Intervention strategies for the prevention or treatment of diabetes, autoimmune diseases, inflammatory diseases, or cardiovascular diseases.

Abstract

This invention relates to an intervention strategy for the prevention or treatment of individuals suffering from inflammation-related diseases such as diabetes, autoimmune diseases, inflammatory diseases, or cardiovascular diseases. The intervention strategy preferably involves the administration of chlorinated, fluorinated, or bromine-substituted tryptophan, preferably 6-bromotryptophan, and / or mono-fatty acid-substituted glycerophosphate choline (GPC) or di-fatty acid-substituted glycerophosphate choline (GPC), preferably selected from 1-myristoyl-2-arachidonicoyl-glycerophosphate choline (MA-GPC) and 1-arachidonicoyl-glycerophosphate choline (A-GPC), or any derivatives or functional equivalents thereof. Alternatively, the intervention involves the application of a species of the genus *Desulfovibrio*, preferably selected from *Desulfovibrio piger*, *Desulfovibrio fairfieldensis*, *Desulfovibrio desulfuricans*, *Desulfovibrio indonensis*, *Desulfovibrio alaskensis*, *Desulfovibrio vulgaris*, *Desulfovibrio vietnamensis*, and *Desulfovibrio gigas*.

Description

Technical Field

[0001] This invention relates to the prevention and / or treatment of inflammatory-related diseases selected from diabetes, autoimmune diseases, inflammatory diseases, and cardiovascular diseases, and more specifically, to the use of compositions comprising specific microorganisms and / or their metabolites in the prevention and / or treatment. Background Technology

[0002] The role of inflammation in both type 1 and type 2 diabetes has generated interest in targeting and enhancing inflammation to improve disease prevention and treatment. Evidence suggests that inflammatory pathways are key mediators in the pathology of diabetes, particularly when stimulated by risk factors including obesity or being overweight.

[0003] Furthermore, the relationship between diabetes and cardiovascular disease is well-established, with diabetic patients having a significantly increased risk of cardiovascular disease. Atherosclerosis is the most common cause of coronary artery disease, and its characteristic status as a chronic low-grade inflammatory state is now widely accepted.

[0004] In addition, inflammation is a typical hallmark of autoimmune diseases. These diseases can have sudden onset, meaning the condition worsens, or they can remit, meaning the symptoms improve or disappear. Treatment for autoimmune diseases depends on the type of disease, but in most cases, an important goal is to reduce inflammation.

[0005] Current therapies for diabetes, cardiovascular disease, autoimmune diseases, and inflammatory diseases, in addition to their primary modes of action, also possess anti-inflammatory properties. Non-pharmacological treatments such as lifestyle interventions reduce inflammatory states, for example, by assessing circulating C-reactive protein (CRP) and interleukin-6 (IL-6) concentrations, and improve cardiovascular and all-cause mortality.

[0006] This makes treatments for inflammation an attractive area of ​​research.

[0007] New treatment strategies are needed to improve the quality of life for patients with inflammation-related diseases, including diabetes, autoimmune diseases, inflammatory diseases, and cardiovascular diseases. There remains a need to develop new or improved prevention and / or treatment strategies. The purpose of this invention is to meet this need. Summary of the Invention

[0008] The inventors investigated whether the application of fecal transplants derived from allogeneic (healthy donors) or autologous (self) sources has beneficial effects on patients with autoimmune diseases. Previous studies have confirmed that the application of autologous fecal transplants can induce an immune reset in patients with autoimmune diseases, thereby reducing the severity of the disease.

[0009] The inventors have surprisingly discovered that this beneficial effect can be traced back to specific components in fecal matter. These components with therapeutic applications are most specifically related to the following:

[0010] Bacteria of the genus *Desulfovibrio* are preferably selected from *Desulfovibrio piger*, *Desulfovibrio fairfieldensis*, *Desulfovibrio desulfuricans*, *Desulfovibrio indonensis*, *Desulfovibrio alaskensis*, *Desulfovibrio vulgaris*, *Desulfovibrio vietnamensis*, and *Desulfovibrio gigas*, with *Desulfovibrio piger* being the most preferred.

[0011] Metabolites 6-bromotryptophan, 1-myristoyl-2-arachidonoyl-glycero-phosphocholine (MA-GPC) and 1-arachidonoyl-glycero-phosphocholine (A-GPC), or more generally, chlorinated, fluorinated, or bromosubstituted tryptophan, or mono- or di-fatty acid-substituted glycero-phosphocholine, or any derivatives or functional equivalents of these metabolites.

[0012] Further research has surprisingly revealed that *Desulfovibrio* species and / or metabolites according to the invention, particularly 6-bromotryptophan, have anti-inflammatory effects, namely reducing inflammatory states and reducing inflammatory markers associated with, for example, type 1 and type 2 diabetes, autoimmune diseases, cardiovascular diseases, and inflammatory diseases such as systemic inflammatory response syndrome (SIRS) or sepsis.

