Indicators of periodontal disease Daniel H. Fine* and irwin D.

iVIandei**
Division of Periodontics* and Preventive
Dentistry**, Columbia University, School of
activity: an evaluation Dentai and Orai Surgery, New York, NY, USA

Fine D H and Mandel ID: Indicators of periodontal disease activity: an evaluation.

J Clin Periodontol 1986; 13: 533-546.

Abstract. It is becoming increasingly apparent that the traditional clinical cri-

teria are inadequate for: (a) determining active disease sites in periodontitis, (b)
monitoring quantitatively the response to therapy or (c) measuring the degree
of susceptibility to future breakdown. In an attempt to develop objective mea-
sures, a wide varity of studies have been undertaken using saliva, blood, plaque
and gingival crevicular fluid (GCF) as the specimen source. Examination has
included: (a) specific bacteria and their products; (b) host cells and their products
(enzymatic and antibacterial, both immunologic and non-immunologic); (c)
products of tissue injury derived from local epithelial and connective tissues
and bone. Although most of the work to date has failed to provide reliable aids
to the clinician, refinements in techniques for sampling and the availability of more
sophisticated analytic techniques give cause for optimism.
Methods proposed for detection of disease-associated bacteria in subgingival
plaque vary in their sensitivity and specificity. Dark field microscopy shows
some correlation with existing disease; however, the limited specificity of this
method imposes severe restrictions on its usefulness. Highly specific polyclonal
and monoclonal antisera to suspected pathogens Bacteroides gingivalis and Acti-
nobacillus actinomycetemcomitans have been developed and improved methods
of identification of these microbes in plaque by ELISA immunofluorescence and
flow cytometry are under development.
With respect to the host response, a strong correlation between antibody patterns
to specific bacteria and periodontal disease categories appears to be emerging.
Although most studies have focused on serum antibody derived from peripheral
blood, a shift to detection of local antibody response appears to be likely.
Techniques of measurement that are exquisitely sensitive have been developed
for detection of major immune recognition proteins such as antibody and
complement in crevicular fluid. Research efforts attempting to correlate local
antibody response to local disease activity are underway.
Measurement of GCF flow rate, endotoxin, H2S, butyrate and a variety of
enzymes (e. g., coUagenase, arylsulfatase, B-glucuronidase) show good corre-
lation with levels of gingivitis. In periodontitis, the most promising markers of
tissue breakdown are prostaglandins of the E series, the enzymes coUagenase and
aspartate aminotransferase, sulfated glycosaminoglycans, osteoclastic activating
factor and bone resorptive capacity of crevicular cells. Assay of the migration of
crevicular leucocytes in vivo can serve as an indicator of a defect in host resistance.
Experimental study in animal models with histological correlates and prospective
Key words: Bacterial products - host celi
studies on humans with standardized clinical correlates are needed to validate products - tissue iniury products - gingivai
these objective measurements. fluid.

Dental cariologists and periodontal re-
searchers face some of the same perlex-
ing problems as we come to the end
of the 20th century. Two of the most
germane to this conference are identifi-
cation of high-risk subjects (disease sus-
ceptibility testing) and determination of
current disease activity and disease pro-
gression. In both diseases, solutions to
these problems will lead to improved
and individualized preventive and treat-
ment strategies. To achieve these goals,
dentistry will require objective and
quantitative methods of analysis of indi-
vidual samples. The cariologist have
placed emphasis on saliva and plaque
and to a very minimal extent, blood.
In periodontal disease an additional
source material, gingival crevicular fluid
(GCF), is available for the evaluation
of a specific site.
Clinical measurements of periodontal
disease have been fully discussed at this
conference, and thus only non-clinical,
objective methods for measuring peri-
odontal disease activity and suscepti-
bility will be considered such as: (a) spe-
534 Fine and Mandel

cific bacteria and their products, (b) hostcharacterize the bacterium by cultural al. 1979, Haffajee et al. 1984, Mandell
cells and their products, (c) products of methods. To further confound the situ- 1984). Similar results have been seen in
tissue injury derived from tocal epithelial ation he was able to isolate the diptheria animal models of periodontal disease
and connective tissues and from bone. bacilli from a small % of normal child- as described by Kornman et al. (1981).
ren. Loeffier thus noted that isolation Taken together, these cultural studies
of C. diphtheria was not therefore by suggest that vulnerable sites must be
itself diagnostic for the disease; and re- colonized by a critical mass of the
(I) Bacteria and their Products commended that diagnosis of diphther- "pathogenic" strain before disease oc-
It is clear that bacteria are the principal ia requires keen clinical judgement curs. If I could guess - Murphy's third
etiologic agents of periodontal disease backed up by careful bacteriology. Con- law of the microbiology of periodontitis
(Socransky 1977, Slots 1979). However, fusion in bacteriologic diagnosis of would suggest (to make our lives more
agreement on the specific nature of the diphtheria exists primarily because difficult) - that critical mass will un-
bacterial infection is not unanimous many members of the pharyngeal resi- doubtedly vary from individual to indi-
(Newman & Socransky 1977, Moore et dent flora may grow particularly well in vidual and from site to site, from pocket
al. 1985). Attempts to use bacterial pro- the steptococcal or diphtheric pseudo- depth to pocket depth and probably
files of patients as indicators of disease membranous sore throat habitat. More- from one time period to another at the
vulnerability or disease activity have over the pharyngeal floral variation same site.
met with mixed success (Listgarten et from individual to individual is so great Definitive answers to these questions
al. 1984, Haffajee et al. 1984). Methods that these secondary invaders vary with require sophisticated indepth cultural
of bacterial analysis have varied in levels respect to types and numbers of organ- analysis of pocket residents, an ap-
of complexity from dark field or phase isms and as a result these normal resi- proach that is currently reserved for the
contrast analysis to elaborate cultural dents often obscure the presence of the research laboratory. Dark field or phase
and biochemical methods of identifi- pathogen C. diphtheriae (Andrewes et contrast microscopy have been suggest-
cation to the species and serotypical al. 1923). ed as alternates to the more cumber-
level (Listgarten & Hellden 1978, This dilemma does not sound to dis- some cultural methods (Listgarten
Moore et al. 1983). Probes used to sim- similar to the one facing those of us & Hellden 1978). The motility of bac-
plify the complex cultural methodolo- searching for microbial markers of peri- terial smears or pocket samples have
gies needed to identify specific bacteria odontal disease. Particularly analagous been used to indicate active disease or
vary in their complexity and include im- to the diphtheria story is the infection to structure maintenance programs
munofiuorence (Fine & Greene 1984), of juvenile periodontitis. Diagnosis of
(Listgarten & Schifter 1982). Of the
enzyme linked immunoassay (ELISA)- the disease is initially made by clinical
potential periodontal pathogens enu-
(Dzink et al. 1983), flow cytometry parameters such as probing. X-ray and
(Kornman et al. 1984) and DNA probe history (Baer 1971). Confirmation can merated thus far most prominent
technology (Strzempko et al. 1985). In be gained by bacteriology of infected among the motile forms observed are
addition a variety of bacterial products sites with some help from patient serum the spirochetes. Sophisticated cultural
have been used to evaluate levels of antibody titers to Actinobacillus acti- methods performed at Virginia Poly-
health and disease. technic Institute by Dr. Robert Smibert
nomycetemcomltans (Aa) (Slots et al. and coworkers suggest that at least 23
1980, Ebersole et al. 1980). However, types of Treponemes can be found in the
even then our knowledge is limited to periodontal environment (Moore et al.
Microbial diagnosis what has already happened. These 1985). Of these 23, only 4 to 6 species
Since bacteria are the initiators of peri- pieces of information merely tell us that have been associated with periodontally
odontal disease it makes good sense to the disease has taken place and it fails diseased areas (Moore et al. 1985).
look for bacteria as indicators of disease to give us any clue as to the current Moreover, most of the principal non-
activity. The precedent for this ap- status of the disease. Would we feel spirochetal "pathogens" suspected as
proach can be found in other areas of more comfortable in our diagnosis of etiologic agents in periodontal disease
medicine. As an example, cultures are activity knowing that (Aa) was found including: Aa, B. gingivalis, B. interme-
routinely used to confirm the diagnosis in the specific site under observation? dius, E. corrodens, Eubacterium genus
of Strep throat (Wanamaker 1973). Probably. If the site is infected with ex- are non-motile (Tanner et al. 1979).
However there are other examples in tremely low levels of Aa does that mean Wolinella species on the other hand are
medicine that question the reliability of that it is currently undergoing break- motile (Socransky et al. 1982). It is high-
this approach. In the case of diphtheria, down? Probably not? A positive identifi- ly unlikely that positive identification to
the causative agent Corynebacterium cation of low levels of S. mutans does the species level could ever be made by
diphtheriae is not always isolated from not necessarily imply caries activity! In the primitive microscopic methodolo-
diphtheric pseudomembranes (Barksda- the diseases we study (caries and peri- gies employed currently. Dark field mi-
le 1970). The classic experiments of Fre- odontal disease(s)), a critical mass of the croscopy cannot differentiate between
derich Loeffler demonstrated the prob- opportunistic pathogen(s) appears to be the suspected pathogens listed above or
lems associated with microbial diag- a necessary prerequisite for disease ac- among the various species of Trepone-
nosis. In smears of 22 cases of clinically tivity. Longitudinal studies should give mes. Therefore darkfield microscopy
diagnosed diphtheria he was able to us the clearest picture of destructive dis- seems an unlikely candidate as a diag-
identify the causative bacilli in only 13 ease and they suggest that identification
nostic test of destructive periodontal
by its characteristic gram stain micro- of an increase in quantity of the "caus-
disease. Use of dark field analysis of
scopic appearance. Furthermore, in ative" bacteria can be helpful in our
only 6 of these 13 was he able to fully assessment of disease activity (Tanner et subgingival plaque is analogous to use
of light microscopic analysis of Gram

stained supragingival plaque samples
for diagnosis of caries activity. There
are at least 20 species of Streptococci
that reside on the proximal tooth sur-
face (Moore et al. 1982). These species
for the most part have classical Gram
staining characteristics of the Strepto-
coccus genus, namely Gram (-h) chains.
Suggestions that the % of Streptococcal
colonies to total colonies by Gram stain
appearance can predict caries activity
are obviously misguided since S. mutans
has a strong association with caries ac-
tivity while S. sanguis has the opposite
association (Loesche et al, 1975). Dis-
tinction among the various Streptococ-
cal species can not be made by micro-
scopic appearance of Gram stained
smears. Why then should we expect
dark field to provide us with infor-
mation for a disease many times more
complicated than caries.
