|Numero di pubblicazione||WO2008092201 A1|
|Tipo di pubblicazione||Richiesta|
|Data di pubblicazione||7 ago 2008|
|Data di registrazione||31 gen 2008|
|Data di priorità||2 feb 2007|
|Numero di pubblicazione||PCT/2008/105, PCT/AU/2008/000105, PCT/AU/2008/00105, PCT/AU/8/000105, PCT/AU/8/00105, PCT/AU2008/000105, PCT/AU2008/00105, PCT/AU2008000105, PCT/AU200800105, PCT/AU8/000105, PCT/AU8/00105, PCT/AU8000105, PCT/AU800105, WO 2008/092201 A1, WO 2008092201 A1, WO 2008092201A1, WO-A1-2008092201, WO2008/092201A1, WO2008092201 A1, WO2008092201A1|
|Inventori||Beverley O'brien, Ronald Cohen|
|Candidato||Tissugen Pty Ltd|
|Esporta citazione||BiBTeX, EndNote, RefMan|
|Citazioni di brevetti (1), Citazioni diverse da brevetti (4), Con riferimenti in (2), Classificazioni (13), Eventi legali (3)|
|Link esterni: Patentscope, Espacenet|
A method for diagnosing prostatic disease
FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
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  and in the urinary tract of adult males  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  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  and has been implicated in several other inflammatory conditions including acne vulgaris, endocarditis, endopthalmitis and osteomyelitis .
A need exists to develop a non-invasive test allowing diagnosis of P. acnes prostatitis in asymptomatic men at a young age prior to the development of benign prostatic hyperplasia or widespread prostatic atrophy, dysplasia and prostate cancer. 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. However, the finding that P. acnes, in particular types IB and II, are common inhabitants of the adult male urethra  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 need therefore exists to develop an alternative, less invasive approach.
In conditions such as acne vulgaris, research has shown that antibody titres to P. acnes are elevated in subjects with severe inflammatory acne [6,8]. This raises the possibility of using immunologic testing for elevated antibody titres against P. acnes as a means to identify men with P. acnes infection of the prostate gland. However, the general population has extremely high natural antibody titres against P. acnes, therefore it is difficult to develop a test which can effectively distinguish patients with P. acnes infection. Since the main antigens of P. acnes are cell surface and/or secreted carbohydrates [9,10], the excess of the human antibody response might be avoided by purifying out more specific protein antigens for use in immunologic tests. This approach has been used to assess immune response in acne patients, using extracts of the total P. acnes cellular proteins or secreted proteins purified from the culture medium [11-13]. Along with secreted proteins, bacterial cell surface proteins are the major bacterial protein targets encountered by the immune system, thus are particularly suitable for use in development of vaccines or immunologic tests .
SUMMARY OF THE INVENTION
In a first aspect of the present invention there is provided 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.
In a second aspect of the present invention there is provided a method according to the first aspect of the invention further comprising the step of measuring the level of prostate specific antigen present. BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows secreted and membrane-anchored cell surface proteins of P. acnes visualized on SDS-PAGE gel and stained with Coomassie Blue.
Figure 2 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).
DETAILED DESCRIPTION OF THE INVENTION
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.
In a first aspect of the present invention there is provided 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.
In a preferred embodiment of the present invention the prostatic disease is selected from the group consisting of prostate cancer, prostatitis and benign prostatic hyperplasia. In a further preferred embodiment of the present invention the body fluid sample is selected from the group consisting of plasma, serum, urine, prostatic secretions and ejaculate.
In a further preferred embodiment 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. In a particularly preferred embodiment, the cell surface proteins include membrane-anchored cell surface proteins.
In a further preferred embodiment of the present invention, the secreted and/or membrane-anchored surface proteins include proteins from P. acnes type IB and II.
In a further preferred embodiment of the present invention the antibodies are IgA. In yet a further preferred embodiment, 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. For example, the enzyme-linked immunosorbent assay (ELISA) 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.
In a further preferred embodiment of the present invention the method comprises quantitating the antibody titre of the body fluid sample to P. acnes protein.
In a second aspect of the present invention there is provided a method according to the first aspect of the invention further comprising the step of measuring the level of prostate specific antigen present.
