Lactobacillus paracasei strain GM-080 for treating allergy related diseases

Isolation of Lactobacillus paracasei GM-080

Sample: A piece of human stomach, intestine or duodenum tissue taken by an endoscope was cultured in 2 mL of Lactobacillus MRS Broth (DIFCO® 0881) containing 100 μg/mL of ampicillin for about two days at 37° C. The broth was plated on MRS agar containing CaCO 3 and incubated at 37° C. for two days. Single colony growing on the plate was selected and subjected to Gram-stain. Gram-positive bacteria were then selected. All of the strains were cultured in Lactobacillus MRS broth at 37° C. to the stationary phase, and collected by centrifuging at 3000 g for 15 minutes and washed with 2 mL and 1 mL PBS (phosphate buffered saline, pH 7.2). The cultures of the strains were re-suspended in 1 mL PBS and then heated at 95° C. for 30 minutes, and were then autoclaved and stored in PBS at −20° C.

Isolation of splenocytes: Five mL blood samples derived from healthy volunteers were added with 5 mL Ficoll-Hypaque (17-1400-02, Pharmacia) and then centrifuged at 500 g for 30 minutes. The splenocytes were taken. In each splenocyte sample, the cell density was adjusted to 5×10 6 cells per sample. The splenocyte samples were incubated in 2 mL RPMI 1640 (pH 7.7) for 6 hours.

Isolation of peripheral blood mononuclear cells: Five mL blood samples derived from healthy volunteers were added with 5 mL Ficoll-Hypaque (17-1400-02, Pharmacia) and then centrifuged at 500 g for 30 minutes. The peripheral blood mononuclear cells (PBMCs) were taken from the interface of the samples, and washed twice with PBS. The PBMCs (10 5 cells/mL) were transferred to the wells of a six-well plate wherein each well contained 2 mL RPMI 1640 medium of pH 7.7.

Stimulating IFN-γ Secretion. The splenocyte or PBMC samples were co-cultured with a given amount of the Gram-positive bacteria. After the 36-hour co-culture, the cells in each sample were collected, respectively. The collected cells were re-suspended and centrifuged at 2000 rpm for 5 minutes. The supernatants were taken for the determination of IFN-γ level in each sample.

Determination of IFN-γ Level: IFN-γ Level was determined by ELISA, comprising the steps of:

adding 30 μL of 2.5 μg/mL purified mouse anti-human IFN-γ antibodies (Cat. No18181D, PharMingen®), USA) in 10 mL of coating buffer (0.1 M Na 2 HPO 4 , pH 9.0) and adding 100 μL of antibody solution into each well of a ELISA plate; shaking the plate at 4° C.; washing each well of the plate with washing buffer (0.05% Tween 20 in PBS); adding 300 μL blocking buffer (1% BSA in PBS) into each well of the plate; shaking the plate at room temperature for at least 2 hours; adding 100 μL of the supernatant of the splenocyte sample to each well of the plate; shaking the plate at 4° C. overnight; washing each well of the plate with washing buffer; adding 150 μL biotin mouse anti-human IFN-γ antibodies (Cat. No18112D, PharMingen®, USA) into each well of the plate; incubating the plate for 1 hour at room temperature; washing each well of the plate with washing buffer; adding 150 μL Streptavidin-AKP diluted with dilute buffer (1:1000) into each well of the plate; shaking the plate for 1 hour at room temperature; washing each well of the plate with wash buffer eight times; adding 200 μL of substrate pNpp was added into each well of the plate; incubating the plates at room temperature until the substrate reaction is completed; measuring the absorbance of each well of the plate at 405 nm (i.e. OD 405 ).

Result: Among the Gram-positive bacteria, GM-080 was selected to have the strongest ability to stimulate IFN-γ secretion in splenocyte cells and PBMCs.