Current research, in fact, supports
this position and work by Listgarten et
al. (1984) demonstrated that minimal
and insignificant differences in coccoid,
motile forms and spirochetes exist when
teeth that were breaking down were
compared to those that were well main-
tained as determined by careful com-
parative differential dark field micro-
scopic monitoring. Savitt & Socransky
(1984) conclude from their studies that
spirochetes were strongly correlated
with pocket depth (not with activity)
and thus that deeper destructive sites
favor colonization by spirochetes. Af-
rica et al. (1985) in a study of a peri-
odontitis-resistant population in Nama-
qualand. South Africa found no differ-
ences in spirochetes when the resistant
and diseased populations were com-
pared.
Microbiologic methods still appear to
be important however, in the assessment
of: (1) patient and site vulnerability; (2)
disease classification; (3) choice of treat-
ment modality. At this time, however,
methods that deal with microbial meth-
ods of identification do not appear to
be likely candidates to stand alone as
indicators of disease activity.
It would appear that methodologies
that are considerably more specific and
sensitive are on the horizon. Immuno-
fiuorescent identification of suspected
periodontal pathogens has already be-
gun (Fine & Greene 1984) as has the
use of enzyme linked immunosorbent
assay (Dzink et al. 1983). Flow cyto-
metry may surpass either of these other
serodiagnostic methods in ease of use
and sensitivity and in fact the use of
DNA probes may prove even more re-
liable.
Bacterial products
A considerable number of bacterial
products can contribute to the in-
itiation and progression of periodontal
disease. These products include endo-
toxin, hydrogen sulfide, butyric and
propionic acid, and a number of en-
zymes. They can be monitored in plaque
extracts, GCF collected on paper strips
and crevicular washings.
Endo toxin
In a number of studies endotoxin ac-
tivity has been shown to be positively
correlated with gingival inflammation.
Simon et al (1969, 1970, 1971) showed
the correlation between GCF levels of
endotoxin activity and inflammation on
both a clinical and histological level.
Shapiro et al. (1972) affirmed the associ-
ation for endotoxin in GCF, dental
plaque, gingival tissue and saliva. More
recently, Tzamouramis et al. (1979)
quantitated endotoxin activity in gin-
gival crevice washings in 5 subjects dur-
ing a 21 day no brushing period of ex-
perimental gingivitis and found a very
high correlation between activity and
gingival index.
Use of the lymulus lysate procedure
allows for rapid quantitation of endo-
toxin activity in very small amounts of
crevicular fluid. Most of the experience
with this indicator, however, has been
in gingivitis rather than periodontitis
and it is not known how the levels of
activity compare. Since the amounts of
endotoxin reflects the numbers of Gram
- bacteria, it may be a better measure
of disease potential rather than disease
activity. This is still valuable infor-
mation to a clinician.
Hydrogen sulfide
This less exotic toxic metabolite of
plaque bacteria was first detected in the
gingival sulcus by Rizzo (1967). Using a
very simple procedure of inserting filter
paper strips impregnated with lead ace-
tate, he showed that H^S is produced in
periodontal pockets of 2 mm or more.
Solis-Gaffar et al. (1980) employed a
highly quantitative method for quanti-
tating the amount of H2S in GCF. On
examination of 240 gingival areas they
found a positive correlation between the
degree of gingival inflammation, GCF
volume and the H2S generating poten-
tial of the fluid. This would appear to
be a lead worth pursuing.
Indicators of periodontal disease 535
Butyrate and proprionate
In a series of experiments Singer &
Buckner (1981) have presented evidence
that butyrate, and to a lesser extent,
proprionate are important toxic com-
ponents of dental plaque. They found
that in vitro the salts of these acids in-
hibited proliferation of both mouse L
929 cells and human gingival fibro-
blasts. Singer et al. (1980) had pre-
viously shown that butyrate and pro-
prionate, at concentrations found in
dental plaque, could induce gingival in-
fiammation in the Beagle dogs. Moore
et al. (1981) found that the incidience
of butyric acid producing bacteria cor-
related with developing gingivitis and
periodontitis. Brickwell et al. (1978)
found that the concentrations of butyr-
ate and proprionate in the subgingival
plaque of patients with periodontitis
was markedly higher than in plaque
from healthy subjects. Carlton et al.
(1979) found a similar relationship in
localized juvenile periodontitis. Since
these acids are common metabolites of
Gram-negative anerobic bacteria the
findings make good sense. And it is not
suprising that Montogomery et al.
(1982) found a high correlation between
reversal of inflammation in Beagle dogs
by antibiotics and the reduction of bu-
tyrate levels in plaque.
Vratsanos & Mandel (unpublished
findings) have confirmed the marked el-
evation in butyrate in supragingival
plaque in gingivitis and the ever higher
level in subgingival plaque in subjects
with infiammation. They have also
shown that butyrate can be readily
measured in crevicular fluid. This
metabolite could serve as a useful
marker of a flora with a ready potential
for active disease. Using a growth inhi-
bition assay of Hela-cells, Levine (1985)
recently showed that butyrate and pro-
prionate, however, only represented a
small fraction of the total plaque tox-
icity toward these cells. Obviously
plaques contain a spectrum of poten-
tially toxic products. Any of these could
probably serve as a marker. The need
is to show the association with disease
activity and to develop a reasonable as-
say. Butyrate is worth keeping on the
list.
Polyamines
The polyamines; putrescine, spermidine
and spermine are rather unique in that
they are metabolic products of plaque
bacteria as well as the constituents of
all mammalian cells. Cadaverine, how-
536 Fine and Mandel
ever, is only produced by bacterial cells
from its pre-cursor, lysine. Polyamines
are of interest to cariologists because
of their role in plaque pH regulation.
Cancer specialists use urinary polyami-
nes as indicators of cell proliferation.
Immunologists are looking at urinary
polyamines as an early warning of
transplant rejection. Periodontists
might do well to examine polyamines in
GCF as both a measure of plaque toxic
potential, T-Lymphocyte proliferation
and tissue breakdown. We will discuss
polyamines and tissue injury a little
later.
Vratsanos & Mandel (1985) have de-
veloped a highly sensitive method for
measurement of the spectrum of poly-
amines in small amounts of plaque and
have adapted the procedure to quantita-
tion of polyamines in GCF. In a recent
cross sectional study of subjects with a
gingival index of 1, 2 and 3, the change
from a score of 1 to 2 was associated
with a 5-6 fold increase in putrescine,
spermidine and spermine and a 10-fold
increase in cadaverine. There was no
futher increase in concentration of poly-
amines with the change from a Gl of 2
to 3. Quantification of cadaverine
would appear to offer the best measure
of increased plaque metabolic activity
associated with initiation of disease.
This increased activity is measurable in
GCF. The failure to see a higher level
in subjects with Gl of 3 may reflect
the dilution effect of the increased fluid
generated by the increasing vasculitis. It
would probably be more appropriate in
future studies to collect GCF for a stan-
dard amount of time (e. g., 30 s) and
measure total amount of the polyamine
collected in that period, total unit ac-
tivity, as suggested by Oshrain et al.
(1984) and Lamster et al. (1985).
Enzymes
Since enzymes are present in bacterial
cells, polymorphonuclear leucocytes
and lysosomes of host tissue cells, the
choice of specific bacterial products is
limited. By choosing very specific sub-
strates, specific inhibitors and working
at specific pH optima, however, it is
possible to differentiate between bac-
terial and mammalian enzymes. This
area has been reviewed by Cimasoni
(1983) in his book on Crevkular Fluid
Updated. Practically speaking, however,
only a few enzymes lend themselves to
ready identification as primarily bac-
terial in origin. Van Palenstein Helder-
man & Hougeveen (1976) collected
crevicular material on absorbent paper
points and found elevated hyaluroni-
dase activity in inflamed crevicular
areas. They found that the increase in
enzyme concentration was correlated
with the increase in the number of
Gram-positive bacteria. Kitawaki et al.
(1983) found that neuraminidase ac-
tivity eluted from filter paper strips
(placed in the crevicular areas for 10
min) was 12-13 times higher in patients
with periodontal disease than from peri-
odontally healthy subjects. Significant
correlations were found between neur-
aminidase activity and debris, calculus
and gingival indices. This would appear
to be an enzyme worth examining more
closely.
Bacterial collagenase
There is considerable interest in quanti-
tating bacterial collagenase as a mea-
sure of Bacteroides gingivalis activity.
To separate bacterial from mammalian
enzymes in samples from gingival fluid,
however, would require analysis of the
breakdown products by acrylamide gel
electrophoresis. The bacterial enzyme
cleaves collagen at multiple sites yield-
ing a variety of peptides (Golub et al.
1976). In contrast, tissue coUagenases
cleave at a single locus. This is not a
practical routine. An immunochemical
method measuring specific enzymes
would be more reasonable. Such a
method would be worth developing.
(II) Host Cells and their Products
Cells counts
It is well established that 95-97% of the
white cells in the gingival crevice are
neutrophils, and that though they in-
crease markedly during inflammation
the proportion of poly to mononuclear
cells does not change. Measurement of
numbers of PMN's would appear to be
a good marker of inflammatory status.
In three studies (Schiott & Loe 1970,
Kowashi et al. 1980, Thurre et al. 1985)
there was a good correlation between
number of cells in gingival washings and
the development of an experimental gin-
givitis. In the study by Kowashi et al.
(1980) it was pointed out that while the
number of PMN's increased by a factor
of 2.1, the volume of gingival fluid in-
creased by a factor of 5.4. This is not
suprising since the vasculitis is gener-
ated by numerous mediators of vascular
permeability while the PMN's are af-
fected mainly by specific chemoattrac-
tants. This duality is supported by the
findings of Golub et al. (1981) in exper-
iments with application of casein as a
specific chemoattractant into the crev-
icular area. There was an eight-fold in-
crease in leucocytes and a two-fold in-
crease in fluid.
There has been some interest in use of
PMN counts in saliva as a quantitative
indicator of disease. In the Orogranul-
ocytic Migratory Rate (OMR) pro-
cedure of Klinkhammer (1968) the
number of PMN's recoverable from sa-
liva per unit of time was related to level
of health or disease (Skougaard &
Klinkhammer 1969). In two studies
(Woolweaver et al. 1972, Cox et al.
1974), however, there was a poor corre-
lation between numbers of leucocytes in
saliva and the status of gingival health
and the fmdings of Klinkhammer were
not confirmed.
