WO2017186571A1 - Biomarker for periodontal disease and peri-implantitis - Google Patents

Biomarker for periodontal disease and peri-implantitis Download PDF

Info

Publication number
WO2017186571A1
WO2017186571A1 PCT/EP2017/059415 EP2017059415W WO2017186571A1 WO 2017186571 A1 WO2017186571 A1 WO 2017186571A1 EP 2017059415 W EP2017059415 W EP 2017059415W WO 2017186571 A1 WO2017186571 A1 WO 2017186571A1
Authority
WO
WIPO (PCT)
Prior art keywords
lps
subgingival
periodontal
periodontal disease
activity
Prior art date
Application number
PCT/EP2017/059415
Other languages
French (fr)
Inventor
Svetislav ZARIC
Original Assignee
University Of Plymouth
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Plymouth filed Critical University Of Plymouth
Publication of WO2017186571A1 publication Critical patent/WO2017186571A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/004—Enzyme electrodes mediator-assisted
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56955—Bacteria involved in periodontal diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/579—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving limulus lysate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/50—Lipopolysaccharides; LPS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/18—Dental and oral disorders

Definitions

  • Periodontal diseases can affect one or more of the periodontal tissues/ structures (e.g. alveolar bone, periodontal ligament, cementum and gingiva). While there are many different periodontal diseases that can affect these tooth-supporting tissues/structures, by far the most common ones are plaque-induced inflammatory conditions, such as gingivitis and periodontitis. Often the term periodontal disease or gum disease is used as a synonym for periodontitis, specifically chronic periodontitis. Periodontal disease ranges from the mildest stage, known as gingivitis, to severe stage, known, as periodontitis. While in some sites or individuals, gingivitis never progresses to periodontitis, data indicate that periodontitis is always preceded by gingivitis.
  • Periodontitis meaning inflammation around the tooth, is a common oral infection which affects the periodontium i.e. the tissues that surround and support the teeth. Periodontitis involves irreversible, progressive loss of the alveolar bone around the teeth and, if left untreated, can lead to the loosening and subsequent loss of teeth.
  • Periodontitis is caused by microorganisms that adhere to and grow on the tooth's surfaces close and below the gum line, along with an over-aggressive immune response against these microorganisms and their products.
  • I bone I bone. Diseased patients would typically exert PPD>3mm, bleeding on probing and apical migration of junctional epithelium (attachment loss), measured by careful periodontal examination and periodontal probes, while their radiographs would show alveolar bone resorption (Parameters of Care, Journal of Periodontology, Volume 71 , Number 5, May 2000 (Supplement)).
  • periodontal treatments must be cost-effective; that is, the treatments should be given to those individuals who need them the most and who can benefit from them. This puts a firm demand on the dentist, who must have the skills and equipment to single out patients with highest needs.
  • Periodontal diseases diagnosis. Ann Periodontol. 1 996 Nov; l ( l ):37-2 l 5 ), periodontal diagnostic procedures should serve five separate but related purposes:
  • the present invention seeks to provide a simple, potentially “chairside” ("near- patient”) test which can be performed on a patient and then used for screening, risk assessment, prevention, early diagnosis, selection of patient-centred point-of-care management options and for monitoring treatment outcomes.
  • a method for aiding the diagnosis and/or monitoring of periodontal disease in a subject comprising: collecting one or more subgingival plaques from one or more sites around one or more teeth; and assaying subgingival LPS activity relating to the or each site.
  • a further aspect provides a method for screening, risk assessment, prevention and early diagnosis of periodontal disease, patient-centred point-of-care periodontal therapy and monitoring treatment outcomes in a subject, the method comprising: collecting subgingival plaque samples from one or more sites around one or more teeth; extracting LPS from the or each sample; and assaying sub-gingival LPS activity after extraction, using recombinant Factor C.
