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Human gingival crevicular fluid contains unidentified proteins which might play a role as markers in periodontal diseases. Therefore, low-molecular-weight proteins found in human gingival crevicular fluid (GCF), but absent from serum, were identified in the present study by means of two-dimensional electrophoresis (2-D PAGE) analysis. GCF, serum, and whole saliva were collected from periodontitis and healthy subjects, as well as from edentulous and newborn subjects. Protein samples were separated by two-dimensional polyacrylamide gel electrophoresis, stained with silver, and compared with reference protein maps in the SWISS-2D PAGE database. In GCF and saliva from periodontitis patients and healthy subjects, four dominant low-molecular-mass (from 8 to 14 kDa) acidic spots were observed. They were not found in serum and were less visible in saliva from edentulous and newborn subjects. From N-terminal amino acid sequencing, the two 2-D protein spots of 8 kDa and isoelectric points between 6.5 and 7.0 were both identified as protein MRP8 (SI00A8), a member of the S100 family of calcium-binding proteins. Using peptide mass fingerprinting and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), we identified the other two protein spots, with mass of 14 kDa and isoelectric points between 5.5 and 6.0, as protein MRP14 (S100A9), also belonging to the S100 family. The presence of MRP8 and MRP14 in GCF was confirmed by Western blot, with monoclonal antibodies. The two polypeptides, MRP8 and MRP14, identified in GCF represent the major difference between the 2-D PAGE patterns of serum and GCF, and we hypothesize that they may play an important role in the gingival sulcus and could represent possible markers for periodontal diseases.

Cigarette smoking is associated with increased incidence of periodontal disease and poor response to therapy. In the present study, we examined the effects of nicotine on several functions of periodontal ligament fibroblasts (PDLF): proliferation, attachment, alkaline phosphatase production and chemotaxis. Nicotine concentrations varying from 5 ng/ml to 250 microg/ml were tested. Proliferation of cells was studied by the incorporation of 3H-thymidine, and a dose-dependent inhibition was observed with concentrations > or =100 ng/ml. Similar results were observed when studying the attachment of the cells on plastic surfaces, using a colorimetric method. The inhibition of attachment was even more evident after 6 h incubation of the cells with nicotine. The activity of alkaline phosphatase, as determined with the substrate p-nitrophenyl phosphate, in both conditioned medium (CM) and cellular extract (CE), was also significantly decreased in a concentration-related fashion. Finally, the chemotaxis of PDLE as examined by a modification of the Boyden's blind-well chamber technique, was inhibited in a dose-dependent manner. The degree of inhibition varied from 15% with the lowest concentration of nicotine (50 ng/ml), to almost 90% with the highest (5 microg/ml). The results show that nicotine can have direct adverse effects on various functions of the periodontal cells.

We describe a method which allows longitudinal measurements of alveolar bone loss in the living rat. The anesthetized animal is kept in a fixed and reproducible position and radiographs of an upper molar arch are taken with a mammography. In a preliminary study, 4 rats were repeatedly radiographed. In the main study, 10 rats were radiographed at day 0 and after 63 days of diet known to cause periodontitis. The radiographs were enlarged and the amount of bone lost calculated by comparing the weights of the interdental areas reproduced on standard paper. In the preliminary study, the coefficient of variation (C.V.) of the average weights of paper were fairly low (4 to 9%) in the first 3 rats, and amounted to 17% in the fourth. A statistically significant average loss of bone (16%) was found in the 10 rats in the main study when comparing the weights of papers at days 0 and 63.

In order to evaluate the contribution of the periodontal sulcus or pocket to the presence of albumin in the mouth, this protein was analysed in whole saliva of 20 completely dentate adults, aged between 63 and 83 years, and in 23 edentulous patients of similar age (51-88 years). In spite of the considerable intra- and interindividual variations revealed by a preliminary trial, the concentration of salivary albumin was significantly higher (range 60-1080 mg/l) in the dentate than in edentulous individuals (range 2-690 mg/l). The low albumin content in saliva of old edentulous people was similar to that in a group of younger individuals with a healthy periodontium.

