JP4847038B2 - Novel microbial strain GM-080 of Lactobacillus paracasei and its use for treating allergy related diseases - Google Patents

Description

  The present invention mainly relates to a novel microbial strain Lactobacillus paracasei GM-080 and its use for treating allergy-related diseases and stimulating IFN-γ secretion.

  Allergy refers to the acquired potential to develop an immunologically mediated adverse reaction to normally harmless substances. Allergic reactions cause symptoms such as itching, coughing, wheezing, sneezing, tear eyes, inflammation and fatigue. Allergic reactions are usually considered to include an early specific immune response and a late inflammatory response. Allergens (eg, pollen and mite dust) have been reported to mediate early allergies by stimulating high affinity immunoglobulin (IgE) receptors. For example, mast cells and basophils release histamine and cytokines when stimulated by allergens. Cytokines released from mast cells and basophils subsequently mediate late allergies by recruiting inflammatory cells. Also, the influx of eosinophils, macrophages, lymphocytes, neutrophils and platelets initiates a severe inflammatory cycle. This late allergy amplifies the initial immune response, which subsequently causes the release of further inflammatory cells (Blease et al. Chemokines and their role in airway hyper-reactivity.Respir Res 2000; 1: 54-61 ).

  Various therapies for treating allergic symptoms are being pursued. Among them, antiallergic drugs and histamine H receptor antagonists (antihistamines) are used. Histamine antagonists are administered to antagonize the action of histamine released from mast cells in response to the presence of allergens. They serve to reduce redness, pruritus and swelling caused by the action of histamine on the target tissue and to prevent or alleviate a number of symptoms resulting from degranulation of mast cells. However, antihistamines are also associated with adverse reactions such as reduced alertness, delayed reaction time, and drowsiness (US Pat. No. 6,225,332).

  There are also several reports of treating allergies by controlling cytokines. Among them, interferon-γ (IFN-γ) inhibits the overexpression of cytokines in Th2 lymphocytes, particularly the secretion of IL-4, and decreases the proliferation of B cells. In addition, IFN-γ may suppress the synthesis of IgE by stimulating the Th1 immune response (Sareneva T et al. Influenza A virus-induced IFN-α / β and IL-18 synergistically enhance IFN-γ gene expression in human T cells. J Immunol 1998; 160: 6032-6038; Shida K, et al. Lactobacillus casei inhibits antigen-induced IgE production through regulation of cytokine production in murine splenocyte cultures.Int Arch Allergy Immunol 1998; 115: 278- 287). Since IFN-γ can inhibit B cell proliferation and IgE secretion, IFN-γ is believed to be effective in treating allergies.

  Lactic acid bacteria, which are Gram-positive bacteria, are commonly used in industrial food fermentation. Recent studies have demonstrated that lactic acid bacteria stimulate cellular IFN-γ secretion (Contractor NV, et al. Lymphoid hyperplasia, autoimmunity, and compromised intestinal intraepithelial lymphocyte development in colitis-free gnotobiotic IL 2 deficient mice. J Immunol 1998: 160: 385-394). Some specific lactic acid bacteria, such as Bifidobacterium lactis and Lactobacillus brevis subspecies, stimulate lymphocyte IFN-γ secretion in blood from mice and humans (US Patent Publication No. US2002 / 0031503 A1; US Pat. No. 5,556,785). Moreover, lactic acid bacteria can stimulate human or mouse-derived lymphocytes, and activate T cells and NK cells to secrete IFN-γ, interleukin-12 (IL-12). (Hessle et al. Lactobacilli from human gastrointestinal mucosa are strong stimulators of IL-12 production. Clin. Exp. Immunol 1999; 116: 276-282).

Lactobacillus paracasei has long been used to make cheddar cheese and Italian ewes cheese. It has been shown to maintain high cheese viability during growth and ripening (Gardiner, G., Ross, RP, Collins, JK, Fitzgerald, G. Stanton, C. Development of a probiotic Cheddar cheese containing human-derived Lactobacillus paracasei strains.Appl Environ Microbiol. 1998; 64: 2192-2199; Angelis, M., Corsetti, A., Tosti, N., Rossi, J., Corbo, MR, Gobbetti, M. Characterization of non-starter lactic acid bacteria from Italian ewes cheeses based on phenotypic, genotipic, cell-wall protein and peptidase analyzes. Appl Environ Microbiol. 2001; 67: 2011-2020). L. paracasei is known to produce antibacterial and anti-yeast compounds such as H ₂ O ₂ and proteinaceous active substances in the human vagina and oral cavity (Atanassova, M., Choiset, Y., Dalgalarrondo , M., Chobert, J.-M., Dousset, X., Ivanova, I., Haertke, T. Isolation and partial biochemical characterization of a proteinaceous anti-bacteria and anti-yeast compound produced by Lactobacillus paracasei subsp. M3 Int J Food Microbiol. 2003; 87: 63-73; Ocana, VS, Holgado, AAP de R. ,, Nader-Macias, ME Growth inhibition of Staphylococcus aureus by H ₂ O _2- producing Lactobacillus paracasei subsp. Paracasei isolated from FEMS Immunol Med Microbiol. 1999; 23: 87-92; Sookkhee, S., Chulasiri, M. Prachyabrued, W. (2001) Lactic acid bacteria from healthy oral cavity of Thai volunteers: inhibition of oral pathogens. Journal of Applied Microbiology 2001; 90: 172-179).