  • the periodontal disease may be: gingivitis; chronic periodontal disease; aggressive periodontal disease; periodontitis as a manifestation of systemic disease; necrotising periodontal disease.
  • the present invention also provides a method for screening, risk assessment, prevention and early diagnosis of peri-implantitis, patient-centred point-of-care therapy and monitoring treatment outcomes in a subject, the method comprising: collecting subgingival plaque samples from one or more sites around one or more dental implants; extracting LPS from the or each sample; and assaying sub-gingival LPS activity after extraction, using recombinant Factor C.
  • LPS activity of a plurality of sites around a tooth/implant may be assayed.
  • the method may further comprise the step of establishing a subgingival endotoxin activity cut-off point to predict the risk of periodontitis or peri-implantitis.
  • the cut-off may be approximately 0.85 EU/ml per site sampled. Treatment may be prescribed based on the cut-off point.
  • the present invention also provides use of recombinant Factor C for measuring subgingival dental plaque endotoxin activity as a biomarker for periodontal disease progression.
  • the present invention also provides use of recombinant Factor C for measuring subgingival dental plaque endotoxin activity as a biomarker for peri-implantitis progression.
  • Endotoxin also known as lipopolysaccharide (LPS)
  • LPS lipopolysaccharide
  • LPS biological activity depends on its chemical structure which could be modified by bacteria involved in the development of periodontitis (Li Y, Powell DA, Shaffer SA, Rasko DA, Pelletier MR, Leszyk JD, Scott AJ, Masoudi A, Goodlett DR, Wang X, Raetz CR, Ernst RK.
  • LPS remodeling is an evolved survival strategy for bacteria. Proc Natl Acad Sci U S A. 201 2 May 29; 109(22)).
  • LPS activity Proc Natl Acad Sci U S A. 201 2 May 29; 109(22)
  • the amoebocyte lysate constituted as the Limulus Amoebocyte Lysate (LAL) test has been used for decades as a tool for detecting trace concentrations of LPS in solution.
  • LPS from gram-negative bacteria induces the amoebocytes of horseshoe crabs to aggregate and degranulate.
  • the molecular mechanism of coagulation in horseshoe crab has been established and it involves a protease cascade.
  • This cascade is based on three kinds of serine protease zymogens: Factor C; Factor B, proclotting enzyme; and one clottable protein, coagulogen.
  • the periodontal disease for which aspects and embodiments of the present invention may be applicable may include: chronic periodontal disease; aggressive periodontal disease; periodontal disease relating to systemic conditions; necrotising periodontal disease; or gingivitis.
  • a further aspect provides a method for aiding the diagnosis and/or prognostic monitoring of periodontal health in a subject, the method comprising: collecting subgingival plaque from one or more sites around one or more teeth; extracting LPS (lipopolysaccharide- endotoxin) from these samples and assaying subgingival LPS activity.
  • a further aspect provides a method for screening, risk assessment, prevention and early diagnosis of periodontal disease, patient-centred point-of-care periodontal therapy and monitoring treatment outcomes in a subject, the method comprising: collecting subgingival plaque from one or more sites around one or more teeth; extracting LPS from samples and assaying sub-gingival LPS activity.
  • Peri-implantitis is a destructive inflammatory process affecting the soft and hard tissues surrounding dental implants.
  • the array of periodontal pathogens found around failing implants are very similar to those found in association with various forms of periodontal disease.
  • a further aspect provides a method for screening, risk assessment, prevention and early diagnosis of peri-implantitis, patient-centred point-of-care therapy and monitoring treatment outcomes in a subject, the method comprising: collecting subgingival plaque from one or more sites around one or more implants; extracting LPS from samples and assaying sub-gingival LPS activity.
  • a further aspect provides a method for aiding the diagnosis and/or monitoring of peri- implantitis in a subject, the method comprising: collecting one or more subgingival plaques from one or more sites around one or more implants; and assaying subgingival LPS activity.
  • the LPS activity may be assayed using recombinant Factor C.
