Even if cranberry is a supplement food, the study has been approved by the different research ethics committees (1/Comité d'éthique Sud Méditerranée III, Nîmes, France; 2/Comité Etico de Investigaciòn Clinica (CEIC) de Fundació Puigvert, Barcelona, Spain; 3/Ethical committee of Jahn Ferenc South-Pest Teaching Hospital, Hungary; 4/Ethics Committee of Medicine and Medical Care, University of Occupational and Environmental Health, Japan), in each country and has been conducted according to the principles expressed in the Declaration of Helsinki. Thirty-two female, sexually-active volunteers over the age of 18, with normal renal function were recruited through four urology departments in Japan (Kyushu University Hospital), Hungary (South-Pest Hospital, Budapest), Spain (Fundació Puigvert, Barcelona), and France (Hôpital Foch, Suresnes) (eight volunteers/department) to participate in this randomized, double-blind, placebo-controlled cross-over study. Volunteers were cleared through ethics committees and provided informed consent. Exclusion criteria included antibiotic use within six months prior to the study, pregnancy, known allergy or intolerance of cranberry products, or routine consumption of any food supplements consisting of vitamins, minerals or trace elements. Throughout the study, volunteers were instructed not to alter their dietary or lifestyle habits in any way. However, during the capsule consumption, volunteers were told to avoid all Vaccinium-containing foods, drinks and supplements including other forms of V. macrocarpon (cranberry), V. myrtillus (bilberry or European blueberry), V. angustifolium (wild or lowbush blueberry), V. corymbosum (highbush blueberry), and V. vitis-ideae (lingonberry). In addition, volunteers were instructed to limit consumption of chocolate, tea, grape and wine. All medications taken during the study were reported to the doctor responsible for following the study at each center.
The study was carried out using commercially available capsules of cranberry powder (Urell®/Ellura™ Pharmatoka, Rueil Malmaison, France) and capsules of placebo composed of colloidal silica, magnesium stearate, cellulose and gelatin. Capsules were opaque to conceal the color of the contents. The PACs in the cranberry powder were quantified by Brunswick Laboratories (Norton, MA) using a colorimetric assay, an updated dimethylaminocinnamaldehyde method (DMAC method), taking advantage of the selective colorimetric reaction between PACs and DMAC after open column gel chromatography on Sephadex® LH-20 (Amersham). The capsule dosages were standardized to deliver 18 or 36 mg of PAC equivalents in the cranberry powder.
The volunteers in Japan and Hungary received 0, 36 or 72 mg PAC equivalents per day and those in France and Spain received 0, 18 or 36 mg PAC equivalents per day. Each volunteer received successively three regimens (always 2 capsules) distributed in random order, consisting of: (1) cranberry (2 PAC dosage levels), or (2) placebo, or (3) 1 capsule of cranberry at each PAC dosage and 1 capsule of placebo, with a washout period of at least one week between each regimen. Volunteers consumed the capsules in the morning at 8:00 AM. The first urines were collected from 9:00 AM-2:00 PM following capsule consumption, and pooled. The second collections were made the following morning (8:00 AM). Pre-cranberry consumption urines collections (0 h) were obtained at the beginning of the study in each volunteer. Various biological and physicochemical parameters of the urine samples (collected at each regimen) were measured using the Multistix® (Bayer) system. Urines with an abundance of leukocytes and nitrites were excluded. Remaining samples were centrifuged at 4000 g for 15 minutes, sterilized by filtration (0.45 μm), separated in 3 aliquots and stored at -20°C.
A uropathogenic E. coli strain previously isolated from a patient with UTI (NECS978323) [12 (link)], with P-fimbriae papG and type-1 pili was utilized. To allow the direct observation of adherent bacteria by fluorescence microscopy, NECS978323 was genetically modified to express green fluorescent protein (GFP) using a pBBR-derived non-mobilizable plasmid carrying a GFP expression cassette [24 (link)]. Bacteria were grown in trypticase soy broth (bioMerieux, Marnes La Coquette, France) or colonization factor antigen agar for 16 h at 37°C to enhance expression of P-fimbriae.