In order to examine the relationship of possible crevicular biochemical parameters to attachment loss (ALOSS), 330 sites from 8 untreated adult patients were monitored longitudinally at 3-month intervals, for up to 1 year. Attachment levels were measured with a force-sensing probe and an acrylic stent in duplicates at each study point. Crevicular samples were collected and used for the determination of the following 11 markers: number of polymorphonuclear leukocytes (PMNs), prostaglandin E2 (PGE2), osteocalcin (OC), alkaline phosphatase (ALP), collagenase (COL), beta-glucuronidase (BG), antigenic and functional elastase (AEL and FEL), alpha-1 antitrypsin (a1AT), alpha-2 macroglobulin (a2M) and aspartate aminotransferase (AST). 10 sites with ALOSS of > or = 1.5 mm per 3 months (active sites) and 43 sites with negligible changes (inactive sites) were identified. Total amounts of ALP, BG and COL were found to be significantly higher in active as compared to inactive sites, prior to significant ALOSS, without any significant differences in crevicular fluid volume and clinical indices. When biochemical parameters were expressed as ratios to the number of PMNs, PGE2/ PMNs was significantly elevated in active sites. The capacity of such individual parameters to distinguish between active and inactive sites was limited. However, linear discriminant analysis using total amounts of PGE2, COL, ALP, a2M, OC and AEL showed more significant diagnostic values (sensitivity: 80%, specificity: 91%). These findings suggest that the combination of several biochemical parameters in crevicular fluid could give more information to predict future clinical ALOSS.

12 buccal or lingual class II furcation lesions were selected in the lower molars of 7 patients after treatment by routine scaling. In each of the 7 patients, 1 lesion was treated by guided tissue regeneration (GTR) with expanded polytetrafluoroethylene membranes, while 5 neighbouring molars underwent conventional flap operations. The index of gingival inflammation, the plaque index, the depths of the pockets, the horizontal and vertical attachment levels were measured before and 6 months, 1 year and 2 years after surgery. Standardized reproducible radiographs were taken before treatment, post-operatively (at the time of membrane removal for the GTR treated sites), at 6 months, 1 year and 2 years after treatment. The density of bone was quantified in several areas by using high-resolution digital analysis. With the exception of the higher gain in horizontal attachment observed in the GTR treated furcations, the improvement of clinical parameters was similar in all sites during the 2 years of follow-up. The radiographical analysis showed no statistically significant changes neither in the GTR nor in the conventionally treated lesions over this period of time, even when they were compared to untreated bone control areas. However, in this study limited to 7 patients, the interdental bone next to the GTR treated furcations showed significant signs of remodeling.

Samples of gingival crevicular fluid (GCF) were collected in 30 volunteers with inflamed gingiva, using either capillary tubes (cGCF) or Durapore strips (sGCF). They were examined, together with samples of serum from the same patients, by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and/or by two-dimensional electrophoresis, followed by silver staining. The results confirmed that the distribution of the major proteins in GCF is similar to that found in serum. However, an 8.5 kDa protein was found in gingival fluid but not in serum. The low molecular-weight protein appears to decrease with time of fluid sampling with both techniques, and does not originate either from blood or from the cellular fraction of GCF. Two-dimensional electrophoretic analysis suggested that it may consist of several polypeptides.

A 17-year-old male patient with localized juvenile periodontitis was treated by subgingival instrumentation with full thickness flap on the lower molars, combined with a 3-week course of systemic tetracycline, and a programme of supervised oral hygiene. The treatment was rapidly followed by dramatic clinical and microbiological improvement. However, despite good oral hygiene, gingival inflammation recurred at regular intervals. It was necessary to maintain the clinical results by periodic subgingival instrumentation with an ultrasonic scaler. Healing of alveolar bone was monitored in the lower 1st molar regions over 3 years by using superimposable radiographs. Quantitative analysis of bone density performed with a high-resolution digitalisation technique showed a considerable improvement 1 year after therapy. However, continuous remodelling, probably related to variations in inflammation, occurred during the 3 postoperative years.