16s rDNA Sequence Determination

DNA extraction: The genomic DNA of GM-080 and other bacteria, CCRC12913, CCRC 14001 and CCRC 16100 were extracted using QIAamp® DNA Stool Mini Kit (Qiagen®, cat No. 51504). The purification was performed according to the steps as listed below:

adding 1.4 mL of ABS buffer in to the culture and vortexing it for 1 min; heating the solution obtained in the previous step at 70° C. for 5 min; vortexing the solution for about 15 sec and then centrifuging it at about 13,000 rpm for 1 min; removing the supernatant into a new centrifuge tube; adding an InhibitEx tablet in the supernatant and shaking it to dissolve the tablet, and then incubating at room temperature for 1 min; centrifuging the solution at about 13,000 rpm for 3 min to make the bacteria attach to InhibitEx; removing the supernatant into a new centrifuge tube and then centrifuging at about 13,000 rpm for 3 min; taking 200 μL of the supernatant to a new centrifuge tube and adding Protease K; adding 200 μL of Buffer AL and vortexing it for 15 min to obtain a homogeneous solution; adding 15 μL of Protease K into the homogenous solution and vortexing it for 15 sec; incubating the solution at 70° C. for 10 min; adding 200 μL of 96–100% ethanol and vortexing; removing the solution into QIAamp spin column and centrifuging it at about 13,000 rpm for 1 min; removing the QIAamp spin column to a new centrifuge tube and adding 500 μL Buffer AW1, and then centrifuging it at about 13,000 rpm for 1 min; removing the QlAamp spin column to a new centrifuge tube and adding 500 μL Buffer AW2, and then centrifuging it at about 13,000 rpm for 1 min; removing the QIAamp spin column into a new centrifuge tube and adding 200 μL Buffer AE, and then incubating it at room temperature for 1 min; and centrifuging at about 13,000 rpm for 1 min to elution DNA.

16s rDNA fragment amplification: The primers for amplifying L region were designed according to Lactobacillus paracasei 16S rRNA VI region, 5′- CAC CGA GAT TCA ACA TGG -3′(SEQ ID No. 1) and Lactobacillus conserved 16S rRNA, 5′- CCC ACT GCT GCC TCC CGT AGG AGT -3′ (SEQ ID No. 2) (Ward, L. J. H. and Timmins, M. J. (1999) Differentiation of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerase chain reaction. Lett. Appl. Microbiol. 29: 90–92). The genomic DNA of GM-080, CCRC12913, CCRC 14001 and CCRC 16100 were taken as the template for performing PCR reaction. The 16s rDNA PCR amplification program is as follows: (1) 95° C. for 10 min; (2) 95° C. for 45 sec; (3) 46° C. for 45 sec; (4) 72° C. for 1 min; (5) 72° C. for 7 min; steps 2 to 5 were repeated for 30 cycles.

16s rDNA sequence determination: The PCR products of GM-080, CCRC 12913, CCRC 14001 and CCRC 16100 were subjected to agarose gel electrophoresis ( FIG. 2 ) and sequenced. The sequences were aligned against the multiple sequence alignment dataset (NCBI blastn, http://www.ncbi.nlm.nih.gov/BLAST) using the ARB sequence editor (release 8.1). It also showed that the 16s rDNA sequences of Lactobacillus paracasei strain PB4, AY186046; F31, AF243147; KLB58, AF243168 were similar to that of GM-080 as shown in FIG. 3 (generated with Vector NTI™, InforMax® Inc.). In addition, 16s rDNA phylogenetic distance tree was generated with EMBL-EBI ClustalW (http://www.ebi.ac.uk/clustalw) as shown in FIG. 4 . According to the 16S rDNA analysis, GM-080 was highly related to Lactobacillus paracasei strain KLB58, but still distinct from KLB58. Given the above, GM-080 belonged to Lactobacillus paracasei.

Randomly Amplified Polymorphic DNA (RAPD Analysis)

DNA extraction of GM-080, Lactobacillus paracasei ATCC 25598, 25302, 335, 11582, and 27216 was performed as described in Example 2.

The primer for random amplification was 5′-ATGTAACGCC-3′ (Gardiner, G., Ross, R. P., Collins, J. K., Fitzgerald, G., Stanton, C. Development of a probiotic cheddar cheese containing human-derived 15 Lactobacillus paracasei strains. Appl Environ Microbiol. 1998; 64: 2192–2199).