To insure product potency, the PAC-standardized cranberry powder was tested for in vitro uropathogenic bacterial anti-adhesion activity prior to consumption by the volunteers, utilizing a MRHA, to detect anti-adhesion activity in uropathogenic P-fimbriated E. coli [11 (link)]. Briefly, the anti-adhesion bioactivity of the powder was tested by measuring the ability of the fractions to suppress agglutination of human red blood cells (HRBC) (A1, Rh+) following incubation with uropathogenic P-fimbriated E. coli. Bacteria were suspended in phosphate-buffered saline (PBS) solution at pH 7.0 at a concentration of 5 × 108 bacteria/mL. The powder was dissolved in PBS at a starting concentration of 60 mg/mL, and the pH adjusted to neutrality with NaOH. A serial 2-fold dilution was prepared, and each dilution (30 mL) was incubated with 10 mL of bacterial suspension on a 24-well polystyrene plate for 10 min at room temperature on a rotary shaker. A 3% v/v suspension of HRBCs in PBS was prepared, and 10 mL of the diluted blood was added to the test suspension. The suspension was incubated for 20 min on a rotary shaker at 21C and evaluated microscopically for the ability to prevent agglutination. The final dilution concentration was recorded at which agglutination suppression by the cranberry fraction occurred. Wells containing bacteria plus PBS, HRBC plus PBS, bacteria plus test fraction, and HRBC plus test fraction served as negative controls for agglutination, and wells containing bacteria plus HRBC served as a positive control for agglutination. Assays were performed in triplicates.
Urines were tested ex vivo for anti-adhesion activity before and after the treatment regimes, utilizing two separate assays. The MRHA assay described above was modified by substituting urine for a cranberry solution. Briefly, a 30-μL drop of each urine was incubated with 10 μL of the bacterial suspension on a 24-well polystyrene plate for 10 min at 21C on a rotary shaker. The HRBCs were added to the urines, incubated for 20 min on a rotary shaker at 21C and evaluated microscopically for the ability to prevent agglutination. If no agglutination was observed, the urine was considered to contain anti-adhesion metabolites and was recorded as possessing Anti-Adhesion Activity (AAA). The results were expressed as a percentage of anti-adhesion activity (0% MRHA = 100% AAA, 50% MRHA = 50% AAA and 100% MRHA = 0% AAA). Assays were repeated 4 times on triplicate urine samples and the standard error calculated.
In the second ex vivo urine assay, bacterial adhesion was evaluated utilizing the human T24 epithelial cell-line (ATCC HTB-4). A new technology was developed using fluorescent NECS978323 to enhance detection of strain adhesion. Monolayers of epithelial cells were grown at 37°C in McCoy's 5a medium containing 10% (v/v) fetal calf serum, 1.5 mM glutamine, and antibiotics (50 U/mL penicillin and 50 mg/mL of streptomycin), on coverslips in 24-well Falcon tissue culture plates. Bacteria were grown overnight in the urine samples containing 5% (v/v) Luria broth. Bacterial were harvested by centrifugation, resuspended at DO600 0.1 in McCoy's medium, added to the tissue culture and incubated for 2.5 h at 37°C. After washes with PBS, the cells were fixed with 4% paraformaldehyde, incubated 20 min at room temperature, and examined under a fluorescent microscope. An adhesion index (AI) corresponding to the mean number of adherent bacteria per cell for 100 cells was calculated. This index was expressed as the mean of at least three independent assays.
