistently lower in the former patient groups. The effects of antibiotic administration were assessed by comparing clinical index scores of immunodeficient patients reporting extensive previous antibiotic experience to the remainder of the immunodeficient population as shown in Table III, No differences were observed in clinical indices between immunodeficient patients with and without extensive antibiotic experience in either plaque accumulation or gingival inflammation. While tiot statistically significant, caries experience of patients with a history of antibiotic therapy, which included four of eight patients with DMF-T scores of zero, was considerably lower than that of the remainder of the immunodeficient population. No correlation was found between caries experience and the nature of the immune abnormality. Oral radiographic examination of im-
DISEASE
41
munodeficient patients was unremarkable and was comparable with examination findings in the matched immunocompetent patients. Eruption sequetice, root morphology, integrity of the lamina dura, width of the periodontal ligament space and osseous architecture were comparable in radiographic surveys of both patient populations. Neither attachment loss, as measured by the P.D.I,, nor radiographic evidence of alveolar bone loss were observed in any immunodeficient patients. One of the immunocompetent patients demonstrated some attachment loss as a function of recession. Crevice measurements, in neither group exceeded 3.0 mm. The mean crevice depth in immunodeficient patients was 1.5 ±0.28 and in immunocompetent patients was 1.6 + 0,30, With one exception, no soft tissue abnormalities were observed on the tongue, lips, nasopharynx or oral mucosa of patients in either group. However, findings in patient number 03-03, who had clinical diagnosis of generalized chronic mucotaneous candidiasis, were so dissimilar to those in other immunodeficient patients that they deserve special comment. The gingiva of this patient was hyperplastic and demonstrated a severe erythema which extended from the gingival margin into alveolar mucosa. Several gingival areas exhibited hemorage upon air drying. The tongue was square shaped with no typical taper on its anterior aspect; it was deeply fissured and displayed a knife-edge lateral margin. All oral mucosal surfaces, particularly the tongue and lips, were covered by white firmly adherent masses which were shown by microbiological analysis to be primarily Candida atbicans. Similar involvement was noted on most skin surfaces, especially the scalp, ears, and nose. The candidiasis affecting the patient's lips, commissures, and perioral tissues severely limited opening the mouth and access to the teeth for adequate oral hygiene. The long
42
R O B E R T S O N ,
W R I G H T ,
I I I ,
M A C K L E R ,
it:
Fig. 1. Oral appearance of a severe combined immunodeficient patient. Severe combined immunodeficient patient exhibiting diffuse, dark, firmly adherent deposits, plaque, and no ciinical signs of inflammation. Photograph was taken through the piastic wall surrounding the patient.
periods of essentially no oral hygiene were reflected in the high caries experience observed (DMF-T = 28), It should be noted that the G.I. of this patient (G.I. = 2.]) was essentially equal to the age-and plaquematched immunocompetent patient (G.I. = 2,3), and no evidence of alveolar bone resorption was observed. One immunodeficient patient not included in the matched population, a child with severe combined immunodeficiency, was delivered germ-free on September 21, 1971, by Caesarian section and has been maintained under gnotobiotic conditions since that date. Oral radiographic surveys revealed a normal eruption pattern and no evidence of dental caries. On clinical examination, diffuse, dark, firmly adherent deposits were noted oa the clinical crowns of all teeth as shown in Figure 1, Portions of these deposits were submitted to Dr. John Ennever for X-ray diffraction. The patterns obtained were typical of biologic apatite, a major crystalline component of dental calculus. Approximately 30 nonpathogen bacterial strains have been cultured from the patient, three of which were isolated from the dental deposits. These oral micro-
L E N E R T Z
A N D
L E V Y
organisms, which were consistent morphologically with Gram positive cocci, demon- strated intracellular calcification when in- ' cubated in buffered metastable calcium phosphate containing essential nutrients and a critical level of mineral salts. Since the calcified deposits plus abundant soft debris generally covered the clinical crowns of all erupted teeth, a PLI, of 3,0 was assigned. G.L, however, was 0, as there were no clinical signs of inflammation, redness or bleeding on probing. The latter observations, particularly with respect to color changes, were tempered by the difficulty in visualization associated with the plastic wall which surrounded the patient. ;
Discussion
