CN112587552B

CN112587552B - Application of bacteroides fragilis839 in preparing medicament for treating or assisting in treating immune related diseases

Info

Publication number: CN112587552B
Application number: CN202010982585.7A
Authority: CN
Inventors: 王蕾
Original assignee: Shenyang New Totem Bioengineering Co ltd; Dalian Totem Life Science Development Co ltd
Current assignee: Shenyang New Totem Bioengineering Co ltd; Dalian Totem Life Science Development Co ltd
Priority date: 2020-09-17
Filing date: 2020-09-17
Publication date: 2023-09-12
Anticipated expiration: 2040-09-17
Also published as: CN112587552A

Abstract

The invention relates to the technical field of application of bacteroides fragilis (Bacteroides fragilis, BF) 839, in particular to application of bacteroides fragilis839 in preparing a medicament for treating or assisting in treating immune related diseases. The invention discovers the key effect of the bacteroides fragilis839 in human intestinal tracts in the treatment or auxiliary treatment of immune related diseases for the first time, and can prepare a pharmaceutical composition by utilizing the key effect of the bacteroides fragilis839 in the immune related diseases, wherein the pharmaceutical composition contains the bacteroides fragilis839, and the pharmaceutical composition can be used for treating the immune related diseases and has very important application value.

Description

Application of bacteroides fragilis839 in preparing medicament for treating or assisting in treating immune related diseases

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to application of bacteroides fragilis839 in preparation of a medicament for treating or assisting in treating immune related diseases.

Background

Immune function is a general term for the body to recognize and eliminate foreign invading antigens, mutate or senescent cells in the body and maintain stable environment in the body. Can be summarized as: (1) immune defenses: preventing invasion of external pathogens and removing invaded pathogens (such as bacteria, viruses, fungi and other microorganisms) and/or other harmful substances, wherein the infection of external pathogens cannot be resisted due to low immune defense function; (2) immune monitoring: the immune monitoring function is low, so that tumors can be generated; (3) immune self-stabilization: stabilization of the in vivo environment is achieved by autoimmune tolerance and immunomodulation. Once immune tolerance is broken, immunomodulation dysfunction can lead to the development of allergic/autoimmune diseases. At present, immune related diseases become common diseases and frequently-occurring diseases in clinic, and the pathogenesis of most diseases relates to abnormal immune functions, immune reaction disorder and inflammatory injury. In particular autoimmune diseases and tumors, are seen in various specialized departments, where lesions may involve focal areas as well as systemic areas. Diseases closely related to immunization are generally considered to include the following: eczema, atopic dermatitis, psoriasis, pemphigus; allergic rhinitis; rheumatoid arthritis, systemic lupus erythematosus, sjogren's syndrome, ankylosing spondylitis; crohn's disease, ulcerative colitis, irritable bowel syndrome, autoimmune hepatitis; glomerulonephritis, nephrotic syndrome, igA nephropathy, interstitial inflammatory nephritis; aplastic anemia, allergic purpura, thrombocytopenic purpura; hyperthyroidism, hashimoto thyroiditis, type I diabetes; idiopathic pulmonary fibrosis, chronic bronchitis emphysema, asthma; multiple sclerosis, myasthenia gravis, neuromyelitis optica, autoimmune epilepsy, autism spectrum disorders, tic disorder, parkinson's disease, alzheimer's disease; autoimmune mastitis; immune infertility, autoimmune hepatitis, tumors, immune side effects of anti-tumor drugs (including but not limited to immune checkpoint inhibitors PD-1/PD-L1 antibodies, chemotherapeutics, autoimmune responses of targeted drugs such as myelosuppression, rash, enteritis, impaired liver and kidney function, etc.); hepatitis and genital tract infection. However, the treatment of these immune-related diseases is very difficult. The methods commonly used at present are: (1) Immunosuppressants such as glucocorticoids, cyclophosphamide, cyclosporine, mycophenolate mofetil, etc.: has strong inhibition effect on various immune cells, but can continuously inhibit immunity after long-term use, has great side effect and increases tumor risk. (2) antibodies: immunoglobulins, monoclonal antibodies, these products have short effective maintenance times, undefined side effects and are expensive. (3) various immune cell infusions and stem cell transplants: has not been used clinically. There is a great clinical need to develop new immunomodulators.