In periodontal surgery, healing after guided tissue regeneration (GTR) may be explained by differences in functional activities of gingival and periodontal ligament fibroblasts (GF and PDLF). Several studies in vitro have supported this hypothesis, but much remains to be defined. In the present work, gingival and periodontal ligament fibroblasts derived from five healthy subjects were isolated and compared in vitro. The morphology of the cells was observed under scanning electron microscopy (SEM). Several extracellular matrix components (ECM) were studied to compare the effects on fibroblast attachment, proliferation, and protein synthesis. Several biochemical markers were examined in both cellular extract (CE) and conditioned medium (CM). We also examined the muscle differentiation markers alpha-smooth muscle actin, desmin, and smooth-muscle myosin. Finally, we studied the effects of epithelial cells on the proliferation and protein synthesis of the two types of fibroblasts. GF and PDLF appeared identical under the SEM. All ECM components enhanced attachment; however, while collagen types I and IV promoted the attachment of GF, gelatin, laminin, and vitronectin promoted that of PDLF. Most ECM components increased the proliferation rate of GF and the biosynthetic activity of PDLF. The biochemical markers were similarly distributed between the two cell types, except for alkaline phosphatase, which was detected only in the CE of PDLF. Both GF and PDLF strongly expressed alpha-smooth-muscle actin and were negative for desmin; only PDLF were positive for smooth-muscle myosin. Epithelial cells increased the proliferation of both GF and PDLF but had no effect on their biosynthetic activity. These in vitro results may better explain the in vivo functional differences between GF and PDLF.

The reliability of a paralleling instrument for dental radiographs was retrospectively evaluated in the course of a 6-month clinical study. The angular variation between successive exposures was precisely quantified. Ninety-one percent of the angular variations were below a 1.4 degree threshold, but some paired images were found unsuitable for quantitative analysis. A mathematic procedure to assess the global angular projection error of any similar coupled x-ray cone-film holder device is proposed. This mathematic procedure should be routinely used to check superimposability of serial radiographs before density quantification or subtraction.

The peripheral distribution of spiramycin and metronidazole, which are combined in the proprietary drug "Rodogyl", has been studied in gingival fluid, saliva and blood after a single administration to 12 healthy volunteers and after repeated administration to 4 patients with recurring severe periodontitis. Analysis of the 2 antibiotics have been performed at regular intervals during the 24-h period immediately following the administration to the volunteers and after the 1st and the 15th days of repeated administration to the patients. The results show that gingival fluid contains concentrations of spiramycin and metronidazole higher than those needed to inhibit the growth of periodontopathic bacteria. Spiramycin was found at higher concentrations in GCF than in blood, although this feature was not found for metronidazole, which was administered simultaneously and showed similar concentrations in both fluid and serum. Such high concentrations persist for a long time, and suggest the potential of this compound in the treatment of severe cases of periodontitis.

The activity of elastase increases significantly in the gingival sulcus during inflammation. The release of this enzyme from crevicular and peripheral polymorphonuclear leucocytes (PMNs) was studied in seven patients with gingivitis, six with adult periodontitis, seven with rapidly progressive periodontitis and in nine healthy subjects. Peripheral PMNs were isolated from blood and crevicular PMNs from gingival washings. After preincubation with cytochalasin B, the same numbers of crevicular and peripheral cells were incubated either in phosphate-buffered saline (spontaneous release) or in the same buffer containing formyl methionyl-leucyl-phenyl-alanine (stimulated release). Elastase activity was measured in the supernatant by a fluorimetric technique. The results confirm that compared to peripheral PMNs, crevicular cells show a higher spontaneous release of elastase and a lower stimulated release. The activity of elastase released either spontaneously or after stimulation of crevicular cells was significantly lower in the controls as compared with cells obtained from patients with gingivitis or periodontitis. Crevicular PMNs collected from patients with deeper pockets appear to release larger amounts of elastase than those from controls.

The present investigation was designed to assess the effects of strips made of different materials on the recovery of enzymes in gingival crevicular fluid (GCF) (Experiment 1), and to examine a possible relationship between lysosomal enzyme activities and number of crevicular polymorphonuclear leukocytes (PMNs) (Experiment 2). In Experiment 1, GCF was collected with capillaries from 14 patients with periodontal disease, and applied on various test strips in microcentrifuged tubes or directly into tubes (controls). Strips were then shaken and centrifuged to elute GCF enzymes. The supernate was used for determinations of alkaline phosphatase (ALP), beta-glucuronidase (BG) and aspartate aminotransferase (AST) activities. The % recoveries were calculated as relative percentages to controls. When using saline as eluent, 90% or more ALP, BG and AST were found to be recovered from strips of durapore and papers. More than 35% of ALP and BG was found to remain on paper points. However, the % recoveries from paper points were improved using eluent with 0.1% (w/v) cetyl pyridinium chloride. In Experiment 2, 54 GCF samples were collected from 3 periodontitis patients by using durapore strips, in order to measure both PMN numbers and BG activities in the same samples. The 2 parameters showed strong and positive correlation with 0.847 (p < 0.001) of the Pearson's correlation coefficient. These findings suggest that durapore is a useful material not only for counting PMNs but also for measuring activities of GCF enzymes. Elevation of BG activities in GCF can be due to increasing numbers of PMNs.