C. elegans has been used to develop an easy model system of host-pathogen interactions to identify basic evolutionarily conserved pathways associated with microbial pathogenesis. This test is based upon the capacity of E. coli to be ingested by C. elegans nematodes leading to infection and ultimately involving the killing death of the worms [25 (link)]. Percentage of killed nematodes in presence of the E. coli strain is an indirect marker of virulence potential of this strain. The C. elegans infection assay was carried out as described by Lavigne et al. [25 (link)] using Fer-15 mutant line, which has a temperature sensitive fertility defect. Fer-15 was provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). Briefly to synchronize the growth of worms, eggs were collected using the hypochlorite method. E. coli strain was grown for overnight in human urine containing 5% (v/v) nematode growth medium (NGM). Bacteria cells were harvested by centrifugation, washed once and suspended in phosphate buffered saline solution (PBS) at pH 7.0 at a concentration of 105 CFU/ml. NGM agar plates were inoculated with 10 μl of strain and incubated at 37°C for 8-10 h. Plates were allowed to cool to room temperature and seeded with L4 stage worms (20-30 per plate). Plates were then incubated at 25°C and scored each day for live worms under a stereomicroscope (Leica MS5). At least three replicates repeated 5 times were performed for each selected clone. A worm was considered dead when it no longer responded to touch. Worms that died as a result of becoming stuck to the wall of the plate were excluded from the analysis. Lethal Time 50% (LT50) and death (LT100) corresponded to time (in hours) required to kill 50% and 100% of the nematode population, respectively. OP50 is an avirulent E. coli strain, an international standard food for nematodes used as control. The number of bacteria within the C. elegans digestive tract was carried out as described by Garsin et al. [26 (link)]. Five C. elegans were picked at 72 h, and the surface bacteria were removed by washing the worms twice in 4 μl drops of M9 medium on a NGM agar plate containing 25 μg/ml gentamicin. The nematodes were placed in a 1.5 ml Eppendorf tube containing 20 μl of M9 medium with 1% Triton X-100 and were mechanically disrupted by using a pestle. The volume was adjusted to 50 μl with M9 medium containing 1% Triton X-100 which was diluted and plated on Luria-Bertani agar containing 50 μg/ml ampicillin. At least three replicates were performed for each assay.
The quantitative variables were described by the median values, the range and the mean, and standard deviation. The qualitative variables were described by figures and percentages. The 95% confidence intervals were assessed by the exact method of Clopper-Pearson. Frequencies between AAA = 0% and AAA > 0% were compared according to the criteria using a Fisher exact test and the index values were compared using a Kruskal-Wallis test. The index value was modelled by the hours, the country and the dose using a Generalized Estimating Equation model. Survival curves of the worms were explored in a univariate analysis (Kaplan-Meier curves). The median values of survival times were given. The survival curves were compared using log-rank test. A multivariate survival analysis was then performed (Cox model). No procedure of variables selection was performed. The assumptions of proportional hazards were checked. A value of p ≤ 0.05 given by the SAS®/ETS software (version 8.1) (SAS Institute Inc, Cary, NC, USA) was considered statistically significant.
The study was carried out using commercially available capsules of cranberry powder (Urell®/Ellura™ Pharmatoka, Rueil Malmaison, France) and capsules of placebo composed of colloidal silica, magnesium stearate, cellulose and gelatin. Capsules were opaque to conceal the color of the contents. The PACs in the cranberry powder were quantified by Brunswick Laboratories (Norton, MA) using a colorimetric assay, an updated dimethylaminocinnamaldehyde method (DMAC method), taking advantage of the selective colorimetric reaction between PACs and DMAC after open column gel chromatography on Sephadex® LH-20 (Amersham). The capsule dosages were standardized to deliver 18 or 36 mg of PAC equivalents in the cranberry powder.