  • Factor C is the endotoxin-specific principal receptor in the LAL enzyme cascade. Being the initial activator of the clotting cascade, Factor C functions as a biosensor that responds to LPS.
  • LPS activity of a plurality of sites around a tooth and/or implant may be assayed, for example two or three sites. For example, a sample from at least the front and rear of a tooth/implant may be taken.
  • the present invention also provides the use of recombinant Factor C for measuring subgingival dental plaque endotoxin activity as a biomarker for periodontal disease progression.
  • the present invention also provides the use of recombinant Factor C for measuring subgingival dental plaque endotoxin activity as a biomarker for peri-implantitis progression.
  • the present invention also provides use of recombinant Factor C as a biomarker for periodontal disease activity.
  • the present invention provides a rapid and reliable test for destructive periodontal activity risk assessment, based on the LAL assay.
  • Figure I Subgingival LPS activity (measured by recombinant factor C in EU/ml) in healthy individuals and patients with chronic periodontitis.
  • Figure 2 Subgingival LPS activity (measured by recombinant factor C in EU/ml) in same patients before ( I ) and after periodontal therapy (2).
  • Figure 3 Subgingival LPS activity (measured by recombinant factor C in EU/ml) in patients who were not treated, at the time of recruitment ( I ) and 3-6 months later (2) ⁇
  • Figure 4 Summary of subgingival LPS activity in healthy patients and chronic periodontitis patients before and after periodontal therapy.
  • Figure 5 Subgingival endotoxin activity as a biomarker for chronic periodontitis. Cutoff point calculations.
  • Picture I Clinical presentation of a patient with healthy periodontal tissues.
  • Picture 2 Clinical presentation of a patient with chronic periodontitis.
  • Picture 3 The same patient after a course of periodontal treatment.
  • Table I test results (EU/ml) from all healthy and diseased patients in a study, and from about half of the patients who have undergone periodontal treatment.
  • Figure I shows the results of LAL assays performed on a panel of diseased and healthy patients. Diseased patients have high endotoxin activity, and healthy patients have low readings.
  • Figure 2 shows the results of assays for subgingival LPS activity after conventional periodontal therapy. In other words, only patients which were determined to have periodontitis after a clinical examination were given full periodontal treatment (such as a scale and polish). Patients deemed to be healthy from a clinical examination were given no treatment.
  • Picture 2 shows a patient with chronic periodontitis. This patient is diagnosed as clearly diseased by clinical examination. The LAL assay result would be high. A course of therapy would be prescribed.
  • Picture 3 shows the patient of Picture 2 following a course of periodontal treatment. This patient now looks healthy and would be cleared by clinical examination. If the LAL test result was negative then the patient would be declared stable (although once an individual has suffered from periodontitis there is a higher chance it will return, so a monitoring programme may be prescribed). However, it is possible that the LAL test would be unexpectedly positive and this would provoke a more aggressive treatment programme.
  • a method comprises the following steps. Collection of subgingival dental plaque, LPS extraction and EndoZyme® assay:
  • Subgingival dental plaque should be collected from a particular site or sites (mesially, distally, buccally or orally) around the tooth which is being assessed. 2.
  • the tooth is isolated and dried with cotton rolls to remove visible saliva and supragingival dental plaque from the gingival margin and tooth surface.
  • Sterile endodontic paper point is inserted into the chosen site (subgingivally) and left there for 30 seconds. Care should be taken it does not get in contact with saliva or oral mucosa.
  • Table I shows test results (EU/ml) from all healthy and diseased patients in a study, and from about half of the patients who have undergone periodontal treatment.
  • Figure 4 shows the Table I results.
  • a cut-off point for the Recombinant Factor C assay has been established in Figures 5 and 6, which would give very good sensitivity and specificity for periodontitis risk assessment.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Toxicology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

