Evaluation of Side Effects and Local Immune Response by Local Administration of Selected Strains
Elisa Vintiñi, Virginia Ocaña and María Elena Nader-Macías
Lactobacilli are the predominant microorganisms in the vaginal tract of human and some homeothermic animals (1). They can maintain the ecological equilibrium of the tract by protecting against pathogenic microorganisms (2). In the last few years, there has been an increased tendency to use probiotic microorganisms to restore the ecological equilibrium and to protect against infections (3). This principle has been widely applied to the gastrointestinal tract (4). More recently, some other studies have reported the application of probiotics in different tracts, for example, the urogenital (5) or respiratory tract (6).
One of the objectives of our group is to design probiotic products for the urogenital tract. With this purpose, lactobacilli were isolated from the human vagina (7), and later some of them were selected for their probiotic characteristics (production of antagonistic substances or adhesion capability) (8-11).
The application of probiotic products in the vaginal tract has been approached empirically; some pharmaceuticals containing these microorganisms (12) are available in the United States or Europe or are protected under the patent process or intellectual property rights (13-15). There are not enough studies in humans or animals to determine whether their administration can produce some type of collateral or adverse effect.
Using Balb/c mice as the experimental model, the object of the present work was to study (1) whether intravaginal administration of human lactobacilli can produce colonization of the tract; (2) whether such administration produces some type of adverse or
From: Methods in Molecular Biology, vol. 268: Public Health Microbiology: Methods and Protocols Edited by: J. F. T. Spencer and A. L. Ragout de Spencer © Humana Press Inc., Totowa, NJ
collateral effect; and (3) whether probiotics are able to stimulate the local immune system. Keeping in mind that hormones can affect the colonization or persistence ability of microorganisms, and with the purpose of having all animals at the same point in the sexual cycle, animals were cycled with estradiol 48 h before inoculation with lac-tobacilli. They were then inoculated im with hormones 48 h before beginning microorganism inoculations. Later they were intravaginally inoculated with the appropriate dose of each Lactobacillus strains. The animals were sacrificed on different days after inoculation to perform the following studies:
1. Microbiological assays: To determine the number of lactobacilli in the tract (in vaginal washes or in organ homogenates), by plating the samples in selective media containing antibiotic (to differentiate the resident flora from those administered experimentally).
2. Histological studies: To evaluate whether the local administration of lactobacilli produced some type of modification at the structural level in the tract.
3. Immunofluoresce assays: To study whether local administration of lactobacilli produced stimulation of cells involved in the immune response at the local level.
1. The different Lactobacillus strains are given in Table 1. They were isolated from human vaginal samples as described before (6) and used for the experiments as described in Subheading 2.3. Their taxonomic identification and relevant characteristic are also shown in Table 1.
2. Storage (in milk-yeast extract): 10% skim milk, 0.5% yeast extract, 1% glucose. Sterilize at 121°C for 15 min and store at refrigerated temperature.
3. Subcultivation (in LAPTg broth): 1.5% meat peptone, 1% tryptone, 1% yeast extract, 1% glucose, 0.1% Tween-80, final pH 6.5. Sterilize by autoclaving and store at refrigeration temperature.
1. Characteristics and conditions.
a. Adult female Balb/c mice were fed ad libitum and kept in metallic cages, with wooden beds.
b. The room temperature was between 19 and 23°C.
c. The light conditions were between 5 and 60 lux in the cages and between 250 and 300 lux in the room.
d. Inbred 7-wk-old female Balb/c mice weighing between 25 and 30 g were used. They came from a closed colony and were randomly paired.
e. All the animals were fed a conventional diet, including the control group.
2. Hormone inoculation: Animals were cycled to the same point of the sexual cycle by using estradiol valerate (0.05 mL Progynon Depot, Schering, Germany).
2.3. Experimentsin Mice
1. Peptone-water agar: 1% peptone, 1.5% agar. Sterilize at 121°C for 15 min (by autoclav-ing) and store at refrigerated temperature.
2. Diluent media (peptone water): 0.1% meat peptone (Britania, Argentina).
Strains and Characteristics of Lactobacilli
L. crispatus 1266
L. paracasei ss. paracasei 1251
L. paracasei ssp paracasei 1289
L. acidophilus 1294
L. salivarius ss. salivarius 1328
L. helveticus 1259
H2O2 producer H2O2 producer H2O2 producer Self- and co-aggregating Bacteriocin producer Lactic acid producer
3. Culture media: de Man-Ragosa-Sharpe (MRS) agar: 1% peptone, 1% meat extract, 0.5% yeast extract, 0.5% sodium acetate, 0.2% ammonium citrate, 0.2% potassium phosphate, 0.02% Mg sulfate, 0.05% Mn sulfate, 2% glucose, 0.1% Tween-80, 1.5% agar, final pH 6.5. Sterilize by autoclaving at 121°C for 15 min and store at refrigeration temperature.
