WO2008092201A1 - Procédé de diagnostic de maladie prostatique - Google Patents

Procédé de diagnostic de maladie prostatique Download PDF

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Publication number
WO2008092201A1
WO2008092201A1 PCT/AU2008/000105 AU2008000105W WO2008092201A1 WO 2008092201 A1 WO2008092201 A1 WO 2008092201A1 AU 2008000105 W AU2008000105 W AU 2008000105W WO 2008092201 A1 WO2008092201 A1 WO 2008092201A1
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WIPO (PCT)
Prior art keywords
acnes
prostate
patients
cancer
serum
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PCT/AU2008/000105
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English (en)
Inventor
Beverley O'brien
Ronald Cohen
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Tissugen Pty Ltd
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Priority claimed from AU2007900486A external-priority patent/AU2007900486A0/en
Application filed by Tissugen Pty Ltd filed Critical Tissugen Pty Ltd
Publication of WO2008092201A1 publication Critical patent/WO2008092201A1/fr

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911Bacteria
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57434Specifically defined cancers of prostate
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854Immunoglobulins
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical 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
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/34Genitourinary disorders
    • G01N2800/342Prostate diseases, e.g. BPH, prostatitis

Definitions

  • the present invention relates to methods for diagnosing prostatic disease in men. hi particular, the present invention provides a method for diagnosing prostatic disease and/or the risk of prostatic disease in men by assaying for the presence of antibodies specific to P. acnes.
  • P. acnes Propionibacterium acnes
  • the inventors have previously cultured Propionibacterium acnes (P. acnes) from the prostate tissue of a considerable proportion of prostate cancer patients and shown a statistically significant association between positive culture of this bacterium and increased levels of inflammation in the prostate tissue [I].
  • the inventors have also showed that the majority of P. acnes in the prostate gland [1] and in the urinary tract of adult males [2] are types IB and II, which differ genetically and phenotypically from the common skin P. acnes of type IA [1-4].
  • Chronic inflammation is strongly implicated in the development of prostate cancer and other prostate diseases [5] and the inventors' findings suggest that P acnes (in particular types IB and II) may be involved in the development of these prostate diseases through an inflammatory mechanism.
  • P. acnes is known to be a potent inflammatory stimulus to the immune system [6] and has been implicated in several other inflammatory conditions including acne vulgaris, endocarditis, endopthalmitis and osteomye
  • Various therapies including antibiotics could then be offered to men with positive test results in the hope of preventing a proportion of prostate tumours and other infection-related prostate diseases.
  • the finding that P. acnes, in particular types IB and II, are common inhabitants of the adult male urethra [2] means that samples such as urine, ejaculate or prostatic secretions may not be suitable for indirect assessment of the prostate gland due to contamination with the urethral flora during collection. Direct quantitation of P. acnes levels by prostatic biopsy is currently too invasive a procedure for testing of asymptomatic men.
  • a method for diagnosing prostatic disease and/or risk of prostatic disease in men comprising obtaining a body " fluid sample from a patient and assaying for the presence of antibodies specific for P. acnes in the body fluid sample.
  • a method according to the first aspect of the invention further comprising the step of measuring the level of prostate specific antigen present.
  • Figure 1 shows secreted and membrane-anchored cell surface proteins of P. acnes visualized on SDS-PAGE gel and stained with Coomassie Blue.
  • FIG. 1 shows P. acnes secreted/cell surface proteins after transfer onto PVDF membrane and staining with Coomassie Blue. All protein bands have transferred well.
  • Figure 3 shows immunoblot of P. acnes secreted/cell surface proteins screened with the serum of 9 prostate biopsy patients. Each set of two lanes represents duplicate samples from a single patient. Only a minority of the protein bands present on the membrane are bound by patient antibodies, and patients show a range of immunoreactivity from strong immunoreactivity (lanes 1, 2 and 8) to weak immunoreactivity (lanes 3-7, 9).
  • the present inventors have developed an immunological test using a mixture of the secreted and cell surface membrane-anchored proteins of P. acnes, with particular emphasis on proteins from P. acnes types IB and II, and used these proteins to assess prostate biopsy patients for immunoreactivity to P. acnes.
  • a method for diagnosing prostatic disease and/or risk of prostatic disease in men comprising obtaining a body fluid sample from a patient and assaying for the presence of antibodies specific for P. acnes in the body fluid sample.
  • the prostatic disease is selected from the group consisting of prostate cancer, prostatitis and benign prostatic hyperplasia.
  • the body fluid sample is selected from the group consisting of plasma, serum, urine, prostatic secretions and ejaculate.
  • the method according to the first aspect of the invention comprises assaying for the presence of antibodies specific for P. acnes secreted proteins or cell surface proteins, or both.
  • the cell surface proteins include membrane-anchored cell surface proteins.
  • the secreted and/or membrane-anchored surface proteins include proteins from P. acnes type IB and II.
  • the antibodies are IgA.