A method is provided for screening, risk assessment, prevention and early diagnosis of periodontal disease and/or peri-implantitis, patient-centred point-of-care periodontal therapy and monitoring treatment outcomes in a subject. The method comprises: collecting subgingival plaque samples from one or more sites around one or more teeth; extracting LPS from the or each sample; and assaying sub-gingival LPS activity after extraction, using recombinant Factor C.

Description

BIOMARKER FOR PERIODONTAL DISEASE AND PERI-IMPLANTITIS
Periodontal diseases can affect one or more of the periodontal tissues/ structures (e.g. alveolar bone, periodontal ligament, cementum and gingiva). While there are many different periodontal diseases that can affect these tooth-supporting tissues/structures, by far the most common ones are plaque-induced inflammatory conditions, such as gingivitis and periodontitis. Often the term periodontal disease or gum disease is used as a synonym for periodontitis, specifically chronic periodontitis. Periodontal disease ranges from the mildest stage, known as gingivitis, to severe stage, known, as periodontitis. While in some sites or individuals, gingivitis never progresses to periodontitis, data indicate that periodontitis is always preceded by gingivitis.
Periodontitis, meaning inflammation around the tooth, is a common oral infection which affects the periodontium i.e. the tissues that surround and support the teeth. Periodontitis involves irreversible, progressive loss of the alveolar bone around the teeth and, if left untreated, can lead to the loosening and subsequent loss of teeth.
Periodontitis is caused by microorganisms that adhere to and grow on the tooth's surfaces close and below the gum line, along with an over-aggressive immune response against these microorganisms and their products.
Current, conventional diagnostic procedures for periodontitis are based on visual and radiographic assessment and reflect only historical disease activity, not current disease state. Clinical diagnosis is made by measuring probing pocket depth (PPD), bleeding on probing and attachment loss (AL), and radiographically by detecting loss of alveolar
I bone. Diseased patients would typically exert PPD>3mm, bleeding on probing and apical migration of junctional epithelium (attachment loss), measured by careful periodontal examination and periodontal probes, while their radiographs would show alveolar bone resorption (Parameters of Care, Journal of Periodontology, Volume 71 , Number 5, May 2000 (Supplement)).
These diagnostic criteria do not assess current disease activity nor identify susceptible individuals who might be at risk of periodontitis in the future. They leave no opportunity to predict future tissue destruction or to formulate the appropriate treatment plan specific to each individual patient.
As with other dental procedures, periodontal treatments must be cost-effective; that is, the treatments should be given to those individuals who need them the most and who can benefit from them. This puts a firm demand on the dentist, who must have the skills and equipment to single out patients with highest needs.
According to Armitage (Armitage GC, Periodontal diseases: diagnosis. Ann Periodontol. 1 996 Nov; l ( l ):37-2 l 5 ), periodontal diagnostic procedures should serve five separate but related purposes:
- Screening,
Diagnosis of specific periodontal diseases,
Identification of sites and subjects at an increased risk of experiencing the progression of periodontal destruction,
Treatment planning,
- Monitoring of therapy. The present invention seeks to provide a simple, potentially "chairside" ("near- patient") test which can be performed on a patient and then used for screening, risk assessment, prevention, early diagnosis, selection of patient-centred point-of-care management options and for monitoring treatment outcomes.
According to an aspect of the present invention there is provided a method for aiding the diagnosis and/or monitoring of periodontal disease in a subject, the method comprising: collecting one or more subgingival plaques from one or more sites around one or more teeth; and assaying subgingival LPS activity relating to the or each site.
By taking subgingival plaque samples from particular teeth it is possible to assess the risk and give valuable prognosis for each individual tooth and particular sites around the teeth.
A further aspect provides a method for screening, risk assessment, prevention and early diagnosis of periodontal disease, patient-centred point-of-care periodontal therapy and monitoring treatment outcomes in a subject, the method comprising: collecting subgingival plaque samples from one or more sites around one or more teeth; extracting LPS from the or each sample; and assaying sub-gingival LPS activity after extraction, using recombinant Factor C.
In embodiments of the present invention the periodontal disease may be: gingivitis; chronic periodontal disease; aggressive periodontal disease; periodontitis as a manifestation of systemic disease; necrotising periodontal disease. The present invention also provides a method for screening, risk assessment, prevention and early diagnosis of peri-implantitis, patient-centred point-of-care therapy and monitoring treatment outcomes in a subject, the method comprising: collecting subgingival plaque samples from one or more sites around one or more dental implants; extracting LPS from the or each sample; and assaying sub-gingival LPS activity after extraction, using recombinant Factor C.
LPS activity of a plurality of sites around a tooth/implant may be assayed.
The method may further comprise the step of establishing a subgingival endotoxin activity cut-off point to predict the risk of periodontitis or peri-implantitis. The cut-off may be approximately 0.85 EU/ml per site sampled. Treatment may be prescribed based on the cut-off point.
The present invention also provides use of recombinant Factor C for measuring subgingival dental plaque endotoxin activity as a biomarker for periodontal disease progression.