[0013] Furthermore, a negative correlation was found between plasma 6-bromotryptophan levels and the presence of type 2 diabetes. This suggests that the *Desulfovibrio* species and / or metabolites according to the invention, particularly 6-bromotryptophan, may contribute to the prevention of type 2 diabetes and may also contribute to the prevention or treatment of cardiovascular complications (microvascular and macrovascular). Additionally, the *Desulfovibrio* species and / or metabolites according to the invention, particularly 6-bromotryptophan, were found to promote insulin secretion from β-cells.

[0014] Mechanistically, the inventors have revealed that the biological effects of 6-bromotryptophan (6-BT) appear to differ from those of tryptophan. 6-BT does not act by activating AhR, but it does inhibit NF-κB activation and enhance mitochondrial metabolism. The latter is typically utilized by cells with an anti-inflammatory phenotype. Due to its broad effects across multiple cell types, its inhibitory effect on NF-κB signaling, and its promoting effects on mitochondrial metabolism and adaptation, 6-BT could be a highly useful therapeutic agent not only in the context of type 1 and type 2 diabetes, but also in many other inflammation-related diseases such as sepsis, systemic inflammatory response syndrome (SIRS), and cardiovascular disease.

[0015] Furthermore, without being bound by any theory, the inventors believe that the Desulfovibrio species and metabolites described according to the present invention can modulate the immune system, for example by resetting B cell clonal function and regulatory T cells, which in turn can suppress autoimmune responses.

[0016] It is believed that in early life, the immune system is trained through continuous crosstalk with the developing gut microbiome. In this way, the gut microbiome plays a crucial role in regulating the development, composition, and function of adaptive immune cells (see, for example, Agace and McCoy Immunity 46, April 18, 2017). It is this process, among other things, that results in a properly functioning immune system without autoimmune factors.

[0017] However, the crosstalk between the immune system and the gut microbiome, or its end result, can be disrupted, leading to the production of autoantibodies (by B cells) and the formation of autoreactive T cells. The treatment according to the invention overcomes this disruption by reactivating the interaction between the immune system and the gut microbiome (including its specific bacteria and / or derivatives such as metabolites) and resulting in suppression of the autoimmune response.

[0018] Therefore, the use of the aforementioned *Desulfovibrio* species and / or metabolites in autoimmune diseases can prevent autoimmune destruction of target tissues and restore immune tolerance. This can be achieved by stimulating the immune system, wherein the *Desulfovibrio* species and / or metabolites are preferably administered into the duodenum (directly or indirectly, e.g., orally). This invention is preferably not intended to alter the gut microbiome, i.e., the composition of the gut microbiota.

[0019] WO2019168401 discloses the use of fecal matter in the prevention and treatment of autoimmune diseases, wherein the fecal matter is autologous to the individual and is preferably applied to the small intestine, preferably the duodenum, where it can initiate immune reset, thereby reducing the severity of autoimmune diseases. However, the therapeutic efficacy of the method in WO2019168401 still has room for improvement, and its treatment is laborious and difficult to scale up.

[0020] Therefore, the present invention aims to prevent or treat inflammation-related diseases selected from the following.

[0021] Type 1 diabetes;

[0022] Type 2 diabetes,

[0023] Cardiovascular diseases, especially coronary artery disease (also known as coronary heart disease and ischemic heart disease), peripheral artery disease, cerebrovascular diseases (such as stroke or transient ischemic attack (TIA)), atherosclerosis, stenosis, renal artery stenosis, aortic disease, aortic aneurysm, cardiomyopathy, hypertensive heart disease, hypertension, heart failure, pulmonary heart disease, arrhythmia, cardiovascular inflammation, inflammatory heart disease, endocarditis, inflammatory cardiac hypertrophy, myocarditis, eosinophilic myocarditis, valvular heart disease, congenital heart disease, or rheumatic heart disease;

[0024] Inflammatory diseases, particularly cardiovascular inflammation such as carditis, endocarditis, myocarditis, pericarditis, vasculitis, arteritis, phlebitis, or capillary vasculitis, or gastrointestinal inflammation such as esophagitis, gastritis, gastroenteritis, enteritis, colitis, enterocolitis, duodenitis, ileitis, cecal inflammation, appendicitis, or proctitis. Further inflammatory diseases include liver inflammation, lung inflammation, bone inflammation, systemic inflammatory response syndrome (SIRS), sepsis; and

[0025] Autoimmune diseases, especially (endocrine) autoimmune diseases (such as Hashimoto's hypothyroidism, Graves' hyperthyroidism, rheumatoid arthritis, celiac disease, asthma / COPD, Addison's disease, IBD (Crohn's disease and ulcerative colitis), systemic lupus erythematosus, vasculitis, Guillain-Barré syndrome and CIDP syndrome, multiple sclerosis, psoriasis (arthritis), vitiligo and Bechterew's disease).

[0026] Alternatively or optionally, the use according to the invention can be used to improve overall health and / or reduce inflammatory conditions.

[0027] Therefore, the present invention also includes the prevention of the aforementioned diseases, namely type 1 or type 2 diabetes, cardiovascular disease, inflammatory disease, or autoimmune disease. Thus, the *Desulfovibrio* species and / or metabolites according to the present invention can be administered to individuals, for example, in whom risk markers associated with the pre- or early stages of the corresponding disease have been detected (before the diagnosis of the corresponding disease), to prevent the onset of any of the aforementioned diseases. This primary or secondary prevention strategy can prevent the development of the disease.

