GB2267753A - Test for periodontitis and susceptibility thereto - Google Patents

Abstract

Periodontitis and susceptibility thereto is tested by measuring the change in morphology of a sample of polymorphonuclear leukocytes (PMNs) from the patient at a predetermined time after incubation thereof with supernatant from a virulent strain of Porphyromonas gingivalis and comparing the result with that from a sample of PMNs from a healthy patient. Since loss of polarisation corresponds to a loss of actin which is bound by phalloidin, the change can be measured by observing a decrease in fluorescence of fluorescently labelled phalloidin in the sample. In serious cases there is a complete loss of fluorescence. Phalloidin may be labelled with Fluorescein isothiocyanate or by conjugation to biotin which is bound to enzyme-labelled avidin or streptavidin. The latter label may be determined by enzyme-linked immunosorbent assay (ELISA). A kit containing a virulent strain of P. gingivalis and labelled phalloidin or components which when reacted will give rise to labelled phalloidin is also disclosed.

Description

TEST FOR PERIODONTITIS AND SUSCEPTIBILITY THERETO Backaround of the invention 1. Field of the invention This invention relates to diagnosis of periodontal disease (periodontitis), a serious gum disease estimated to affect 10% of the population. The invention aims to diagnose susceptibility to the disease, thereby giving an early warning to patients most at risk.

2. Description of the related art So far as is known, there is no known way of predicting susceptibility to periodontal disease. Prior art, the relevance of which becomes apparent only with knowledge of the invention, is referred to below.

Summary of the invention It is known that polymorphonuclear leukocytes (PMNs) change shape (become non-polarised) when these cells are exposed to products from virulent strains of Porphvromonas aingivalis, a bacterium implicated in destructive periodontitis. This morphological change is manifested by an increase in large non-polar cells of diameter at least 18 micrometres and a decrease in polarised and of small non-polar cells less than 18 micrometres in diameter. In simple terms, the cells become large and flattened. See M. A. Scragg, L. R. Turton and D. M.

Williams, Archives of Oral Biology 35, 167-173 (1990). The effect is less pronounced when the strain of P. ainaivalis is a variant of reduced pathogenicity : M. A. Scragg, L. R. Turton and D. M. Williams, Archives of Oral Biology 36, 709-714 (1991).

The change of shape has been correlated with alterations in the polymerisation of globular actin present in the PMNs to filamentous actin (F-actin). See e.g. T. D. Coates et al., J.

Cell Biology 117 (4), 765-774 (May 1992).

It has now surprisingly been found that the interaction between PMNs and virulent strains of P. ainaivalis can be used to test for the susceptibility of dental patients to periodontal disease. The PMNs of patients suffering from periodontal disease exhibit a much more marked shape change than those from non-sufferers and since this phenomenon is detectable, especially by a known binding reaction of phalloidin to F-actin, it forms the basis of an assay. In view of the involvement of a non-localised cell, the test is presumed to reflect host response to the disease, i.e. to be a measure of the capability of the individual's immune and inflammatory systems to resist the disease, and therefore to be a test for susceptibility.As the change in shape of the PMNs to large non-polarised forms is correlatable with loss of F-actin and therefore a reduction in the observed binding of phalloidin to F-actin, which can be measured fluorescently (or in other ways), the test can be practised by measuring loss of phalloidin binding over a pre-determined time.

The change in shape of PMNs has to be determined at an appropriate time interval since, in the end, both affected patients and controls will show a loss of shape of PMNs as will be explained below by reference to the drawing.

According to the invention, therefore, there is provided a method of testing for periodontitis or a susceptibility thereto, the method comprising measuring the change in morphology of polymorphonuclear leukocytes of a sample taken from a dental patient, after they have been caused to interact with supernatant from a culture of a periodontitis-associated strain of Porphvromonas ai nai val i s or of a periodontitis-associated component of said culture, and for a time period sufficient to reveal a difference in morphological change of polymorphonuclear leukocytes between patients suffering from or susceptible to periodontitis and patients free from periodontitis.

A kit for carrying out the test of the invention comprises (consists of or includes) (1) supernatant from a culture of P.

aingivalis or of a component of said culture, as defined above, conveniently supplied in a freeze-dried form, and (2) labelled phalloidin or components which when interacted will give rise to labelled phalloidin.

Brief description of the drawings Figure 1 illustrates schematically the changes in morphology of PMNs which occur when they have lost polarity through incubation with virulent P. gingivalis.

Figure 2 illustrates the decrease in fluorescence the PMNs incubated with P. gingivalis with respect to time, for samples of PMNs from a patient suffering from periodontitis and a healthy patient.