The present invention also provides use of recombinant Factor C for measuring subgingival dental plaque endotoxin activity as a biomarker for peri-implantitis progression. Endotoxin, also known as lipopolysaccharide (LPS), is an integral component of the gram-negative bacterial cell membrane and is responsible for many, if not all, of the toxic effects that occur during gram-negative bacterial sepsis and is one of major bacterial products involved in the development of periodontitis. It causes alveolar bone resorption directly by activating osteoclasts (bone resorbing cells) and indirectly by triggering an inflammatory response within the periodontal tissues. LPS biological activity depends on its chemical structure which could be modified by bacteria involved in the development of periodontitis (Li Y, Powell DA, Shaffer SA, Rasko DA, Pelletier MR, Leszyk JD, Scott AJ, Masoudi A, Goodlett DR, Wang X, Raetz CR, Ernst RK. LPS remodeling is an evolved survival strategy for bacteria. Proc Natl Acad Sci U S A. 201 2 May 29; 109(22)). By measuring LPS activity in subgingival areas, it is possible to successfully identify and treat the teeth which are at high risk for periodontal tissue destruction before it occurs (something we are not able to do currently). The amoebocyte lysate constituted as the Limulus Amoebocyte Lysate (LAL) test has been used for decades as a tool for detecting trace concentrations of LPS in solution.
LPS from gram-negative bacteria induces the amoebocytes of horseshoe crabs to aggregate and degranulate. The molecular mechanism of coagulation in horseshoe crab has been established and it involves a protease cascade. This cascade is based on three kinds of serine protease zymogens: Factor C; Factor B, proclotting enzyme; and one clottable protein, coagulogen.
The periodontal disease for which aspects and embodiments of the present invention may be applicable may include: chronic periodontal disease; aggressive periodontal disease; periodontal disease relating to systemic conditions; necrotising periodontal disease; or gingivitis.
A further aspect provides a method for aiding the diagnosis and/or prognostic monitoring of periodontal health in a subject, the method comprising: collecting subgingival plaque from one or more sites around one or more teeth; extracting LPS (lipopolysaccharide- endotoxin) from these samples and assaying subgingival LPS activity. A further aspect provides a method for screening, risk assessment, prevention and early diagnosis of periodontal disease, patient-centred point-of-care periodontal therapy and monitoring treatment outcomes in a subject, the method comprising: collecting subgingival plaque from one or more sites around one or more teeth; extracting LPS from samples and assaying sub-gingival LPS activity.
Peri-implantitis is a destructive inflammatory process affecting the soft and hard tissues surrounding dental implants. The array of periodontal pathogens found around failing implants (those affected by peri-implantitis) are very similar to those found in association with various forms of periodontal disease.
A further aspect provides a method for screening, risk assessment, prevention and early diagnosis of peri-implantitis, patient-centred point-of-care therapy and monitoring treatment outcomes in a subject, the method comprising: collecting subgingival plaque from one or more sites around one or more implants; extracting LPS from samples and assaying sub-gingival LPS activity. A further aspect provides a method for aiding the diagnosis and/or monitoring of peri- implantitis in a subject, the method comprising: collecting one or more subgingival plaques from one or more sites around one or more implants; and assaying subgingival LPS activity.
In some aspects and embodiments the LPS activity may be assayed using recombinant Factor C. Factor C is the endotoxin-specific principal receptor in the LAL enzyme cascade. Being the initial activator of the clotting cascade, Factor C functions as a biosensor that responds to LPS.
In aspects and embodiments of the present invention LPS activity of a plurality of sites around a tooth and/or implant may be assayed, for example two or three sites. For example, a sample from at least the front and rear of a tooth/implant may be taken.
The present invention also provides the use of recombinant Factor C for measuring subgingival dental plaque endotoxin activity as a biomarker for periodontal disease progression.
The present invention also provides the use of recombinant Factor C for measuring subgingival dental plaque endotoxin activity as a biomarker for peri-implantitis progression. The present invention also provides use of recombinant Factor C as a biomarker for periodontal disease activity.
The present invention provides a rapid and reliable test for destructive periodontal activity risk assessment, based on the LAL assay.
Different aspects and embodiments of the invention may be used separately or together. Further particular and preferred aspects of the present invention are set out in the accompanying independent and dependent claims. Features of the dependent claims may be combined with the features of the independent claims as appropriate, and in combination other than those explicitly set out in the claims. The present invention will now be more particularly described, by way of example, with reference to the accompanying drawings, in which:
Figure I : Subgingival LPS activity (measured by recombinant factor C in EU/ml) in healthy individuals and patients with chronic periodontitis.
Figure 2: Subgingival LPS activity (measured by recombinant factor C in EU/ml) in same patients before ( I ) and after periodontal therapy (2). Figure 3: Subgingival LPS activity (measured by recombinant factor C in EU/ml) in patients who were not treated, at the time of recruitment ( I ) and 3-6 months later (2)· Figure 4: Summary of subgingival LPS activity in healthy patients and chronic periodontitis patients before and after periodontal therapy.
Figure 5: Subgingival endotoxin activity as a biomarker for chronic periodontitis. Cutoff point calculations.
Figure 6: Optimal cut-off value using method "outcome significance": 5. 1 14 EU/ml.
Picture I : Clinical presentation of a patient with healthy periodontal tissues. Picture 2: Clinical presentation of a patient with chronic periodontitis.
Picture 3: The same patient after a course of periodontal treatment.
Table I : test results (EU/ml) from all healthy and diseased patients in a study, and from about half of the patients who have undergone periodontal treatment.