Microbial agents are also a common immunomodulator, such as bacillus calmette-guerin. Imbalance in the composition and function of the intestinal microbiota (dysbiosis) is the root cause of a number of diseases including: tumors, neuropsychiatric diseases, rheumatic immune diseases, skin diseases, chronic infections caused by hypoimmunity, etc.

There are studies found that: bacteroides fragilis NCTC9343 can regulate the host immune system in health and disease, has a key effect on the development of the mammalian immune system and on the stimulation of CD4+ T cells that produce IL-10, can regulate the Th1/Th2 balance, confers host benefits in experimental autoimmunity, inflammation and infectious disease far beyond the gastrointestinal tract, and can affect almost all of the body systems. As is well known, the efficacy of probiotics is strain-specific, and the presently discovered bacteroides fragilis has tens of strains, as well as virulent bacteroides fragilis strains. The bacteroides fragilis839 (totem probiotics) is a Chinese patent strain (homologous patent number: CN 1029857C) cultured from neonatal feces by a microbiologist Zhang Jijie in China, 9 months in 1983, and is found to have the functions of improving immune T cells and preventing and treating intestinal tract and respiratory tract diseases through preliminary researches, and is a non-toxic and harmless bacteroides fragilis. However, studies in other immune-related diseases have not been reported. The unit is the only company which can produce and sell the bacteroides fragilis839 (totem probiotics), and the unit is found to have therapeutic effect on the immune related diseases applied by the unit in the long-term use process.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides application of bacteroides fragilis839 in preparing medicines and foods for treating or assisting in treating immune related diseases and provides a medicine combination for treating or assisting in treating immune related diseases.

In a first aspect, the invention provides the use of bacteroides fragilis839 (Bacteroides fragilis839, BF 839) in the manufacture of a medicament for the treatment or co-treatment of an immune related disorder.

As a preferred embodiment of the present invention, the bacteroides fragilis839 is any one of the following: bacteroides fragilis839 live bacteria; bacteroides fragilis839 subjected to gene recombination, transformation or modification, attenuation, chemical treatment, physical treatment or inactivation; bacteroides fragilis839 lysate; and/or bacteroides fragilis839 culture supernatant.

In a second aspect, the invention provides a pharmaceutical composition for the treatment or adjuvant treatment of immune related diseases, which is characterized in that the pharmaceutical composition comprises a pharmaceutically effective dose of bacteroides fragilis839 and a pharmaceutically acceptable carrier thereof.

As a preferred embodiment of the present invention, the immune-related disease is any one of the following diseases: eczema, atopic dermatitis, psoriasis; allergic rhinitis, pemphigus disease; rheumatoid arthritis, systemic lupus erythematosus, sjogren's syndrome, ankylosing spondylitis; crohn's disease, ulcerative colitis, irritable bowel syndrome, autoimmune hepatitis; glomerulonephritis, nephrotic syndrome, igA nephropathy, interstitial inflammatory nephritis; aplastic anemia, allergic purpura, thrombocytopenic purpura; hyperthyroidism, hashimoto thyroiditis, type I diabetes; idiopathic pulmonary fibrosis, chronic bronchitis emphysema, asthma; multiple sclerosis, myasthenia gravis, neuromyelitis optica, autoimmune epilepsy, autism spectrum disorders, tic disorder, parkinson's disease, alzheimer's disease; autoimmune mastitis; immune infertility; autoimmune hepatitis; immune side effects of tumor and antitumor drugs (including but not limited to immune checkpoint inhibitor PD-1/PD-L1 antibodies, chemotherapeutics, autoimmune responses of targeted drugs such as myelosuppression, rash, enteritis, liver and kidney function impairment, etc.); hepatitis, genital tract infection, helicobacter pylori infection.

As a preferred embodiment of the present invention, the pharmaceutical composition is a tablet, a capsule, an oral liquid or a lyophilized powder.

As a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is one or more of skimmed milk, lactose, glucose, sucrose, sorbitol, mannose, trehalose, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil.