PMN products
Leucocytes contain a wide spectrum of
acid hydrolases, neutral proteases and
antibacterial agents to help in the pro-
cess of phagocytosis. Unfortunately, on
stimulation, much of the intracellular
content is released into the crevicular
area and the adjacent tissue to the detri-
ment of the host. Its presence in the
crevicular fluid, however, provides
many possibilities for monitoring the
PMN response and indirectly the in-
flammatory state.
Lactoferrin
Lactoferrin is an antimicrobial agent
found within the azurophil granules of
the PMN and is probably the only un-
ambigous marker for PMN activity
(Lefell & Spitznagel 1972). It has a high
affinity for iron and can aid in killing
of microorganisms by competing for es-
sential iron required by many bacteria.
Against some species it also has cidal
properties independent of iron binding
(Arnold et al. 1982). Since serum levels
of lactoferrin are extremely low, and sa-
liva, which does contain lactoferrin,
does not enter the crevicular area, lacto-
ferrin levels in crevicular fluid should be
a good indicator of PMN activity at a
specific site.
In the only reported study to date
Friedman et al. (1983) found a greater
than two fold increase in GCF lactofer-
rin (P < 0.01) in gingivitis (G1 > 3), peri-
odontitis and LJP when compared to
normal subjects. The values were calcu-
lated on a concentration basis. Futher
study is indicated with different levels of

gingivitis in an experimental gingivitis
model and calculation of lactoferrin as
amount generated by unit of time, the
total unit activity as suggested by Lam-
ster et al. (1985).
Enzymes
Lysozyme
This enzyme is found in both the azur-
ophil and specific granules of the PMN
and in mononuclear phagocytes. Since
the latter cell comprises only about 2%
of the crevicular leucocyte population
and serum and tissue levels of lysozyme
are very low, lysozyme should be a good
indicator of PMN activity. This enzyme
not only contributes to the phagocytic
activity in the crevice but on release me-
diates microbial aggregation and ag-
glutination and affects bacterial growth
(Iacona et al. 1982). It may also play a
regulatory role inhibiting PMN chemo-
taxis and depressing superoxide gener-
ation by PMN's (Gordon et al. 1979).
The findings on the relation of lyso-
zyme concentration in GCF to peri-
odontal disease have been contradic-
tory. Brandtzaeg & Mann (1964) found
increased levels in patients with severe
disease. Nord et al. (1971) noted an in-
crease in concentration during experi-
mental gingivitis. Van Palenstein-Hel-
derman (1976), on the other hand,
found no significant differences in lyso-
zyme concentration between subjects
with non inflammed gingiva and vari-
ous degrees of gingivitis.
In the study on lactoferrin referred
to above, Friedman et al. (1983) also
monitored lysozyme in the normal sub-
jects and in those with gingivitis, peri-
odontitis and LJP. They found no differ-
ence between the normal, gingivitis and
periodontitis subjects. They did find,
however, that there was a two fold in-
crease in the LJP patients (P<0.01)
relative to the three groups. Although it
is not clear why lysozyme should be
elevated in this population it has been
shown by McArthur et al. (1982) that
in the presence of Actinohacillus acti-
nomycetemcomitans and serum from
LJP patients, human PMN's released a
higher level of lysozyme than with nor-
mal serum. Whether this response was
due to a specific antigen-antibody com-
plex or to other factors was not deter-
mined.
It is of interest that although all
studies have indicated comparable lev-
els of lysozyme and lactoferrin in PMN
granules, the crevicular fluid concen-
tration of lysozyme is always much low-
er than lactoferrin in the same sample.
This could reflect the much greater
tendency for lysozyme to bind to bac-
terial and host cells electrostatically be-
cause of its very high isolectric point
and the presence of hydrophobic forces
as well. This differential availability of
lysozyme versus lactoferrin is reflected
in the much higher concentration of the
latter in extracts of supragingival plaque
(Di Paola et al. 1984).
Friedman et al. (1983) suggested that
the ratio of lysozyme to lactoferrin
could be of value as a diagnostic test
for LJP. This is a possibility that should
be explored.
Acid and alkaline phosphatases
Acid phosphatase, a commonly em-
ployed marker for lysosomal activity,
has not shown any consistent relation-
ships with periodontal disease (Cima-
soni 1983). This is not suprising since it
is not only present in PMNs, but in
desquamated epithelial cells and many
bacteria as well. Alkaline phosphatase,
on the other hand, has been found to
be significantly correlated with pocket
depth (Ishikawa & Cimasoni 1970) and
with increasing infiammation in an ex-
perimental gingivitis model (Baehni et
al. 1975). Binder et al. (1985) recently
showed that alkaline phosphatase levels
varied inversely with GCF volume, hen-
ce total activity per unit time would be
the most appropriate way of expressing
the alkaline phosphatase value. Al-
though the major source of the alkaline
phosphatase is probably the PMN, shed
cells and bacteria probably contribute
as well. Serum levels are only about half
that of GCF, hence it has a diluting
effect. Since increases in alkaline phos-
phatase in serum have been associated
with bone disease, local elevations in
GCF could also reflect local destruction
in active disease. Analysis for total alka-
line phosphatase in GCF should be en-
couraged in prospective studies.
B-galactosidase
In a survey of crevicular fluid enzymes
Winer et al. (1970) found no marked
differences between normal and dis-
eased states for acid and alkaline phos-
phatases. They did, however, find a sig-
nificant elevation in B-galactosidase in
disease.
B-glucwonidase and arylsuljatase
Although B-glucuronidase is often used
as a marker for lysosomal release from
Indicators of periodontal disease 537
phagocytosing cells, its presence in
GCF is probably due to contributions
from numerous host cells in addition
to PMN's. Bang et al. (1970) found a
positive statistical correlation between
GCF levels of the enzyme and depth
of periodontal pockets and mean % of
bone loss. In recent studies of enzymatic
profiles in GCF Lamster et al. (1985)
examined B-glucuronidase (BG) and ar-
ylsulfatase (AS), another lysosomal en-
zyme, in a 4 week experimental gingi-
vitis model. These enzymes were select-
ed as a measure of breakdown of
connective tissue ground substance. In
the catabolism of acid mucopolysac-
charides BG is thought to degrade the
oligosaccharides generated by the ac-
tion of hyaluronidase. AS catalyses the
hydrolysis of sulfate esters. It had pre-
viously been shown by Oshrain et al.
(1984) that arylsulfatase activity in GCF
was much higher in gingivitis and peri-
odontitis than in normal subjects, es-
pecially when calculated as total unit
activity per unit time rather than on a
concentration basis. In the experimental
gingivitis study, Lamster et al. (1985)
demonstrated that B-glucuronidase and
arylsulfatase were significantly higher
than baseline, 2 to 4 weeks following
initiation of inflammation. These differ-
ences in patterns of activity between BG
and AS, however, suggest differences in
the source of enzyme and futher exam-
ination is in order.
The concept of simultaneous exam-
ination of different markers to yield a
profile of activity is an attractive one
and many permutations and combi-
nations are possible. Selecting the best
choices will require extensive research
and some negative findings are to be
expected.
Proteolytic enzymes
Major interest has been in Cathepsin
D, elastase and coUagenase as the most
important lysosomal proteases.
Cathepsin D. Using gingival wash-
ings, collected with individual ap-
pliances, Tzamouranis et al. (1979)
examined the free and total activities of
Cathepsin D at regular intervals during
a period of experimental gingivitis.
There was a close correlation between
the concentrations of free and total en-
zymes and the clinical signs of gingivitis.
This study supported an earlier one by
Ishikawa et al. (1972) which showed a
positive correlation with periodontal
breakdown.
Recently Eisenhauer et al. (1983) re-
538 Fine and Mandel
ported the presence of significant levels
of Cathepsin B activity in GCF in pa-
tients with gingivitis. This cysteine pro-
tease, also of lysosomal origin has been
shown to degrade soluble monomeric
collagen and insoluble polymeric col-
lagen in vitro. Its possible role in peri-
odontal disease and its value as an indi-
cator remains to be established.
Elastase. Cimasoni & Kowashi (1980)
have shown that crevicular fluid pos-
sesses neutral protease activity most
probably derived from the azurophil
granules of PMNs. This protease ac-
tivity, presumably elastase, rose signif-
iciantly during an experimental gingi-
vitis period and then returned to base-
line levels when tooth brushing was
resumed. The assay employed hemoglo-
bin, however and hence specificity was
not established. More specific substrates
are now available as well as elastase
kits and this enzyme activity should be
studied thoroughly.
Collagenase. This has been the most
widely studied of the crevicular fluid
proteases because of the centrality of
collagen breakdown in the periodontal
disease process. Several groups have
demonstrated a correlation between
coUagenolytic activity of GCF and se-
verity of periodontal disease (Robertson
et al. 1973, Golub et al. 1976, 1979,
Kowashi et al. 1979, Villela & Birkedal-
Hansen 1985). Of particular pertinence
to the consideration of collagenase as
an indicator of disease was the obser-
vation by Golub et al. (1976) that the
collagenase activity was more highly
correlated with pocket depth than with
severity of inflammation and hence
"may reflect the degradative activity of
the gingival tissues lining the pocket and
could, therefore, be of diagnostic
value." This study also demonstrated
that most of the collagenase was of
mammalian orgin (PMNs, epithelial
cells, fibroblasis, macrophages) and that
its activity level was increased by the
addition of sodium thiocyanate. Tissue
collagenases (but not bacterial) are in-
hibited by binding to a-2-macroglobu-
lins from serum. The thiocyanate acti-
vates this latent collagenase by selec-
tively denaturing the macroglobulin.
The study by Villela & Birkedal-Han-
sen (1985) also noted the association
of coUagenolytic activity with probeable
pocket depth. Clearly this is a fmding
that is worth pursuing vigorously. Col-
lagenase is an attractive enzyme to in-
clude in a biochemical profile for dis-
ease activity.
Future research on collagenase ac-
tivity in crevicular fluid has to consider
simpler procedures for quantitating the
activity and standardization of the
measurement of latent as well as active
collagenase. Recently Lamster et al.
(1985) introduced the use of a synthetic
peptide and a spectrophotometric meth-
od for assaying collagen. This should be
compared to the more conventional but
complex use of radioactively labeled
collagen fibrils.
Host factors: immune response
Antibody
Serum antibody titers to periodontal
pocket inhabitants were first reported
by Mergenhagen et al. (1965), by Evans
et al. (1966), by Lehner & Clarry (1966)
and by Steinberg & Gershoff (1968).