  The present invention provides a novel microbial strain Lactobacillus paracasei GM-080.

  In another aspect, the present invention provides a composition comprising the microbial strain Lactobacillus paracasei GM-080.

  In another aspect, a method for treating an allergy-related disease in a subject comprising administering to said subject a composition comprising the microbial strain Lactobacillus paracasei GM-080; The complications are preferably airway hypersensitivity and inflammation, atopic dermatitis, allergic conjunctivitis, rhinitis, sinusitis, irritable pneumonia, exogenous allergic alveolitis, hives, eczema, anaphylaxis, angioedema, A method selected from the group consisting of allergic and migraine headaches, certain gastrointestinal disorders, and asthma is provided.

  In yet another aspect, the present invention is a method for stimulating IFN-γ secretion in a subject, comprising administering to the subject a composition comprising the microbial species Lactobacillus paracasei GM-080. A method comprising:

  The present invention provides a novel microbial strain Lactobacillus paracasei GM-080 capable of treating allergies. The stock GM-080 was deposited with the CCTCC (the China Cernter for Type Culture Collection) on February 19, 2004 under the accession number of CCTCC M 204012.

  Lactobacillus paracasei GM-080 is isolated from the healthy human gastrointestinal tract. Tissue samples taken from stomach, intestine or duodenum are suspended in culture medium of MRS broth containing 100 μg / mL ampicillin cultured at 37 ° C. for 2 days and streak plated on agar plates. The lactic acid bacteria colonies growing on the plate can be preliminarily screened by microscopic examination. The candidate strain is subsequently co-cultured with splenocytes. The amount of IFN-γ produced by the splenocytes in the broth is determined. Subsequently, GM-080 is selected for its high productivity of IFN-γ.

The mycological characteristics of GM-080 are shown below:
(A) Morphological characteristics (1) Cell shape and size: Aspergillus having a rod-like shape with rounded edges when observed under a microscope after overnight culture at 37 ° C. in MRS broth.
(2) Motility: Motor type (3) Flagella: None (4) Spore formation: No sporulation (5) Gram staining: Positive

(B) Culture characteristics:
(1) Medium: MRS broth (DIFCO ™ 0881), final pH 6.5 ± 0.2
(2) Culture conditions: anaerobic or aerobic culture at 37 ° C. (3) Antibiotic resistance: Ampicillin 100 μg / mL

(C) Physiological properties:
(1) Catalase: positive (2) Oxidase: negative (3) API 50 CHL test: API 50 CHL system is used for identification of lactic acid bacteria. By assaying the response of a series of enzymes, the properties of the lactic acid are established. The API 50 CHL test results of GM-080 are listed in Table 1.

(D) Genetic characteristics:
Determine the 16s rDNA sequence analysis of GM-080. The results indicate that GM-080 is highly homologous to other Lactobacillus paracasei strains (as shown in FIG. 2). Furthermore, the phylogenetic tree of distance is shown in FIG. In addition, random amplified polymorphic DNA (RAPD analysis) was performed. It indicates that GM-080 belongs to Lactobacillus paracasei but has a specific 16s rDNA sequence. Considering the above, GM-080 is a novel Lactobacillus paracasei strain.

(E) Cell wall protein of GM-080:
The cell wall protein of GM-080 shows a similar pattern when compared to other common Lactobacillus paracasei strains. The SDS-PAGE pattern of the cell wall protein of GM-080 is shown in FIG.

(F) Standardized detection system for identifying GM-080 A standard detection system for identifying microorganisms is US patent application Ser. No. 10 / 446,781, filed May 29, 2003. Which uses the difference in gene expression cultured in test cell lines with or without a given microorganism as a marker for identification. The tested genes are listed in Table 2.

  A standard detection system for identifying GM-080 employs the Jurkat cell line as the test cell line. When comparing the expression patterns of Jurkat cell lines cultured with and without GM-080, the genes listed in Table 3 are significantly different. Furthermore, the detection results of other Lactobacillus paracasei strains CCRC12193 and CCRC12188 are also shown in Table 3. This indicated that these strains are all Lactobacillus paracasei but belong to different strains.

+: Gene expression increased 2-fold-: Gene expression decreased 2-fold

  GM-080 is active after treatment with HCl solution (pH 2.0) for 3 hours followed by bile for 4 hours. Therefore, GM-080 is recognized to remain active upon digestion. GM-080 has been isolated from healthy individuals and is safe and free of natural toxicity, and R. A. S. (Generally recognized as safe).

  Furthermore, GM-080 adheres strongly to intestinal epithelial cells. In view of the above, GM-080 can remain in the intestine for extended periods of time, and thus acts to regulate physiological functions. In addition, by occupying the adhesion site of intestinal epithelial cells, GM-080 prevents other pathogenic bacteria from adhering to the intestine. GM-080 is recognized as a good probiotic bacterium.

  According to the present invention, GM-080 has been shown to stimulate IFN-γ secretion and can be used to treat allergy related diseases.

  In one aspect, the present invention provides a composition comprising GM-080. More preferably, a composition comprising GM-080 is used to stimulate IFN-γ secretion, which is useful for treating allergy related diseases.