The beneficial effects of the invention are as follows: the key role of the bacteroides fragilis839 (totem probiotics) in human intestinal tracts in immune related diseases is discovered for the first time in the invention. Bacteroides fragilis839 (totem probiotics) can effectively treat immune related diseases. Has great application prospect in the medical and large health fields. Bacteroides fragilis839 (totem probiotics) is a microorganism normally existing in intestinal tracts, is a non-toxic harmless probiotics after being researched, has no side effect on bodies, has been marketed for 20 years in China, and has incomparable advantages of other new medicines. The key role of the bacteroides fragilis839 (totem probiotics) in immune related diseases can be utilized to prepare a pharmaceutical composition, wherein the pharmaceutical composition contains the bacteroides fragilis839 (totem probiotics), can be used for treating immune related diseases, and has very important application value.

Drawings

FIG. 1 is a schematic diagram of the distance along the edge and center of the movement of the mouse.

Figure 2 is a schematic of the edge and center residence time of two groups of mice.

Fig. 3 is a diagram of an open field experimental trajectory.

FIG. 4 is a graph showing the number of times two groups of mice entered the open arm and the closed arm.

Figure 5 is a schematic of residence time in open arm, closed arm for two groups of mice.

Fig. 6 is a schematic diagram of the movement distance of two groups of mice on an open arm and a closed arm.

Fig. 7 is a diagram of an overhead plus maze experimental trace.

FIG. 8 is a graph showing the number of contacts between two groups of mice and each region during the first and second stages of the experiment.

Fig. 9 is a schematic diagram showing the contact time of two groups of mice with each zone during the first and second stages of the experiment.

Fig. 10 is a schematic diagram of a first stage three-box experimental trajectory (top left metal pellet, bottom right familiar mouse).

FIG. 11 is a schematic diagram of a second stage three-box experimental trace (strange mice in the upper left corner, familiar mice in the lower right corner).

FIG. 12 is a schematic of escape latency(s) for two groups of mice in a pilot voyage experiment for 4 consecutive days.

Fig. 13 is a graph showing the number of passes across the platform on day 5 for two groups of mice in a space search experiment.

Fig. 14 is a trace diagram of a water maze experiment.

Fig. 15 is a schematic diagram of the skin of patient 1 prior to intervention.

Fig. 16 is a schematic diagram of the skin after patient 1 intervention.

Fig. 17 is a schematic diagram of the skin comparison of patient 2 before and after intervention.

Fig. 18 is a schematic diagram of the skin comparison of patient 3 before and after intervention.

Fig. 19 is a schematic diagram of the skin comparison of patient 4 before and after intervention.

Fig. 20 is a schematic diagram of the skin comparison of patient 5 before and after intervention.

Fig. 21 is a schematic diagram of the skin comparison of patient 6 before and after intervention.

Fig. 22 is a pre-treatment electroencephalogram for patient 1.

Fig. 23 is an electroencephalogram of patient 1 after treatment.

Fig. 24 is a pre-treatment electroencephalogram for patient 2.

FIG. 25 is an electroencephalogram of patient 2 after treatment

Detailed Description

In order to more clearly demonstrate the technical scheme, objects and advantages of the present invention, the technical scheme of the present invention is described in detail below with reference to the specific embodiments and the accompanying drawings.

The Bacteroides fragilis839 used in the following examples is Bacteroides fragilis839 BF839 (bacteroides fragilis 839), which is preserved in China general microbiological culture collection center (CGMCC) of 5 months and 3 days in 1990, and has a preservation number of CGMCC No.0157 and a preservation address of North Xicilu No. 1, 3 in the Chaoyang area of Beijing city. The product of the bacterial liquid (totem probiotics) of the bacteroides fragilis839 is provided by the company of development of biological sciences of Dalian totem, the content of each bacterial liquid is 20ml, and the viable bacterial content of the bacteroides fragilis839 is 60 multiplied by 10 ⁸ CFU/branch.