[0028] Some of the autoimmune diseases mentioned in this article are currently treated with immunotherapy, for example, by using antibodies against TNFα. However, these expensive immunotherapies may only be effective in a subset of patients. This is attributed to differences in the gut microbiota (Kolho et al 2015 Am J Gastroenterol.110(6):921-30). The inventors envision that treatment with TNFα antagonists or anti-TNFα can be synergistic with treatments according to the invention, such as the administration of Desulfovibrio species and / or metabolites according to the invention. Detailed Implementation

[0029] This invention relates to the prevention or treatment of inflammatory diseases selected from type 1 or type 2 diabetes, autoimmune diseases, cardiovascular diseases, and inflammatory diseases by using one or more agents selected from the following:

[0030] Species of the genus *Desulfovibrio*, preferably selected from *Desulfovibrio piger*, *Desulfovibrio fairfieldii*, *Desulfovibrio desulfurization*, *Desulfovibrio sinica*, *Desulfovibrio alaskaii*, *Desulfovibrio commonis*, *Desulfovibrio vinifera*, and *Desulfovibrio giantii*, with *Desulfovibrio piger* being the most preferred; and

[0031] The compounds (metabolites) 6-bromotryptophan, 1-myristoyl-2-arachidonico-glycerophosphate choline (MA-GPC) and 1-arachidonico-glycerophosphate choline (A-GPC), or more generally, chlorinated, fluorinated, or bromosubstituted tryptophan, or mono- or di-fatty acid-substituted glycerophosphate choline, or any derivatives or functional equivalents thereof.

[0032] Therefore, the present invention provides a method for preventing or treating an individual in need, particularly an individual suffering from an inflammation-related disease (e.g., type 1 or type 2 diabetes), an autoimmune disease (e.g., an endocrine autoimmune disease), a cardiovascular disease, or an inflammatory disease, the method comprising the step of administering one or more of the above-mentioned agents.

[0033] As described in WO2019168401, the method according to the invention has been found to have improved therapeutic efficacy in autoimmune diseases compared to the application of autologous fecal matter. It is less labor-intensive, easier to apply, and easier to produce, for example, under a certified quality management system (QMS) or good manufacturing practice (GMP), and / or easier to scale up.

[0034] The present invention is at least a priori preferably not intended to alter the gut microbiota, i.e., the composition of the gut microbiota or, in particular, the composition of the colonic microbiota.

[0035] In the context of this invention, an autoimmune disease can be any autoimmune disease, including systemic and local (organ-specific) autoimmune diseases, particularly those selected from endocrine autoimmune diseases (e.g., type 1 diabetes, Hashimoto's disease, Graves' disease, or Addison's disease); cutaneous autoimmune diseases (e.g., psoriasis or vitiligo); rheumatoid autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus, vasculitis, or Bekhterev disease); gastrointestinal autoimmune diseases (e.g., celiac disease, inflammatory bowel disease); nervous system diseases (Guillain-Barré syndrome, CIDP disease, and multiple sclerosis); and lung diseases (COPD / asthma).

[0036] In the context of this invention, cardiovascular disease can be any cardiovascular disease, such as coronary artery disease (also known as coronary heart disease and ischemic heart disease), peripheral artery disease, cerebrovascular disease (e.g., stroke or transient ischemic attack (TIA), atherosclerosis, stenosis, renal artery stenosis, aortic disease, aortic aneurysm, cardiomyopathy, hypertensive heart disease, hypertension, heart failure, pulmonary heart disease, arrhythmia, cardiovascular inflammation, inflammatory heart disease, endocarditis, inflammatory cardiac hypertrophy, myocarditis, eosinophilic myocarditis, valvular heart disease, congenital heart disease, or rheumatic heart disease (optionally, any of these diseases may also be excluded from this application).

[0037] Furthermore, in the context of this invention, inflammatory disease can be any inflammatory disease, particularly cardiovascular inflammation, such as carditis, endocarditis, myocarditis, pericarditis, vasculitis, arteritis, phlebitis, or capillary vasculitis, or gastrointestinal inflammation, such as esophagitis, gastritis, gastroenteritis, enteritis, colitis, enterocolitis, duodenitis, ileitis, appendicitis, or proctitis. Inflammatory disease according to the invention can further refer to liver inflammation, lung inflammation, or bone inflammation. Alternatively, inflammatory disease can refer to systemic inflammatory response syndrome (SIRS) or sepsis (optionally, any of these diseases may also be excluded from this application).

[0038] diabetes

[0039] Type 1 diabetes

[0040] Type 1 diabetes is a chronic endocrine autoimmune disease in which the pancreas produces too little or no insulin. It is generally believed to be associated with progressive β-cell destruction and is associated with an increased risk of morbidity and mortality compared to healthy individuals. Since β-cell function can also deteriorate in type 2 diabetes, this invention can also relate to the prevention and / or treatment of type 2 diabetes.