This report deals with clinical survey findings obtained in the initial phase of an ongoing investigation of the oral status of patients with immune deficiencies. Results of immunological characterization studies and interrelationships of immunological parameters to clinical observations in both normal and immunodeficient patients are in preparation. Clinical results clearly suggest that, while immune system abnormalities may have major consequences with respect to other organ systems, young immunodeficient patients do not manifest more gingival inflammation, early periodontal destruction, or dental caries than immunocompetent patients of similar age and oral hygiene. Indeed, in this investigation, immunodeficient patients with primarily humoral defects demonstrated significantly lower levels of gingival inflammation and dental caries than matched patients with normal immune systems. The observations are in concert with findings by Robertson and Cooper (1974) which suggested that abnormalities of the IgA system did not necessarily lead in any direct way to destruction to the
I M M U N O D E F I C I E N C I E S
A N D
teeth or their supporting structures. The results with respect to gingival inflammation are consistent with findings by Lindstrom and Folke (1973) and Orstavik and Brandtzaeg (1975) who reported a significant positive correlation between periodontal inflammation and salivary IgA concentrations. In the present study, immunodeficient patients in groups 01-02 who demonstrated low to absent serum and salivary IgA had less gingival inflammation than that observed in patients in groups 03-04 with normal IgA levels. The Plaque Index, however, was also lower in groups 01-02 than 03-04 and interpretation of these results must be viewed in the context of the strong correlation between plaque accumulation and gingival inflammation found in both immunodeficient and immunocompetent patients. The inflammatory response of gingiva, however, to essentially identical plaque scores in immunodeficient and immunocompetent matched patient pairs showed a consistent difference in that the mean ratio of G.L: PLI. in the immunodeficient group was 0.80 and in the immunocompetent group was 1.2. These observations suggest that basic differences are present either in the microbiological constitution of plaque and/or in the gingival response to similar plaques. The age of the patient population precludes any conclusions related to loss of periodontal supporting structures. Although antibiotic therapy in the immunodeficient population did not appear to affect the severity of gingival inflammation, it was correlated with caries experience. It should be noted that, in general, the reported antibiotics are active in the gram positive spectrum. Comparable results in humans were observed in dental studies of longterm systemic penicillin therapy for rheumatic fever or chronic respiratory disease (Handelman, Mills & Hawes 1966, Littleton & White 1964). The caries ex-
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43
perience of patients receiving penicillin was approximately two-thirds less than that in the untreated groups. This caries protection persisted for several years after cessation of therapy, Littleton and White (1964) also assessed the clinical status of periodontal tissues using the Periodontal Index and found no statistically significant differences between groups with and without histories of extensive penicillin therapy. Clinical observations on the severe combined immunodeficient patient appear to confirm earlier findings in germ-free animal models (Fitzgerald & McDaniel 1960) and suggest that calcified deposits in the absence of an organized plaque will not induce clinical gingival inflammation. The finding of some gingival inflammation in the immunodeficient patients would appear to question the concept that periodontal disease is mediated by host immune responses to dental plaque antigens. However, this early gingival inflammation (G,I, = 1,05) may have represented nonimmune host inflammatory responses to plaque. In the absence of specific host immunity, plaque can be inflammatory (Mackler 1974), chemotactic for polymorphonuclear leukocytes (Ward et al. 1968, Tempel et al. 1970, Lindhe & Hellden 1972), destroy fibroblasts (Baboolal et al. 1970, Mlinek et al. 1974), and its lipopolysaccharide components can activate the alternate complement pathway (Snyderman et al. 1968). In immunocompetent patients, the higher levels of gingival inflammation may have reflected the additional contribution of their functional immune responses to plaque during the early stages of the disease. Three major observations were made throughout the course of the investigation. Firstly, the presence and severity of early periodontal disease in both groups was directly related to the accumulation of microbial plaque. Indeed, in a patient main-
44
R O B E R T S O N ,
W R I G H T ,
III, M A C K L E R ,
L E N E R T Z
A N D
L E V Y