  As used herein, the term “allergy-related disease” is a systematic response to a normally innocuous environmental antigen that interacts between an antigen and an antibody or T cell produced by initial exposure to the same antigen. It means the disease of the reaction resulting from the action. The term “allergic reaction” as used herein means a response to an innocuous environmental antigen or an antigen caused by an existing antibody or T cell. Although there are various immune mechanisms for allergic reactions, the most common type is allergen binding to IgE antibodies on mast cells that cause asthma, hay fever, and other common allergic reactions. Allergic-related diseases include airway hypersensitivity and inflammation, atopic dermatitis, allergic conjunctivitis, rhinitis, sinusitis, irritable pneumonia, exogenous allergic alveolitis, hives, eczema, anaphylaxis, angioedema, allergy Includes sexual and migraine headaches, certain gastrointestinal disorders, and asthma. In another aspect, allergy-related diseases are associated with exposure to allergens in the air (aeroallergens), such as pollen, fungi, animal dander, and insects.

  As used herein, the term “aeroallergen” is defined as having at least the following characteristics: a specific antigen class that elicits an active reggin response, and obvious tissue changes in susceptible subjects Ambient exposure level until it can cause. Aeroallergens are particles in the air that can cause respiratory, skin, or conjunctival allergies. The water-soluble part of ragweed pollen can affect, for example, the respiratory and conjunctival mucosa, and the fat-soluble allergen of ragweed pollen can cause typical contact dermatitis to exposed areas of the skin.

  GM-080 is selected to have the ability to stimulate IFN-γ secretion when incubated together with splenocytes and peripheral blood mononuclear cells (PBMC) in vitro. Furthermore, in a model according to the invention, animals sensitized with aeroallergens and then treated with GM-080 are observed to increase IFN-γ secretion. Furthermore, the amount of aeroallergen-specific IgE is significantly reduced after treatment. On the other hand, the amount of allergen-specific IgG does not show a significant difference between before and after treatment. Furthermore, the number of eosinophils in bronchoalveolar lavage fluid (BALF) is greatly reduced; however, the number of macrophages and lymphocytes in BALF is greatly increased. Proven to reduce inflammation.

  According to the present invention, GM-080 for use in the treatment of allergies may be live or inactive. Preferably GM-080 is inactive. For example, live strains can be treated with a heating step or other treatment commonly used in the art to kill lactic acid bacteria as inactive strains.

  According to the present invention, the lactic acid strain may be included in a pharmaceutical composition, dietary supplement, food, health food, medical food, or components thereof, which are usually administered to humans. In a preferred embodiment of the present invention, the lactic acid strain may be carried in the form of a food product, for example, a coagulated and dairy product prepared via fermentation of lactic acid bacteria in milk. The food prepared according to the present invention can be easily administered to infants or children.

  In another aspect, the present invention is a method for treating an allergy related disease in a subject comprising administering to said subject a composition comprising isolated microorganism GM-080. A method is provided.

  In yet another aspect, the invention provides a method for stimulating IFN-γ secretion in a subject comprising administering to the subject a composition comprising an isolated microorganism GM-080. A method comprising comprising is provided.

  The following examples are given for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: Isolation of Lactobacillus paracasei GM-080 Sample: Endoscopically harvested human stomach, intestine or duodenum tissue section with 2 mL of Lactobacillus MRS broth containing 100 μg / mL ampicillin ( The cells were cultured at 37 ° C. in DIFCO ™ 0081) for about 2 days. The broth was plated on CaCO ₃ containing MRS agar and incubated at 37 ° C. for 2 days. A single colony growing on the plate was selected and subjected to Gram staining. Gram positive bacteria were subsequently selected. All of the strains were grown to stationary phase in Lactobacillus MRS broth at 37 ° C. and recovered by centrifugation at 3000 g for 15 minutes and resuspended in 1 mL PBS (phosphate buffered saline, pH 7.2). Turbid and then heated at 95 ° C. for 30 minutes and subsequently autoclaved and stored at −20 ° C. in PBS.

  Splenocyte isolation: A 5 mL blood sample from a healthy volunteer was added to 5 mL Ficoll-Hypaque (17-1400-02, Pharmacia) and then centrifuged at 500 g for 30 minutes. Splenocytes were collected. The cell density in each splenocyte sample was adjusted to 5 × 10 6 cells / sample. Splenocyte samples were incubated in 2 mL RPMI 1640 (pH 7.7) for 6 hours.

Isolation of peripheral blood mononuclear cells: A 5 mL blood sample from a healthy volunteer was added to 5 mL Ficoll-Hypaque (17-1400-02, Pharmacia) and then centrifuged at 500 g for 30 minutes. Peripheral blood mononuclear cells (PBMC) were collected from the sample interface and washed twice with PBS. PBMC (10 ⁵ cells / mL) were transferred to wells of a 6-well plate, each well containing 2 mL of RPMI 1640 medium, pH 7.7.

  Stimulation of IFN-γ secretion: Splenocytes or PBMC samples were co-cultured with a predetermined amount of gram positive bacteria. After 36 hours of co-culture, the cells in each sample were collected individually. The collected cells were resuspended and centrifuged at 2000 rpm for 5 minutes. Supernatants were collected for determination of IFN-γ levels for each sample.