The volunteers in Japan and Hungary received 0, 36 or 72 mg PAC equivalents per day and those in France and Spain received 0, 18 or 36 mg PAC equivalents per day. Each volunteer received successively three regimens (always 2 capsules) distributed in random order, consisting of: (1) cranberry (2 PAC dosage levels), or (2) placebo, or (3) 1 capsule of cranberry at each PAC dosage and 1 capsule of placebo, with a washout period of at least one week between each regimen. Volunteers consumed the capsules in the morning at 8:00 AM. The first urines were collected from 9:00 AM-2:00 PM following capsule consumption, and pooled. The second collections were made the following morning (8:00 AM). Pre-cranberry consumption urines collections (0 h) were obtained at the beginning of the study in each volunteer. Various biological and physicochemical parameters of the urine samples (collected at each regimen) were measured using the Multistix® (Bayer) system. Urines with an abundance of leukocytes and nitrites were excluded. Remaining samples were centrifuged at 4000 g for 15 minutes, sterilized by filtration (0.45 μm), separated in 3 aliquots and stored at -20°C.
A uropathogenic E. coli strain previously isolated from a patient with UTI (NECS978323) [12 (link)], with P-fimbriae papG and type-1 pili was utilized. To allow the direct observation of adherent bacteria by fluorescence microscopy, NECS978323 was genetically modified to express green fluorescent protein (GFP) using a pBBR-derived non-mobilizable plasmid carrying a GFP expression cassette [24 (link)]. Bacteria were grown in trypticase soy broth (bioMerieux, Marnes La Coquette, France) or colonization factor antigen agar for 16 h at 37°C to enhance expression of P-fimbriae.
To insure product potency, the PAC-standardized cranberry powder was tested for in vitro uropathogenic bacterial anti-adhesion activity prior to consumption by the volunteers, utilizing a MRHA, to detect anti-adhesion activity in uropathogenic P-fimbriated E. coli [11 (link)]. Briefly, the anti-adhesion bioactivity of the powder was tested by measuring the ability of the fractions to suppress agglutination of human red blood cells (HRBC) (A1, Rh+) following incubation with uropathogenic P-fimbriated E. coli. Bacteria were suspended in phosphate-buffered saline (PBS) solution at pH 7.0 at a concentration of 5 × 108 bacteria/mL. The powder was dissolved in PBS at a starting concentration of 60 mg/mL, and the pH adjusted to neutrality with NaOH. A serial 2-fold dilution was prepared, and each dilution (30 mL) was incubated with 10 mL of bacterial suspension on a 24-well polystyrene plate for 10 min at room temperature on a rotary shaker. A 3% v/v suspension of HRBCs in PBS was prepared, and 10 mL of the diluted blood was added to the test suspension. The suspension was incubated for 20 min on a rotary shaker at 21C and evaluated microscopically for the ability to prevent agglutination. The final dilution concentration was recorded at which agglutination suppression by the cranberry fraction occurred. Wells containing bacteria plus PBS, HRBC plus PBS, bacteria plus test fraction, and HRBC plus test fraction served as negative controls for agglutination, and wells containing bacteria plus HRBC served as a positive control for agglutination. Assays were performed in triplicates.
Urines were tested ex vivo for anti-adhesion activity before and after the treatment regimes, utilizing two separate assays. The MRHA assay described above was modified by substituting urine for a cranberry solution. Briefly, a 30-μL drop of each urine was incubated with 10 μL of the bacterial suspension on a 24-well polystyrene plate for 10 min at 21C on a rotary shaker. The HRBCs were added to the urines, incubated for 20 min on a rotary shaker at 21C and evaluated microscopically for the ability to prevent agglutination. If no agglutination was observed, the urine was considered to contain anti-adhesion metabolites and was recorded as possessing Anti-Adhesion Activity (AAA). The results were expressed as a percentage of anti-adhesion activity (0% MRHA = 100% AAA, 50% MRHA = 50% AAA and 100% MRHA = 0% AAA). Assays were repeated 4 times on triplicate urine samples and the standard error calculated.