[0041] The agents according to the present invention have been found to be useful for the prevention and / or treatment of type 1 diabetes. This treatment can also prolong the honeymoon phase of type 1 diabetes, a period after diagnosis during which the pancreas continues to produce sufficient insulin to limit the body's need for exogenous insulin and maintain glycemic control. Prolonging this period can significantly improve the patient's quality of life. This treatment can also be used to reduce the severity of type 1 diabetes symptoms, such as symptoms or complications associated with impairment of eye, kidney, nerve, and / or brain function.

[0042] More specifically, the treatment can inhibit the decline of β-cell function and / or suppress the production of autoantibodies associated with type 1 diabetes, such as islet (β) cell autoantibodies, insulin autoantibodies, GAD (GAD65) autoantibodies, tyrosine phosphatase IA-2 and IA-2β autoantibodies and / or zinc transporter 8 (ZnT8) autoantibodies.

[0043] Symptoms of type 1 diabetes may include frequent urination, excessive thirst, increased appetite, weight loss, fatigue, nausea, and blurred vision. The onset of symptomatic illness can be sudden. In this respect, diabetic ketoacidosis (DKA) is not uncommon in people with type 1 diabetes. The following diagnostic criteria can be used for both type 1 and type 2 diabetes (American Diabetes Association, ADA):

[0044] Fasting plasma glucose (FPG) level ≥126 mg / dL (7.0 mmol / L), or

[0045] During a 75-gram oral glucose tolerance test (OGTT), a 2-hour blood glucose level ≥200 mg / dL (11.1 mmol / L), or

[0046] Patients with typical symptoms of hyperglycemia or hyperglycemic crisis have a random blood glucose level ≥200 mg / dL (11.1 mmol / L).

[0047] In addition and / or alternatively, as described in the examples and / or Lachin et al. (2011 PLoS ONE Vol.6(11)e26471), the C-peptide response after a mixed diet trial can be evaluated.

[0048] Type 1 diabetes and / or its prior symptoms can be confirmed by the presence of one or more autoimmune markers, including islet (β) cell autoantibodies, insulin autoantibodies, GAD (GAD65) autoantibodies, tyrosine phosphatase IA-2 and IA-2β autoantibodies, zinc transporter 8 (ZnT8) autoantibodies, as well as elevated HbA1c and altered glucose tolerance.

[0049] Type 2 diabetes

[0050] Type 2 diabetes is a common metabolic disease that occurs when the body cannot produce enough insulin or when insulin does not function properly—a condition known as insulin resistance. Insulin is a hormone that stimulates cells to take up glucose from the blood to obtain energy. In this situation, insulin does not instruct cells to take up glucose from the blood, which means that blood sugar levels rise (a condition called hyperglycemia).

[0051] People typically develop type 2 diabetes after age 40, although people of South Asian descent are at higher risk and may develop diabetes as early as age 25. This is also becoming increasingly common in children and adolescents across all populations. Type 2 diabetes is often caused by being overweight, obese, and lack of physical activity, and as these problems become more prevalent, the prevalence of diabetes is rising worldwide. Type 2 diabetes accounts for approximately 90% of all diabetes cases (the other form being type 1 diabetes), and treatment options include lifestyle modifications and medication.

[0052] The agents according to the invention have been found to be useful for the prevention and / or treatment of type 2 diabetes. This treatment can be used to reduce the severity of symptoms of type 2 diabetes, such as symptoms or complications related to polyuria, polydipsia, etc. More specifically, this treatment can reduce the need for exogenous hormone supplementation.

[0053] Cardiovascular disease

[0054] Coronary artery disease

[0055] Coronary artery disease is the most common cardiovascular disease. It involves reduced blood flow to the heart muscle due to the buildup of plaque (atherosclerosis) in the heart's arteries. A common symptom is chest pain or discomfort that may radiate to the shoulder, arm, back, neck, or jaw. In many cases, the first sign is a heart attack. Other complications include heart failure or abnormal heartbeats.

[0056] Risk factors include high blood pressure, smoking, diabetes, lack of exercise, obesity, high cholesterol, poor diet, depression, and excessive alcohol consumption. Several tests can aid in diagnosis, including: electrocardiogram (ECG), cardiac stress testing, coronary computed tomography (CT) angiography, and coronary angiography, among others.

[0057] Inflammatory diseases

[0058] Individuals with or at risk of developing inflammatory diseases can be identified using methods known in the art, such as gross examination of tissue or detection of relevant inflammation in tissues or blood. Symptoms of inflammation include pain, redness, and swelling in the affected tissues.

[0059] Systemic inflammatory response syndrome (SIRS) and sepsis

[0060] Systemic inflammatory response syndrome (SIRS) is an excessive defensive response of the body to factors such as infection, trauma, surgery, acute inflammation, local ischemia or reperfusion, or malignancy. It involves the release of acute-phase reactants, which are direct mediators of widespread autonomic, endocrine, hematological, and immunological changes in an individual. Although defensive in nature, a dysregulated cytokine storm can lead to a massive inflammatory cascade, resulting in reversible or irreversible end-organ dysfunction and even death. SIRS with a suspected infectious agent is termed sepsis. Sepsis with one or more end-organ failures is termed severe sepsis, and sepsis with hemodynamic instability despite intravascular volume replenishment is termed septic shock.