4. MRS agar with antibiotic: chloramphenicol (Sigma, St. Louis, MO) stored at refrigerated temperature.
5. Agarized peptone water: 1% peptone, 1.5% agar. Sterilize by autoclaving and stored at refrigerated temperature.
6. Distilled water.
7. Other materials.
a. Glass tubes.
b. Nitrocellulose filter membranes (type GS, Sigma), pore size 0.22 ^m, diameter 25 mm.
c. Automatic pipets.
d. Povidone iodine (10% topical solution of iodated povidone).
e. Petri dishes.
f. Tissue homogenizer with Teflon pestle (MSE, Sussex, UK).
g. Glass tubes.
h. Glass slides.
i. Water bath (Vicking S.R.L. Masson, Argentina). j. Sterile tips for pipets.
k. Dissection board.
o. Dissection pliers.
p. Glass bottles.
2.4. Microbiological Assays
1. Vaginal washes.
a. Sterile saline solution (0.85% sodium chloride), sterilized by autoclaving and stored at refrigeration temperature.
b. Automatic micropipets.
c. Glass tubes with sterile saline solution (sterilized by autoclaving at 121°C for 15 min and stored at refrigeration temperature).
d. Glass slides.
e. Petri dishes.
g. Crystal violet solution.
h. Iodine solution.
i. Safranin solution (Gram staining kit. Biopack, Argentina).
a. Glass tubes with sterile saline solution (sterilized by autoclaving at 121°C for 15 min and stored at refrigeration temperature).
b. Petri dishes.
d. Glass tubes containing peptone water (sterilized by autoclaving at 121°C for 15 min and stored at refrigeration temperature).
e. Tissue homogenizer with Teflon pestle (MSE, Sussex, UK).
f. Glass slides.
3. MRS agar (described before in Subheading 2.3., item 3.).
4. MRS agar with chloramphenicol (described before in Subheading 2.3., item 4).
5. Incubator jars for microaerophilic conditions.
6. Gram stain evaluation.
a. Glass slides.
b. Crystal violet solution.
c. Iodine solution.
d. Safranin solution (Gram staining kit, Biopack) prepared according to the manufacturer's instructions.
7. Optical microscope.
2.5. Histological Technique
1. Fixation: Ethyl alcohol 96° maintained at refrigeration temperature (Biopack).
2. Dehydration: Ethyl alcohol 100°, always at 10°C.
3. Clearing step: Cold pure xylol (Biopack). Store at room temperature
4. Paraffin baths: Paraffin (Cicarelli, Argentina). Melting point 56-58°C, melted at 60°C for 48 h before use and filtered for 24 h before tissues addition.
5. Other materials: Wood pieces for mounting, paper boxes, spatulas, boxes.
6. The tissues were cut into 2-^m-wide slides, by using disposable microtome blades (Leica)
7. Hematoxylin-eosin stain.
a. Mayer's activated hematoxyline: Stabilized solution for histological use (Biopur).
c. Ethyl alcohol at concentrations of 50°, 70°, 80°, 96°, and 100° (Biopack) prepared by dilution of absolute alcohol in distilled water. The different ethanol dilutions are stored in screw-top flasks at room temperature.
d. Xylol: Pure, pro-analysis stored at 4°C (Cicarelli).
8. Optical microscope.
2.6. Indirect Immunofluorescence
1. Phosphate-buffered saline (PBS): 6% disodium phosphate, 1% potassium phosphate, 68% sodium chloride, pH 7.2. Store at 4°C.
2. Anti-IgA monospecific antiserum.
a. a-Chain obtained from goat, labeled with fluorescein isothiocyanate (FITC; Sigma).
b. A bottle containing 1 mL antiserum was fractioned in Eppendorf tubes (20 ^L in each one) and stored in the freezer.
c. At the time of use, an adequate dilution was prepared (previously titrated in our experimental conditions, by diluting in saline solution).
d. Once diluted, the antiserum was stored at 4°C for no more than 48 h.
3. Anti-IgM monospecific antiserum.
a. a-Chain obtained from goat, labeled with FITC (Sigma).
b. Prepare as just described in step 2. Preparation and dilution of the fluorescent labeled antiserum must be in the dark.