These studies were completed long be-
fore the specific plaque hypothesis was
developed and thus the bacteria selected
for study may not have had a prinicipal
role in the disease process.
The recent explosion of information
regarding serum antibody levels in peri-
odontal disease was stimulated for the
most part by the work of Newman &
Socransky (1977) who first demon-
strated a strong association between a
specific group of subgingival bacteria
[(Aa) and LJP]. As the association of
Aa with LJP gained support in other
laboratories the relationship of other
suspected pathogens and other forms of
periodontal disease was pursued with
renewed vigor. Suspicion of the involve-
ment of Bacteroides melaninogenicus in
adult periodontitis can be tracted to the
work of Hemmons & Harrison (1942)
and then MacDonald et al. (1956). Ef-
forts to confirm the relationship of Bac-
teroides with periodontitis have been
frustrated for the most part by:
(a) heterogeneity of periodontal disease
in the adult periodontitis category;
(b) heterogeneity of the species within
the Bacteroides group.
Stronger correlations have been made
between Bacteroides species and peri-
odontitis in recent years as a result of
the clarification of species types in the
Bacteroides group (Coykendall et al.
1980), and the subsequent emergence of
serologically specific reagents (Dzink et
al. 1983). Thus in the last several years a
stronger albeit not absolute correlation
has been made between B. gingivalis and
adult periodontitis (Zambon et al. 1981)
while B. melaninogenicus intermedius
has been implicated in Acute Necroti-
zing Ulcerative Gingivitis (ANUG)
(Loesche et al. 1982).
The last 5 years has seen a dramatic
rise in studies showing correlations be-
tween specific disease categories and el-
evated antibody titers to the pathogen
suspected of initiating that form of peri-
odontal disease as follows:
(1) LJP - Antibody to Actinobacillus
actinomycetemcomitans (Listgar-
ten et al. 1981, Ebersole et al. 1982,
Ranney et al. 1982).
(2) ANUG - Antibody to Bacteroides
intermedius and intermediate size
spirochetes (Chung et al. 1983).
(3) Adult periodontitis - Antibody to
Bacteroides gingivalis (Mouton et al.
1981,Taubmanetal. 1980).
Nevertheless, the relationship of these
elevated antibody titers to periodontal
disease activity or pathogenesis of dis-
ease remain enigmatic.
Results of animal experiments and
longitudinal human studies of peri-
odontal treatment shed some light on
the relationship of antibody titers to dis-
ease.
Animal experiments
Periodontal bone loss was induced in
rats by ovalbumin sensitization and
feeding. In these experiments periodon-
tal bone loss was greater in ovalbumin
sensitized rats than in rats not pre-
viously exposed to the antigen. The
plasma IgG response peaked in 14 days
and remained high throughout the 128
day experiment. Bone loss increased in
the sensitized group over the experimen-
tal period and thus the peak IgG re-
sponse preceded the major periods of
bone loss in these animals and remained
high throughout (Taubman et al. 1983).
Similar results were seen in germ-free
rats immunized with Aa and then in-
fected with Aa strain Y-4. In these ex-
periments peak IgG response was seen
30 days after infection with Aa whereas
bone loss appeared 90 days after infec-
tion (Taubman et al. 1983).
Results of experiments of Nagahata
et al. (1982) demonstrated that implan-
tation of B. assacharolyticus and E.
corrodens could be accomplished in a
hamster model with the assistance of a
ligature. Bone loss was greater in ham-
sters implanted with B. assacharolyticus
and with titers to B. assacharolyticus
than in control animals or animals re-
ceiving E. corrodens having no serum
titer.
In experiments by Chung et al.
(1983), beagle dogs with pre-existing
 Indicators of periodontal disease 539

gingivitis had ligature induced peri- crease in antibody activity to Aa and B. that in general serum titers to Aa, Pep-
odontitis, Bacteriologic studies demon- gingivalis. All patients were well main- tostreptococcus micros or B. gingivalis
strated significant levels of Gram ( - ) tained, were higher relative to crevicular titers.
bacteria including various Bacteroides Ebersole et al, (1985) have followed However, examination of specific sites
species. serum IgG antibody titers after root revealed that while most sites were low-
Results pointed to general lack of planing and scaling. For the most part er than serum titers some were elevated.
change of antibody titers over the ex- IgG levels where elevated shortly after Thus the work of Tew et al, (1985) sup-
perimental period. However since the scaling and returned to preexisting ports the concept of local production of
animals started with a pre-existing gin- levels over time. These studies suggest antibody and suggests that elevations in
givitis elevated antibody levels had al- that scaling may boost the systemic im- local antibody titer may serve as a signal
ready existed. If animals were divided mune response initially. of local disease activity,
into those with severe bone loss vs, Taken together studies from animal A variety of other parameters can be
those with moderate bone loss, animals experiments and longitudinal human used to measure the host response.
in the severe group appeared to have a therapy studies suggest that antigenic Among these are: complement levels,
elevated IgG to Gram (—) bacteria in challenge whether it be in the form of prostaglandins and interleukins.
the gingivitis stage and before ligature intentional (animal) or unintentional
induced periodontitis. Taken together (therapy) antigen deposition cause a Complement
the animal experiments suggest that boost in the humoral response that pla- The complement system is a complex
peak serum IgG levels can preceed bone teaus over time. Successful elimination set of nine functional units consiting of
loss and can remain at plateau levels of the antigen by means of full mouth 11 discrete serum porteins that act in a
(or high) throughout the period of bone treatment causes a drop in the antigenic cascading fashion. After activation of
loss. In fact these experiments suggest load and a subsequent drop in serum the system several antimicrobial as well
that serum IgG levels may be predictors antibody levels. as host tissue destructive phases may
of disease vulnerability but do not show Therefore, serum antibody levels ap- follow.
any clear correlation with bursts of dis- pear to reflect total oral exposure. Since Most reports on the role of com-
ease activity. multiple sites may be undergoing chal- plement in periodontal disease implicate
lenge; local elevation of antibody may complement factors as immunoreactive
Post treatment experiments be more relevant to studies of current tissue destructive components (Snyder-
Tolo et al, (1982) monitored IgG, A and local disease activity. man 1972, Kahnberg et al, 1976), For
M response in serum by ELISA to a The complexity of using local anti- the present complement levels still need
battery of eight oral micro-organisms in body production as a measure of disease to be assessed relative to our newer con-
a group of 12 periodontitis and 24 nor- activity can be illustrated by a case pre- cepts of the episodic nature of periodon-
mal patients. All periodontitis patients sented by Haffajee et al, 1984, Shallow tal disease activity. Moreover activated
(12 of 12) showed an elevated IgG re- (1 mm) sites in that case demonstrated or functional forms of complement
sponse to B. gingivalis prior to treat- elevated antibody levels to Aa and a should be detected at the local level and
ment and 7 of 12 showed a decreased high proportion of Aa (74%) in pocket compared to serum levels if correlations
IgG titer to B. gingivalis after treatment. sites at a period within 2 months prior to local burst of disease activity are to
The remaining subjects showed no to bone loss. Moderate sites (4 to 6 mm be made accurately. The groundwork
change in titer, pocket depth) showed elevated antibody for these studies has been laid by Schen-
Haffajee et al, (1984) investigated one levels to Aa at a time 10 months before kein& Genco (1978),
LJP patient bimonthly for a period of detection of active destruction, Aa was Complement cleavage has been as-
26 months using combined clinical mi- detected in these sited before destruc- sessed in gingival crevicular fluid in two
crobiological and immunological meth- tion. Since the local antibody response recent studies by Patters et al, (1985)
ods of monitoring. Initial antibody was not elevated prior to active destruc- and Niekrash (1985), Filter paper strips
levels were elevated to Aa and E. cor- tion it is possible that the breakdown taken directly from sites being studied
rodens and were low to C. sputigena may have been due to a second burst of were analysied for cleavage products of
and B. intennedius. Immediately before activity perhaps due to a different spe- C3, C4 and B by multilayer crossed-
therapy antibody levels to C. sputigena cies of bacteria. immunoelectrophoresis. An experimen-
rose while Aa remained high and E. In the case of the deepest pocket (8 tal gingivitis study demonstrated in-
corrodens fell. Post-treatment Aa fell mm) a biopsy revealed antibody to Aa creasing quantities of C3 cleavage over
and remained low as did that of C, sputi- in the tissue but the organism could not time and thus percent C3 cleavage in-
gena while E. corrodens antibody levels be found in the pocket site nor was there creased significantly with plaque ac-
rose, B. intermedius levels remained low an elevated antibody in the crevice. cumulation and developing gingivitis.
throughout the experimental period, Thus Aa or antibody to Aa could not While B cleavage was seen, conversion
Vincent et al, (1985) studied antibody be conclusively demonstrated to corre- of C4 to C4c was never seen. In a second
responses 3 to 4 years post therapy in a late with disease activity at the local site study, complement conversion was ana-
group of 9 JP patients, ELISA titers to in all cases however, more work in this lysed in healthy, gingivitis adult peri-
Eusobacterium nucleatum, B. gingivalisarea appears warranted. odontitis, and juvenile periodontitis
and Aa were evaluated. Immediately af- Recent work by Tew et al, (1985) sup- sites (Niekrash 1985), Classical path-
ter therapy antibody titers to B. gin- port the work of Haffajee et al, (1984) way (antibody associated) activation
givalis and E nucleatum rose while titers and once again demonstrates site to site (conversion of C4 to C4c) occured only
to Aa fell. Sera collected 3 to 4 years variations in serum titers to supected in JP sites (6 of 11), B and its cleavage
post therapy showed a significant de- pathogens. These investigators found product Bb (as a measure of alternate
540 Fine and Mandel
or non-antibody mediated cleavage)
was consistently present in gingival fluid
obtained from inflamed sites. Percent
C3 conversion (a measure of total acti-
vation - classical and alternate) corre-
lated signiflcantly with pocket depth,
bleeding and gingivitis.
Indeed a complement activation in-
hibitor trans-4-aminomethylcyclohexa-
ne carboxylic acid (an inhibitor of alter-
nate pathway activation) has been
shown to reduce gingivitis formation in
dogs (Gaffar et al. 1985).
Prostaglandin
Mammalian cells can synthesize prosta-
glandins from arachadonic acid, a fatty
acid occuring in mammalian cell mem-
branes and derived from linoleic acid.