  • the IgA is secretory IgA.
  • Detecting for the presence of antibodies specific to P. acnes proteins can be achieved using routine assays known in the art.
  • the enzyme-linked immunosorbent assay ELISA
  • ELISA enzyme-linked immunosorbent assay
  • the enzyme-linked immunosorbent assay can be used to detect the presence and immunoreactivity of a particular antibody for its antigen by detecting the amount of chromogenic or fluorogenic substrate (hence "enzyme-linked”) conjugated to a secondary antibody which binds the antibody-antigen complex.
  • the method comprises quantitating the antibody titre of the body fluid sample to P. acnes protein.
  • a method according to the first aspect of the invention further comprising the step of measuring the level of prostate specific antigen present.
  • surface protein extraction buffer (1 mL of 1.0 M Tris pH 7.5, lO ⁇ L of 0.5 M EDTA, 10 ⁇ L of Sigma P8849 protease inhibitor cocktail
  • the secreted/cell surface proteins from each P. acnes type were compared and characterized by SDS-PAGE analysis using protein molecular weight standards and staining with Coomassie Blue.
  • sample buffer (62.5mM Tris-HCL pH 6.8, 2% SDS, 25% glycerol, 0.01% bromothymol blue, with 5% 2-mercaptoethanol added just before use), with frequent mixing by pipette.
  • the protein was loaded onto a 4-15% polyacrylamide gel (BioRad Readygel) at a concentration of 20 ⁇ L per lane and run in a mini-Protean 3 electrophoresis cell (BioRad) at 200V in standard running buffer (25mM Tris, 192mM glycine, 0.1% SDS, pH 8.3). Proteins were visualized by staining with the BioSafe Coomassie Blue stain (BioRad) according to the manufacturers instructions.
  • BioRad Readygel a concentration of 20 ⁇ L per lane and run in a mini-Protean 3 electrophoresis cell (BioRad) at 200V in standard running buffer (25mM Tris, 192mM glycine, 0.1% SDS, pH 8.3). Proteins were visualized by staining with the BioSafe Coomassie Blue stain (BioRad) according to the manufacturers instructions.
  • the mixture yielded at least 25 discrete protein bands ranging in size from 10 kDa to 150 kDa.
  • Protein extractions from P. acnes type IA and IB had a similar pattern whereas the protein extraction from type II differed slightly and showed significantly higher amounts of two large proteins approximately 10OkDa and 15OkDa in size (Fig 1). These two large proteins were produced by types IA and IB in small amounts only (Figs 1 and 2).
  • each channel was then washed 6 X with wash solution and loaded with 600 ⁇ L of secondary antibody solution (Goat Anti-Human Ig (H + L) AP conjugate diluted 1 :3000 in antibody buffer) for a 2 hour incubation, with mixing by pipette halfway through this time.
  • the membrane was removed from the multiscreen apparatus and immersed in TBS for 2 X 5 min washes to remove all Tween-20.
  • colour development solution (2mL of 25X colour development buffer plus 48mL filtered de-ionised water, with addition of colour reagent A (0.5mL) and colour reagent B (0.5mL) just before use) for 30 - 40 minutes. Finally the membrane was rinsed in de-ionised water (2 X 5 mins) and air-dried.
  • the major antigenic protein band common to all positive serum samples was around 25kDa in size, with other commonly positive bands being 10, 15, 50 and 100 kDa in size (Fig 3).
  • raised PSA levels can be caused by prostatic infection and inflammation [16,17] it is possible that infection of the prostate with P. acnes had caused both the raised PSA levels and the high degree of P. ⁇ cwes-specific immune reactivity in this subset of patients. However, the number of cases analyzed is too small for a definite conclusion to be reached and a larger study is needed to either confirm or disprove this trend.
  • Enzyme-linked immunoabsorbent assay (ELISA) tests can give a more sensitive and quantitative assessment of immune response to an antigen, plus they are less labour intensive than immunoblotting.
  • ELISA Enzyme-linked immunoabsorbent assay
  • Each 96-well polystyrene ELISA plate was used to analyse duplicate samples of serum from 4 patients, using 15 ⁇ g of surface/secreted proteins in total (7.5 ⁇ g from P. acnes type EB mixed with 7.5 ⁇ g from P. acnes type II).
  • the proteins were purified by precipitation as described in Example 2, pre-treated with 2 ⁇ L of 0.2M NaOH for 5 mins then resolubilized in 45 ⁇ L of 0.05M carbonate buffer (15mM Na 2 CO 3 , 35mM NaH CO 3 , pH 9.6) by incubating at room temperature for 2 hours with frequent mixing by pipette. This was stored at 4 0 C until use later in the day.
  • the protein mixture was diluted to a concentration of 1.5 ⁇ g/mL with carbonate buffer and lOO ⁇ L was added to each well of the ELISA plate except for the negative (no antigen) control wells, which received only carbonate buffer.
  • the ELISA plate was sealed with an adhesive sealing sheet, wrapped in plastic wrap and incubated at 4°C for 16-18 hours (overnight) to allow protein coating of the wells.
  • the wells were washed 2 X with TBS, filled with blocking solution and incubated for 1 hour, then washed 4 X with wash solution.
  • the first well of every row of 12 wells was filled with 200 ⁇ L of the appropriate patient serum (diluted 1 :32 in antibody buffer) while the next 9 wells received 100 ⁇ L of antibody buffer only. 100 ⁇ L of the 1 :32 dilution was then withdrawn to make serial two-fold dilutions of the patient serum in the next 9 wells, hi the first 6 rows, wells 11 and 12 were negative controls with no serum and no protein respectively, hi the final 2 rows, wells 11 and 12 were positive controls (receiving a 1 :64 serum dilution from a strongly reactive patient).
  • the specificity of the assay was confirmed by testing the serum of two rabbits after immunization with the antigen mixture (three doses of 0.5mg antigen given at 21 day intervals). Pre-immunization titres were negative and 1 :2 whereas post-immunization titres were 1 :32 768 and 1 :8192 respectively, confirming that the ELISA can detect serum antibodies against these P. acnes surface proteins.