4. Ethyl alcohol at different gradations: 50°, 70°, 80°, 96°, and 100° (Biopack). Store at 4°C for not more than 1 wk.
6. Mounting liquid: 9 parts neutral glycerine and 1 part buffered saline solution, pH adjusted to 9.0 with 0.5 M Na2CO3. Store at room temperature.
7. Incubation: In humid chamber.
8. Other materials.
a. Glass flasks to perform fixation of the material.
d. Filter papers.
e. Glass slides.
g. Canada balsam.
h. Optical microscope.
i. Thermostatized water bath.
1. The lactic acid bacteria were placed in sterile tubes containing 1 mL of milk-yeast extract and stored at -20°C for 6 mo.
2. The microorganisms were transferred from the milk-yeast extract tubes to tubes containing 5 mL of LAPTg broth. Three subcultures were performed, incubating each one for 12 h at 37°C (see Note 1).
1. Hormone inoculation: The animals were cycled to the same stage of the sexual cycle; by im inoculation (in the internal part of the leg) with 0.5 mg of estradiol valerate (0.05 mL Progynon Depot). All animals from all experimental groups were inoculated in this fashion 48 h before beginning the experiments (inoculation of lactobacilli by the intravaginal route) (see Notes 2 and 3).
1. The third subculture of lactobacilli in LAPTg broth was centrifuged for 10 min at 2000g and washed with 1 mL of saline solution. These suspensions were centrifuged again and resuspended in 50 ^L of agarized peptone water melted previously and cooled at 45°C (see Note 4).
2. The final concentration of microorganisms was determined by the plate dilution method using serial dilutions of peptone water. Aliquots were plated in MRS agar and incubated at the adequate temperature. The suspension of microorganisms was between 107 and 108 CFU/mL.
3. To differentiate the inoculated lactobacilli from those of the normal microbial flora, their resistance to different antibiotics was determined. The antibiotic sensitivity of the different lactobacilli strains was assayed by the disc diffusion agar method and also the minimal inhibitory concentration (MIC) method (16). For each strain of lactobacilli, one antibiotic was selected, according to the sensitivity/resistance pattern. The method employed to determine these patterns of sensitivity is the one recommended by the National Committee for Clinical and Laboratory Standards (NCCSL) (16). After having the previous results available, the following assays were performed at the laboratory level (for space reasons, only one example will be described). Different concentrations were used in order to determine the antibiotic quantities that allow the growth of the lactic bacteria inoculated in the culture media. The antibiotic was prepared at different concentrations (2.5, 5, 10, 50, and 100 ^g/mL) and added to the culture media. Control experiments were performed to determine whether lactobacilli grow in the same numbers in media with and without antibiotic. The antibiotic was prepared by dissolving different amounts of the adequate solvent and was later sterilized by filtration. They were divided into small tubes and stored in the freezer.
4. Lactobacili inoculation: The lactobacilli strains were inoculated intravaginally to different experimental groups of mice. Inoculations were performed twice every day (8 h in between) for 2 d (according to the scheme shown in Table 2). The microorganisms were inoculated in agarized peptone water (prepared as described in Subheading 2.3.). The agarized bacterial suspension was introduced into the mouse vaginas by loading automatic pipetor tips with the desired amount of agarized suspension.
5. Sacrifice: Mice were sacrificed after 24 h and 2, 5, 7, and 11 d post lactobacilli inoculation and used for the different studies. They were sacrificed by cervical dislocation and placed on the dissection board.
a. The mice were dissected to obtain the vaginas by using specific dissection material.
b. Once killed, they were immobilized on the dissection board and their extremities were fixed with metallic pins.
c. The skin was first decontaminated with povidone iodine and later cut with scissors in aseptic conditions.
d. The different tissue layers were cut or moved to separate the vagina, which was extracted (The extraction was made by cutting just above the endocervix and the distal extremity).
e. The organ was transferred to a glass bottle containing alcohol 96°C (for histological and immunological techniques) or cut longitudinally with sterile scissors and transferred to sterile tubes containing 1 mL of peptone water (for microbiological assays).
7. Microbiological assays; samples from washes and homogenates. Determination of the number of lactobacilli in the mice vagina (in both vaginal washes and homogenates) was made by plating aliquots of the samples (or their dilutions) in selective media containing antibiotic (to differentiate the resident flora from those administered experimentally), as explained in Subheading 2.3.
8. Histological assays were performed to determine whether the local administration of lac-tobacilli produces some type of modification at the histological structural level in the tract, as described in Subheading 3.5.
Inoculation Schedule Used for the Colonization Studies in BALb/c Mice
Inoculation Schedule Used for the Colonization Studies in BALb/c Mice
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