Determination of IFN-γ levels: IFN-γ levels were determined by ELISA, which was −30 μL of 2.5 μg / mL purified mouse anti-human IFN-γ antibody (Cat # 18181D, PharMingen ™, USA) Adding 10 mL of coating buffer (0.1 M Na ₂ HPO ₄ , pH 9.0) and adding 100 μL of antibody solution to each well of the ELISA plate;
-Shaking the plate at 4 ° C;
-Washing each well of the plate with wash buffer (0.05% Tween 20 / PBS);
-Adding 300 [mu] L blocking buffer (1% BSA / PBS) to each well of the plate;
Shaking the plate at room temperature for at least 2 hours;
-Adding 100 μL supernatant of splenocyte sample to each well of the plate;
Shaking the plate at 4 ° C. overnight;
-Washing each well of the plate with wash buffer;
Adding 150 μL of biotin mouse anti-human IFN-γ antibody (Cat. No. 18112D, PharMingen ™, USA) to each well of the plate;
Incubating the plate at room temperature for 1 hour;
-Washing each well of the plate with wash buffer;
Adding 150 μL of streptavidin-AKP (1: 1000) diluted in dilution buffer to each well of the plate;
Shaking the plate for 1 hour at room temperature;
-Washing each well of the plate 8 times with wash buffer;
Adding 200 μL of substrate pNpp to each well of the plate;
Incubating the plate at room temperature until the substrate reaction is complete;
Measuring the absorbance at 405 nm (ie OD ₄₀₅ ) of each well of the plate;
Comprising.

  Results: Among gram positive bacteria, GM-080 was selected, which has the strongest ability to stimulate IFN-γ in spleen cells and PBMC.

Example 2: Sequencing of 16s rDNA DNA extraction: Genomic DNA of GM-080 and other bacteria, CCRC12913, CCRC14001 and CCRC16100 using the QIAamp ™ Stool Mini Kit (Qiagen ™, catalog number 51504) Extracted. Purification was performed according to the steps listed below:
-Adding 1.4 mL of ABS buffer to the medium and vortexing it for 1 minute;
Heating the solution obtained in the previous step at 70 ° C. for 5 minutes;
Vortexing the solution for about 15 seconds and then centrifuging it for about 1 minute at about 13,000 rpm;
-Transferring the supernatant to a new centrifuge tube;
-Adding the InhibitEx tablet to the supernatant and shaking it to dissolve the tablet and then incubating for 1 hour at room temperature;
-Centrifuging the solution at about 13,000 rpm for 3 minutes to attach bacteria to InhibitEx;
Transferring the supernatant to a new centrifuge tube and then centrifuging at about 13,000 rpm for 3 minutes;
Transferring 200 μL of supernatant to a new centrifuge tube and adding protease K;
Adding 200 μL of buffer AL and vortexing it for 15 minutes to obtain a homogeneous solution;
Adding 15 μL of protease K to the homogeneous solution and vortexing it for 15 seconds;
Incubating the solution at 70 ° C. for 10 minutes;
Adding 200 μL of 96-100% ethanol and vortexing;
-Transferring the solution to a QIAamp spin column and centrifuging it at about 13,000 rpm for 1 minute;
Transferring the QIAamp spin column to a new centrifuge tube and adding 500 μL of buffer AW1 and then centrifuging it at about 13,000 rpm for 1 minute;
Transferring the QIAamp spin column to a new centrifuge tube and adding 500 μL of buffer AW2 and then centrifuging it at about 13,000 rpm for 1 minute;
Transferring the QIAamp spin column to a new centrifuge tube and adding 200 μL of buffer AE and then incubating it for 1 minute at room temperature;
-Eluting the DNA by centrifugation at about 13,000 rpm for 1 minute;
Comprising.

  16s rDNA fragment amplification: Primers for amplifying the L region include Lactobacillus paracasei 16S rRNA V1 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, LJH and Timmins, MJ (1999). Differentiation of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerase chain reaction. Lett Appl Microbiol 29, 90-92). Genomic DNAs of GM-080, CCRC12913, CCRC14001 and CCRC16100 were collected as templates for carrying out the PCR reaction. The 16s rDNA PCR amplification program is as follows: (1) 95 ° C for 10 minutes; (2) 95 ° C for 45 seconds; (3) 46 ° C for 45 seconds; (4) 72 ° C for 1 minute; (5 ) 7 minutes at 72 ° C; steps 2-5 were repeated 30 cycles.

  Sequencing of 16s rDNA: PCR products of GM-080, CCRC12913, CCRC14001 and CCRC16100 were subjected to agarose gel electrophoresis (FIG. 2) and sequenced. Sequences were aligned using a ARB sequence editor (release 8.1) against a number of sequence alignment data sets (NCBI blastn http: //www/ncbi.nlm.nih.gov/BLAST). Further, the 16s rDNA sequences of Lactobacillus paracasei strains PB4, AY186060; F31, AF243147; KLB58, AF243168 were the same as those of GM-080 as shown in FIG. 3 (VectorNTI (trademark), InforMax (trademark) Inc. Created with. Furthermore, a phylogenetic tree at a distance of 16s rDNA was prepared by EMBL-EBI ClustalW (http://www.ebi.ac.uk/clustalw) as shown in FIG. According to the 16S rDNA analysis, GM-080 was highly related to Lactobacillus paracasei strain KLB58, but is still different from KLB58. Considering the above, GM-080 belongs to Lactobacillus paracasei.