In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following non- limiting examples. EXAMPLE 1
Extraction of P. acnes secreted/cell surface proteins
Extraction of membrane-anchored surface proteins and secreted proteins from P. acnes was performed using a previously published method developed for Listeria monocytogenes .
P. acnes was grown at 37°C in brain heart infusion broth supplemented with 5% horse serum to late logarithmic growth phase (OD600 = 1.9 to 2.0). 20OmL was harvested by centrifugation (4000 x g for 12mins), the cells were then washed in 6mL of phosphate- buffered saline and resuspended by gently pipetting up and down in ImL of surface protein extraction buffer (1 mL of 1.0 M Tris pH 7.5, lOμL of 0.5 M EDTA, 10 μL of Sigma P8849 protease inhibitor cocktail) to make a thick suspension. After 30mins incubation at 370C with moderate agitation, the cells were pelleted by centrifuging (4000 x g for 12 mins) and the supernatant containing the proteins was carefully drawn off into a clean tube. Protein was quantitated using a standard Bradford assay and stored in small aliquots at minus 8O0C.
20OmL of culture typically yielded protein concentrations of around:
350-450 μg/mL (P. acnes type IB) 650-750 μg /mL (P. acnes type IA) 850-950 μg /mL (P. acnes type II). EXAMPLE 2
Characterization of P. acnes secreted/cell surface proteins by molecular weight
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.
An aliquot of protein mixture containing 180μg of total protein was thawed. After adding one-fifth of a volume of 100% Trichloroacetic acid and vortex mixing, the mixture was cooled at -20° for 5 mins then left at 4°C overnight for protein precipitation to occur. Proteins were pelleted by centrifugation (6000 x g for 10 mins) then given 4 washes with 500μL of ice-cold acetone (analytical grade), remaining in acetone at 0°C for at least 30 minutes. A large amount of carbohydrates precipitated at this step and were removed along with the acetone supernatant. Finally the protein pellet was dried briefly at 37°C, taking care not to overdry, and pretreated with 22.5 μL of 0.2M NaOH for 5 mins. The protein sample was then resolubilized for 2 hours at room temperature in 200μL of 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.
After heating at 95°C for 7 mins, the protein was loaded onto a 4-15% polyacrylamide gel (BioRad Readygel) at a concentration of 20μL 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.
For all three different P. acnes types, 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).
Testing immune reaction of prostate biopsy patients to P. acnes secreted/cell surface proteins by immunoblot
Patient immunoreactivity to P. acnes surface proteins was tested initially by transfer of the proteins to a membrane (western blot) followed by screening with patient serum (immunoblot) to identify which specific protein bands were recognized by patient antibodies.
For immunoblots, 180 μg of total protein was purified by precipitation and run on an SDS-PAGE gel as described in Example 2, except that a 4-15% polyacrylamide gel (BioRad Readygel) with a prep-well format was used. Following electrophoresis the gel was equilibrated for 1 hour in transfer buffer (48mM Tris, 39mM glycine, 0.05% SDS, 20% v/v analytical grade methanol, pH 9.2). PVDF membrane (BioRad Immun- B lot PVDF) was wet in methanol for a few seconds then equilibrated in transfer buffer for 10 mins prior to use. Western blotting was performed in a Mini-Transblot cell (BioRad) for 1 hour and 40 minutes at 100V (constant), after which the membrane was rinsed (3 x 5min with agitation) in de-ionised water and air-dried on Whatman paper overnight. Transfer of proteins was confirmed by use of pre-stained protein molecular weight standards and by Coomassie Blue staining of the gel after transfer.
Immunoblotting was performed with the BioRad Immun-blot Assay kit according to the manufacturers instructions with minor variations as described below. All of the following steps were performed at room temperature, using gentle agitation for steps where the membrane was immersed in liquid. The next morning the membrane was wet in methanol for a few seconds, equilibrated for 10 mins in high-salt Tris-buffered saline (TBS; 2OmM Tris, 50OmM NaCl, pH 7.5) then immersed for 1 hour in blocking solution (TBS with 5% w/v BioRad blotting-grade non-fat milk powder). After 10 mins immersion in wash solution (TBS with 0.05% blotting-grade Tween-20) the membrane was placed into a Mini-Protean 2 Multiscreen Apparatus (BioRad) for screening with patient serum. Each channel was loaded with 600 μL of a patient serum diluted 1:1000 in antibody buffer (wash solution containing 1% w/v BioRad blotting- grade non-fat milk powder) and incubated for 2 hours, with mixing by pipette halfway through this time. For this step the negative control lane (no primary antibody) received only antibody buffer. Each channel was then washed 6 X with wash solution and loaded with 600 μL 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. After washing each channel 6 X with wash solution, the membrane was removed from the multiscreen apparatus and immersed in TBS for 2 X 5 min washes to remove all Tween-20. For colour development the membrane was immersed in 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.