The result of RAPD was shown in FIG. 5 . According to the RAPD analysis, GM-080 was distinct from the conventional Lactobacillus paracasei strains. Given the above, GM-080 was a novel Lactobacillus paracasei strain.

Cell Wall Proteins Extraction and Analysis of GM-080

The cell wall proteins were purified according to the method described by Angelis (Angelis, M. D., Corsetti, A., Tosti, N., Rossi, J., Corbo, M. R., and Gobbetti, M. (2001) Characterization of Non-Starter Lactic Acid Bacteria from Italian Ewe Cheeses Based on Phenotypic, Genotypic, and Cell Wall Protein Analyses. Appl. Environ. Microbiol. 67: 2011–2020). The cells cultured overnight in MRS broth (Difco®) were harvested and then washed twice with 0.05 M Tris-HCl (pH 7.5) containing 0.1 M CaCl 2 , and resuspended in 1 ml of the same buffer at an OD 600 of 10.0. After centrifugation at 8,000×g for 5 min, cell wall proteins were extracted from the pellets with 1.0 ml of extraction buffer (pH 8.0) containing 0.01 M EDTA, 0.01 M NaCl, and 2% (wt/vol) SDS. Suspensions were stored at room temperature for 60 min, heated at 100° C. for 5 min, and centrifuged at 11,600×g for 10 min at 4° C. The supernatants were analyzed by 12% SDS-PAGE and stained with Comassie blue.

The result was shown in FIG. 6 . The pattern of GM-080 had three specific bands, P1, P2 and P3 that similar to those of Lactobacillus paracasei reported in the prior study (Angelis et al. 2001). Therefore, GM-080 was evidenced to belong to Lactobacillus paracasei.

A Standardized Detection System for Identifying GM-080

Stimulation: The Jurkat cells were refreshed by adding a fresh medium and cultured for 16 hours. Subsequently, the cells were divided into two groups, one for the culture with the lactic acid bacteria and the other for the culture without the lactic acid bacteria. When the cell concentration reached 1×10 7 /10 mL, cells were stimulated for 24 h with or without 1×10 7 different lactic acid bacteria (CCRC12193, GM-080 or CCRC12188). After stimulation, the cells were collected, washed twice with PBS, and used for RNA isolation.

RNA isolation and labeling: RNA was extracted from cell by using Trizol Reagent (Life Technologies®, Gaithersburg, Md.) according to the manufacturer's instructions. 8 L of the RNA (10 μg) and 2 L oligo poly-dT (12–18 mer, 1 g/L) were well mixed and kept at 70° C. for 10 minutes and then were cooled with ice for 2 minutes. Mixed the RNA with reverse transcription labeling mixture and 3 L Cy3-dUTP (1 mM), 2 L SuperScript III (200 U/L), and RNasin (1 L) in dark. The mixture was incubated at 50° C. for 2 hours for reverse transcription, and the reaction was terminated by adding 1.5 L 20 mM EDTA. After the labeling, RNA was removed by NaOH treatment and neutralized by HCl. cDNA was immediately purified with a YM30 purification kit.

Microarray fabrication: Hundreds of genes chosen were amplified through polymerase chain reaction and quantified by spectrophotometry at 260 nm. All purified PCR products were adjusted to a concentration of 0.1 μg/μl in 50% dimethyl sulfoxide and spotted in duplicate on UltraGAPSTM coated slides (Corning®, Inc., Coming, N. Y.). After printing, the microarrays were UV cross-link at 700 mJoulesand stored in the slide container in a desiccator at room temperature. The genes were listed in Table 2 as mentioned above.

Microarray hybridization: Fluorescently labeled cDNA was denatured in the hybridization solution (5×SSC, 0.1% SDS and 25% formamide) at 100° C. for 5 min, cooled to ambient temperature, and deposited onto slides. The hybridization was carried out for 18 h at 55° C. After hybridization, the slides were successively washed in low-stringency (1×SSC and 0.1% SDS), medium-stringency (0.1×SSC and 0.1% SDS), high-stringency (0.1×SSC) buffer and finally were dried by compressed N 2 .