Example 1 Effect of Bacteroides fragilis839 on fragile X syndrome related autism behavior

1. Design of experiment

The mice of Fmr KO with 3 weeks age of weaning are selected as experimental subjects and divided into two groups, namely a blank control group (Fmrl KO group) and an experimental group (BF 839 group), wherein each group comprises 15 mice, 5 mice are one cage, and each group comprises 3 cages. Wherein the experimental group (BF 839 group) mice are Fmr1 gene knockout mice which drink Bacteroides fragilis839 BF839 bacterial liquid for 4 weeks in a free drinking mode, the blank control group (Fmrl KO group) mice are Fmr gene knockout mice which drink autoclaved tap water for 4 weeks in a free drinking mode, and the two other feeding modes are consistent. Animal behavioural experiments were started at 7-8 weeks of age in mice.

1. Open field experiment

The open field experimental analysis box is a square open box with the height of 30cm and the bottom side of 70 cm. Each mouse was placed in the central position within the box and allowed to move freely for 5 minutes while the mouse motion profile was tracked using a camera and the movement distance and residence time of the mouse in the peripheral and central areas of the box were recorded using SMART software (Smart v 2.5.21; panlab, spain) for 5 minutes.

1.2 overhead maze test

The elevated plus maze is cross-shaped, the instrument is 50cm in height and consists of four arms, each arm is 30cm long and 5cm wide, two sides of one opposite side are covered by a baffle plate with the height of 15 cm, and the two sides are closed areas, namely closed arms; the other side is not covered by a baffle plate, and is called an open area and an open arm. The arms are connected by a central platform of 5 x 5 cm. Each mouse was placed in the central area facing the open arm at the beginning of the test and allowed to freely move for 5 minutes. Meanwhile, a camera is used for tracking the movement track of the mouse, SMART software is used for recording the movement distance and the residence time of the mouse in the open arm and the closed arm, and the movement distance and the residence time are recorded for 5min.

1.3 three-box social experiments

The three boxes comprise three transparent plastic cabins, the three cabins are divided into a left cabin, a middle cabin and a right cabin by two partition boards, the size of each cabin is 20 multiplied by 40 multiplied by 23cm, and 1 rectangular (6 multiplied by 6 cm) opening is arranged below each partition board, so that experimental animals can freely enter the three cabins. Before the experiment starts, an empty metal cage is placed in each of the left and right cabins, and the test mice are placed in three boxes along the side wall of the middle cabin, so that the test mice are adapted to 10min. In the first stage of the experiment, the test mice are taken out, a ball is placed in a metal cage in a left cabin, a mouse with the same identity with the cage is placed in a metal cage in a right cabin, and finally the test mice are placed in three boxes along the side wall of a middle cabin again, so that the test mice can freely move in the three cabins for 10min. In the second stage of the experiment, the test mice were taken out, in a metal cage with balls, one different cage mice were used to replace the balls, and the test mice were placed in three boxes again along the side wall of the middle compartment, so that the test mice were free to move in the three compartments for 10min. The movement track of the mice was tracked simultaneously using cameras during the first and second stages of the experiment and the number and time of contact of the test mice with the metal cage (4.0 cm around the metal cage defined as the contact range) was recorded with SMART software for 10min.

1.4 Water maze experiment

The water maze pool is round, the diameter is 120cm, the height is 50cm, the using platform is round, the diameter is 5cm, and the height is 30cm. The water depth just goes over the platform by 1cm, and the temperature is 21-22 ℃. The experiment is divided into two parts: (1) in the positioning navigation experiment stage, the mice are trained continuously for 4 days, 4 times a day, 30s each time, and the time for the mice to find the platform by entering water from different points in different quadrants respectively in sequence, namely the time from entering water to climbing the platform is called as escape latency. The movement track of the mice is tracked by using a camera, the escape latency of the mice is recorded by using SMART software, and the average value of the score of 4 escape latencies is used as the final score of the day and used as statistics. During the experiment, if the mouse does not find the platform within 60s, it is led to the platform and placed for 10s, and the escape latency is calculated as 60 s. (2) During the experimental phase of space exploration, the platform was removed on day 5 and the mice were placed in water from the opposite side of the original platform quadrant. The movement track of the mice was tracked using a camera and the swimming track of the mice within 60s was recorded with SMART software and the number of times the mice traversed the hidden platform within 60s was analyzed. After each experiment, the animals were removed, wiped dry and returned to the cages.