Inclusion of methanol and SDS in the transfer buffer gave good transfer of all protein bands to the membrane for screening (Fig 2). For immunob lotting, a 1 : 1000 dilution of patient serum allowed discrimination of patients into two groups based on strong or weak immunoreactivity to P. acnes surface proteins (Fig 3). Reaction to the different P. acnes types
Each person screened gave a similar reaction to all three P. acnes types, although in most people the reaction to type IA was slightly stronger. In each person screened, the serum antibodies mostly recognized the same-sized protein bands from each of the three P. acnes types, suggesting that the most antigenic surface/secreted proteins are produced by all three types. It is therefore impossible to tell which type(s) of P. acnes had caused the patient antibody response.
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).
Of the 26 prostate biopsy patients screened by this method, 11 (42%) had strong reactivity to P. acnes and the remaining 15 (58%) had weak reactivity. These two groups did not differ significantly in age, prostate volume, percentage with cancer in the biopsy specimens, total amount (mm) of cancer present in the biopsy specimens, or the degree of inflammation in the biopsy specimens. However, the group with strong reactivity to P. acnes did have a statistically significantly higher mean serum prostate specific antigen (PSA) level (12.1 ng/mL compared to 6.9 ng/mL in the group with weak reactivity to P. acnes, p = 0.003).
Since 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. αcwes-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.
Furthermore, similar high levels of reactivity to P. acnes were seen in 2 of the 4 female controls analyzed to date, showing that this high level of antibodies is not specific to prostate biopsy patients only, it can clearly also reflect exposure to P. acnes in other regions in the body.
Assessment of immune reaction to P. acnes surface/secreted proteins by ELISA test
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. We therefore developed an ELISA test using the mixture of P. acnes surface/secreted proteins. Although we used a mixture of proteins from P. acnes type EB and II, the immunoblot results (Example 3) showed that all 3 types of P. acnes produce the same main antigenic proteins, thus the ELISA will detect antibodies raised against any P. acnes type, it is not specific for types IB and II.
Each 96-well polystyrene ELISA plate was used to analyse duplicate samples of serum from 4 patients, using 15μg of surface/secreted proteins in total (7.5 μg from P. acnes type EB mixed with 7.5 μg from P. acnes type II). The proteins were purified by precipitation as described in Example 2, pre-treated with 2μL of 0.2M NaOH for 5 mins then resolubilized in 45 μL of 0.05M carbonate buffer (15mM Na2CO3, 35mM NaH CO3, pH 9.6) by incubating at room temperature for 2 hours with frequent mixing by pipette. This was stored at 40C until use later in the day. Ln the afternoon the protein mixture was diluted to a concentration of 1.5 μg/mL with carbonate buffer and lOOμL 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.
All of the following steps were carried out at room temperature, with reagents warmed to room temperature. Except for the colour development solution (BioRad alkaline phosphatase substrate kit), all reagents were the same as those used for immunoblot in Example 3. Washes were performed by filling the wells by pipette and emptying them by flicking out over the sink then tapping upside down on paper towels.