Signal detection and data analysis: N 2 -dried slides were immediately scanned on a GenePix 4000B scanner (Axon Instruments®), Inc.) at the same laser power and sensitivity level of the photomultiplier for each slide. Raw fluorescence data were acquired (10-nm resolution), and subsequent processing and data visualization were performed in Microsoft Excel™. In order to compare the results of independent hybridization experiments, the local background signal was subtracted from the hybridization signal of each separate spot, and then divided by the housekeeping gene, β-actin. The final expression of each gene was represented in a mean of duplicates. The gene expression profiles of the Jurkat cell cultured with and without the lactic acid bacteria were then obtained. A group of genes upregulated or down-regulated more than 2 fold in Jurkat cell cultured with lactic acid bacteria (CCRC12193, GM-080 or CCRC12188) to that cultured without the bacteria were selected. The results were shown in Table 3. The difference indicated that different species or strain can turn on or turn off different genes of the cell. Hence, from the gene expression profile, it indicated that CCRC12193, GM-080 and CCRC12188 are L. paracusei but belong to different strains.

Adhesion of GM-080 to The Epithelial Cells in The Intestine

Caco-2 cells were taken as the epithelial cells in the example. Caco-2 cells had functional microvilli and hydrolase attached thereon, it exhibited differentiated morphology and functions of a mature epithelial cell in the intestine in vitro.

Cells: Caco-2 were cultured in Mineral essential medium (MEM, GIBCO®) supplemented with 5% FBS at 37° C. in an 5% CO 2 /95% air. For adhesion assay, 2 ml of monolayer of Caco-2 cells (3×10 5 cells/ml) were prepared on glass cover slips that were placed in 6-well plate. The culture medium was replaced every second day and the monolayers were used in the adhesion assay after 2 weeks incubation. Just before use, the monolayer was wash twice with PBS and 1.5 ml of MEM was added to each well and incubated at 37° C. for 1 h before inoculation of bacteria.

Adhesion: 1.5 ml of (4×10 8 CFU/ml) of GM-080 washed once with PBS and resuspended in 1.5 ml MEM medium was added to the Caco-2 cells. After 1 h of incubation at 37° C., monolayer of cells were washed four times with PBS buffer, fixed with 3 ml of methanol and incubated for 5 to 10 min at room temperature, wash three times with PBS, dried in air and Gram stained. Adherent bacteria were detected microscopically under oil immersion (×100) by counting 15 random fields per coverslip and mean ±SD of adhering bacteria per field was determined.

Result: After counting, there were 102±23.6 GM-080 bacteria adhered to the Caco-2 cells. Therefore, GM-080 was regarded to have strongly adhesion to Caco-2 cells according to the standard established by Jacobsen et al. (Jacobsen, C. N., Nielsen, R. V., Hayford, A. E., Moller, P. L., Michaelsen, K. F., Paerregarrd, A., Sandstrom, B., Tvede, M. and Jakobsen, M. Screening of probiotic activities of forty-seven strains of Lactobacillus spp . by in vitro techniques and evaluation of the colonization ability of five selected strains in human. Appl. Environ. Microbiol. 1999; 65: 4949–4956).

Activities of GM-080 and Other Lactic Acid Bacteria in An Environment Mimicking GI Tract

Acid: The overnight-cultured GM-080, L. plantarum, L. acidophilus, L. casei , and L. bulgaricus were added with 9 mL of PBS with different pH values of 2.0, 2.5 and 3.2 and then further cultured at 37° C. for 3 hours. After culturing, 1 mL cells were serially diluted with 9 mL of pH 7.4 PBS. The cell counts before and after acid treatment were estimated and shown in Table 4 as listed below.

gall. The overnight-cultured GM-080, L. plantarum, L. acidophilus, L. casei , and L. bulgaricus were added with 9 mL of PBS with different pH values of 2.0 and then further cultured at 37° C. for 3 hours. After culturing, the 1 mL cells were centrifuged at 6,000 rpm for 10 min. The pellet was re-suspended with 100 μL of PBS (pH 7.2). The solution was further added with 10 mL of MRS broth containing 0.3% (w/v) of ox gall. The cells were cultured and 1 mL of sample was taken at 3, 12 and 24 hours. The samples were serially diluted with 9 mL of pH 7.4 PBS. The cell counts before and after gall treatment were estimated and also shown in Table 4. It shows that these lactic acid bacteria remain active in the environment mimicking the GI tract.