Description of the preferred embodiments In a preferred embodiment, the morphological change is determined by loss of F-actin, the presence of which is detectable by binding of phalloidin thereto. Typically, the phalloidin is labelled and the amount of bound label determined. Most preferably, the F-actin phalloidin binding is measured by conjugating the phalloidin to the residue of a molecule which fluoresces when bound to phalloidin, e.g.

fluorescein isothiocyanate (FITC). Figure 1 represents schematically highly fluorescent (+++) bipolar PMN cells, predominating in the cell population, at the start of the assay (before addition of the sample). After the exposure to the sample containing the P. aingivalis supernatant, the number of small non-polarised cells (SNP), showing only patchy fluorescence (+), and the non-fluorescent (0) large non-polar cells increase.

Alternatively, the phalloidin is conjugated to biotin or a bindable derivative thereof and the presence of phalloidin is then detected by use of labelled, preferably enzyme-labelled, avidin or streptavidin.

Other ways of detecting bound phalloidin will be apparent to those skilled in the art, including direct conjugation of an enzyme thereto.

In the test of the invention, the change in morphology must be measured after a suitable period of time, because it is the rate of change of shape of cells which is critical in this invention. Accordingly, the PMN cells are preferably fixed, e.g. by a treatment with formaldehyde or paraformaldehyde in manner known per se, after a pre-determined time interval which, for the majority of patient samples, will maximise the observed difference in morphology. Simple incubation with the supernatant at about 37"C in a humidified atmosphere for 15 minutes, washing for 15 minutes and then fixing has been found particularly suitable. The change to a non-polarised form begins during incubation and continues during washing.The time of incubation may be varied considerably, especially between 1 minute and 1 hour, most preferably between 10 and 30 minutes, and the time of washing can also be varied depending on the time of incubation, so that the overall time taken to allow the morphological changes to take place will range from 10 minutes to 2 hours. Thus the time of washing might typically be from 30 seconds to 90 minutes, preferably from 5 to 30 minutes. Figure 2 of the drawings is a schematic diagram representing the time course of F-actin disappearance from PMNs in healthy and diseased subjects following exposure to P. ainaivalis supernatant. Healthy subjects are represented by the upper, dotted line, diseased subjects or disease-susceptible subjects by the lower, continuous line. Time X represents a time point, determined by observation, at which the difference in F-actin levels in the PMNs of healthy and susceptible subjects is maximal. This would allow discrimination between healthy and disease susceptible subjects. This time is dependent firstly on the time of incubation of PMNs with the P. ainaivalis supernatant and secondly on the time allowed for washing.

The temperature of incubation in the supernatant can be varied from 4 to 40"C but will usually be in the range 2 to 38"C. Washing is conveniently done at room temperature, but other temperatures may be suitable so long as the cells remain viable. Conveniently the cells are attached to a surface but they could be in a suspension in the supernatant and subsequently in the washing solution. This would enable washing by centrifuging.

In an alternative embodiment, the cells can be treated first with the P. gingivalis supernatant and then with a chemotactic agent such as FMLP, N-formyl-methionyl-leucylphenylalanine.

Cells will become large non-polar when treated with P.

gingivalis supernatant and rapid polymerisation of F-actin will not occur subsequently. This treatment with a chemotactic agent may enhance the discrimination between levels of fluorescence in healthy and diseased subjects depicted in Figure 2.

The strain of e. gingivalis used in this invention must be virulent (pathogenic) in the sense that it is or corresponds to an isolate from a patient suffering from severe periodontal disease. A number of strains are suggested in the Examples herein, but the invention does not require any one particular strain for its performance.

Although the invention has been described with reference to a culture supernatant of P. gingivalis, it is only a matter of time before the component thereof responsible for the effect is isolated from P. ginoivalis and purified. Thereafter, it may confidently be expected that the binding site of the said protein will be identified, enabling a synthetic compound to be used in its place. The invention includes such a synthetic compound within the above terminology "component of said culture".

Preferably the kit described above also contains at least one material useful as a calibration standard, e.g. a sterile culture medium. Any other appropriate components can also be present, including those components known to be useful in the particular assay method selected.

The following Examples illustrate the invention.

Example 1 Bacteroides ainaivalis strain W50 has been described by H. N. Shah and M. D. Collins, J. Systematic Bacteriology 38, 128-131 (1988) as similar to American Type Culture Collection (ATCC) strain 33277. W50 is available from the inventor at the Department of Oral Microbiology, London Hospital Medical College, Turner St., London El 2AD. It has been widely distributed. It is also believed similar to Slot's Strain 2561. W50 was grown for 3-6 days and culture supernatant taken, as described by M. A. Scragg et al. (1990) supra for strain W83, with one exception: the BM culture medium method used by H. N. Shah et al., J. Appl. Bact. 41, 473-492 (1976) was varied by adding the haemin/menadione solution before autoclaving.

Blood was taken from two groups of patients, a test group of 10 aged 16-54 suffering from advanced periodontal disease and a control group of 11 healthy subjects aged 22-33. These samples were used to prepare PMNs as described by M. A. Scragg et al.

(1990) supra.