Mammalian cell membranes contain the
ester of arachadonic acid which is un-
available for enzymatic conversion to
its acid. A number of initiators of in-
flammation are capable of activating
enzymes such as phospholipase which
can liberate arachadonate and initiate
either the PG cycloxygenase or lipoxyg-
enase pathways. One of the products of
the cycloxygenase pathway, Prostaglan-
din E2 (PGE2) has been the focus of
recent work. Elevated quantities of
PGE2 were found in gingival crevicular
fluid in patients with periodontitis as
compared to patients with gingivitis
(Offenbacher et al. 1981). A second
study showed a correlation between gin-
gival crevicular fluid (GCF), PGE2
levels and levels in the adjacent tissue.
In addition samples from JP patients
were 3 fold higher than these from adult
periodontitis (Offenbacher et al. 1984).
The last study in this series was longi-
tudinal in nature. 41 adult periodon-
titis patients were followed every 3
months for a period of 3 years. Moni-
toring of crevicular fluid continued until
a site demonstrated attachment loss.
Retrospective analysis of the GCF-
PGE2 levels at the sites losing attach-
ment showed five fold elevations in
PGE levels compared to contralateral
stable control sites. One month follow-
ing treatment PGE levels dropped sig-
nificantly. This method appears to have
a high degree of sensitivity, speciflcity
and predictive value (Offenbacher et al.
1985).
Moreover, recent longitudinal studies
in beagle dogs have shown that admin-
istration of ibuprofen a prostaglandin
inihibitor can cause significant re-
duction of bone loss in these animals
(Williams et al. 1984).
Cytokines - Interleukin 1 measurement of crevicular fluid volume
Interleukin 1 is derived as a soluble se- per unit time (flow rate) has been widely
cretion product from macrophages and studied as a measure of gingival inflam-
was formerly known as lymphocyte ac- mation. This area has been extensively
tivating factor. IL-I augments B-cell reviewed by Cimasoni (1983). He noted
antibody production, promotes T cell that "a positive correlation was always
helper function, stimulates fibroblast found between the clinical appreciation
growth, collagenase and prostaglandin of gingival inflammation and the
production. As a result of cloning tech- amount of gingival fluid. This corre-
nologies it has been possible to charac- lation was highly significant in most in-
terize IL-1 as a macrophage derived vestigations, while it was more moder-
polypeptide of 12 to 15,000 daltons. IL- ate in others." Cimasoni went on to
1 can be regarded as an augmenting as point out, however, that the correlation
opposed to a primary signal, it does not between flow and histological inflam-
stimulate T-cells directly; nor does it matory changes was poor. This is not at
induce the production of IL-2 by acti- all suprising since fluid flow reflects
vated T-cells. only one aspect of the inflammatory
By means of a bioassay it was pos- process, capillary permeability, which
sible to detect levels of IL-1 in gingival is difficult is measured histologically.
fluid using the Skapski & Lehner (1975) Since the clinical changes, redness and
washout technique. Gingival fluid levels bleeding on probing reflect the same tis-,
of IL-1 were detected and in all cases sue alteration, the usually good corre-
and inflamed sites harbored more IL-1 lation between the two is consistent.
than normal sites (Charon et al. 1982). In early disease the change in per-
meability in the crevicular area may be a
Host factors: response to chemotactic forerunner of clinical changes and hence
challenge can serve as an early warning system.
In addition to assessment of disease ac- Fluid flow offers the advantage of ob-
tivity per se future diagnostic p/o- jectivity and quantitation. If this is to
cedures should also consider the pres- be clinically useful, a reproducible, stan-
ence of defects in the ability to mount dardized methodology has to be em-
an appropriate protective response. De- ployed. Such procedures using filter pa-
fects in PMN chemotaxis in LJP have per strips have been described by a num-
figured prominently in such consider- ber of investigators (see Cimasoni, 1983
ations (Clark et al. 1977, Van Dyke et for review). Recently Schifter & Chilton
al. 1980). Until recently the measures of (1985) reported a low level of reproduci-
defect have been in vitro. Golub et al. bility, however, and the need for at least
(1981) demonstrated that the response duplicate samples. The best method for
of human sulcular leucocytes to a sampling still remains to be established.
chemotactic challenge could be mea- The new Periotron 6000, appears to be
sured in vivo. Singh et at. (1984) ap- a very reliable device for measuring
plied the method to examination of sub- the fluid once an appropriate sample is
jects with gingivitis, chronic periodonti- secured (Henrichs et al. 1984, Bickel &
tis and LJP. In gingivitis and Cimasoni 1984).
periodontitis the response was similar In considering the use of crevicular
to normal subjects except that the peak fluid fiow in relation to disease activity
cell count was much greater. LJP sub- it is important to note that in contrast to
jects, however, showed an abnormal the high correlation with inflammation
pattern with two leucocyte peaks. Osh- there was a poor correlation with pocket
rain & Telsey (unpublished obser- depth in cross sectional studies (Valazza
vations) recently studied a group of re- et al. 1972). In longitudinal sampling
fractory subjects with advanced peri- increases in fluid volume could signal
odontal disease and found a similar the development of inflammatory
abnormal pattern of a double peak. change before the occurence of bleeding
This type of evaluation should be very on probing or suppuration. The patient
useful to the chnician in determining serves as his own control. Just what de-
which subjects require intense, on going gree of change would be predictive re-
therapies. mains to be established.
(Ill) Products of Tissue Injury
Electrolytes
GCFflow
Since vasculitis is the initial host re- With breakdown of tissue intracellular
sponse in all inflammatory disease. ions should admix with the fluid trans-

udates and affect the ionic concen-
tration of GCF. Most of the reports on
sodium, potassium and calcium in GCF
in relation to level of disease were per-
formed 15-20 years ago (see Cimasoni
(1983) for a review). In general they
found higher levels of sodium and po-
tassium than in serum; calcium was
variable. Sodium and calcium were
positively correlated with inflammation;
potassium was not. Circadian rythm
was a confounding factor and the ana-
lytical procedures had limited sensi-
tivity. New technologies for direct
measurement of ions, such as ion sensi-
tive micro-electrodes should stimulate
more research in this area.
Intracellular enzymes
Lactate dehydrogenase
A number of investigators have exam-
ined lactate dehydrogenase activity
(LDH) as an indicator of disease ac-
tivity since intracellular enzymes be-
come extracellular with cell death. Bang
et ai. (1972) found LDH to be much
higher in GCF than in serum but could
find no quantitative relationship be-
tween LDH activity and clinical par-
ameters. Weinstein et al. (1972) also
found elevated LDH levels in GCF
and greater intensity of staining of iso-
enzymes in GCF from inflammed sulci
than from normal. Lamster et al. (1985)
using a standardized filter paper strip
method (rather than the capillary tube
employed by previous investigators)
found that LDH was much higher in
GCF than previously reported. When
calculated on the basis of concentration
(volume activity) there was no corre-
lation with inflammation. When calcu-
lating LDH on the basis of total unit
activity (total activity per unit of time),
however, there was a significant re-
lationship to inflammation. This is an-
other situation in which the serum
transudate has a diluting effect and use
of the conventional concentration cal-
culation underestimates the signifi-
cance.
Snyder & Wolf (1983) also found that
areas that exhibited the greatest degree
of periodontal disease also exhibited the
highest levels of LDH in GCF. Mukher-
jee et al. (1985), in a study of ligature
induced periodontal disease in dogs
found that the LDH concentration was
unrelated to any of the clinical indices
of attachment loss. It would be inter-
esting to see if recalculation as total unit
Indicators of periodontal disease 541
activity (Lamster et al. 1985) would af- a much higher ratio of spermine to sper-
fect the relationship. midine, however, in periodontitis. In
LJP not only was the total concen-
tration markedly elevated and the
Aspartate aminotransferase
spermine to spermidine ratio extremely
Chambers et al. (1984) investigated an- high, but a series of additional amines
other intracellular enzyme as a possible were clearly evident on the chromato-
marker for tissue destruction in peri- grams. These are probably acetyl de-
odontitis, Aspartate Aminotransferase rivatives of the polyamines and are of-
(AST), which is routinely used as a diag- ten seen in urine when breakdown of
nostic indicator of myocardial infarc- transplants occur. Whether the organ
tion. They employed a ligature-induced undergoing necrosis or the destructive
periodontitis model in the beagle and cells are the source of the polyamine
examined AST levels in crevicular fluid metabolites, is not clear. This appears
before and after ligation. They found to be a fertile field of investigation. In
that two weeks after ligation the enzyme addition to serving as markers, polyami-
level was 10-100 times higher in crevicu- nes should be examined in relation to
lar fluid than in serum. This is a promis- collagenase activity. Huang et al. (1982)
ing start and certainly should be exam- reported that polyamines produced in
ined in human clinical situations. cultures of both human and rat tumor
tissues were able to bind a-2-macroglo-
bulin and reduce its anti-collagenase ac-
Polyamines tivity.
In addition to quantitating polyamines
in GCF in subjects with different levels
Glycosaminoglycans
of gingivitis, Vratsanos & Mandel (un-
published observations) also examined The extracellular ground substance of
GCF from filter paper strips in several connective tissue contains a complex
subjects with adult periodontitis and mixture of polysaccharides termed gly-
LJP. The concentration of total poly- coaminoglycans (GAG), which are link-
amines in adult periodontitis was com- ed to specific proteins. Various GAG
parable to that in gingivitis. There was have been found in GCF obtained from
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Arnold, R. R., Russell, J. E., Champion, W. J., Brewer, M. & Gautier, J. J. (1982) Bactericidal
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Baehni, P., Sudan, J. M., Steube, W. & Cimasoni, G. (1975) Enzymes lysosomiales dans les
prodvits de rincage de la region parodontale marginale au cours de la gingivite experimen-
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Baer, P. N. (1971) The case for periodontosis as a clinical entity. Journal of Periodontology
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Bang, J., Cimasoni, G. & Held, A. J. (1970) Beta-glucuronidase correlated with inflammation
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Bang, J., Rosenbusch, C, Ahmad-Zadeh, C. & Cimasoni, G. (1972) Isoenzymes of lactic
dehydrogenase in human gingival fluid. Helvetica Odontological Aeta 16, 89-93.
Barksdale, L. (1970) Coryrebacterium diphtherial and its relatives. Bacteriology Reviews 34,
378-401.
Beicknell, K., Grinenko, U., Carlton, D. & Newman, M. (1978) GLC rapid analysis of
bacteria in plaque. Journal of Dental Research 57, 351.
Bickel, M. & Cimasoni, G. (1984) Reliability of volume measurements with the new Periotron
6000. Journal of Periodontal Research 19, 313-316.
Binder, T. A., Goodson, J. M. & Durham, S. L. (1985) Volume and phosphatase levels with
repeated sampling of crevicular fluid. Journal of Dental Research (Special Issue) 64, 374.