Animal Model

Animals. Female BALB/c mice were obtained from the National Laboratory Animal Center in Taiwan and raised for 2 weeks in a room where light and temperature were both controlled.

Allergen purification: The dust mite allergen, Der p 5, was expressed in Escherichia coli comprising PGEX-2T expression vector as a recombinant Der p 5-Glutathione S-transferase fusion protein that can be purified with a glutathione-agarose binding chromatography. The specific E. coli strain which is able to express the desired allergen was cultured and induced. The bacteria were collected and washed with TBS (pH 7.5) and added with 0.1 M phenylmethylsulfony fluride. The cells were broken by adding DNase I, Tween 20 and lysozyme, and by freeze-thaw method. The mixed solution was added with EDTA and the residues were removed by centrifugation to obtain the supernatant containing recombinant Der p 5-Glutathione S-transferase fusion protein. The supernatant was subjected to a glutathione-agarose affinity column for absorbing the fusion protein. The column was then washed with TBS buffer at 4° C. and then with reduced glutathion in Tris base (pH 8.0) for separating the protein from column. The molecular weight of the protein was estimated by SDS-PAGE and the concentration was also assayed.

Sensitization: Mice were actively sensitized by intraperitoneal injection of 10 μg of Der p 5 with 4 mg of aluminium hydroxide. 14 and 21 days after the initial sensitization, the mice were exposed to an aerosol of 0.1% of the purified Der p 5 for 30 min to perform inhalation challenge.

Treatment: The sensitized mice were divided into three groups for the experiments. The mice of Group A were fed ten times in two weeks with MRS broth as a control group. The mice of Group B were administered with Lactobacillus casei ten times in two weeks and 109 CFU of bacteria were administered every time. Group B was taken as a positive control, because L. casei had been evidenced to be effective on inhibiting IgE secretion. The mice of Group C were administered with GM-080 ten times in two weeks and 10 9 CFU of bacteria were administered every time.

IgG and IgE Secretion

Determination of Derp 5-specific IgG and IgE: Eighteen hours after last inhalation challenge, 500 μL of blood sample was taken from the tail. The blood samples were kept at room temperature for 1 hour and then subjected to centrifugation. The sera were stored at −80° C. The amounts of Der p 5-specific IgG2a, and IgE were determined by ELISA. Protein high-binding plates with 96 wells were coated with 200 μL of purified Der p 5 diluted in coating buffer (0.1 M NaHCO 3 , pH 9.6) at a concentration of 10 μg/mL. After overnight incubation at 4° C., the plates were washed with PBS-Tween 20 and then added with 300 μL blocking buffer (3% BSA). After shaking for 2 hours at room temperature, the plates were washed again with PBS-Tween 20. Sera were used at 1:10 dilution for IgG measurement and 1:4 dilution for IgE measurement. The samples were shaken at room temperature for 2 hours. After overnight incubation at 4° C., the plates were washed with PBS-Tween 20, and added with 200 μL biotinylated rat anti-mouse IgE monoclonal antibody, or rat anti-mouse IgG mAb. The sample was shaken at room temperature for 2 hours and then washed with PBS-Tween 20. 200 μL of Streptavidin-alkaline phosphatase (1:1000) was then added and shaking the sample at room temperature for 1 h. After 6 washes, color reaction was imitiated with the addition of 200 μL phosphatase substrate p-nitrophenyl phosphate, di-sodium salt (pNPP) (Sigma® N-2770, USA). Plates were read in a microplate autoreader (Metertech®, Taiwan) at 405 nm.

Statistical analysis: To assay the changes of IgE and IgG levels, repeated measures for analysis of One-way ANOVA were performed to compare the differences between the groups. After analysis of variance, Duncan multiple range tests were used to differentiate differences between experimental and control groups. A value of p<0.05 was used to indicate a statistically significant difference.