1.5 statistical analysis

All results are expressed in (x±s) and analyzed by SPSS 17.0 statistical software; the comparison between the two groups of experiments adopts independent sample t test, and the test p is less than 0.05, thus having statistical significance.

2. Results and analysis

1. Open field experiments: the two groups of mice were differentiated in distance of movement and residence time along the edge and in the center. In contrast to the Fmrl KO group, BF839 group mice had shorter edgewise movement distances (t=3.631, p=0.001), longer central movement distances (t=4.657, p=0.000), and the results were statistically significant (fig. 1); in comparison to the Fmrl KO group, BF839 group mice had less edge residence time (t=5.959, p=0.000), and longer residence time in the center (t=6.024, p=0.000); the results were statistically significant (FIG. 2); the results are combined with an open field experimental trace diagram (figure 3) to intuitively show that the bacteroides fragilis839 BF839 can strengthen the autonomous exploration behaviors of Frl KO mice on new environments and reduce anxiety.

2. Overhead maze test

In the 5-minute experiment, two groups of mice were different in the number of entries, residence time and movement distance between the open arm and the closed arm. In comparison to the Fmrl KO group, BF839 group mice had a reduced number of open arms (t=2.506, p=0.018), an increased number of closed arms (t=2.499, p=0.021), and as a result, there was a statistical difference (fig. 4); in comparison to the Fmrl KO group, BF839 group mice had a reduced residence time in the open arm (t=2.610, p=0.014) and an increased residence time in the closed arm (t=5.845, p=0.000), resulting in a statistical difference (fig. 5). In comparison to the Fmrl KO group, BF839 group mice had a statistically different result from the decrease in open arm movement distance (t=2.825, p=0.010) and the increase in closed arm movement distance (t=3.553, p=0.002). The results are intuitively displayed by combining an overhead plus maze experimental trace diagram (figure 7), so that BF839 mice are more willing to stay in a safer closed arm, dangerous environments are avoided, and the Bacteroides fragilis839 BF839 can improve the environmental cognitive ability of Fmrl KO mice and increase the dangerous consciousness.

3. Three-box social experiment

In the first stage of the experiment, the BF839 group was exposed to the familiar mice with a trend of increasing times and times, but no statistical difference (p < 0.05) was observed, and at the same time, the metal balls were exposed to increasing times (t=2.841, p=0.011) and times (t=2.148, p=0.046) compared to the Fmrl KO group, and the results were significantly different (fig. 8, 9); in the second phase of the experiment, BF839 group was contacted with strange mice more times (t=4.718, p=0.000), longer time (t=3.419, p=0.002) compared to Fmrl KO group, resulting in significant differences (fig. 3a, 3 b). The above results are visually displayed in combination with the first stage three-box experimental trace plot (fig. 10, 11): when facing the same kind of familiarity and complete strangeness, the Bacteroides fragilis839 BF839 group mice are more willing to contact with strange individuals for longer frequency and time, show social novelty preference, and suggest that Bacteroides fragilis839 BF839 can increase the social behavior of Fmr gene knockout mice.

4. Water maze test

In the positioning voyage experiment, the two groups of mice were observed to have no difference in escape latency, and the BF839 group was significantly lower in escape latency than the Fmrl KO group (t=4.05, p=0.001) by day 4 of the experiment, resulting in statistical differences (p < 0.05) (fig. 12). In the space search experiment, after removing the platform, BF839 group was observed to pass through the platform significantly more times than Fmrl KO group (t=2.80, p=0.012) within 1 minute, resulting in statistical differences (p < 0.05) (fig. 13); the above results are combined with a water maze experimental trace graph (FIG. 14)

Intuitively, the speed of finding the hidden platform position of the BF839 group mice is obviously faster than that of the FrlKO group mice, and the difference is more obvious along with the time, which indicates that the Bacteroides fragilis839 BF839 can improve the cognitive function and the learning and memory capacity of the Fmr1 gene knockout mice.