After protein coating was completed 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 μL of the appropriate patient serum (diluted 1 :32 in antibody buffer) while the next 9 wells received 100 μL of antibody buffer only. 100 μL 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 sera were incubated (covered) for 2 hours, with mixing by pipette after 1 hour. Following 6 X washes with wash solution, each well received 100 μL of secondary antibody solution for 2 hours incubation (covered), with mixing by pipette after 1 hour. Wells were washed 6 X with wash solution, then 2 X with TBS before addition of 100 μL of colour development solution (add 2mL of 5X buffer to 8mL of filtered de-ionised water and dissolve in this two p-Npp tablets just before use) and incubation for 30 mins. The reaction was stopped by addition of 100 μL stop solution (0.4M NaOH) and plates were read at 405nm in a microplate reader. All samples were done in duplicate on the same plate, the mean absorbance readings for each dilution were calculated and the mean absorbance of the blanks was subtracted. Absorbance readings were then multiplied by a standardization factor (the amount required to adjust the positive control to its mean value of 0.280) to allow accurate comparison of samples from different runs. The antibody titre was then determined as the highest dilution giving an absorbance reading greater than 0.100. Results
Initial optimisation steps showed that a P. acnes surface protein concentration of 1.5μg/mL gives a protein coating suitable for ELISA assay of human sera. Two-fold serial serum dilutions from 1 :32 to 1 : 16 384 are suitable for determining antibody titres in most prostate biopsy patients and controls at this concentration of protein coating.
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.
Sera from 68 prostate biopsy patients (mean age of 64.5 years, range 46 to 84 years) were analysed by ELISA. The results by ELISA closely reflected the results obtained by immunoblot for individual patients. Patient antibody titres ranged between 1 :8 to 1:8192 (median = 1:256), showing that the most strongly reactive patients have anti-P. acnes antibody levels around 1000 times higher than the levels found in the most weakly reactive patients. We also analyzed sera from 16 healthy male volunteers (mean age of 55.2 years, range 48 to 70 years) with low serum PSA levels (< 1.5 ng/mL) and no past or present symptoms of prostate disease. These controls had a similar distribution of antibody titres, ranging from 1:64 to 1:8192 (median = 1:256).
Patients were divided into two groups based on anti-P. acnes immune reactivity: a high-titre group with antibody titres equal to or greater than 1:1024 (range 1:1024 to 1:8192) and a low-titre group with antibody titres below 1:1024 (range 1:8 to 1:512). Of the 68 patients analysed, 22 (32%) were in the high-titre group and 46 (68%) were in the low-titre group. The healthy male controls again showed a similar distribution, with six men (37.5%) being in the high-titre group.
Comparison between the high and low-titre patient groups showed that cancer was diagnosed in 13/22 (59%) of high-titre compared to 24/46 (51%) of low-titre patients (p = 0.62). No differences were detected when these groups were compared for multiple other parameters commonly associated with prostate disease, including patient age, serum PSA level, prostate volume (a surrogate indicator of benign prostatic hyperplasia), the extent of inflammation and the grade of aggressive inflammation (data not shown).
Separate analysis was then conducted for patients with a diagnosis of prostate cancer (cancer group, Table 1) and patients with only benign tissue detected on biopsy (biopsy-negative group, Table 2). Prostate volume was significantly larger in the biopsy-negative group (median = 51.0 cm3, range 20.0 to 89.2 cm3) compared to the cancer group (median = 38.0 cm3, range 18.8 to 71.3 cm3, p = 0.004). Anti-P. acnes antibody titres tended to be higher in the cancer group, which contained all patients with titres of 1:4096 (3 cases) and 1:8192 (1 case) although there was no overall difference (median titre = 1 :256 for both cancer and biopsy-negative groups, p = 0.23).
Within the cancer group, comparison of high and low-titre patients again did not show significant differences in any of the parameters commonly associated with prostate disease, including the length of cancer (a surrogate for total cancer volume) or the percent of high grade (Gleason Grade 4/5) cancer present in the biopsy cores (Table 1). However in the biopsy-negative group, a trend towards higher PSA levels was apparent in high-titre patients (p = 0.07). In particular, high-titre patients were almost 5 times more likely to have a PSA level of 10.0 ng/L or greater (p = 0.04) (Table 2). TABLE 1: Cancer group; comparison of parameters associated with prostate disease in patients with high or low titres of antibodies against P. acnes
TABLE 2: Biopsy-negative group; comparison of parameters associated with prostate disease in patients with high or low titres of antibodies against P. acnes
Since high antibody titres were associated with high PSA levels in biopsy-negative patients only, we used regression analysis to determine if different factors were influencing the PSA levels in the cancer group compared to the biopsy-negative patient group. In the cancer group, simple linear regression showed that cancer length (R = 0.40) and patient age (R = 0.40) were statistically significant predictors of PSA (Table 3). In multiple linear regression, a stepwise model identified cancer length as the only significant independent predictor of PSA, explaining 13.7% of the total variance in serum PSA in cancer patients (Table 3).