[0061] SIRS can be diagnosed by meeting any two of the following criteria:

[0062] Body temperature exceeding 38 degrees Celsius or below 36 degrees Celsius;

[0063] Heart rate greater than 90 beats per minute;

[0064] A respiratory rate greater than 20 breaths / minute or a CO2 partial pressure less than 32 mmHg;

[0065] White blood cell count greater than 12,000 / μL or less than 4,000 / μL or more than 10% immature form or bands.

[0066] Alternatively or additionally, the application according to the invention can be used to improve overall health and / or reduce inflammatory states, the latter preferably measured by a higher erythrocyte sedimentation rate (e.g., ESR values ​​of at least 35 mm / h, 40 mm / h, 45 mm / h, 50 mm / h, 55 mm / h, 60 mm / h) and / or a decrease in C-reactive protein levels in the blood (plasma) compared, for example, to healthy individuals who have not been given the composition according to the invention. C-reactive protein can be measured, for example, 1-12 weeks, 1-4 weeks, 2-8 weeks, 4-12 weeks after administration of the composition of the invention, or 1-12 months or 1-12 years later. C-reactive protein (CRP) is a protein produced by the liver. When a condition causing inflammation occurs somewhere in the body, the level of CRP in the blood increases. CRP tests measure the amount of CRP in the blood to detect inflammatory states.

[0067] Alternatively or alternatively, the application according to the invention can be used to induce weight loss in an individual or to reduce body mass index (BMI).

[0068] Autoimmune diseases

[0069] Autoimmune diseases are a group of illnesses in which the immune system reacts inappropriately to an individual's own cells, tissues, and / or organs. This can lead to inflammation, damage, and loss of function. Common autoimmune diseases include Hashimoto's hypothyroidism, Graves' hyperthyroidism, rheumatoid arthritis, celiac disease, asthma / COPD, Addison's disease, IBD (Crohn's disease and ulcerative colitis), systemic lupus erythematosus, vasculitis, Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy (CIDP), multiple sclerosis, psoriasis (arthritis), vitiligo, type 1 diabetes, and Bekhterev disease.

[0070] The causes of autoimmune diseases are not fully understood. However, factors such as infection and genetic predisposition may play a role in their development. Autoimmune diseases are usually diagnosed by combining clinical history with blood tests (detecting autoantibodies or markers of inflammation or organ function, etc.).

[0071] Although there are various treatment options depending on the stage and type of autoimmune disease, there is no definitive cure for autoimmune diseases.

[0072] Treatment strategies typically aim to relieve symptoms, minimize organ or tissue damage, and protect organ function. For example, treatment options may include organ function replacement (e.g., insulin in type 1 diabetes, thyroxine in Hashimoto's hypothyroidism), nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroid anti-inflammatory drugs (e.g., prednisolone), TNFα inhibitors, immunosuppressive drugs, or immunoglobulin replacement therapy.

[0073] Endocrine autoimmune diseases

[0074] Among various autoimmune diseases, autoimmune endocrine disorders are the most common. The endocrine system includes glands that produce hormones and transport them directly to the circulatory system, as well as feedback loops that maintain homeostasis. Organs of the endocrine system can be affected by several autoimmune diseases, characterized by varying degrees of impact and severity. Sometimes multiple organs are involved, such as in polyglandular autoimmune syndromes.

[0075] Among various autoimmune endocrine disorders, type 1 diabetes, Hashimoto's disease, Graves' disease, and Addison's disease are particularly common in clinical practice.

[0076] Hashimoto's disease

[0077] Hashimoto's disease is the most common organ-specific autoimmune disease. It is also known as Hashimoto's thyroiditis, or chronic lymphocytic thyroiditis, and is considered an autoimmune disease in which the thyroid gland is progressively destroyed. The etiology of Hashimoto's disease is unclear, although it is generally believed to be related to inappropriate cell-mediated immune responses and the production of autoantibodies against the thyroid gland. Both B (CD20+ and CD79α+) cells are seen in mononuclear lymphoid infiltration (destruction of thyroid follicles and thyroid cells) and hyperstimulated T cells CD4+ (helper T cells type 2 Th2 cells lead to hyperstimulation and the production of B cells that produce antibodies against thyroid antigens, which subsequently lead to thyroiditis in the thyroid gland, Marazuela et al J Clin Endocrinol Metab. 2006 Sep; 91(9):3639-46).

[0078] Goiter is often the only symptom before hypothyroidism becomes apparent. However, the condition can progress to hypothyroidism, which frequently leads to symptoms including edema, weight gain, and fatigue (easily tired), sensitivity to cold and diarrhea, as well as physical manifestations such as dry skin, hoarseness, bradycardia, and / or prolonged relaxation phase of the Achilles tendon reflex.

[0079] Hashimoto's disease can be diagnosed by the presence of anti-thyroid peroxidase (TPO) antibodies and anti-thyroglobulin (Tg) antibodies in the patient's serum. Furthermore, elevated thyroid-stimulating hormone (TSH) levels, decreased free T4 (FT4) levels, decreased free T3 levels, and / or elevated anti-microsomal antibody levels compared to the average level in healthy individuals can help in obtaining a positive diagnosis.