Prostaglandin E2 (PGE2) and alkaline phosphatase (ALP) have often been measured in gingival crevicular fluid (GCF) as possible indicators of gingival inflammation and bone metabolism. Osteocalcin (OC), a major component of bone matrix, is mainly produced by osteoblasts, and could also be considered as a marker of bone turnover. The aims of this preliminary study were to examine if OC was present in GCF and to assess the relationships of OC, PGE2 and ALP in GCF to periodontal conditions. GCF samples were collected with durapore strips from 34 healthy, 72 gingivitis and 118 periodontitis sites in 17 subjects. ELISA techniques were used for the determinations of OC and PGE2. ALP was measured spectrophotometrically by using p-nitrophenyl phosphate as substrate. Total amounts and concentrations of PGE2 and ALP were significantly higher in periodontitis as compared to healthy and gingivitis sites, and were significantly and positively correlated with probing depth (PD) and gingival index (GI). OC was present in GCF from both healthy and diseased sites with mean concentrations more than ten times greater than normal serum levels. Total OC amounts from strips soaked with GCF from periodontitis sites were significantly higher than those found in healthy and gingivitis sites. When the data were expressed as concentrations, OC showed significantly positive correlations with GI, but not with PD. However, total amounts of OC significantly correlated with both clinical parameters. OC, PGE2 and ALP were found to have significantly positive correlations with each other, both when expressed as total amounts and concentrations. These data suggest that a significant amount of OC present in GCF is produced locally by periodontal tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

The frequency of gingival overgrowth is increasing, due to the more and more frequent use of cyclosporin and calcium antagonists. Such lesions are the same as those known to occur in patients treated with phenytoin. They appear after a few months of treatment and are favoured by inflammation. Marginal gingiva starts to grow in excess, especially in the anterior region, and might end up covering the teeth entirely, with major occlusal problems. The hyperplasia occurs in about 50% of the phenytoin treated patients and in about 20% of those treated with cyclosporin or calcium antagonists. A genetic predisposition has been thought to be present in such susceptible people. However, no precise pathogenic pathway, possibly common for the three types of drugs, has been clearly proposed. As for therapy, the most severe lesions are treated by surgery but, in general, the dentist can be very helpful by simply stressing gingival hygiene.

The techniques presently available for counting crevicular polymorphonuclear leucocytes (CPMNs) require special equipment and are only semi-quantitative. The present new and simple method is based on the property that Durapore strips have to harvest a maximum number of crevicular cells and to release them when shaken in a suitable buffer. Durapore strips were inserted into periodontal crevices or pockets, shaken in PBS and the liberated PMNs counted in a Neubauer chamber. The validity of the method was ascertained in 5 experiments. First, it was shown that 90% of the harvested cells were released from Durapore strips after being shaken in buffer. Second, decreasing numbers of CPMNs were found when performing sequential samplings. Third, when counting CPMNs before and after therapy, a significant decrease in the number of cells was found. Fourth, we could confirm that sites with bleeding on probing did contain a much higher number of CPMNs as compared to non-bleeding sites. Finally, our counting technique confirmed that deeper pockets contain a significantly higher number of CPMNs.

Many antibiotics are utilized in dentistry, particularly in periodontology, to treat aggressive forms of periodontitis. In order to be efficient, both on a clinical and on a microbiological point of view, these substances must be present in sufficient concentrations in the gingival sulcus. The aim of the present literature review was to summarize studies concerning the passage of the various families of antibiotics into the gingival fluid, together with their spectrum and clinical efficacy. Whereas certain antibiotics, such as natural or semisynthetic tetracyclines or spiramycin, are able to accumulate in gingival fluid comparatively to serum, others, like metronidazole, tinidazole and rifampicin, have been found in similar concentrations in the fluid as in serum. Some other antibiotics, such as erythromycin and ampicillin, are, on the contrary, less concentrated in crevicular fluid as compared to blood. One has to remember that any systemic antibiotic treatment will be useful, clinically and microbiologically speaking, only when a mechanical therapy is also applied.