However, in the biopsy-negative patient group, simple linear regression showed that anti-P. acnes antibody titre (R = 0.40) was a statistically significant predictor of PSA level while patient age (R = 0.36), prostate volume (R = 0.23) and aggressive inflammation grade (R = 0.20) had lower positive predictive values (Table 4). hi multiple linear regression, a stepwise model confirmed anti-P. acnes antibody titre as the only significant independent predictor of PSA, explaining 13.2% of the total variance in serum PSA in biopsy-negative patients (Table 4).
The relationship between patient age, prostate volume, aggressive inflammation grade and P. acnes antibody titre was further explored with a multiple regression model using the former three variables to predict PSA: adding anti-P. acnes titre to this model reduced the predictive values of age by 1.3 times, of prostate volume by 1.4 times and of aggressive inflammation grade by 4.8 times (not shown), to give the final coefficients seen in the block entry model (Table 4). These results suggest that anti-P. acnes titre is correlated to serum PSA partly through the mechanisms of increasing age, prostate volume and inflammation, which indicate the development of subclinical benign prostatic hyperplasia (BPH). TABLE 3: Cancer group; simple and multiple linear regression analysis to determine predictors of serum PSA level
TABLE 4: Biopsy-negative group; simple and multiple linear regression analysis to determine predictors of serum PSA level
We have shown that 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. The presence of antibody titre in the regression model reduced the predictive values of age 1.3-fold, prostate volume 1.4-fold and aggressive inflammation grade 4.8- fold, indicating that the antibody titre is linked to PSA levels partly through these factors. Since age-associated increases in both prostate volume and serum PSA mainly reflect subclinical BPH  which is associated with marked inflammation , our results suggest that P. acnes infection of the prostate gland may be related to development of BPH through generation of inflammation. This is in accord with our finding of significantly larger prostate volumes in the biopsy-negative patient group, indicating the presence of subclinical BPH.
Although the volume of cancer was the main factor influencing PSA levels in the cancer group, we did note a tendency for higher anti-P. acnes antibody titres in the cancer patients compared to the biopsy-negative patients. Certainly all patients with titres of 1 :4096 (3 cases) and 1:8192 ( 1 case) were in the cancer group. It is possible that screening of a larger patient group may confirm a significant association between high anti-P. acnes antibody titres and development of prostate cancer.
While our ELISA assay has shown some evidence of relationships between high anti-P. acnes antibody titres and inflammation-related prostate diseases such as BPH and prostate cancer, the assay is clearly not specific for detecting P. acnes infection of the prostate gland. High antibody titres were also found in some subjects without infection of the prostate gland (for example in female controls who do not have a prostate gland) and in some healthy male controls with very low PSA levels, who are unlikely to have infection of the 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. Alternately they may indicate individuals with a hypersensitive immune response to normal levels of P. acnes antigen exposure. These sensitized individuals may be likely to develop a strong inflammatory reaction in response to infections involving P. acnes, including infection of the prostate gland, thus may be at increased risk of developing inflammation-related prostate diseases such as BPH and/or prostate cancer.
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]. We propose that 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.
Assessment of local immune reaction to P. acnes surface/secreted proteins using IgA ELISA to test urine, prostatic secretions or ejaculate.
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 .
In contrast, the serum contains 5 -fold lower levels of IgA antibodies compared to IgG . IgA is locally produced within mucosal lymphoid tissues , 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 . Similarly ELISA to detect Chlamydia trachomatis-specific IgA in ejaculate has been used to assess the role of these bacteria in prostatic infection . Development of an ELISA that is more highly specific for infection in a particular organ may be possible using secondary antibodies that detect secretory IgA, which comprises only 1.6% of the total IgA in serum but is the predominant antibody produced by local mucosal tissue . 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 . ELISA to detect coliform-specifϊc secretory IgA in urine has been used to assess the role of these bacteria in urinary tract infections , while detection of total secretory IgA in prostatic secretions and prostate tissue by radioimmunoassay has been used to assess the role of this antibody in prostatic infections .