Example 3: Random 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, RP, Collins, JK, Fitzgerald, G. and Stanton, C. Development of a probiotic Cheddar cheese containing human-derived Lactobacillus paracasei strains. App Environ Microbiol. 1998; 64, 2192-2199).

  The results of RAPD are shown in FIG. According to RAPD analysis, GM-080 was different from regular Lactobacillus paracasei. Considering the above, GM-080 was a novel Lactobacillus paracasei strain.

Example 4: Extraction and analysis of cell wall protein of GM-080 Cell wall protein was obtained by the method described by Angelis (Angelis, MD, Corsetti, A., Tosti, N., Rossi, J., Corbo, MR, and Gobbetti, M. (2001) Characterization of Non-starter Lactic acid bacteria (NSLAB) from Italian ewe cheeses based on phenotypic, genotipic, cell-wall protein analyzes.Appl.Environ Microbiol. 2001; 67: 2011-2020) It was done. Cells cultured overnight in MRS broth (Difco ™) were collected and then washed twice with 0.05 M Tris-HCl (pH 7.5) containing 0.1 M CaCl ₂ and 1.0 ml of Resuspended at OD ₆₀₀ of 10.0 in the same buffer. After 5 minutes centrifugation at 8,000 × g, the cell wall protein was extracted in 1.0 ml extraction buffer (pH 8.0) containing 0.01 M EDTA, 0.01 M NaCl, and 2% (wt / vol) SDS. Was used to extract from the pellet. The suspension was stored at room temperature for 60 minutes, heated at 100 ° C. for 5 minutes, and centrifuged at 11,600 × g for 10 minutes at 4 ° C. The supernatant was analyzed by 12% SDS-PAGE and stained with Coomassie blue.

  The results are shown in FIG. The pattern of GM-080 had three specific bands P1, P2 and P3 similar to those of Lactobacillus paracasei reported in a previous study (Angelis et al. 2001). Therefore, GM-080 proved to belong to Lactobacillus paracasei.

Example 5: Standardized detection system to identify GM-080 Stimulation: Jurkat cells were refreshed by adding fresh media and cultured for 16 hours. Next, the cells were divided into two groups, one being a culture solution containing lactic acid bacteria and the other being a culture solution not containing lactic acid bacteria. When the cell concentration reached 1x10 ^7/10 mL, cells with 1x10 ⁷ different lactic acid bacteria (CCRC12193, GM-080 or CCRC12188), or were stimulated for 24 hours without. After stimulation, cells were harvested, washed twice with PBS and used for RNA isolation.

  RNA isolation and labeling: RNA was extracted from cells using Trizol reagent (Life Technologies ™, Gaithersburg, Md.) As per instructions. 8 L RNA (10 g) and 2 L oligopoly-dT (12-18 mer, 1 g / L) were mixed well and maintained at 70 ° C. for 10 minutes and then cooled with ice for 2 minutes. The RNA was mixed with reverse transcription labeling mixture and 3L Cy3-dUTP (1 mM), 2L SuperScript III (200 U / L), and RNasin (IL) in the dark. The mixture was incubated for reverse transcription at 50 ° C. for 2 hours and the reaction was terminated by adding 1.5 L of 20 mM EDTA. After labeling, RNA was removed with NaOH treatment and neutralized with HCl. The cDNA was immediately purified with the YM30 purification kit.

  Microarray generation: Hundreds of selected genes were amplified by polymerase chain reaction and quantified spectrophotometrically at 260 nm. All purified PCR products were adjusted to a concentration of 0.1 μg / μl in 50% dimethyl sulfoxide and twice on UltraGAPS ™ coated slides (Corning ™, Inc., Corning, NY) Spotted. After printing, the microarray was UV crosslinked with 700 m Joulesand and stored at room temperature in a slide container in a desiccator. The genes are listed in Table 2 as mentioned above.

Microarray hybridization: Fluorescently labeled cDNA was denatured in hybridization solution (5 × SSC, 0.1% SDS and 25% formamide) at 100 ° C. for 5 minutes, cooled to ambient temperature, and deposited on slides. Hybridization was performed at 55 ° C. for 18 hours. After hybridization, slides are subjected to low stringency buffer (1 × SSC and 0.1% SDS), moderately stringent buffer (0.1 × SSC and 0.1% SDS), highly stringent buffer Washed continuously in liquid (0.1 × SSC) and finally dried with compressed N ₂ .

Signal detection and data analysis: N ₂ dried slides were immediately scanned on a GenePix 4000B scanner (Axon Instruments ™, Inc.) with the same photomultiplier laser power and sensitivity level for each slide. Raw fluorescence data was obtained (10 nm resolution) and the following processing and data visualization was 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 distinct spot and then separated with the housekeeping gene β-actin. The final expression of each gene was expressed as an average of two sets. The resulting gene expression profile of Jurkat cells cultured with and without lactic acid bacteria was obtained. Gene groups that were up-regulated or down-regulated more than 2-fold in Jurkat cells cultured with lactic acid bacteria (CCRC12193, GM-080 or CCRC12188) were selected against those cultured without lactic acid bacteria. The results are shown in Table 3. This difference indicated that different species or strains could switch on or off different genes in the cell. Therefore, the gene expression profile indicated that CCRC12193, GM-080 and CCRC12188 are L. paracusei but belong to different strains.