From the above results, the bacteroides fragilis839 provided by the invention can improve abnormal exploration behaviors, anxiety, social behaviors and cognitive functions of the Frl KO mice, and provides a safe and effective treatment method for autism spectrum disorder diseases including but not limited to fragile X syndrome.

EXAMPLE 2 Single arm, open, self-control test of the therapeutic Effect of Bacteroides fragilis839 BF839 on psoriasis

1. Inclusion criteria:

1. age 6-65 years (including 6-65 years), and sexual intercourse is unlimited;

2. patients with psoriasis vulgaris clinically and pathologically diagnosed (diagnosis standard reference Zhao Bian, chinese clinical dermatology): rash is manifested by erythema and scales, and has the characteristics of thin film phenomenon, punctate bleeding and symmetrical distribution mainly on the extending side of the body; point-like, small sphere-like, hairpin-like, annular blood vessels and diffusely distributed white scales can be seen under the skin scope;

3. the PASI score is less than or equal to 12 points, and is light-medium psoriasis.

2. Exclusion criteria:

1. age < 6 years, > 65 years;

2. hormone (excluding external use) or immunosuppressant drugs and other probiotic foods or drugs for oral administration for one month;

3. other clinical trials were accepted within one month;

4. women who are pregnant or in lactation, or women of child bearing age who cannot take effective contraceptive measures (condoms, oral contraceptives, intrauterine devices, etc.) during the test period.

3. Exit criteria

1. Voluntary termination test for patients

2. Not according to the scheme, missed dose > 20%/month

3. The original treatment scheme is obviously changed, such as stopping or adding hormone ointment and adding other probiotics foods or medicines.

4. And (3) judging curative effect: PASI score was performed before and after the group, and PASI decrease rate was calculated.

Table 1: PASI scoring table (Psoriasis Area and Severity Index, psoriasis skin lesion area and severity)

PASI score= (E _Head +I _Head +D _Head )×A _Head ×0.1+(E _{Upper limb} +I _{Upper limb} +D _{Upper limb} )×A _{Upper limb} ×0.2+(E _{Trunk body} +I _{Trunk body} +D

_{Trunk body} )×A _{Trunk body} ×0.3+(E _{Lower limb} +I _{Lower limb} +D _{Lower limb} )×A _{Lower limb} ×0.4

1. Scoring according to the score of 0-6 given by the size of the skin damage area (A)

Reference area: the area of the palm of the patient was 1%.

2. Skin lesion severity score

(1) Erythema (E):

1-pale red 2-red 3-deep red 4-red extremely deep

(2) Infiltration (I):

0-skin loss is flush with normal skin;

1-skin lesions are slightly elevated from normal skin surface;

2-moderately raised, the edge of the plaque being round or sloped;

3-the skin is damaged and fattened, and the bulge is obvious;

the 4-skin loss is highly thickened and the bulge is very obvious.

(3) Scale (D):

0-no visible scale on the surface;

1-most of the surfaces of the skin lesions are covered with scales, and the fine scales are the main part;

2-most of the skin lesions are completely or incompletely covered with scales, and the scales are flaky;

3-almost all the surface of the skin damage is covered with scales, and the scales are layered thicker;

4-the whole surface of the skin is covered with scales, and the scales are layered in a very thick way

4. The scheme is implemented as follows:

the patient took bacteroides fragilis839 BF839 orally (powder/dose, taken within 20 minutes after meals, <18 years old, 1 dose per day, > 18 years old and PASI < 6 doses per day, and > 18 years old and PAS >6 doses per day for two doses per day, continuously taken for 3 months, and the PASI score was performed monthly to calculate the PASI decline rate.

5. And (3) result statistics:

recovery, PASI score reduction >90%;

the effect is obvious, and the PASI score reduction rate is 60 percent to 89 percent;

effectively, the PASI score decrease rate was 20% to 59%;

ineffective, PASI score decrease rate <20%.

Total effective rate (number of cure cases + number of significant cases + number of effective cases)/total number of cases 100%.

Statistical treatment was performed using the statistical software SPSS (version 18.0), using paired sample t-test based on the nature of the data, with P <0.05 as the significance level.