Brandtzaed, P. & Mann, W. V. (1964) A comparative study of the lysozyme activity of human
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542 Fine and Mandel
areas with chronic periodontal disease
(Embery et al. 1982). In a recent study
Last et al. (1985) explored the electro-
phoretic separation and identification
of GAG as a possible indicator of active
periodontal disease. Hyaluronic acid, a
non-sulfated GAG, was the only major
band in sites of chronic gingivitis. In
untreated, advanced periodontitis, an
additional band, identified as chrondoi-
tin-4-sulfate was also detected. This
band was also found in some samples
of early periodontitis and LJP, but not
in samples collected after completion of
surgical treatment or daily subgingival
irrigation with chlorhexidine. The sul-
phated GAG was found, however, in
teeth undergoing orthodontic move-
ment and from post extraction wounds,
situations involving bone resorption.
Chrondoitin-4-sulfate, predominates in
bone; dermatan sulfate is its counterpart
in non-mineralized connective tissues,
hence theoretically, it is a reasonable
marker for active periodontal destruc-
tion of bone. Development of a more
sensitive technology than cellulose-ace-
tate electrophoresis is needed for identi-
fication and hopefully quantitation. The
procedure employed for sample collec-
tion, a micropipette at the gingival mar-
gin for 15 minutes would also have to
be modified for clinical use, but its a
good start.
Bone resorption assay
Feldman et al. (1984) described a bone
resorption assay that could be corre-
lated with clinically progressive disease
severity. Subjects had been under obser-
vation in a longitudinal aging study
over a nine year period and control and
active sites were selected, the latter on
the basis of increasing pocket depth and
radiographic bone loss. Subjects were
recalled at 2-3 week intervals over a
three month period for assessment of
clinical indices and bone resorption as-
says. The assay system involved curet-
ting of gingival and periodontal cells,
centrifugation in an appropriate me-
dium and the incubation with ''^Ca la-
beled devitalized rat bone over a 72-h
period. Data was recorded as % calcium
released. Clinically progressive disease
was accompanied by elevated ^'Ca re-
lease relative to control while remissive
sites exhibited a decrease. This is poten-
tially a very useful laboratory method
for clinical studies and conceivably even
for treatment evaluation.
Comparison of chromatographic profiles of plaque from idiopathie juvenile periodontitis.
Journal of Denial Research (Special Issue A) 58, 177.
Chambers, D. A., Crawford, J. M., Mukherjee, S. & Cohen, R. L. (1984) Aspartate amino-
transferase increases in crevicular fluid during experimental periodontitis in beagle dogs.
Journal of Periodontology 55, 526-530.
Charon, J. A., Luger, T. A., Mergenhagen, S. E. & Oppenheim, J. J. (1982) Increased
thymosyte-activating factor in human gingival fluid during gingival inflammation. Infection
and Immunily 38, 1190-1195.
Chung, C. P., Nisengard, R., Slots, J. & Ciancio, S. (1983) Bacterial antibody titers in Ligature-
induced periodontitis in beagle dogs. Journal of Periodontology 54, 236-246.
Chung, C. P., Nisengard, R. J., Slots, J. & Genco, R. J. (1983) Bacterial IgG and IgM
antibody titers in acute necrotizing ulcerative gingivitis. Journal of Periodontology 54,
557-562.
Cimasoni, G. & Kowashi, Y. (1980) Proteinases of the gingival crevice and their inhibitors.
In Borderland between earies and periodontal disease, eds. Lehner, T. & Cimasoni, G. vol.
II, pp. 31^9. London: Academic Press.
Cimasoni, G. (1983) Crevieular fluid updated. Basel Krager.
Clark, R. A., Page, R. C. & Wilde, G. (1977) Defective neutrophil chomotaxis in juvenile
periodontitis. Infection and Immunity 18, 694-700.
Cox, M. a , Crawford, J. J., Lundblad, R. L. & McFall, W. T. Jr. (1974) Oral leucocytes and
gingivitis in the primary dentition. Journal of Periodontal Research 9, 23-26.
Coykendall, A. L., Kaczmareck, F. S. & Slots, J. (1980) Genetic heterogeneity in Bacteroides
assaraccharolyticus (Holdemand & Moore 1970) (Finegold & Barnes 1977) (approved lists
1980) and proposal Bacteroides gingivalis sp. nov. and Bacteroides macacae (Slots and
Genco) comb. nov. International Journal of Systematic Bacteriology 30, 559-564.
Di Paola, G., Herrera, M. S. & Mandel, 1. D. (1984) Immunochemical study of host proteins
in human supragingival plaque compared with denture plaque. Archives of Oral Biology 9,
161-163.
Dzink, J. L., Socransky, S. S., Ebersole, J. L. & Frey, D. E. (1983) ELISA and conventional
techniques for identification of black-pigmented Bacteroides isolated from periodontal
pockets. Journal of Periodontal Research 18, 369-374.
Ebersole, J. L., Frey, D. E., Taubman, M. A. & Smith, D. J. (1980) An ELISA for measuring
scrum antibodies to Actinobacillus actinomycetemcomitans. Journal of Periodontal Research
15, 621-632.
Ebersole, J. L., Taubman, M. A., Smith, D. J., Genco, R. J. & Frey, D. E. (1982) Human
immune responses to oral microorganisms. I. Association of localized juvenile periodontitis
(JP) with serum antibody responses to Actinobacillus actinomycetemcomitans. Clinical and
Experimental Immunology 46, 43-52.
Ebersole, J. L., Taubman, M. A., Smith, D. J. & Haffajee, A. D. (1985) Effect of subgingival
scaling on systemic antibody responses to oral microorganisms. Infection and Immunity 48,
534-539.
Eisenhaver, D. A., Hutchinson, R., Javid, T. & McDonald, J. K. (1983) Identification of a
Cathepsin B-like protease in the crevicular fluid of gingivitis patients. Journal of Dental
Research 62, 917-921.
Embery, G., Oliver, W. M., Stanbury, J. B. & Purvis, J. A. (1982) The electrophoretic detection
of acidic glycosaminoglycans in human gingival sulcus fluid. Archives of Oral Biology 27,
177-179.
Evans, R. T, Spaeth, S. & Mergenhagen, S. J. (1966) Bacteriocidal antibody in mammalian
serum to obligatorily anaerobic gram-negative bacteria. Journal of Immunology 97, 112-119.
Feldman, R. S., Carnes, D. L. & Key, L. L., Jr. (1984) Determination of periodontal disease
activity by a bone resorption assay. Journal of Periodontal Research 19, 628-632.
Fine, D. H. & Greene, L. S. (1984) Microscopic evaluation of root surface associations in
vivo. Journal of Periodontal Research 19, 152-167.
Friedman, S. A., Mandel, I. D. & Herrera, M. S. (1983) Lysozyme and lactoferrin quantitation
in the crevicular fluid. Journal of Periodontology 54, 347-350.
Gaffar, A., Coleman, E. J. & Niles, H. P. (1985) Effects of a complement activation inhibitor
on gingivitis formation in beagles. Journal of Dental Research (Special Issue A) 64, abstract
#1676, 361.
Golub, L. M., Siegel, K., Ramamurthy, N. S. & Mandel, 1. D. (1976) Some characteristics
of coUagenase activity in gingival crevicular fluid and its relationship to gingival disease in
humans. Journal of Dental Research 55, 1049-1057.
Golub, L. M., Iacono, V. J., Nicoll, G., Ramamurthy, N. & Kaslick, R. S. (1981) The response
of human sulcular leucocytes to a chemotactic challenge. Journal of Periodontal Research
16, 171-179.
Goodson, J. M., Tanner, A. C. R., Haffajee, A. D., Sornberger, G. C. & Socransky, S. S.
(1982) Patterns of progression and regression of advanced destructive periodontal disease.
Journal of Clinical Periodontology 9, 472-481.
Gordon, L. I., Douglas, S. D., Kay, N. E. Yamado, O., Osserman, E. F & Jacob, H. S. (1979)
Modulations of neutrophil function by lysozyme. Journal of Clinical Investigation 64,
226-232.
 Indicators of periodontal disease 543

All three of the tissue breakdown Haffajee, A. D., Socransky, S. S., Ebersole, J. L. & Smith, D. J. (1984) Clinical, microbiological
assays, polyamines, GAG and bone and immunological features associated with the treatment of active periodontosis lesions.
resorption require sophisticated, Journal of Clinical Periodonlology 9, 600-618.
Hemmens, E. S. & Harrison, R. W. (1942) Studies on the anaerobic bacterial flora of
time consuming laboratory procedures. suppurative periodontitis. Journal of Infectious Diseases 70, 131-146.
However, so do many tests currently in Hinrichs, J. E., Bandt, C. L., Smith, J. A. & Golub, L. M. (1984) A comparison of 3 systems
use in medical practice. With enough for quantifying gingival crevicular fluid with respect to linearity and the effects of qualitative
interest on the part of clinicians our differences in fluids. Journal of Clinical Periodonlology 11, 652-661.
laboratory enterpreneurs will rise to the Huang, C. C , Blitzer, A. & Abramson, M. (1982) Effects of polyamines on the anticollagenase
occasion. The dental office is a major activity of 2-macroglobulin. Federation Proceedings 41, 637.
marketing opportunity. But before we Iacona, V. J., Mackay, B. J., Pollock, J. J., Boldt, P. R., Ladenhaeim, S., Grossbard, G.
reach the stage of routinizing the diag- L. & Roehon, M. I. (1982) Role of lysozyme in the host response to periodontopathic
nostic procedures we have to plan for microorganisms. In Host-parasile interactions in periodontal diseases, eds. Genco, R. J. &
experimental studies in animal models Mergerhagen, S. E., pp. 318-342. Washington, D. C:, American Society of Microbiology.
with histological correlates and a num- Ishikawa, I. & Cimasoni, G. (1970) Alkaline phosphatase in human gingival fluid anf its
relation to periodontitis. Archives of Oral Biology 15, 1401-1404.
ber of prospective studies on humans Ishikawa, I., Cimasoni, G. & Ahmad-Zadeh, C. (1972) Possible role of lysosomal enzymes in
with standardized clinical correlates to the pathogenesis of periodontitis. A study on Catheasin D in human gingival fluid. Archives
validate the objective measurements. of Oral Biology 17, 111-117.
Kahnberg, K. E., Lindhe, J. & Attstrom, R. (1976) The role of complement in initial gingivitis.