Example 6: Adhesion of GM-080 to intestinal epithelial cells
Caco-2 cells were employed as epithelial cells in this example. Caco-2 cells have functional microvilli and have hydrolytic enzymes attached to it, which showed the differentiated morphology and function of intestinal mature epithelial cells in vitro.

Cells: Caco-2 was cultured at 37 ° C., 5% CO ₂ /95% air in minimal essential medium (MEM, GIBCO ™) supplemented with 5% FBS. For adhesion assays, 2 ml monolayer Caco-2 cells ((3 × 10 ⁵ cells / ml) were prepared on glass coverslips placed in 6-well plates. The monolayer was used for adhesion assay after 2 weeks of incubation, just prior to use, the monolayer was washed twice with PBS, and 1.5 ml of MEM was added to each well to inoculate the bacteria. Incubated 1 hour before.

Adhesion: 1.5 ml (4 × 10 ⁸ CFU / ml) GM-080 washed once with PBS and resuspended in 1.5 ml MEM medium was added to Caco-2 cells. After incubation at 37 ° C. for 1 hour, monolayer cells were washed 4 times with PBS buffer, fixed with 3 ml of methanol and incubated for 5-10 minutes at room temperature, washed 3 times with PBS, air dried, and Gram stained. Adherent bacteria were detected under microscope-soaked conditions (x100) by counting 15 random fields per coverslip and the mean ± SD of bacteria adhering per field was determined.

  Results: After counting, 102 ± 23.6 GM-080 bacteria adhered to Caco-2 cells. Therefore, GM-080 is a standard established by Jacobsen et al. (Jacobsen, CN, Nielsen, RV, Hayford, AE, Moller, PL, Michaelsen, KF, Parregaard, A., Sandstrom, B., Tvede, M. and According to 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 Humans.Appl.Environ.Microbiol. 1999; 65: 4949-4956). Strong adhesion to Caco-2 cells was observed.

Example 7: Activity of GM-080 and other lactic acid bacteria in an environment mimicking the gastrointestinal tract Acid: GM-080, L. Plantarum, L. Acidophilus, L. et al. Casei and L. To the bulgarcus was added 9 mL PBS with different pH values of 2.0, 2.5 and 3.2 and then this was further incubated at 37 ° C. for 3 hours. After incubation, 1 mL of cells was serially diluted with 9 mL of pH 7.4 PBS. The number of cells before and after acid treatment was estimated and shown as listed in Table 4 below.

  Bile: GM-080, L. Plantarum, L. Acidophilus, L. et al. Casei and L. To the bulgarcus was added 9 mL PBS with a pH value of 2.0 and then this was further incubated at 37 ° C. for 3 hours. After culturing, 1 mL of cells was centrifuged at 6,000 rpm for 10 minutes. The pellet was suspended in 100 μL PBS (pH 7.2). To the solution was further added 10 mL of MRS broth containing 0.3% (w / v) bull bile. Cells were cultured and 1 mL samples were taken at 3, 12, and 24 hours. Samples were serially diluted with 9 mL of pH 7.4 PBS. The number of cells before and after bile treatment was estimated and is also shown in Table 4. This indicates that these lactic acid bacteria remain active in an environment that mimics the gastrointestinal tract.

Example 8: Animal model Animals: Female BALB / c mice were obtained from the National Laboratory Animal Center in Taiwan and were housed for 2 weeks in a room where both light and temperature were controlled.

  Allergen purification: The dust mite allergen Der p 5 is an Escherichia coli comprising a PGEX-2T expression vector as a recombinant Der p 5-glutathione S-transferase fusion protein that can be purified by glutathione-agarose binding chromatography. Expressed in Escherichia coli). Specific E. coli capable of expressing a desired allergen Coli strains were cultured and induced. The bacteria were harvested and washed with TBS (pH 7.5) and 0.1 M phenylmethylsulfonyl fluoride was added. Cells were disrupted by the addition of DNase I, Tween 20 and lysozyme, and the freeze-thaw method. EDTA was added to the mixed solution, and the residue was removed by centrifugation to obtain a supernatant containing the recombinant Der p 5-glutathione S-transferase fusion protein. The supernatant was applied to a glutathione-agarose affinity column to adsorb the fusion protein. The column was subsequently washed with TBS buffer at 4 ° C. and then reduced with Tris base glutathione (pH 8.0) to separate the protein from the 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 Der p 5 and 4 mg aluminum hydroxide. 14 and 21 days after the initial sensitization, mice were exposed to 0.1% purified Der p 5 aerosol for 30 minutes to induce inhalation.

Treatment: Sensitized mice were divided into three groups for this experiment. Group A mice were fed MRS broth 10 times within 2 weeks as a control group. Group B mice received Lactobacillus casei 10 times within 2 weeks and 10 ⁹ CFU bacteria each time. Group B is treated as a positive control. This is because casei has been proven to be effective in inhibiting IgE secretion. Group C mice received 10 doses of GM-080 within 2 weeks and 10 ⁹ CFU of bacteria each time.