Human gingival keratinocytes were cultured and, after the first passage, subjected to cell detachment assays with polymorphonuclear leucocytes (PMNs) and/or sonic extracts from Actinobacillus actinomycetemcomitans Y4, and Eikenella corrodens 1073. The effector-to-target cell ratio was 30:1. Bacterial extracts alone caused no disruption of keratinocyte monolayers. PMNs alone also caused only minimal detachment after 14 h incubation. Adding A. actinomycetemcomitans to the PMN-keratinocyte co-cultures at the concentration of 100 micrograms/ml caused dramatic cell detachment. The effect of A. actinomycetemcomitans was heat labile and not inhibited by polymyxin B. Cell detachment was inhibited by alpha 1-antitrypsin, whereas catalase and superoxide dismutase could not prevent it. No lysis of keratinocytes was observed after incubation, as judged by 51Cr release. E. corrodens had little effect even at the concentration of 1000 micrograms/ml. H2O2 and partially purified PMN elastase also caused detachment of keratinocytes. These data indicate that PMNs can cause non-lytic detachment of keratinocytes when interacting with certain bacteria.

The relative concentrations and absolute amounts of neutrophil elastase and its two inhibitors, alpha 2-macroglobulin (alpha 2-M) and alpha 1-antitrypsin (alpha 1-AT), were determined in gingival crevicular fluid (GCF) collected from six dental students who refrained from brushing the upper left or right quadrant during three weeks. Plaque and gingival indices and flow of GCF were measured before, during, and after the three weeks of no brushing. Functional elastase, representing the enzyme complexed with alpha 2-M, was measured by use of a low-molecular-weight fluorogenic substrate. Elastolytic activity in GCF was also assayed by use of elastin as substrate. Antigenic elastase, representing the enzyme complexed with alpha 1-AT, as well as the inhibitors alpha 2-M and alpha 1-AT were measured by ELISA. After three weeks of plaque accumulation, the concentrations of both functional and antigenic elastase increased by a factor of about 3, whereas the concentrations of the inhibitors increased in a much higher proportion. No free elastase could be detected in GCF, as evidenced by the Sephadex G-75 elution profile of GCF, by the negative results obtained when elastin was used as substrate, and by the demonstration that pure enzyme kept its activity against the low-molecular-weight substrate after being saturated by alpha 2-M.

Photodensitometry is known to provide high spatial resolution and continuous measurement of optical density for the analysis of dental radiographs, whereas digitization allows powerful image manipulations but, when using conventional video cameras, gives less spatial resolution and fewer grey levels. The aim of this study was therefore to develop a technique of high-resolution digital analysis for the measurement of bone density following the same principles as those of photodensitometry and based upon the use of a CCD Scanner Camera which provides up to 4096 grey levels and a spatial resolution of 4096 x 4096 pixels. Twenty-four zones were analysed with both techniques in five serial dental radiographs taken before and after periodontal therapy in eight patients. Statistical comparison of the results obtained by digital analysis and photodensitometry shows that the two techniques have the same accuracy.

Dentin hypersensitivity is a painful manifestation resulting from a response of the openings of dentin tubules to oral environment. It is probably caused by movements of intratubular fluids, under the influence of different types of stimuli at the dentin surface. Hypersensitivity has been known for a long time and its treatment has always been difficult. Sometimes, by a kind of natural reaction to aggression, dentin is able to increase its degree of mineralisation but, most of the time, an active treatment has to be performed. Unfortunately the mechanism of dentinal pain is not well known and the symptoms of sensitivity are unspecific and subjective, so that the ideal compound for treatment does not exist. Many products have been tested, but the results are most of the time contradictory and the placebo effect is often very significant. In all cases the aim of the treatment remains the obliteration of dentin tubules, in order to obtain a decrease of dentin permeability.

Peripheral PMNs were collected from blood, and crevicular PMNs separated by filtration from gingival washings in 13 patients, aged 22-75 y, who had varying degrees of gingivitis and periodontitis. After pre-incubation with cytochalasin B, the same number of crevicular and peripheral cells were incubated either in PBS (with Ca2+ and Mg2+) (spontaneous release) or in the same buffer containing increasing concentrations of FMLP (stimulated release); elastase activity was measured in the supernatant by a fluorometric technique. There was a higher spontaneous release of enzyme from crevicular than from peripheral neutrophils. The average elastase activity in the supernatant of 1 x 10(4) crevicular cells was more than five times higher than that obtained from the same number of peripheral cells. However, stimulated crevicular PMNs liberated smaller amounts of enzyme than did stimulated peripheral PMNs. These results suggest that crevicular PMNs are already releasing elastase, and are consistent with the possibility that lysosomal enzymes contribute to tissue damage during gingivitis and periodontitis.