I. The effect of decomplementation by cobra venom factor. Journal of Periodontal Research
II, 269-278.
Conclusions Kitawaki, M., Lijima, K. I., Nakashizuka, T. & Hayakawa, T. (1983) Neuraminidase activity
in human crevicular fluid. Journal of Periodontal Research 18, 318-320.
In a recent review, Cimasoni (1983) con- Klinkhammer, J. M. (1968) I. The orogranulocytic migratory rate. Periodontics 6, 207-211.
cluded that "none of the multiple com- Kornman, K. S., Holt, S. C. & Robertson, P. B. (1981) The microbiology of ligature-induced
ponents analysed in (gingival) fluid has periodontitis in the cynomolgous monkey. Journal of Periodontal Research 16, 363-371.
improved clinical judgement of the rate Kornman, K. S., Patters, M. S., Kiel, R. & Marucha, P. (1984) Detection and quantitation
of progress of gingivitis and periodonti- of Baeteroides gingivalis in bacterial mixtures by means of flow cytometry. Journal of
tis or the rate of repair of these con- Periodontal Research 19, 570-573.
ditions" and this conclusion is under- Kowashi, Y., Jaccard, F. & Cimasoni, G. (1979) Increase of free collagenase and neutral
standable since some of the leads have protease activities in the gingival crevice during experimental gingivitis in man. Archives of
not paid off. Cimasoni's conclusions Oral Biology 24, 645-650.
were based, to a great extent, on results Kowashi, Y., Jaccard, F. & Cimasoni, G. (1980) Sulcular polymorphonuclear leucocytes and
obtained from static models of disease gingival exudate during experimental gingivitis in man. Journal of Periodontal Research 5,
151-158.
and should therefore be re-examined in
Lamster, I. B., Hartley, L. J. & Vogel, R. I. (1985) Development of a biochemical profile
the light of the newer concepts of dis- for gingival crevicular fluid: methodological considerations and evaluation of collagen-
ease activity (Goodson et al. 1982, So- degrading and ground substance-degrading enzyme activity during experimental gingivitis.
cransky et al. 1984) and some of the Submitted for publication to Journal of Periodontology.
newer methodological approaches in- Lamster, I. B., Vogel, R. 1., Hartley, L. J., De George, C. A. & Gordon, J. M. (1985) Lactate
cluding: local antibody analysis; PGE dehydrogenase, B-Glucuronidase and arylsulfatase activity in gingival crevicular fluid as-
analysis; and enzyme and tissue product sociated with experimental gingivitis in man. Journal of Periodontology 56, 139-147.
analysis etc. In addition, the advantage Last, K. S., Stanbury, J. B. & Embery, G. (1985) Glycosaminoglycans in human gingival
of simultaneous evaluation of different crevicular fluid as indicators of active periodontal disease. Archives of Oral Biology 30,
markers to obtain a profile of local dis- 275-281.
Lefell, M. S. & Spitznagel, J. K. (1972) Association of lysozyme and lactoferdn in granules
ease activity has been highlighted in this
of human polymorphonuclear leucocytes. Infection and Immunity 6, 761-765.
discussion as an approach that may Lehner, T. & Clarry, E. D. (1966) Acute ulcerative gingivitis. An immunofluorescent investiga-
provide a more reliable picture of the tion. British Dental Journal 121, 366-368.
local destructive disease. These new ap- Levine, M. (1985) The role for butyrate and proprionate in mediating Hela-cells growth
proaches are currently being tested in inhibition by human dental plaque fluid from adult periodontal disease. Archives of Oral
either animal models or in human thera- Biology ifi, 155-159.
peutic trials and we anxiously await the Listgarten, M. A. & Hellden, L. (1978) Relative distribution of bacteria at clinically healthy
results. and periodontally diseased sites in humans. Journal of Clinical Periodontology 5, 115-132.
Listgarten, M. A., Lai, C. H. & Evian, C. I. (1981) Comparative antibody titers to Actinobacil-
Most conferences on periodontal dis- lus aetinomyeetemcomitans in juvenile periodontitis, chronic periodontitis and periodontally
ease usually end with a call for new healthy subjects. Journal of Clinical Periodontology 8, 155-164.
therapeutic or preventive strategies, for Listgarten, M. A., Levin, S., Schifter, C. C , Sullivan, P., Evian, C. I. & Rosenberg, E. S.
a magic bullet. In this conference our (1984) Comparative differential dark-field microscopy of subgingival bacteria from tooth
need is less flambuoyant: what we surfaces with recent evidence of recurring periodontitis and from non-affected surfaces.
would like is a magic marker, to tell us Journal of Periodontology 55, 398^01.
when a site is threatened or vulnerable Listgarten, M. A. & Schifter, C. (1982) Differential dark field microscopy of subgingival
to attack and when active disease is oc- bacteria as an aid in selecting recall intervals: Results after 18 months. Journal of Clinical
Periodontology 9, 305-316.
curing. This is relatively simple when
Loesche, W. J., Syed, S. A., Murray, R. J. & Mellberg, J. R. (1975) Effect of topical acidulated
the entity is gingival inflammation, but phosphate fluoride on percentage of Streptococcus mutans and Streptococcus sanguis in
more difficult for periodontitis. It may plaque. Caries Research 9, 139-145.
not be possible to differentiate the in- Loesche, W. J., Syed, S. A., StoU, J. & Laughon, B. E. (1982) The bacteriology of acute
flammatory burst in periodontitis from necrotizing ulcerative gingivitis. Journal of Periodontology 53, 223-230.
544 Fine and Mandel
its gingival counterpart until the bone
resorption stage is reached. Since this
stage follows attachment loss our
marker is one step too late. We may
have to be content with measuring sus-
ceptibility at a site and here the spec-
trum of possibilities is broader. In prag-
matic terms, it should be possible to
determine when intervention is called
for and when it is not and if we
do intervene, whether we have ac-
complished anything.
Zusammenfassung
Indikatoren parodontaler Krankheitsaktivitdt:
Eine Auswertung
Es wird immer deuUicher, dass die traditio-
ncllen klinischen Kritcrien nicht ausrcichen,
wenn es sich um (a) die Bestimmung aktiver
Krankheilsregionen handelt, (b) um die
kvantitative Bestimmung des Therapieerfol-
ges Oder (c) um ein Mass uber die Anfalligkeit
gegenliber zukiinftiger Parodontolyse. Um
objektive Masseinheiten zu erarbeiten, hat
man eine Vielzahl verschiedenster Studien
konzipiert, die vom Spcichcl, Blut, der Pla-
que und dem Zahnfleischsekret (GCF) aus-
gingen. Die Untersuchung umfasste: (a) spe-
zifischc Bakterien und ihre Produkte; (b)
Wirtzellen und ihre Produkte (enzymatisch
und antibakteriell, sowohl immunologisch als
auch nicht-immunologisch); (c) Produkte ge-
weblichcr Schadcn, lokalcr Epithel- und Bin-
degewebe sowie des Knochens. Obwohl die
meisten Arbeiten den Klinikcr bis heute keine
verlassliche Hilfc leisten konnten, so lassen
doch die technischen Verbesserungen bei der
Entnahme von Abstrichen und Gewebepro-
ben und die hcutc zur Verfugung stehende
sophistische Analysentechnik einen gewissen
Optimismus bcrechtigt erscheinen.
Vorgeschlagene Methoden zur Entdeckung
subgingivaler Plaquebakterien sind hinsicht-
lich ihrer Empfindlichkeit und ihrer Spezifi-
tat unterschiedlich. Mittels Dunkelfeldmi-
kroskopie hat man einigc Zusammenhange
mit beslehendcr Krankheit nachweisen kon-
nen, jedoch lasst die nur begrenzte Spezifitiit
auch nur begrenzte Brauchbarkeit zu. Es sind
hochspezifische polyclonale und monoclona-
le Antiseren gegen vermutete Pathogene wie
den Bacteroides gingivalis und den Actinoba-
cillus actinomycetemcomitans cntwickelt wor-
den und vcrbesserte Methoden zur IdentiTi-
kation dieser Plaquemikroben durch Im-
munofluoreszenz und Fliess-Zytometrie be-
finden sich in der Erprobung.
Was nun die Antwort des Wirtsorganismus
angeht, so scheint cine starkc Bindung
zwischen den Bildungsmustern von Antikor-
pern und parodontalen Krankheitskatego-
rien deutlich zu werdcn. Obwohl sich die mei-
sten Studien auf Serumantikorper aus dcm
pcriphcren Blut konzentrieren, so scheint
eine Verschiebung des Interesses in Richtung
der lokalen Antikorperreaktion wahrschcin-
MacDonald, J. B., Sutton, R. M., Knole, M. L., Madlener, E. M. & Grainger, R. M.
(1956) The pathogenic components of an experimental fusospirocetal infection. Journal of
Infectious Diseases 70, 131-146.
McArthur, W. P., Tsai, C. C , Baehni, P., Shenker, B. J. & Taichman, N. S. (1982) Non
cytolytic effects of actinobacillus actinomycetemcomitans on leukocyte function. In Host-
parasite interactions in periodontal diseases, eds. Genco, R. J. & Mergenhagen, S. E., pp.
179-192. Washington, D. C , American Society of Microbiology.
Mandell, R. L. (1984) A longitudinal microbiological investigation oi actinobacillus actinom-
ycetemcomitans and Eikcnella corrodens in juvenile periodontitis. Infection and Immunity
45, 778-780.
Mergenhagen, S. E., deAraujo, W. C. & Varah, E. (1965) Antibody to Leptotrichia buccalis
in humans sera. Archives of Oral Biology 10, 29-33.
Montgomery, R. E., Singer, R. E., Ryan, L. D , Leedy, D. W, Keough, T. W. & De Stefano,
A. J. (1982) Relation between plaque butyrate production and reversal of gingivitis. Journal
of Dental Research 61, 260.
Moore, W. E. C , Cato, E. P. & Holdeman, L. V. (1981) Butyric acid bacteria in periodontal
disease. Journal of Dental Research (Special Issue A) 60, 414.
Moore, W. E. C , Holdeman, L. V., Cato, E. P , Smibert, R. M., Burmeister, J. A. & Ranney,
R. R. (1983) Bacteriology of moderate (chronic) periodontitis in mature adult humas.
Infection and Immunity 42, 510-515.
Moore, W. E. C , Holdeman, L. V, Cato, E. P, Smibert, R. M., Burmeister, J. A., Palcanis,
K. G. & Ranney, R. R. (1985) Comparative Bacteriology of juvenile periodontitis. Infection
and Immunity 48, 507-519.