vitro correlation with periodontal disease. J. Periodontal Res. 43: 522-527, Ivanyi, L, & Lehner, T, 1971, Stimulation of lymphocyte transformation by bacterial antigens in patients with periodontal disease. Arch. Oral Biol. 16: 1117-1121, Lindhe, J, & Hellden, L, 1972, Neutrophil chemotactic activity elaborated by human dental plaque, /, Periodontal Res. 7: 297-303. Lindstrom, F, D. & Folke, L E, 1973. Salivary IgA in periodontal disease, Acta Odont. Scand. 31: 31-34. Littleton, N, W, & White, C, L, 1964, Dental findings from a preliminary study of children receiving extended antibiotic therapy, /, Am. Dent. Assoc. 68: 520-525, Loe, H, & Silness, 1. 1963, Periodontal disease in pregnancy, I. Prevalence and severity. Acta Odont. Scand. 21: 533-551, Mackler, B. F., Altman, L, C , Rosenstreich, D, L., Oppenheim, J, 1, & Mergenhagen, S, E. 1974, Blastogenesis and lymphokine synthesis by T and B lymphocytes from patients with periodontal disease. Infect, lmnnm. 10: 844-850, Mackler, B, E, 1975, Plaque dialyzate effects on human lymphocyte blastogenesis and inflammatory responses. Arch. Oral Biol. 20: 423^28. Mackler, B, E,, Waldrop, T. C , Schur, P., Robertson, P. B. & Levy, B, M. Submitted 1977. IgG subclasses in human periodontal References disease, /. Periodontal Res. Baboolal, R,, Mlinek, R, & Powell, R, N, 1970, Mlinek, A,, Buckem, A,, Hennig, S. & Begleitter. A, 1974. Cytotoxic effects of soluble A study of the effects of gingival plaque plaque extract on cells in vitro J. Periodontal extracts on cells cultured in vitro. J. PerioRes. 9: 342-348, dontal Res. 5: 248-254, Brandtzaeg, P, 1973, Immunology of inflam- Nisengard, R, 1, & Beutner, E, H. 1970, Immunologic studies of periodontal disease, V. matory periodontal lesions. Int. Dent. J. IgG type antibodies and skin test responses Lond. 23: 438-454, to actinomyces and mixed oral flora. J. Ellison, S, A, 1970, Oral bacteria and perioPeriodontol. 41: 149-152, dontal disease, J. Dent. Res. 49: 198-202, Fitzgerald, R, J, & McDaniel, E, G, 1960, Oppenheim, 1, J, & Horton, J, E, Comparative Dental calculus in the germ free rat. Arch. Immunology of the Oral Cavity. DHEW Oral Biol. 2: 239-244, Publication No. 73-438, U.S. Government Printing Office, Washington, D, C, Genco, R, J,, Evans, R. T. & Ellison, S, A. 1969, Dental research in microbiology with Orstavik, D, & Brandtzaeg, P. 1975, Secretion emphasis on periodontal disease, J. Am. of parotid IgA in relation to gingival inDent. Assoc. 78: 1016-1024. flammation and dental caries experience in man. Arch. Oral Biol. 20: 701-704. Handelman, S, L,, Mills, J. R. & Hawes, R, R, 1966. Caries incidence in subjects receiving Ramfjord, S,P, 1967. The periodontal disease long term antibiotic therapy, /, Oral Therap. index (PDI). J. Periodontol. 38: 602-610. and Pharmacol. 2: 338-345, Robertson, P. B, & Cooper, M. D. 1974. Oral Horton, J, E,, Leiken, S, & Oppenheim, 1, 1, manifestations of IgA deficiency. In The Im1972. Human lymphoproliferative reaction munoglobulin A System. Eds. 1, Mestecky of saliva and dental plaque deposits: An in and A, R. Lawton. Adv. Exp. Med. & Biot.,
tained in a controlled environment conducive to the absence of a mature oral microflora, no signs of disease were observed under the most adverse systemic conditions. Secondly, young immunodeficient patients manifested less gingival inflammation and caries than age and plaque matched immunocompetent patients. Extensive antibiotic experience appeared to be responsible for some reduction in caries scores but no appreciable effect was observed with respect to indices of gingival inflammation. In general, no oral developmental abnormalities or defects in supporting structure integrity were noted as a consequence of immune deficiency disease. Finally, the results supported the speculation that effector mechanisms of plaque-induced gingivitis are multifactorial in nature. While clinical scores for early periodontal disease were consistently greater in the immunocompetent group, none of the pair-matched immunodeficient patients were completely free of gingivitis.
I M M U N O D E F I C I E N C I E S
A N D
Plenum Publishing, New York 45: 497-503, Silness, J. & Loe, H, 1964. Periodontal disease in pregnancy, II, Correlation between oral hygiene and periodontal condition, Acta Odont. Scand. 22: 121-135, Snyderman, R,, Gewurz, H. & Mergenhagen, S. E, 1968, Interaction of the complement system with endotoxic lipopolysaccharide. Generation of a factor chemotactic for polymorphonuclear leukocytes, 7. Exp. Med. 128: 259-275, Address: D e p t . of P e r i o d o n t i c s
.
' . "
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School of Dental Medicine University of Connecticut Health Center Farmington, Cl. 06032 U.S.A.
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P E R I O D O N T A L
D I S E A S E
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Tempel, T, R., Snyderman, R,, lordan, H. V, & Mergenhagen, S, E, 1970, Eactors in saliva and oral bacteria, chemotactic for polymorphonuclear leukocytes: their possible role in gingival inflammation, / . Periodontol. 41: 71-80, Ward, P, A,, Lepow, I. H. & Newman, L, J. 1968, Bacterial factors chemotactic for polymorphonuclear leukocytes. Am. J. Path. 52: 725-736, • ' '• ' •' ,
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