This is based on three teeth (sites) sampled. So, per tooth (site) it would be 1 .7 EU/ml. Given the fact that the extracted LPS was re-suspended in 500μΙ of endotoxin free water, the optimal endotoxin cut-off point that would successfully predict the risk of periodontitis is 0.85 EU/ml per site sampled. Example embodiments are described below in sufficient detail to enable those of ordinary skill in the art to embody and implement the systems and processes herein described. It is important to understand that embodiments can be provided in many alternate forms and should not be construed as limited to the examples set forth herein.
Accordingly, while embodiments can be modified in various ways and take on various alternative forms, specific embodiments thereof are shown in the drawings and described in detail below as examples. There is no intent to limit to the particular forms disclosed. On the contrary, all modifications, equivalents, and alternatives falling within the scope of the appended claims should be included. Elements of the example embodiments are consistently denoted by the same reference numerals throughout the drawings and detailed description where appropriate.
The terminology used herein to describe embodiments is not intended to limit the scope. The articles "a," "an," and "the" are singular in that they have a single referent, however the use of the singular form in the present document should not preclude the presence of more than one referent. In other words, elements referred to in the singular can number one or more, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises," "comprising," "includes," and/or "including," when used herein, specify the presence of stated features, items, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, items, steps, operations, elements, components, and/or groups thereof. Unless otherwise defined, all terms (including technical and scientific terms) used herein are to be interpreted as is customary in the art. It will be further understood that terms in common usage should also be interpreted as is customary in the relevant art and not in an idealised or overly formal sense unless expressly so defined herein.
In the following description, all orientational terms, such as upper, lower, mesially and distally, are used in relation to the drawings and should not be interpreted as limiting on the invention.
Figure I shows the results of LAL assays performed on a panel of diseased and healthy patients. Diseased patients have high endotoxin activity, and healthy patients have low readings. Figure 2 shows the results of assays for subgingival LPS activity after conventional periodontal therapy. In other words, only patients which were determined to have periodontitis after a clinical examination were given full periodontal treatment (such as a scale and polish). Patients deemed to be healthy from a clinical examination were given no treatment.
Majority of patient who underwent periodontal therapy exhibited a significant reduction in subgingival LPS activity. Contrary, four patients did not attend their periodontal treatment appointment and visited 3 to 6 months later without any therapy. Subgingival LPS activity in all of these patients went up (Figure 3). Picture I shows a patient with healthy periodontal tissues as diagnosed by a clinical examination. If this patient was tested with the subgingival LAL assay of the present invention and the result was low endotoxin levels it would be possible to say that there was no periodontitis risk. However, if the patient showed a high level they would be more likely to be predisposed to periodontal problems and a course of treatment and/or monitoring could be prescribed.
Picture 2 shows a patient with chronic periodontitis. This patient is diagnosed as clearly diseased by clinical examination. The LAL assay result would be high. A course of therapy would be prescribed.
Picture 3 shows the patient of Picture 2 following a course of periodontal treatment. This patient now looks healthy and would be cleared by clinical examination. If the LAL test result was negative then the patient would be declared stable (although once an individual has suffered from periodontitis there is a higher chance it will return, so a monitoring programme may be prescribed). However, it is possible that the LAL test would be unexpectedly positive and this would provoke a more aggressive treatment programme. In one embodiment of the present invention a method comprises the following steps. Collection of subgingival dental plaque, LPS extraction and EndoZyme® assay:
I . Subgingival dental plaque should be collected from a particular site or sites (mesially, distally, buccally or orally) around the tooth which is being assessed. 2. The tooth is isolated and dried with cotton rolls to remove visible saliva and supragingival dental plaque from the gingival margin and tooth surface.
3. Sterile endodontic paper point is inserted into the chosen site (subgingivally) and left there for 30 seconds. Care should be taken it does not get in contact with saliva or oral mucosa.
4. After 30 seconds, remove the paper point and place it in a sterile container ( 1 5ml tube or 1 .5ml Eppendorf).
5. Extract LPS from the paper point using either the Tri-reagent or the phenol- water method. Lyophilise the extract and re-dissolve the powder in 500microliters of LPS free water. 6. Perform the EndoZyme® assay with this solution in I / 10 dilution.
Table I shows test results (EU/ml) from all healthy and diseased patients in a study, and from about half of the patients who have undergone periodontal treatment. Figure 4 shows the Table I results.
A cut-off point for the Recombinant Factor C assay has been established in Figures 5 and 6, which would give very good sensitivity and specificity for periodontitis risk assessment. Optimal cut-off value using method "outcome significance": 5. 1 14 EU/ml. This is based on three teeth (sites) sampled. So, per tooth (site) it would be 1.7 EU/ml. Given the fact that the extracted LPS was re-suspended in 500μΙ of endotoxin free water, the optimal endotoxin cut-off point that would successfully predict the risk of periodontitis is 0.85 EU/ml per site sampled.
Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiments shown and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention.