ELISAs to detect IgA and secretory IgA are carried out as described in Example 4, with the following exceptions:
1. Instead of patient serum, the primary antibody solution contains either urine, expressed prostatic secretions, or ejaculate.
2. Instead of anti -human IgG, the secondary antibody used is anti-human IgA (for detection of IgA) or anti-human secretory component (for detection of secretory IgA). Overall Conclusions
In patients undergoing prostate biopsy due to either elevated serum PSA level or abnormal DRE examination, there is a significant positive association between high PSA level (> 8.0 ng/mL) and high P. acnes-specific antibody titre (> 1:1024). However the prostate biopsy patients with high P. αc/zes-specific antibody titres (group 1) 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.
Since 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. αcwes-specifϊc immune reactivity in this subset of patients (group 1).
High levels of P. αcwes-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). We propose that 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.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
1. Cohen RJ, Shannon BA, McNeal JE, Shannon T, Garrett KL.
Propionibacterium acnes associated with inflammation in radical prostatectomy specimens: a possible link to cancer evolution? J Urol 2005: 173:1969-1974.
2. Shannon BA, Cohen RJ, Garrett KL. PCR-based identification of
Propionibacterium acnes types isolated from the male urinary tract: evaluation of adolescents, normal adults and males with prostate pathology. BJU International 2006: 98:388-392.
3. McDowell A, Valanne S, Ramage G, et al. Propionibacterium acnes types I and II represent phylogenetically distinct groups. J Clin Microbiol 2005: 43:326-
4. Valanne S, McDowell A, Ramage G, et al. CAMP factor homologues in Propionibacterium acnes: a new protein family differentially expressed by types I and II. Microbiology 2005: 151:1369-1379.
5. deMarzo AM, Platz EA, Sutcliffe S, et al. Inflammation in prostate carcinogenesis. Nature Reviews 2007: 7:256-269.
6. Webster G F. Inflammation in acne vulgaris. J Am Acad Dermatol 1995:
7. Funke G, von Graevenitz A, Clarridge III JE, Bernard KA. Clinical microbiology of coryneform bacteria. Clin Microbiol Rev 1997: 10:125-159.
8. Burkhart CG, Burkhart CN, Lehman PF: Acne: a review of immunologic and microbiological factors. Postgrad. Med. J 1999: 75:328-331.
9. Webster GF, Indrisano JP, Leyden JJ. Antibody titres to Propionibacterium acnes cell wall carbohydrate in nodulo-cystic acne patients. J Invest Dermatol 1985: 84:496-500. 10. Burkhart CG, Cantrill JL, Butcher CL, Lehmann PF. Propionibacterium acnes: interaction with complement and development of an enzyme-linked immunassay for the detection of antibody. Int J Dermatol 1999: 38:200-203.
11. Holland KT, Holland DB, Cunliffe WJ, Cutcliffe AG. Detection of Propionibacterium acnes polypeptides which have stimulated an immune response in acne patients but not in normal individuals. Experimental Dermatology 1993: 2:12-16.
12. Basal E, Jain A, Kaushal GP. Antibody response to crude cell lysate of Propionibacterium acnes and induction of pro-inflammatory cytokines in patients with acne and normal healthy subjects. J Microbiology 2004: 42:117-
13. Lodes MJ, Secrist H, Benson DR, et al. Variable expression of immunoreactive surface proteins of Propionibacterium acnes. Microbiology 2006: 152: 3667- 3681.
14. Rodriguez-Ortega MJ, Norais N, Bensi G, Liberatori S, Capo S, Mora M, et al. Characterization and indentification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome. Nature Biotechnology 2006: 24:191-197.
15. Schaumberg J, Diekmann O, Hagendorff P, Bergmann S, Rohde M, Hammerschmidt S, Jansch L, Wehland J, Karst U. The cell wall subproteome of
Listeria monocytogenes. Proteomics 2004, 4:2991-3006.