[0080] Hashimoto's disease is currently treated with thyroid hormone replacement therapy, such as levothyroxine (to supplement FT4), triiodothyronine (to supplement T3), or dried thyroid extract. The inventors have discovered that the pharmaceutical agents according to the invention, optionally combined with the thyroid hormone replacement agents described above, can be used for the prevention and / or treatment of Hashimoto's disease. The treatment according to the invention can also be used to reduce the severity of Hashimoto's disease symptoms, such as one or more of the symptoms or complications described above.

[0081] Graves' disease

[0082] Graves' disease is an autoimmune disorder affecting the thyroid gland and is the most common cause of hyperthyroidism. The disease is characterized by the presence of autoantibodies in the serum that bind to the thyroid-stimulating hormone (TSH) receptor. These anti-TSH receptor antibodies (TBII) overstimulate the thyroid gland, which can lead to goiter and signs of thyrotoxicosis, as well as ocular muscle involvement in some patients (Graves' eye disease).

[0083] Symptoms include hyperthyroidism, goiter, and orbital edema. Other major symptoms include weight loss (increased appetite), fatigue, shortness of breath, excessive sweating, finger tremors, diarrhea, periodic paralysis (in men), and muscle weakness. Regarding Graves' eye disease, patients may experience bulging eyes, blurred vision, and dry eye / pink eye (which, in rare cases, can lead to blindness). Two signs are characteristic of Graves' disease and are not found in other hyperthyroid disorders: bulging eyes and pretibial myxedema.

[0084] Graves' disease can be diagnosed by low serum TSH levels (sometimes undetectable) and / or elevated free T3 and free T4 levels compared to healthy individuals. Patients typically test positive for anti-TSH receptor antibodies (TBII) in their serum.

[0085] Current treatment for Graves' disease may involve the administration of antithyroid drugs (blockade and replacement therapy), radioactive iodine (radioactive iodine I-131); and / or thyroidectomy (surgical removal of the gland). Typically, the prescription consists of methimazole (strumazol) and methimazole (PTU), followed by thyroid hormone replacement therapy such as levothyroxine (FT4 supplement), triiodothyronine (T3 supplement), or dried thyroid extract.

[0086] Optionally or in combination with the above treatments, the inventors have found that the agents according to the invention can be used for the prevention and / or treatment of Graves' disease, including eye diseases. The treatments according to the invention can also be used to reduce the severity of symptoms of Graves' disease, such as one or more of the symptoms or complications described above.

[0087] Eddie is sick

[0088] Addison's disease is a chronic endocrine autoimmune disorder in which the adrenal glands fail to produce sufficient steroid hormones. The disease is caused by the destruction of the adrenal glands (both the cortex and medulla produce hormones). It can be a manifestation of a multi-glandular autoimmune syndrome, including complications arising from other organ-specific autoimmune diseases such as type 1 diabetes, Hashimoto's disease, and vitiligo.

[0089] Hyperpigmentation due to increased ACTH secretion is a characteristic clinical symptom of Addison's disease. Other symptoms include abdominal pain, orthostatic hypotension, and weight loss.

[0090] Medical examinations will typically determine the presence of orthostatic hypotension, hypoglycemia, hyponatremia, hyperkalemia, and peripheral blood eosinophilia. To confirm Addison's disease, low adrenal hormone levels are usually observed after stimulation with the synthetic pituitary hormone tetracosactide (ACTH) (called the ACTH stimulation test or synacthen test).

[0091] Treatment typically involves oral hydrocortisone and / or mineralocorticoid replacement therapy such as fludrocortisone (if the adrenal medulla is also involved). The inventors have discovered that, optionally in addition to treatment with hydrocortisone, the agents according to the invention can be used for the prevention and / or treatment of Addison's disease. The treatment according to the invention can also be used to reduce the severity of Addison's disease symptoms, such as one or more of the symptoms or complications described above.

[0092] Cutaneous autoimmune diseases

[0093] Psoriasis (arthritis)

[0094] Psoriasis is a chronic autoimmune disease that causes skin cells to regenerate rapidly. The underlying cause is that T cells attack healthy skin cells, causing the skin cell production process to accelerate. New cells are pushed to the skin's surface, where they accumulate. This leads to plaques and red, inflamed areas on the skin, which are commonly associated with psoriasis. Subtypes of psoriasis include...

[0095] (1) Plaque psoriasis, which is the most common type of psoriasis. It is characterized by red, inflamed plaques covering areas of skin, usually on the elbows, knees, and scalp. These plaques are usually covered with silvery-white scales or patches;

[0096] (2) Guttate psoriasis, a form of psoriasis, is common in children and causes small pink spots that usually appear on the trunk, arms and legs;

[0097] (3) Pustular psoriasis, which is the more common form of adult psoriasis, causes white, pus-filled blisters and red, inflamed areas of skin, usually on the hands or feet;

[0098] (4) Inverse psoriasis, which causes bright, red, shiny, inflamed areas of skin. Inverse psoriasis plaques typically appear in the armpits or under the breasts, in the groin, or around skin folds;

[0099] (5) Erythrodermic psoriasis: This is a severe and rare type of psoriasis. This form typically covers large areas of the body where the skin may become sunburned. People with this type of psoriasis may experience fever or become very ill, and this form of psoriasis can be life-threatening.

[0100] (6) Psoriatic arthritis involving joints.