  • biopsy-negative group Table 2
  • the main factor predicting serum PSA levels in the cancer patient group is the volume of cancer present, whereas the main factor predicting serum PSA levels in the biopsy-negative patient group is their serum titre of antibodies against P. acnes.
  • Previous studies investigating the causes of elevated PSA in biopsy-negative patients without symptoms of prostatitis have identified patient age, prostate volume and the aggressiveness of inflammation as significant predictors, however multivariate analysis consistently identified prostate volume as the predominant predictor of serum PSA [18- 20].
  • Our results also identified patient age, prostate volume and aggressiveness of inflammation as positive predictors of PSA in this patient group, but multiple regression revealed anti-P. acnes antibody titre as the predominant independent predictor.
  • Serum PSA level is currently the main diagnostic test used to screen for prostate cancer, but it has low specificity for detecting prostate cancer because numerous other factors can also cause raised serum PSA levels, including BPH and prostatitis (prostatic infection and/or inflammation)[16,17].
  • ELISA testing for immune reactivity to P. acnes may be a useful supplement to PSA screening and prostate biopsy, capable of identifying patients who either have P. acnes infection of the prostate gland or are hypersensitve to P. acnes antigens, and who therefore have increased risk of developing prostate diseases including cancer even if their biopsy results are currently negative for cancer. This may be particularly relevant for patients with both high PSA levels and high P. acnes titres, but with biopsy results negative for cancer. These patients could then be given treatment with antibacterial agents such as antibiotics which may prevent the development of prostate disease including cancer.
  • ELISA testing of serum to detect antibodies against P. acnes can identify individuals with raised antibody titres, suggesting the presence of a current or recent P. acnes infection. However, this method does not specifically identify men with P. acnes infection of the prostate gland.
  • ELISA testing of samples such as urine, expressed prostatic secretions or ejaculate can be conducted using standard secondary antibodies that detect human IgG, however these methods are also not specific to urinary tract or prostate gland infection because a large proportion of the IgG antibodies in bodily fluids such as urine is derived from the serum [23].
  • the serum contains 5 -fold lower levels of IgA antibodies compared to IgG [24].
  • IgA is locally produced within mucosal lymphoid tissues [25], thus IgA levels in bodily fluids may be more representative of local infections.
  • ELISA to detect coliform- specific IgA in urine has been used to assess the role of these bacteria in urinary tract infections [26].
  • ELISA to detect Chlamydia trachomatis-specific IgA in ejaculate has been used to assess the role of these bacteria in prostatic infection [27].
  • Secretory IgA is comprised of IgA polymers attached to secretory component and is formed during transport of IgA through the epithelial cells lining mucosal surfaces and out into the lumen of the mucosa [25].
  • the primary antibody solution contains either urine, expressed prostatic secretions, or ejaculate.
  • the secondary antibody used is anti-human IgA (for detection of IgA) or anti-human secretory component (for detection of secretory IgA).
  • prostate biopsy patients with high P. ⁇ c/zes-specific antibody titres did not have significantly higher values for any of the traditional parameters known to contribute to raised PSA levels, including age, prostate volume, presence of cancer in the biopsy specimen, amount of cancer in the biopsy specimen or grade of cancer in the biopsy specimen.
  • raised PSA levels can be caused by prostatic infection and inflammation [18-20] it appears likely that infection of the prostate with P. acnes may have caused both the raised PSA levels and the high degree of P. ⁇ cwes-specif ⁇ c immune reactivity in this subset of patients (group 1).
  • P. acnes High levels of P. ⁇ cwes-specific antibodies are clearly not specific to patients with prostate disease, since they can also be found in subjects without infection of the prostate gland (for example in female controls who do not have a prostate gland). In these cases, high immune reactivity to P. acnes may reflect infection in other regions of the body known to be inhabited by P. acnes , for example in the urinary tract, intestinal tract, genital tract or on the skin.
  • Serum PSA level is currently the main diagnostic test used to screen for prostate cancer, but it has low specificity for detecting prostate cancer because numerous other factors can also cause raised serum PSA levels, including benign prostatic hyperplasia and prostatitis (prostatic infection and/or inflammation)[19].
  • testing for immune reactivity to P. acnes may be a useful supplement for PSA screening and prostate biopsy, capable of identifying patients with P. acnes infection of the prostate gland and therefore with increased risk of developing prostate diseases including cancer even if the biopsy results are currently negative for cancer. This may be particularly relevant for patients with both high PSA levels and high P. acnes titres (as in our patient group 1), but with biopsy results negative for clinically significant cancer. These patients could then be given treatment with antibacterial agents such as antibiotics which may prevent the development of prostate disease including cancer.
  • Propionibacterium acnes associated with inflammation in radical prostatectomy specimens a possible link to cancer evolution? J Urol 2005: 173:1969-1974.