Ten interproximal sites, with periodontal pockets deeper than 5 mm and showing loss of bone on standard dental radiographs, were treated by subgingival instrumentation in 10 patients properly motivated and given thorough hygiene instructions. The index of gingival inflammation, the plaque index, the degree of mobility, the depth of the pockets, and the loss of attachment were measured before starting the treatment, and 2 months, 6 months, and 1 year after treatment. Standardized reproducible radiographs of the 10 sites were taken by using a recently developed paralleling instrument before treatment, immediately after and 2 months, 6 months, and 1 year afterwards. The density of the interdental bone was measured on the radiographs by a computer assisted densitometric technique at three levels of the interdental septum: the most occlusal or "superficial" level; the "deep" level, arbitrarily chosen 1.5 mm below; and the "control" level, in a much deeper area of interproximal bone. As further control, the density of the superficial crestal bone was also followed in 5 healthy untreated sites from 5 of the patients. As expected, a significant improvement of the clinical parameters was observed during the year following therapy. The results of the radiographical analysis showed statistically significant increases of both the superficial and deep average densities of interproximal bone at 6 months and 1 year after treatment. The superficial bone density was, on the average, 13% higher at 6 months and 16% higher at 1 year, as compared to that measured immediately after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumin and alpha 2-macroglobulin were analysed in gingival fluid collected by filter paper strips from 6 volunteers before, during and after 10 days of dental plaque accumulation. The two proteins were also determined in samples of venous blood at the beginning of abstention from tooth brushing and 10 days later. A nephelometric technique was used for the analysis of albumin, but alpha 2-macroglobulin was determined by ELISA. The plaque and gingival indices, as well as the flow of gingival fluid, increased significantly during the period of plaque accumulation and then returned to baseline values. The concentrations of the two proteins in blood remained within normal limits. In gingival fluid, however, the concentration of albumin increased about two-fold at the peak of inflammation, while that of alpha 2-macroglobulin increased as much as 6 times. The enhanced passage of alpha 2-macroglobulin into the gingival sulcus in comparison to the entirely plasma-derived albumin was consistent with the concept that the greater excretion of globulin during inflammation must be due to an increased local synthesis.

In a group of 5 women taking oral contraceptives, the concentration of polymorphonuclear leucocytes along the gingival margin and the activities of cathepsin D, beta-glucuronidase and elastase were found to increase significantly during the intermenstrual phases.

A simple, disposable paralleling device to produce identical radiographs of the maxillary and mandibular molar regions is described. Its precision was determined by taking serial radiographs of several patients. No deformations were found when enlargements of the radiographs were made. Identical tracings were produced during analysis with a computer-assisted densitometric technique. The new device is inexpensive and could be used advantageously for qualitative and quantitative longitudinal studies.

Small samples of hydroxyapatite (HA) beads were coated with 10 microL of either saliva, serum, human crevicular fluid, or a preparation of lysosomal enzymes from human polymorphonuclear leucocytes (PMN), before being added to suspensions of Treponema denticola. The beads were then observed with the scanning electron microscope. Abundant T. denticola were found to adhere to HA or HA coated with saliva, serum, or crevicular fluid. Coating the beads with lysosomal enzymes consistently prevented the adhesion of T. denticola. Adhesion of treponemes to hard surfaces, modulated by enzymes from PMNs, could represent a new virulence factor for these bacteria.

In the course of the extensive tissue remodeling occurring after flap surgery and during the subsequent gingival reattachment in man, numerous intracellular vacuoles were observed in fibroblasts of the lamina propria. These vacuoles contained one or several typical collagen fibrils and had either an electron-lucent matrix or contained an electron-dense material obscuring the fibrillar outlines and cross-striations.

Two sets of radiographs, taken at an interval of 4-14 years in 105 patients of a dental school, were examined in order to determine the rate of resorption of alveolar bone. The percentage loss of maximum alveolar bone height was measured in the interproximal and interradicular sites by means of the Schei ruler. The average percentage of interproximal bone resorption was 0.51% (0.07 mm) per year. Age, sex, professional status, type of periodontal treatment and general state of health did not seem to affect the annual resorption rate. However, crowned teeth had a significantly greater rate of resorption than uncrowned teeth. Loss of bone tended to be more rapid in interdental as compared to interradicular areas. A positive and statistically significant correlation was found between initial resorption and annual tooth loss.