[0101] Symptoms of psoriasis vary from person to person. Common symptoms include red plaques of skin covered with thick, silvery scales; small scaly patches (common in children); dry, cracked skin that may bleed; itching; burning or pain; thickened, pitted or raised nails; and / or swollen and stiff joints. Most types of psoriasis can go through cycles, with flare-ups lasting weeks or even months, followed by periods of remission or even remission. Psoriatic arthritis is a condition that accompanies psoriasis, causing swollen and painful joints.

[0102] For mild cases affecting only a small area of ​​the body, topical treatments (applied to the skin), such as creams, lotions, and sprays, are usually prescribed. Sometimes, direct topical injection of steroids into hard or resistant isolated plaques of psoriasis may be helpful.

[0103] Tumor necrosis factor (TNF) antagonists (or anti-TNFα therapy) have become first-line drugs for the treatment of moderate to severe psoriasis or psoriatic arthritis. Examples include infliximab, etanercept, and adalimumab. Anti-TNFα therapy has been found to be effective in treating psoriasis and psoriatic arthritis and can also reduce the risk of cardiovascular events. The inventors have discovered that, additionally or optionally, the agents according to the invention can be used for the prevention and / or treatment of psoriasis and / or psoriatic arthritis. Furthermore, the treatment according to the invention can also be used to reduce the severity of symptoms of psoriasis and psoriatic arthritis, such as one or more of the symptoms or complications described above. In particular, combination therapy with TNF antagonists or anti-TNFα and the treatment according to the invention can be synergistic.

[0104] vitiligo

[0105] Vitiligo is a condition characterized by white patches on the skin in various parts of the body. It is generally believed to be caused by an autoimmune process that destroys pigment-producing cells in the skin, namely melanocytes. Vitiligo can also occur in mucous membranes (such as inside the mouth and nose) and in the eyes.

[0106] Recent studies have revealed an ecological imbalance in the diversity of microbial community structure in the skin microbiota of vitiligo patients. Although individual-specific microbiome characteristics dominate in vitiligo-specific microbiota, a significant decrease in taxonomic richness and evenness can be observed in lesion patches (Ganju et al Sci Rep. 2016 Jan 13; 6:18761).

[0107] The white patches of vitiligo are more commonly found in areas of skin frequently exposed to sunlight. Patches may appear on the hands, feet, arms, face, and lips, but occasionally also in the armpits and groin, around the mouth, eyes, nostrils, navel, genitals, and rectum. Additionally, people with vitiligo often experience premature graying of hair (e.g., before age 35).

[0108] Ultraviolet (UV) light is particularly useful for the early diagnosis of vitiligo and for determining the effectiveness of UV treatment. Skin with vitiligo typically emits blue light when exposed to UV radiation. In contrast, healthy skin does not react.

[0109] Vitiligo can be divided into segmental vitiligo (SV) and non-segmental vitiligo (NSV), with NSV being the most common type of vitiligo.

[0110] In non-segmental vitiligo (NSV), the depigmented patches are usually symmetrically located. In extreme cases, almost no pigmented skin remains, a condition known as universal vitiligo. NSV can develop at any age, while segmental vitiligo is more common during adolescence.

[0111] Segmental vitiligo (SV) tends to affect skin areas associated with the dorsal root of the spinal cord and is usually unilateral. It is more stable / static during the course of the disease. UV phototherapy typically does not improve SV, but surgical treatments such as cell transplantation may be effective.

[0112] There is no definitive cure for vitiligo, but several treatment options are available, including ultraviolet (UV) light and / or creams. Topical formulations (i.e., creams) of immunosuppressive drugs, including corticosteroids or glucocorticoids (such as clobetasol and / or betamethasone) and calcineurin inhibitors (such as tacrolimus and / or pimecrolimus), are considered first-line treatments for vitiligo, while UV(B) therapy is considered a second-line treatment.

[0113] The inventors have discovered that, in addition to or as an alternative to the treatments described above, the agents according to the invention can be used to prevent and / or treat vitiligo. Furthermore, the treatments according to the invention can also be used to alleviate the severity of vitiligo symptoms, such as one or more of the symptoms or complications described above.

[0114] Rheumatoid diseases

[0115] rheumatoid arthritis

[0116] Rheumatoid arthritis (RA) can be considered an autoimmune disease in which the immune system attacks the joints. This causes inflammation, leading to thickening of the tissue inside the joint (synovium), which in turn causes joint pain.

[0117] If left untreated, rheumatoid arthritis (RA) can damage cartilage, the elastic tissue that covers the ends of bones in a joint, and even the bone itself. Eventually, the cartilage may be lost, and the joint can become loose, unstable, painful, and lose mobility, or even deformed. Unfortunately, joint damage is usually irreversible, so early diagnosis and treatment are recommended to manage RA.

[0118] Rabies (RA) most commonly affects the hands, feet, wrists, elbows, knees, and ankles. RA can also affect other body systems, such as the cardiovascular or respiratory systems, hence the term systemic RA. In the early stages, RA patients may experience joint tenderness and pain.