Abstract

L'invention concerne un procédé qui permet de diagnostiquer une maladie prostatique et/ou le risque de maladie prostatique chez les hommes en prélevant un liquide corporel chez un patient et en analysant ledit liquide corporel à la recherche d'anticorps spécifiques de Propionibacterium acnes (P. acnes).
PCT/AU2008/000105 2007-02-02 2008-01-31 Procédé de diagnostic de maladie prostatique WO2008092201A1 (fr)

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AU2007900486 2007-02-02
AU2007900486A AU2007900486A0 (en) 2007-02-02 A method for diagnosing prostatic disease

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2945125A1 (fr) * 2009-05-04 2010-11-05 Ingen Biosciences Methode de diagnostic des infections a propionibacterium acnes

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005087929A1 (fr) * 2004-03-15 2005-09-22 Tissugen Pty Ltd Etiologie infectieuse de maladie prostatique et procede d'identification d'agents etiologiques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005087929A1 (fr) * 2004-03-15 2005-09-22 Tissugen Pty Ltd Etiologie infectieuse de maladie prostatique et procede d'identification d'agents etiologiques

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
COHEN R.J. ET AL.: "PROPIONIBACTERIUM ACNES ASSOCIATED WITH INFLAMMATION IN RADICAL PROSTATEECTOMY SPECIMENS: A POSSIBLE LINK TO CANCER EVOLUTION", THE JOURNAL OF UROLOGY, vol. 173, no. 6, 2005, pages 1969 - 1974, XP005375240 *
SHANNON B.A. ET AL.: "Links between Propionibacterium acnes and prostate cancer", FUTURE ONCOLOGY, vol. 2, no. 2, 2006, pages 225 - 232 *
SHANNON B.A. ET AL.: "Polymerase chain reaction-based identification of Propionibacterium acnes types isolated from the male urinary tract: evaluation of adolescents, normal adults and men with prostatic pathology", BJU INTERNATIONAL, vol. 98, no. 2, 2006, pages 388 - 392 *
SHANNON B.A. ET AL.: "The antibody response to Propionibacterium acnes is an independent predictor of serum prostate-specific antigen levels in biopsy-negative men", BJU INTERNATIONAL, vol. 101, no. 4, 10 September 2007 (2007-09-10), pages 429 - 435 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2945125A1 (fr) * 2009-05-04 2010-11-05 Ingen Biosciences Methode de diagnostic des infections a propionibacterium acnes
WO2010128232A3 (fr) * 2009-05-04 2011-01-06 Ingen Biosciences Méthode de diagnostic des infections à propionibacterium acnes

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