16. MacLennan GT, Eisenberg R, Fleshman RL, Taylor JM, Fu P, Resnick MI, Gupta S. The influence of chronic inflammation in prostatic carcinogenesis: a 5- year followup study. The Journal of Urology 2006: 176:1012-1016. 17. Battikhi MN, Ismail H, Battikhi Q. Effects of chronic bacterial prostatitis on prostate specific antigen levels total and free in patients with benign prostatic hyperplasia and prostate cancer, hit Urol Nephrol 2006: 38:21-26.
18. Nadler RB, Humphrey PA, Smith DS, et al. Effect of inflammation and benign prostatic hyperplasia on elevated serum prostate specific antigen levels. J Urol
19. Okada K, Kojima M, Naya Y, et al. Correlation of histological inflammation in needle biopsy specimens with serum prostate specific antigen levels in men with negative biopsy for prostate cancer. Urology 2000: 55:892-898.
20. Kwak C, Ku JH, Kim T, et al. Effect of subclinical prostatic inflammation on serum PSA levels in men with clinically undetectable prostate cancer. Urology 2003: 62:854-859.
21. Oesterling JE, Jacobsen SJ, Chute CG, et al. Serum prostate-specific antigen in a community-based population of healthy men: establishment of age-specific reference ranges. JAMA 1993: 270:860-864.
22. Nickel JC. Inflammation and benign prostatic hyperplasia. Urol Clin N Am 2007: 35:109-115.
23. Vazquez S, Cabezas S, Perez AB, et al. Kinetics of antibodies in sera, saliva and urine samples from adult patients with primary or secondary dengue 3 virus infections. Int J Infect Dis 2007: 11 :256-262.
24. Andreu-Ballester JC, Perez-Griera J, Ballester F, et al. Secretory immunoglobulin A (slgA) deficiency in serum of patients with GALTectomy (appendectomy and tonsillectomy). Clin Immunol 2007: 123:289-297.
25. Giraldo PC, Goncalves AK, Eleuterio J(Jr). Secretory immunoglobulin A: a protective factor in the genital mucosa. Braz J Infect Dis 2006: 10:232-234. 26. Ethel S, Bhat GK, Hegde BM. Bacterial adherence and humoral immune response in women with symptomatic and asymptomatic urinary tract infection. Ind J Med Microbiol 2006: 24:30-33.
27. Mazzoli S, Cai T, Rupealta V, et al. Interleukin 8 and anύ-Chlamydia trachomatis mucosal IgA as urogenital immunologic markers in patients with C. trachomatis prostatic infection. Eur Urol 2007: 51:1385-1393.
28. Yi Fx, Wei Q, Li H, et al. Risk factors for prostatic inflammation extent and infection in benign prostatic hyperplasia. Asian J Androl 2006: 8: 621-627.
|Brevetto citato||Data di registrazione||Data di pubblicazione||Candidato||Titolo|
|WO2005087929A1 *||15 mar 2005||22 set 2005||Tissugen Pty Ltd||Infectious aetiology of prostatic disease and methods to identify causative agents|
|1||*||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|
|2||*||SHANNON B.A. ET AL.: "Links between Propionibacterium acnes and prostate cancer", FUTURE ONCOLOGY, vol. 2, no. 2, 2006, pages 225 - 232|
|3||*||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|
|4||*||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|
|Brevetto con rif.||Data di registrazione||Data di pubblicazione||Candidato||Titolo|
|WO2010128232A2 *||21 apr 2010||11 nov 2010||Ingen Biosciences||Method for diagnosing propionibacterium acnes infections|
|WO2010128232A3 *||21 apr 2010||6 gen 2011||Ingen Biosciences||Method for diagnosing propionibacterium acnes infections|
|Classificazione internazionale||C12Q1/04, G01N33/53, G01N33/50, G01N33/68|
|Classificazione cooperativa||G01N33/6893, G01N2800/342, G01N33/56911, G01N33/6854, G01N33/57434|
|Classificazione Europea||G01N33/68B, G01N33/569D, G01N33/68V, G01N33/574C14|
|17 set 2008||121||Ep: the epo has been informed by wipo that ep was designated in this application|
Ref document number: 08700401
Country of ref document: EP
Kind code of ref document: A1
|4 ago 2009||NENP||Non-entry into the national phase in:|
Ref country code: DE
|31 mar 2010||122||Ep: pct application non-entry in european phase|
Ref document number: 08700401
Country of ref document: EP
Kind code of ref document: A1