The PMNs, adherent to glass coverslips, were incubated with the culture supernatant for 15 minutes, washed for 15 minutes and fixed as described by M. A. Scragg et al. (1990) supra.

The PMNs were then studied under the microscope and classified as described by M. A. Scragg et al. (1990) supra.

Decrease in P. ginaivalis supernatant on the PMNs causes a change in the bipolar (BP) cells, resulting in small non-polarised (SNP) cells and then large non-polarised (LNP) cells. The reduction in number of bipolar cells and increase in the number of large non-polar cells were measured. The definition of these cells is given by the M. A. Scragg et al.

(1990) paper supra. As shown in the Table below, the PMNs of the diseased group showed significantly reduced polarisation on exposure to the P. ainaivalis supernatant compared with the controls, with an associated increase in large, non-polar cells.

Table Control Group Diseased Group Patient % Bipolar % Large Patient % Bipolar % Large No. Cells Non-polar No. Cells Non-polar Cells Cells 1 10.5 77.5 12 5.0 84.1 2 9.0 76.3 13 6.6 55.1 3 25.4 21.7 14 9.2 61.2 4 41.8 23.9 15 1.9* 93.5 5 18.7 52.4 16 8.4 63.5 6 15.7 55.7 17 8.4 58.5 7 12.6 64.6 18 15.9 41.8 8 26.1 20.2 19 10.0 62.7 9 23.0 53.1 20 13.3 57.2 10 30.8 46.5 21 5.7 64.5 11 40.2 46.1 Average 23.1 48.9 8.4 64.2 SD 11.2 20.2 4.1 14.7 SE 3.4 6.1 1.3 4.6 n = 11 n = 10 * This patient was aged 54, significantly above the average age of the group.

Example 2 In this Example P. gingivalis strain W83 was used in place of W50. It is available from the same source and has also been widely distributed.

The procedure for obtaining P. ainaivalis culture supernatant and PMNs and incubating them was the same as in Example 1, except that in some instances the P. ginoivalis was grown in only 20 ml of the BM medium. The cells were fixed for 5 min in phosphate buffered saline (PBS), pH 7.4, containing 3.7 per cent paraformaldehyde. This step is critical to allow the next step to work, but would also allow preparations to be stored for batch processing if desired.

A 3.3M methanolic solution of F-phalloidin, i.e. linked to fluorescein isothiocyanate, from Sigma Chemical Co. Ltd., diluted 1:25 with PBS, was added and the cells allowed to stand for 90 min, washed extensively with several changes of PBS for 30 minutes and then mounted in "Immunomount" and viewed under ultra-violet light The fluorescent signal was evaluated by eye using fluorescence microscopy (but spectrofluorometry could be used instead).

The former is quick, but the latter is more objective, and could be adapted for either automated batch processing or for single samples. Preparations from diseased subjects fluoresced less intensely than those from healthy subjects or not at all.

The following claims define some important aspects of the invention, but do not purport to include every conceivable aspect for which protection might be sought in subsequent continuing and foreign patent applications, and should not be construed as detracting from the generality of the inventive concepts hereinbefore described.

Claims (10)

1. A method of testing for periodontitis or a susceptibility thereto, the method comprising measuring the change in morphology of polymorphonuclear leukocytes of a sample taken from a dental patient, after they have been caused to interact with supernatant from a culture of a periodontitis-associated strain of Porphvromonas ainaivalis or of a periodontitisassociated component of said culture, for a time period sufficient to reveal a difference in morphological change of polymorphonuclear leukocytes between patients suffering from or susceptible to periodontitis and patients free from periodontiti s.

2. A method according to claim 1 wherein the morphological change is measured as reduction of F-actin content in the polymorphonuclear leukocytes.

3. A method according to claim 2 wherein the reduction of F-actin content is measured by the binding of F-actin to phalloidin.

4. A method according to claim 3 wherein the actin-phalloidin binding is measured by labelling the phalloidin and determining the amount of bound label.

5. A method according to claim 4 wherein the phalloidin is conjugated to the residue of a molecule which fluoresces when bound to phalloidin.

6. A method according to claim 5 wherein the molecule is fluorescein isothiocyanate.

7. A method according to claim 4 wherein the phalloidin is labelled by conjugation to biotin which is bound to enzymelabelled avidin or streptavidin.

8. A method according to claim 7 wherein the determination of label is by enzyme-linked immunosorbent assay (ELISA).

9. A kit for testing for periodontitis or susceptibility thereto, comprising: (1) a strain of P. ainaivalis as defined in claim 1, and (2) labelled phalloidin or components which when interacted will give rise to labelled phalloidin.

10. A kit according to claim 9 wherein component (2) consists of (a) phalloidin conjugated to the residue of a molecule which fluoresces when bound thereto or (b) phalloidin conjugated to biotin and, separately therefrom, enzyme-labelled avidin or streptavidin.