Free gingival grafts were placed in the lower anterior vestibular region of 30 patients each of whom had a gingival recession. According to the width of the recession and to the surgical procedure, the patients were divided into four groups. Group 1 showed a "narrow" (less than 3 mm) type of recession, while Group 2 had a "wide" type. In both of these groups the graft was placed over the denuded root. In Group 3 (narrow recession) and Group 4 (wide), a collar of vestibular gingiva was left in place on the lower margin of the denuded root at the moment of grafting. Measurements and photographs taken before and 1 month after surgery, 6 months and 2 years postoperatively, showed varying degrees of root covering by the phenomenon of creeping attachment. An optimal recovery of the gingival profile was obtained in the three patients of Group 1. In the other categories of patients the creeping of the attachment apparatus was relatively small (between 0 and 33%) within the 2 years of observation. The factors which seem to have a definite influence upon the phenomenon of creeping attachment are the following: width of the recession, position of the graft, bone resorption, position of the tooth and hygiene of the patient.

Superposable radiographs of lower molar areas were obtained by means of a specially designed film holder and a gnathostat maintained in a constant position with respect to long-cone radiological equipement. A quantitative evaluation of the bone mass in the interradicular area was obtained by scanning the area under a photodensitometer. A photodensitometric scan of the image of an aluminium wedge adapted to each radiograph was also performed. This allowed the transformation, with the aid of a computer, of the photodensitometric recording of bone into a tracing of mm of aluminium equivalents. The reproducibility and precision of the method were verified by comparing magnifications of repeated radiographs, by photodensitometric readings of radiographs developed in different batches and by repeated quantitative readings of the same film. The method allowed one, for instance, to follow quantitatively the healing of a radiographically visible periapical lesion. It was also used to evaluate quantitatively, in the interradicular area, bone changes which could not be seen with the marked eye.

Most of the previous histological studies devoted to free gingival grafts in humans were limited to the description of the various phases of healing between the graft and the subjacent tissues. The present long-term study illustrates the histological findings 1 to 36 months after grafting along the zone of passage between graft and surrounding tissues. In the transitional zone between graft and alveolar mucosa it was found that the structural characteristics of the palatal mucosa, with its dense network of collagen fibres and its orthokeratinized superficial layer were conserved even after an interval of time as long as 16 months. These features contrasted with the loose network of collagen fibres and the presence of numerous elastic fibres in the alveolar mucosa. As for the transitional zone between graft and gingiva the findings confirmed that these two types of mucosa are similar, except that the gingival epithelium showed, in epon embedded sections, a layer of parakeratinization, as opposed to the dense layer of orthokeratin found on the grafted palatal epithelium. A constant feature in the present investigation was the change in orientation of the collagen fibres along the zones of healing, both between graft and alveolar mucosa and between graft and gingiva.

A case of chronic neutropenia in a 12-year-old boy is reported. The patient presented with severe gingival inflammation and alveolar bone loss. Immunologic analysis of the patient's serum revealed the presence of precipitating antibodies against antigenic components of Actinobacillus actinomycetemcomitans Y4 and 652. It was also found that the serum neutralized the leukotoxic activity of Actinobacillus actinomycetemcomitans Y4. The etiology and the pathogenesis of periodontal disease in neutropenic patients are discussed in view of these findings.

The concentration of alpha 2-macroglobulin (alpha 2-M) in human gingival sulci has been investigated in two studies: first, in gingival washings during a 21-day period of experimental gingivitis in eight human volunteers and, second, in crevicular fluid collected with filter paper strips before and after initial periodontal therapy in 11 patients. The concentration of total alpha 2-M was found to increase in the washings of the volunteers throughout the period of experimental gingivitis. In the group of patients receiving periodontal therapy, the absolute amount of alpha 2-M in the fluid showed a significant decrease after therapy. The gingival index of inflammation and the crevicular fluid flow also decreased significantly. The specific content of the inhibitor (micrograms of alpha 2-M per mg of fluid per min), however, was found to increase in the fluid with decreasing inflammation. As detected by crossed immunoelectrophoresis, the fluid collected in these patients before therapy, in the presence of severe inflammation, invariably showed peaks of both free and complexed alpha 2-M. In contrast, the fluid collected from the same sites after healing of the inflammation contained no detectable free alpha 2-M.