Claims

I . A method for screening, risk assessment, prevention and early diagnosis of periodontal disease, patient-centred point-of-care periodontal therapy and monitoring treatment outcomes in a subject, the method comprising: collecting subgingival plaque samples from one or more sites around one or more teeth; extracting LPS from the or each sample; and assaying sub-gingival LPS activity after extraction, using recombinant Factor C.
2. A method as claimed in claim I , in which the periodontal disease is: gingivitis; chronic periodontal disease; aggressive periodontal disease; periodontitis as a manifestation of systemic disease; necrotising periodontal disease.
3. A method for screening, risk assessment, prevention and early diagnosis of peri-implantitis, patient-centred point-of-care therapy and monitoring treatment outcomes in a subject, the method comprising: collecting subgingival plaque samples from one or more sites around one or more dental implants; extracting LPS from the or each sample; and assaying sub-gingival LPS activity after extraction, using recombinant Factor C.
4. A method as claimed in any preceding claim, in which LPS activity of a plurality of sites around a tooth/implant are assayed.
5. A method as claimed in any preceding claim, further comprising the step of establishing a subgingival endotoxin activity cut-off point to predict the risk of periodontitis or peri-implantitis.
6. A method as claimed in claim 5, in which the cut-off is approximately 0.85 EU/ml per site sampled.
7. A method as claimed in claim 5 or claim 6, in which treatment is prescribed based on the cut-off point.
8. Use of recombinant Factor C for measuring subgingival dental plaque endotoxin activity as a biomarker for periodontal disease progression.
9. Use of recombinant Factor C for measuring subgingival dental plaque endotoxin activity as a biomarker for peri-implantitis progression.
PCT/EP2017/059415 2016-04-24 2017-04-20 Biomarker for periodontal disease and peri-implantitis WO2017186571A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1607106.0 2016-04-24
GB1607106.0A GB2549712A (en) 2016-04-24 2016-04-24 Biomarker

Publications (1)

Publication Number Publication Date
WO2017186571A1 true WO2017186571A1 (en) 2017-11-02

Family

ID=58668851

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2017/059415 WO2017186571A1 (en) 2016-04-24 2017-04-20 Biomarker for periodontal disease and peri-implantitis

Country Status (2)

Country Link
GB (1) GB2549712A (en)
WO (1) WO2017186571A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2773275C1 (en) * 2021-07-26 2022-06-01 Федеральное государственное бюджетное образовательное учреждение высшего образования "Дальневосточный государственный медицинский университет" Министерства здравоохранения Российской Федерации (ФГБОУ ВО ДВГМУ Минздрава России) Method for predicting the severity of periodontitis by the composition of conditionally periodontopathogenic species of the microbiome of the root of the tongue
CN117169521A (en) * 2023-08-25 2023-12-05 暨南大学附属第一医院（广州华侨医院） Mass spectrum negative ion mode metabonomics biomarker for peri-implant inflammation and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990000620A1 (en) * 1988-07-15 1990-01-25 The Trustees Of Columbia University Method for monitoring periodontal disease by monitoring endotoxins and inflammatory agents
US20040175388A1 (en) * 1998-12-24 2004-09-09 National University Of Singapore Recombinant proteins and peptides for endotoxin biosensors, endotoxin removal, and anti-microbial & anti-endotoxin therapeutics

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ZA896085B (en) * 1988-09-07 1990-05-30 Minnesota Mining & Mfg Monoclonal antibodies specific for elkenella corrodens
AU2006892A (en) * 1991-05-16 1992-12-30 Associates Of Cape Cod, Inc. Endotoxin binding and neutralizing protein and uses thereof
WO1995013094A1 (en) * 1993-11-10 1995-05-18 Bristol-Myers Squibb Company Treatment of bacterially-induced inflammatory diseases
JP2005140618A (en) * 2003-11-06 2005-06-02 Lion Corp Method for estimating fluctuation of biochemical parameter, and method for predicting onset risk of lifestyle-related disease