6. Results

Table 2: bacteroides fragilis839 BF839 therapeutic effect on psoriasis

The results of a total of 27 cases, 1 case actively exited for its own cause, and 4 cases successively exited for self-disabling hormone ointment are shown in Table 2. From the results, the bacteroides fragilis839 provided by the invention can effectively treat psoriasis, and provides a safe and effective treatment method for psoriasis.

Example 3 effect of probiotic bacteroides fragilis839 BF839 on treatment of refractory autoimmune epilepsy-one, group-entering criteria:

1. diagnosis of chronic epilepsy: at least 2 non-evoked or non-reflex seizures with a phase separation time of >24h, and an electroencephalogram suggests epileptiform abnormality, which accords with the definition of 2014 epilepsy.

2. Patients diagnosed with seizures with partial seizures have historically been insufficiently controlled with at least 2 AEDs. At the time of group entry, the patient receives a steady dose of 1-5 standard AEDs and has at least 4 local attacks in the 4 week baseline phase, with a course of disease of at least >4 months

3. Meets any one of the following conditions:

(1) The past diagnosis of "autoimmune encephalitis" including various neuroantibody positive postlimbic encephalitis epilepsy, autoimmune disease combined epilepsy, primary autoimmune Resamussen encephalitis

(2) Once diagnosed as "viral encephalitis but no specific virus detected"

(3) Seizures of unknown cause, with one of the following symptoms prior to onset:

(3.1) prodromal symptoms of infection such as fever or sore throat;

(3.2) vaccine injection within 2 months;

(3.3) taking more cold drink before onset of illness or having gastrointestinal symptoms such as constipation, diarrhea, abdominal pain, dyspepsia, etc

(3.4) there are life stresses, such as insufficient sleep, stress and other diseases, before illness

(3.5) examination to find a cervical or celiac lymphadenectasis

(3.6) epilepsy with psychotic symptoms, character changes, cognitive decline, memory decline

(3.7) blood routine discovery of neutrophil or lymphocyte or monocyte abnormalities

(3.8) examination of lymphocyte subpopulations for any one of abnormalities

(3.9) electroencephalogram background slow, or increase in slow waves

(3.10) blood or cerebrospinal fluid autoimmune antibody positivity

(3.11) autoimmune diseases such as allergic rhinitis, eczema, urticaria, hyperthyroidism or hypothyroidism, systemic lupus erythematosus, etc.

2. Exclusion criteria:

1. structural etiologies such as trauma, tumor (paraneoplastic syndrome or nodular sclerosis), cortical developmental deformity (FCD), stroke, epilepsy caused by hypoxic ischemic encephalopathy.

2. Hereditary etiology mainly refers to that the patient has mutation which has obvious effect on individual epilepsy, including Dravet syndrome, childhood absence epilepsy, adolescent myoclonus epilepsy and independent comprehensive tonic clonic epilepsy; self-limiting focal epilepsy, e.g. benign epilepsy in children with spike waves in the central temporal region

3. The infectious etiology is directly caused by known infection, wherein epileptic seizure is the core symptom 1 of the disease, such as cerebral cysticercosis, tuberculosis, AIDS, cerebral malaria, toxoplasmosis, suppurative meningoepithymia, and viral encephalitis of disease origin, such as herpes simplex viral encephalitis, etc

4. Etiology of metabolism: epilepsy directly caused by known or presumed metabolic disorders, including porphyria, uremia, amino acid disorders or epileptic seizures dependent on pioglycerol.

3. Method of

1. On the basis of not changing the original medicine, 2 bacteroides fragilis839 BF839 (totem probiotics) are taken after meals for 3 months.