In the second ex vivo urine assay, bacterial adhesion was evaluated utilizing the human T24 epithelial cell-line (ATCC HTB-4). A new technology was developed using fluorescent NECS978323 to enhance detection of strain adhesion. Monolayers of epithelial cells were grown at 37°C in McCoy's 5a medium containing 10% (v/v) fetal calf serum, 1.5 mM glutamine, and antibiotics (50 U/mL penicillin and 50 mg/mL of streptomycin), on coverslips in 24-well Falcon tissue culture plates. Bacteria were grown overnight in the urine samples containing 5% (v/v) Luria broth. Bacterial were harvested by centrifugation, resuspended at DO600 0.1 in McCoy's medium, added to the tissue culture and incubated for 2.5 h at 37°C. After washes with PBS, the cells were fixed with 4% paraformaldehyde, incubated 20 min at room temperature, and examined under a fluorescent microscope. An adhesion index (AI) corresponding to the mean number of adherent bacteria per cell for 100 cells was calculated. This index was expressed as the mean of at least three independent assays.
C. elegans has been used to develop an easy model system of host-pathogen interactions to identify basic evolutionarily conserved pathways associated with microbial pathogenesis. This test is based upon the capacity of E. coli to be ingested by C. elegans nematodes leading to infection and ultimately involving the killing death of the worms [25 (link)]. Percentage of killed nematodes in presence of the E. coli strain is an indirect marker of virulence potential of this strain. The C. elegans infection assay was carried out as described by Lavigne et al. [25 (link)] using Fer-15 mutant line, which has a temperature sensitive fertility defect. Fer-15 was provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). Briefly to synchronize the growth of worms, eggs were collected using the hypochlorite method. E. coli strain was grown for overnight in human urine containing 5% (v/v) nematode growth medium (NGM). Bacteria cells were harvested by centrifugation, washed once and suspended in phosphate buffered saline solution (PBS) at pH 7.0 at a concentration of 105 CFU/ml. NGM agar plates were inoculated with 10 μl of strain and incubated at 37°C for 8-10 h. Plates were allowed to cool to room temperature and seeded with L4 stage worms (20-30 per plate). Plates were then incubated at 25°C and scored each day for live worms under a stereomicroscope (Leica MS5). At least three replicates repeated 5 times were performed for each selected clone. A worm was considered dead when it no longer responded to touch. Worms that died as a result of becoming stuck to the wall of the plate were excluded from the analysis. Lethal Time 50% (LT50) and death (LT100) corresponded to time (in hours) required to kill 50% and 100% of the nematode population, respectively. OP50 is an avirulent E. coli strain, an international standard food for nematodes used as control. The number of bacteria within the C. elegans digestive tract was carried out as described by Garsin et al. [26 (link)]. Five C. elegans were picked at 72 h, and the surface bacteria were removed by washing the worms twice in 4 μl drops of M9 medium on a NGM agar plate containing 25 μg/ml gentamicin. The nematodes were placed in a 1.5 ml Eppendorf tube containing 20 μl of M9 medium with 1% Triton X-100 and were mechanically disrupted by using a pestle. The volume was adjusted to 50 μl with M9 medium containing 1% Triton X-100 which was diluted and plated on Luria-Bertani agar containing 50 μg/ml ampicillin. At least three replicates were performed for each assay.
The quantitative variables were described by the median values, the range and the mean, and standard deviation. The qualitative variables were described by figures and percentages. The 95% confidence intervals were assessed by the exact method of Clopper-Pearson. Frequencies between AAA = 0% and AAA > 0% were compared according to the criteria using a Fisher exact test and the index values were compared using a Kruskal-Wallis test. The index value was modelled by the hours, the country and the dose using a Generalized Estimating Equation model. Survival curves of the worms were explored in a univariate analysis (Kaplan-Meier curves). The median values of survival times were given. The survival curves were compared using log-rank test. A multivariate survival analysis was then performed (Cox model). No procedure of variables selection was performed. The assumptions of proportional hazards were checked. A value of p ≤ 0.05 given by the SAS®/ETS software (version 8.1) (SAS Institute Inc, Cary, NC, USA) was considered statistically significant.
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