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990000620A1 (en) * 1988-07-15 1990-01-25 The Trustees Of Columbia University Method for monitoring periodontal disease by monitoring endotoxins and inflammatory agents
US20040175388A1 (en) * 1998-12-24 2004-09-09 National University Of Singapore Recombinant proteins and peptides for endotoxin biosensors, endotoxin removal, and anti-microbial & anti-endotoxin therapeutics

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2773275C1 (en) * 2021-07-26 2022-06-01 Федеральное государственное бюджетное образовательное учреждение высшего образования "Дальневосточный государственный медицинский университет" Министерства здравоохранения Российской Федерации (ФГБОУ ВО ДВГМУ Минздрава России) Method for predicting the severity of periodontitis by the composition of conditionally periodontopathogenic species of the microbiome of the root of the tongue
CN117169521A (en) * 2023-08-25 2023-12-05 暨南大学附属第一医院（广州华侨医院） Mass spectrum negative ion mode metabonomics biomarker for peri-implant inflammation and application thereof

Also Published As

Publication number Publication date
GB2549712A (en) 2017-11-01

Similar Documents

Publication Publication Date Title
Doornewaard et al. How do peri‐implant biologic parameters correspond with implant survival and peri‐implantitis? A critical review
Ng et al. Outcome of primary root canal treatment: systematic review of the literature–Part 2. Influence of clinical factors
Monje et al. Diagnostic accuracy of clinical parameters to monitor peri‐implant conditions: a matched case‐control study
Timmerman et al. Untreated periodontal disease in Indonesian adolescents: Longitudinal clinical data and prospective clinical and microbiological risk assessment
Thierbach et al. Peri-implant sulcus fluid (PISF) matrix metalloproteinase (MMP)-8 levels in peri-implantitis
de Oliveira Silva et al. A 3‐year longitudinal prospective study assessing microbial profile and clinical outcomes of single‐unit cement‐retained implant restorations: zirconia versus titanium abutments
Sarlati et al. Receptor activator of nuclear factor kappa B ligand (RANKL) levels in peri-implant crevicular fluid
Czerniuk et al. Plasmatic NT-proBNP concentrations in patients with coexistent periodontal disease and congestive heart failure: pilot studies
Zehnder et al. Comparison of vehicles to collect dentinal fluid for molecular analysis
DeAngelo et al. Early soft tissue healing around one‐stage dental implants: clinical and microbiologic parameters
Tervit et al. Proportion of healed teeth with apical periodontitis medicated with two percent chlorhexidine gluconate liquid: a case-series study
WO2017186571A1 (en) Biomarker for periodontal disease and peri-implantitis
Golbasi et al. Comparison of ADAMTS levels in pulp tissue samples of healthy and symptomatic irreversible pulpitis teeth
Vengerfeldt Apical periodontitis: prevalence and etiopathogenetic aspects
Hirsch et al. Pulpal pathosis and severe alveolar lesions: a clinical study
Ranjith Raj et al. Role of Salivary PH on the Prevalence of Periodontal Disease: A Cross Sectional Pilot Study
Doan et al. Evaluation of host β-globin gene fragment lengths in peri-implant crevicular fluid during the wound healing process: a pilot study.
Hadjieva et al. Stomatitis prosthetica-a polyetiologic disorder
Costa et al. Is there bacterial infection in the intact crowns of teeth with pulp necrosis of sickle cell anaemia patients? A case series study nested in a cohort
RU2799012C1 (en) Method of predicting the risk of developing chronic generalized periodontitis
Almeida et al. Gingival crevicular fluid volume evaluation in patients with controlled periodontal disease submitted to orthodontic treatment
A Qasim et al. Assessment of periodontal status among premenopausal and postmenopausal women in Mosul city
Salman et al. Effect of ZnO Nanoparticles on AST activity in gingival cervicular fluid of smokers and nonsmokers chronic periodontitis patients: in vitro study
Janošević et al. Nitric oxide as prediction factor of gingival inflammation in orthodontic patients
Khairani et al. The Effect of Scaling and Root Planing on Total Antioxidant Status of Chronic Periodontitis Patients

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17721073

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 17721073

Country of ref document: EP

Kind code of ref document: A1