Example 9: IgG and IgE secretion
Determination of Der p 5 specific IgG and IgE: After 18 hours from the last inhalation induction, a 500 μL blood sample was taken from the tail. The blood sample was maintained at room temperature for 1 hour and then centrifuged. Serum was stored at -80 ° C. The amount of Der p5-specific IgG2a and IgE was determined by ELISA. A 96 well protein high binding plate was coated with 200 μL of purified Der p 5 diluted to a concentration of 10 μg / mL in coating buffer (0.1 M NaHCO ₃ , pH 9.6). After overnight incubation at 4 ° C., the plate was washed with PBS-Tween 20, and 300 μL blocking buffer (3% BSA) was added thereto. After shaking for 2 hours at room temperature, the plate was washed again with PBS-Tween20. Serum was used for IgG measurement at 1:10 dilution and for IgE measurement at 1: 4 dilution. The sample was shaken for 2 hours at room temperature. After overnight incubation at 4 ° C., the plate was washed with PBS-Tween 20 and 200 μL of biotinylated rat anti-mouse IgE monoclonal antibody or rat anti-mouse IgG mAb was added thereto. Samples were shaken for 2 hours at room temperature and then washed with PBS-Tween20. 200 μL of streptavidin-alkaline phosphatase (1: 1000) was subsequently added and the sample was shaken for 1 hour at room temperature. After 6 washes, the color reaction was initiated by the addition of 200 μL of the phosphatase substrate p-nitrophenyl phosphate disodium salt (pNPP) (Sigma ™ N-2770, USA). Plates were read at 405 nm in a microplate autoreader (Metertech ™, Taiwan).

  Statistical analysis: To assay for changes in IgE and IgG levels, repeated measures of a one-way ANOVA analysis were performed to compare differences between the groups. After analysis of variance, Duncan's multiple range test was used to distinguish differences between experimental and control groups. A value of p <0.05 was used to indicate statistical significance.

  Results: The results are shown in FIG. IgE secretion in the sera of animals treated with GM-080 was greatly reduced and only 25% of those without treatment. On the other hand, IgG secretion in the serum of animals treated with GM-080 was increased 2-fold. Since IgG secretion represents a Th1 T cell response, GM-080 is a target for eliminating IgE secretion associated with allergy related diseases.

Example 10: Cell count of bronchoalveolar lavage fluid Sample preparation: Mice were placed in 5 x 0.5 ml aliquots of 0.9% sterile saline through polyethylene tubes introduced via tracheostomy 18 hours after sensitization. Washed. The lavage fluid was centrifuged (500 g for 10 min, 4 ° C.) and the cell pellet was resuspended in 1 ml PBS solution. Differentiated cells were counted with cytospin preparations stained with Leu staining.

  Statistical analysis: To assay for changes in cell number, repeated measures for one-way ANOVA analysis were performed to compare differences between the groups. After analysis of variance, Duncan's multiple range test was used to distinguish differences between experimental and control groups. A value of p <0.05 was used to indicate statistical significance.

  Results: The results are shown in FIG. The blood cell type contribution in BALF represents the degree of inflammation. Furthermore, the main symptoms of allergic asthma are chronic inflammation of the respiratory tract and eosinophil infiltration. Eosinophils in BALF of animals treated with GM-080 were greatly reduced from 5% to 1%. On the other hand, macrophages and lymphocytes in BALF of animals treated with GM-080 were significantly elevated.

Example 11: IFN-γ secretion in bronchoalveolar lavage fluid Sample preparation: 5 x 0.5 ml 0.9% sterile saline through a polyethylene tube introduced via tracheostomy 24 hours after sensitization Mice were washed with aliquots. The washings were centrifuged (500 g for 10 min, 4 ° C.) and the supernatant was subjected to IFN-γ quantitative analysis as described in Example 1.

  Results: The results are shown in FIG. It was shown that animals fed GM-080 produced 100 pg / mL IFN-γ in BALF. On the other hand, the control group produced only 20-40 pg / mL IFN-γ in BALF. GM-080 was effective in inhibiting allergic inflammation.

Example 12: Inactive GM-080 for treating allergies
Preparation of inert GM-080: Lyophilized GM-080 powder was suspended in distilled water and autoclaved (121 ° C., 15 minutes) before feeding the mice.

Mice and sensitization: Female BALB / c mice (6-8 weeks old) were purchased from the National Laboratory Animal Center (Taipei, Taiwan). All animals were individually maintained in cages controlled at temperature (24 ± 2 ° C.) and humidity (60 ± 5%) and maintained in a 12 hour light-dark cycle under conditions free of specific pathogens. BALB / c mice were injected intraperitoneally with 10 g of recombinant Dermatophagoides pteronyssinus allergen Der p5-6xHis fusion protein adsorbed on 4 mg of aluminum hydroxide. The mice were fed 10 ⁷ , 10 ⁹ and 10 ¹¹ CFU GM-080 per mouse per day. The mice were boosted on day 14 with the same amount of allergen as the sensitization and exposed to 0.1% Der p5-6xHis diluted in PBS 21 days after sensitization. Inhalation induction was performed in a 1 L chamber connected to a DeVilbiss ™ pulmosonic nebulizer (model 2512; DeVilbiss ™ Corp., Somerset, PA) that generates aerosol mist. After 18 hours, serum was collected by tail vein bleeding and IgE was determined by ELISA as described in Example 9.