Moore, W. E. C , Holdeman, L. V, Smibert, R. M., Hash, D. E., Burmeister, J. A. & Ranney,
R. R. (1982) Bacteriology of severe periodontitis in young adult humans. Infection and
Immunity 3S, 1137-1148.
Mouton, C , Hammond, P. G., Slots, J. & Genco, R. J. (1981) Serum antibodies to oral
Bacteroides asaccharolyticus (Bacteroides gingivalis). Relationship to age and periodontal
disease. Infection and Immunity 31, 182-192.
Mukherjee, S. Crawford, J. M., Chambers, D. A. & Cohen, R. L. (1985) Lactate dehydrogen-
ase activity in crevicular fluid during induced periodonlitis in dogs. Journal of Dental
Research (Special Issue) 64, 374.
Nagahata, T, Kiyoshige, T., Tomono, S., Abe, R., Sasaki, S. & Takazoe, I. (1982) Oral
implantation of Bacteroides asaccharolyticus and Eikenella corrodens in conventional ham-
sters. Infeetion and Immunity 36, 304-309.
Newman, M. G. & Socransky, S. S. (1977) Predominant cultivable microbiota in periodontosis.
Journal of Periodontal Research 12, 120-128.
Niekrash, C. E. (1985) Assessment of complement cleavage in gingival fluid with and without
periodontal disease. Journal of Dental Research (Special Issue A) 64, Abstract #H15, 164.
Offenbacher, S., Farr, D. H. & Goodson, J. M. (I98I) Measurement of Prostaglandin E in
crevicular fluid. Journal of Clinical Periodontology 8, 359-367.
Offenbacher, S., Odel, B. M., Gray, R. C. & VanDyke, T. E. (1984) Crevicular fluid prostaglan-
din E levels as a measure of the periodontal disease status of adult and juvenile patients.
Journal of Periodontal Research 19, 1-13.
Offenbacher, S., Odle, B. & VanDyke, T. E. (1985) Crevicular fluid Prostaglandin E2 levels
predict periodontal attachment loss. Journal of Dental Research (Special Issue A) 64,
Abstract #1797, 374.
Oshrain, R. L., Lamster, I. B., Hartley, L. J. & Gordon, J. M. (1984) Arylsulphalase activity
in human gingival crevicular fluid. Archives of Oral Biology 29, 399-402.
Patters, M. R., Niekrash, C. E. & Lang, N. P. (1985) Assessment of complement cleavage in
gingival fluid during experimental gingivitis in humans. Journal of Dental Research (Special
Issue A) 64, Abstract #1674, 360.
Ranney, R. R., Yamni, N. R., Burmeister, J. A. & Tew, J. G. (1982) Relationship between
attachment loss and precipitating serum antibody to Actinobacillus actinomycetemcomitans
in adolescents and young adults having severe periodontal destruction. Journal of Periodon-
tology 53, 1-7.
Rizzo, A. A. (1967) The possible role of hydrogen sulfide in human periodontal disease. I.
Hydrogen sulfide production in periodontal pockets. Periodontics 5, 233-236.
Robertson, P B., Grupe, H. E., Taylor, R. E., Shyu, K. W. & Fullmer, H. M. (1973) The
effect of collagenase-inhibition complexes on collagenolytic activity of normal and inflamed
gingival tissue. Journal of Oral Pathology 2, 28-32.
Savitt, E. D. & Socransky, S. S. (1984) Distribution of certain subgingival mecrobial species
in selected periodontal conditions. Journal of Periodontal Research 2, 111-123.
Schenkein, H. A. & Genco, R. J. (1977) Gingival fluid and serum in periodontal disease. 11.
Evidence for cleavage of complement composents C3, C3 proactivator (Factor B), and C4
in gingival fluid. Journal of Periodontics 48, 778-784.
Schifter, C. S. & Chilton, N. W (1985) Reproducibility of gingival crevicular fluid. Journal
of Dental Research (Special Issue) 64, 375.
Schiott, C. R. & Loe, H. The origin and variation in number of leucocytes in human saliva.
Journal of Periodontal Research 5, 36-41.

lich. Ausserordentlich empfindliche Messver-
fahren zur Isolicrung des hauptsiichlichen
Immun-Erkcnnungsproteincs, wie z. B. Anti-
korper und Komplement in der krevikularen
Fliissigkcit, sind entwickelt wordcn. For-
schungseinsalze mit dem Ziel lokale Antikor-
pcrreaktion mil lokalcr Krankheitsaktivitat
zu korrcliercn, sind im Kommcn.
Messungcn der Menge des Zahnfleischse-
kretes, von Endoxin, H2S, Butirat und unter-
schiedlichen Enzymen (z. B. KoUagenase,
Arylsulfatase, B-Glukuronidase) korrelieren
gut mit den Gingivitisniveaus. Bei der Paro-
dontitis sind die Prostaglandine der E-Serie,
die Enzyme KoUagenase und Aspartate-Ami-
notransferase, sulfurisierte Glykosamino-
glycane, der die osteoklastisehe Aktivitat ak-
tivierende Faktor und die Knochenresorp-
tionskapazitat der krevikularen Zellen, die
vielversprechendsten Indikatoren der ge-
weblichen Lysis. Die in-vivo Probe der Mi-
gration krevikularer Leukozyten kann als In-
dikator eines Defektes der Resistenz des Wir-
tes aufgefasst werden. Experimentelle
Studien an Tiermodcllen mit histologischen
Korrelaten, sowie prospektive Studien am
Menschen mit standardisierten klinisehen
Korrelaten sind notwendig, um diese objekti-
ven Messungen zu bestatigen.
Resume
Indicateurs de I'activite de la matadieparodon-
tale: evaluation
II apparait de plus en plus nettement que les
critercs cliniques elassiques sont inadequats
pour: (a) determiner les localisations avec ac-
tivite pathologique dans les parodontites, (b)
faire une surveillance quantitative de la re-
ponse a un traitement et (c) mesurer le degre
de susceptibilite a une destruction ulterieure.
Pour chereher a trouver des mesures objecti-
ves, de nombreuses etudes ont etc entrepriscs,
ou on utilisait la salive, le sang, la plaque et
le fluide gingival (GCF) comme prelevement.
L'examen comprenait: (a) des bactcries speci-
fiques et leurs produits; (b) des eellules de
riiote et leurs produits (enzymatiques et anti-
microbiens, tant immunologiques que non
immunologiques); (c) les produits provenant
de lesions aux tissus dans les tissus epithe-
liaux et conjonctifs locaux et dans l'os. Bien
que la plupart des travaux aient jusqu'a pre-
sent cchoue a fournir au clinicicn une aide
fiable, un raffinement dans les techniques d'e-
chantillonnage et le fait qu' on dispose main-
tenant de methodes analytiques perfec-
tionnees permet d'etre optimiste.
Les methodes proposees pour mettre en
evidence les bacteries associees a la maladie
dans la plaque sous-gingivale sont de sensibi-
lite ct de specificite variables. En microscopie
a fond noir, on constate une certaine correla-
tion avec la maladie existant; cependant la
spccifieite limite de cette methode impose de
graves restrictions a son utilite. Des antisera
specifiques monoclonaux et polyclonaux en-
vers Bacteroides gingivalis et Aetinobacillus
actinomycetemcomitans, suspects de patho-
Indicators of periodontal disease 545
Shapiro, L., Lodato, F. M., Courant, P. R. & Stallard, R. E. (1972) Endotoxin determinations
in gingival inflammation. Journal of Periodontology 43, 591-596.
Simon, B., Goldman, H., Reuben, M. & Baker, E. (1969) The role of endotoxin in periodontal
disease. L A reproducible quantitative method for determining the amount of endotoxin
in human gingival exudate. Journal of Periodontology 40, 695-701.
Simon, B., Goldman, H., Reuben, M. & Baker, E. (1970) IL Correlation of the quantity of
endotoxin in human gingival exudate with the clinical degree of inflammation. Journal of
Periodontology 41, 81-86.
Simon, B., Goldman, H., Reuben, M. & Baker, E. (1971) III. Correlation of the quantity of
endotoxin in human gingival exudate with the histologic degree of inflammation. Journal
of Periodontology 42, 210-216.
Singer, R. E., Buckner, B. A., Meckel, A. H. & Leonard, G. J. (1980) Proprionate and
butyrate induction of gingival inflammation in the Beagle. Journal of Dental Research 59
(Special Issue A), 435.
Singer, R. E. & Buckner, B. A. (1981) Butyrate and proprionate: Important components of
toxic dental plaque extracts. Infection and Immunity 32, 458-463.
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546 Fine and Mandel
genicite, ont ete prepares et des methodes
pcrfectionnees d'identification de ces micro-
bes dans la plaque par immunofluorescence
ELISA ct cytometrie du Oux sont en voic de
preparation.
En ce qui concerne la reaction de l'hote,
une forte correlation semble apparaitre entre
les types d'anticorps envers les bacteries spe-
cifiques et les categories de la maladie paro-
dontale. Bien que la plupart des etudes se
soient concentrees sur les anticorps du serum
provenant du sang peripherique, il semble
probable qu'on en vienne a la recherche de
la reponse locale aux anticorps. Des techni-
ques de mesures extremement sensibles ont
ete elaborees pour rechercher les proteines
de reconnaissance immunitaire tells que les
anticorps et le complement dans le fluide gin-
gival. Des efforts de recherche tentant de
trouver une correlation entre la reponse des
anticorps locaux et l'activite locale de la ma-
ladie sont en cours.
Les mesures du taux de flux gingival, de
l'endotoxine, de H2S, de butyrate, et d'une
variete d'enzymes (par exemple: coUagenase,
arylsulfatase, B-glucuronidase) presentent
une bonne correlation avec les niveaux de la
gingivite. Dans la parodontite, les marqueurs
les plus prometteurs pour la constatation de
la destruction des tissus sont les prostaglandi-
nes de la serie E, les enzymes collagenase et
aspartate aminotransferase, glucosaminogly-
cans sulfates, le facteur d'aetivite osteoclasti-
que et la capacite de resorption osseuse des
cellules des silions. Des tests de migration des
leucocytes du sillon in vivo peuvent servir
d'indicateurs de defauts de resistance de
l'hote. Des etudes experimentales sur un mo-
dele animal, avec correlations histologiques
et des etudes humaines avec correlations cli-
niques standardisces sont necessaires pour
confirmer la valeur de ces mesures objectives.
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caries-susceptible adults. Journal of Denial Research 64, 422-424.
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