Result. The result was shown in FIG. 7 . It evidenced that IgE secretion in sera of the animal treated with GM-080 was enormously lowered and only 25% of that of without treatment. On the other hand, IgG secretion in sera of the animal treated with GM-080 was raised to two fold. Because IgG secretion represents Th1 T cell reaction, GM-080 is directed to eliminate IgE secretion that correlated to allergy related disease.

Bronchoalveolar Lavage Fluid Cell Count

Samples preparation: Eighteens hours after sensitization, the mice were lavaged with 5×0.5-ml aliquots of 0.9% sterile saline through a polyethylene tube introduced through a tracheostomy. Lavage fluid was centrifuged (500 g for 10 min at 4° C.), and the cell pellet was resuspended in 1 ml of PBS solution. Differentiated cell counts were made from cytospin preparations stained by Leu's stain.

Statistical analysis: To assay the changes of cell counts, repeated measures for analysis of One-way ANOVA were performed for comparing the differences between the groups. After analysis of variance, Duncan multiple range tests were used to differentiate differences between experimental and control groups. A value of p<0.05 was used to indicate a statistically significant difference.

Result: The result was shown in FIG. 8 . The blood cell type contribution in the BALF represents the degrees of inflammation.

Furthermore, the main symptoms of allergenic asthma are chronic inflammation in airway and eosinophils infiltration. It evidenced that the eosinophils in the BALF of the animal treated with GM-080 was enormously lowered from 5% to 1%. On the other hand, the macrophages and lymphocytes in the BALF of the animal treated with GM-080 were significantly raised.

IFN-γ Secretion in The Bronchoalveolar Lavage Fluid

Samples preparation: After 24 hours of sensitization, the mice were lavaged with 5×0.5-ml aliquots of 0.9% sterile saline through a polyethylene tube introduced through a tracheostomy. Lavage fluid was centrifuged (500 g for 10 min at 4° C.), and the supernatant was subjected to IFN-γ quantitative analysis as described in Example 1.

Result: The result was shown in FIG. 9 . It showed that the animals fed with GM-080 produced about 100 pg/mL of IFN-γ in the BALF. On the other hand, the control group produced only 20 to 40 pg/mL of IFN-γ in the BALF. GM-080 was effective on inhibiting allergenic inflammation.

Inactive GM-080 for Treating Allergy

Inactive GM-080 preparation: Lyophilized GM-080 powder was suspended in distilled water and autoclaved (121° C., 15 min) before feeding mice.

Mice and sensitization: Female BALB/c mice (6–8 week-old) were purchased from National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). All animals were maintained individually in cages with controlled temperature (24±2° C.) and humidity (60±5%) and maintained on a 12-h light-dark cycle under specific-pathogen-free conditions. BALB/c mice were i.p. with 10 g recombinant Dermatophagoides pteronyssinus allergen Der p5–6×His fusion protein adsorbed to 4 mg of alumium hydroxide. The mice were fed with 10 7 , 10 9 and 10 11 CFU GM-080 per mouse per day for three weeks. The mice were boosted with the same dosage of allergen as sensitization at 14th day and were challenged with 0.1% of Der p5–6×His diluted in PBS 21 days after sensitization. The inhalation challenge was performed in 1-L chamber connected to a DeVilbiss™ pulmosonic nebulizer (Model 2512; DeVilbiss® Corp., Somerset, Pa.), which generated an aerosol mist. After 18 hours, serum was collected by tail vein bleeding and IgE was determined by ELISA as described in Example 9.

Result: The result was shown in FIG. 10 . It showed that BALB/c mice challenged with dust allergy Der p-5 had significantly elevated serum IgE levels compared to naive group (p<0.05). It suggested that allergic sensitized mice model could be successfully set up. After feeding of different dosage of GM-080 per day for 21 days, the serum IgE in GM-080 group had significantly decreased (p<0.05) compared with control group. The results showed that inactive GM-080 could decrease the allergic responses by reducing the allergen-specific IgE.

While embodiments of the present invention have been illustrated and described, various modifications and improvements can be made by persons skilled in the art. It is intended that the present invention is not limited to the particular forms as illustrated, and that all the modifications not departing from the spirit and scope of the present invention are within the scope as defined in the appended claims.