2. Issuing records to the patient, re-diagnosing the patient once a month after entering the group, issuing bacterial liquid and registering the number of attacks

3. And (3) conducting electroencephalogram examination within 3 months before and after the group entering.

4. Efficacy index

The primary efficacy endpoint is remission rate, defined as the proportion of patients with a 30-day seizure rate (all partial seizures) reduced by ≡50% compared to baseline throughout the 12-week maintenance phase. Secondary efficacy evaluation 1, proportion of secondary tonic clonic (SGTC) seizures reduced by ≡50% compared to baseline; 2. electroencephalogram report rate of improvement (including reduction or disappearance of discharge and slow waves)

4. Results

Group-in patients 25, 6 of which did not complete for 3 months, 1 of 6 failed visits, 5 exited, for the following reasons: 2 are patient choices, 1 is a change in treatment regimen, 1 is an increase in episodes, and 1 is a side effect.

In 19 patients, 8 out of >50% of the episodes were reduced (42%) and the total number of episodes was reduced on average to 42%, 9 out of >50% of the 12 patients with tonic clonic episodes and tonic episodes (75%), 5 patients reached 30 days without episodes (26%) at 3 months after the group, and 12 out of 15 patients with pre-and post-electroencephalographic results (80%) were improved. There were 3 reported side effects, 2 vomiting, 2 cases were withdrawn, 1 case were diarrhea, and diarrhea was ameliorated after decrement. The electroencephalogram of 2 patients before and after treatment is shown in figures 23-25.

Example 4 Effect of Bacteroides fragilis839 BF839 on treatment of pregnancy complicated with vaginitis

1. Clinical data

1.1 general data collection hospital pregnant women who are documented and examined, patients complain of pruritus vulvae, and vaginal secretions are taken for examination. The patient voluntarily accepts the use of the sanitary pad containing the bacteroides fragilis839 BF839, and the vaginal secretion is reviewed by taking one month (two pieces per day) as a period, and whether the sanitary pad BF839 containing probiotics is continued or not is judged according to the condition of the pregnant woman to be followed. The complications of pregnancy (gestational diabetes, gestational hypertension, gestational complicated blood system diseases, etc.), the treatment with antibiotics or other vaginal cleansing lotion were eliminated at the time of the visit or the first 1 week. A total of 4 pregnant women who were not interviewed and failed to use sanitary napkins as prescribed were included in the study. The age of 4 cases of pregnancy complicated with vaginitis was 18-45 years, and all subjects with heart, brain, liver, kidney and hematopoietic diseases were denied. See table 3 for general cases.

Table 3: clinical data

1.2 laboratory checks: index in cervical secretion examination: cleanliness, pus cells, clue cells, spores, hyphae, gardnerella, and leukocyte esterases. The vaginal secretion examination was completed at the time of the labor examination, and the results are shown in Table 4.

Table 4: laboratory related index of 4 cases of pregnancy complicated with vaginitis

The results are shown in Table 4, 4 pregnant women have pruritus vulvae discomfort in the conventional production and examination, vaginal secretion examination is examined, the results are shown in Table 2, spores, hyphae and trichomonas in 4 pregnant women are negative, sanitary pads containing probiotics are free, and the pregnant women are ordered to have discomfort following diagnosis and next re-diagnosis time in the using process. After 4 pregnant women use the sanitary pad containing probiotics, the pruritus vulvae symptom is obviously improved. The cleanliness and the pus cells in secretion examination in case 1 and case 2 are obviously improved compared with the first time, and leucocyte esterase turns to negative; although the cleanliness is unchanged in case 3, gardnerella turns to yin, the vaginal secretion results of case 4 are not changed obviously, and the symptoms of pruritus vulvae of the patient are improved obviously.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. Use of bacteroides fragilis839 (Bacteroides fragilis) 839 in the preparation of a pharmaceutical composition for the treatment or co-treatment of immune related diseases;

the immune related diseases are selected from autism caused by fragile X syndrome, refractory autoimmune epilepsy and pregnancy complicated vaginitis.

2. The use of claim 1, wherein the pharmaceutical composition comprises a pharmaceutically effective dose of bacteroides fragilis839 and a pharmaceutically acceptable carrier thereof.

3. The use according to claim 1 or 2, wherein the pharmaceutical composition is in the form of a tablet, capsule, oral liquid or lyophilized powder.

4. The use according to claim 2, wherein the pharmaceutically acceptable carrier is one or more of skim milk, lactose, glucose, sucrose, sorbitol, mannose, trehalose, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate or mineral oil.