  Results: The results are shown in FIG. This indicates that BALB / c mice exposed to dust allergy Der p-5 had significantly elevated serum IgE levels when compared to the untreated group (p <0.05). This suggests that allergic sensitized mouse models can be successfully induced. After feeding different amounts of GM-080 per day for 21 days, serum IgE in the GM-080 group was significantly reduced compared to the control group (p <0.05). This result indicated that inactive GM-080 could reduce the allergic response by reducing allergen-specific IgE.

  While embodiments of the present invention have been illustrated and described, various modifications and improvements can be made by those skilled in the art. The invention is not limited to the specific forms as illustrated, and all modifications that do not depart from the spirit and scope of the invention are intended to be within the scope as defined in the claims. .

FIG. 1 illustrates a 1000 × photomicrograph of GM-080 subjected to Gram staining. FIG. 2 illustrates the results of agarose gel analysis of PCR amplified 16s rDNA fragments of GM--080 and lactic acid strains CCRC129313, CCRC14001 and CCRC16100; M represents a molecular marker; 1 represents GM-080; Represents CCRC12913; 3 represents CCRC14001; and 4 represents CCRC16100. FIG. 3A illustrates 16s rDNA sequence alignment of GM-080 and lactic acid strains CCRC129313, CCRC14001, CCRC16100, KLB58, PB4, and F31. FIG. 3B illustrates 16s rDNA sequence alignment of GM-080 and lactic acid strains CCRC129313, CCRC14001, CCRC16100, KLB58, PB4, and F31. FIG. 3C illustrates 16s rDNA sequence alignment of GM-080 and lactic acid strains CCRC129313, CCRC14001, CCRC16100, KLB58, PB4, and F31. FIG. 3D illustrates the 16s rDNA sequence alignment of GM-080 and lactic acid strains CCRC129313, CCRC14001, CCRC16100, KLB58, PB4, and F31. FIG. 3E illustrates the 16s rDNA sequence alignment of GM-080 and lactic acid strains CCRC129313, CCRC14001, CCRC16100, KLB58, PB4, and F31. FIG. 4 illustrates a 16s rDNA distance phylogenetic tree comparing GM-080 of the present invention with related lactic acid bacteria. FIG. 5 illustrates RAPD analysis of GM-080 and common lactic acid strains; M: 100-bp ladder, lane 1: GM-080; lane 2: Lactobacillus paracasei ATCC 25598; lane 3: Lactobacillus paracasei ATCC 25302; Lane 4: Lactobacillus paracasei ATCC 335; Lane 5: Lactobacillus paracasei ATCC 11582; Lane 6: Lactobacillus paracasei ATCC 27216. FIG. 6 illustrates the SDS-PAGE pattern of cell wall proteins of GM-080, conventional Lactobacillus paracasei and Lactobacillus fermentum strains; where M represents the molecular weight of the protein; lane 1 Represents Lactobacillus paracasei; Lane 2 represents Lactobacillus paracasei GM-080; Lane 3 represents Lactobacillus fermentum; F1 represents a Lactobacillus fermentum specific band; and P1, P2 And P3 represents a Lactobacillus paracasei specific band. FIG. 7 illustrates Der p 5 specific IgG (white bars) and IgE (black bars) in the serum of Der p 5 sensitized BALB / c mice induced by inhalation of Der p 5; A is MRS broth B represents the group treated with L. casei; and C represents the group treated with GM-080. FIG. 8 illustrates the number of macrophage, lymphocyte and eosinophil cells in the bronchoalveolar lavage fluid of Der p 5 sensitized mice; A represents the group treated with MRS broth; B treated with L. casei And C represents the group treated with GM-080. FIG. 9 illustrates IFN-γ secretion in bronchoalveolar lavage fluid of Der p 5 sensitized mice; A represents the group treated with MRS broth; B represents the group treated with L. casei; and C represents Represents the group treated with GM-080. FIG. 10 illustrates the effect of inactive GM-080 on IgE production in Der p 5 sensitized BALB / c mice. Der p 5 sensitized mice were orally dosed with different amounts of GM-080 or distilled water (control) daily for three weeks. Serum Der p5-specific IgE levels were determined by ELISA. Compared to the control group, ^* (p <0.1) and ^** (p <0.05) are significantly different in Kruskal-Wallis H test and inductive comparison is used in Dunnett t test It was.

Claims (9)

  The isolated microorganism strain Lactobacillus paracasei GM-080 deposited with CCTCC (the China Cernter for Type Culture Collection) under CCTCC number: CCTCC M 204012.   A composition comprising the microbial strain of claim 1.   The composition according to claim 2, which is used for stimulating IFN-γ secretion.   The composition according to claim 2, which is used for treating an allergy-related disease. The composition according to claim 4, wherein the allergy-related disease is inflammation .   5. The composition of claim 4, wherein the allergy related disease is associated with exposure to aeroallergens.   The composition of claim 2, wherein the microbial strain is alive or inactivated. 8. A composition according to claim 7 , wherein the microbial strain is inactivated.   The composition according to claim 2, in the form of a pharmaceutical composition, a dietary supplement, a food, a health food, or components thereof.