[0119] Symptoms of rheumatoid arthritis (RA) include stiffness and joint pain, especially in the small joints (certain joints in the wrist, hand, and foot), and usually last for six weeks or longer. In addition to pain, many people also experience fatigue, loss of appetite, and a mild fever.

[0120] No single test can definitively diagnose RA, but blood tests can be performed to measure inflammation levels and look for biomarkers, such as antibodies associated with RA.

[0121] Compared to healthy individuals, elevated erythrocyte sedimentation rate (ESR) and elevated C-reactive protein (CRP) levels are biomarkers of inflammation. While elevated ESR or CRP is not specific to RA, it can be diagnosed when combined with the presence of RA-associated antibodies.

[0122] Rheumatoid factor (RF) is an antibody found in most RA patients. Because RF can occur in other inflammatory diseases, it is not a definitive marker of RA. However, a different antibody—anti-cyclic citrullinated peptide (anti-CCP)—is predominantly present in RA patients. This makes a positive anti-CCP test a stronger indicator of RA. Furthermore, X-ray, ultrasound, or MRI scans can be performed to look for joint damage, such as erosion and narrowing of the joint space.

[0123] Treatment typically involves prescribing nonsteroidal anti-inflammatory drugs (NSAIDs), which relieve arthritis pain and inflammation. Examples of NSAIDs include ibuprofen, ketoprofen, and naproxen sodium. Additionally, corticosteroids, including prednisone, prednisolone, and methylprednisolone, can be administered as anti-inflammatory medications.

[0124] DMARDs, or disease-modifying antirheumatic drugs, are used to slow the progression of disease. DMARDs include methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide, and azathioprine. A subclass of DMARDs is called "JAK inhibitors," which block the Janus kinase, or JAK pathway. An example is tofacitinib.

[0125] Biologics may act faster than traditional DMARDs and are administered via injection or infusion. In many RA patients, biologics can slow, improve, or halt the disease. Tumor necrosis factor (TNF) antagonists (anti-TNFα therapy) are particularly preferred.

[0126] The inventors have discovered that, in addition to or as an alternative to the treatments described above, the agents according to the invention can be used to prevent and / or treat rheumatoid arthritis and / or one or more of the aforementioned symptoms. Combined treatment according to the invention with TNF antagonists or anti-TNFα agents can be synergistic.

[0127] Bechterew's disease

[0128] Bekhterev disease (or ankylosing spondylitis) is a chronic autoimmune rheumatoid disease that primarily affects the axial skeleton. It typically occurs in adult men aged 20-30.

[0129] The most severe symptoms are neck and lower back pain. Typical symptoms include nighttime pain and sacroiliac joint inflammation. In some patients, spinal deformities may occur, which can lead to limited mobility. In addition to these spinal conditions, inflammation of the peripheral joints is also common.

[0130] To diagnose Bekhterev disease, a spinal examination is required to assess limitations in cervical and lumbar spine mobility. The Schober test helps assess the degree of limitation in lumbar flexion. Diagnosis is confirmed by detecting the HLA-B27 antigen in the patient's blood.

[0131] Treatment regimens include the use of NSAIDs, sulfasalazine, methotrexate, leflunomide, corticosteroids, and TNFα inhibitors. The inventors have discovered that, in addition to or as an alternative to the above treatments, the agents according to the invention can be used for the prevention and / or treatment of Bekhterev disease and / or one or more of its symptoms. In particular, the treatment according to the invention can be synergistically combined with TNF antagonists or anti-TNFα agents.

[0132] Systemic lupus erythematosus

[0133] Systemic lupus erythematosus (SLE), also known simply as lupus, is an autoimmune disease in which the body's immune system mistakenly attacks healthy tissues in many parts of the body. Symptoms vary from person to person and can range from mild to severe. SLE significantly increases the risk of cardiovascular disease, the most common cause of death. With modern treatments, approximately 80% of patients survive more than 15 years after diagnosis. Common symptoms include joint pain and swelling, fever, chest pain, hair loss, mouth ulcers, swollen lymph nodes, fatigue, and a rash most commonly found on the face. There are typically periods of illness known as flares, as well as periods of remission during which there are few or no symptoms. SLE is incurable. Treatments may include NSAIDs, corticosteroids, immunosuppressants, hydroxychloroquine, and methotrexate. While corticosteroids are fast-acting, long-term use can cause side effects. The inventors have discovered that, in addition to or as an alternative to the treatments described above, the pharmaceutical agents according to the present invention can be used to prevent and / or treat SLE disease and / or one or more of the symptoms described above.

[0134] Vasculitis

[0135] Vasculitis can occur in both large and small blood vessels. Large vessel vasculitis includes giant cell arteritis (or temporal arteritis) and Takayasu's disease (Takayasu arteritis). Medium- and large vessel vasculitis includes polyarteritis nodosa (PAN) and Kawasaki's disease. Small vessel vasculitis includes microscopic polyangiitis, GPA (granulomatous disease with polyangiitis, also known as Wegener's disease), and EGPA (eosinophilic granulomatous disease with polyangiitis, also known as Churg-Strauss syndrome). Syndrome, anti-GBM (Goodpasture's syndrome) and cryoglobulinemia-related vasculitis. The inventors have discovered that the agents according to the invention can be used for the prevention and / or treatment of vasculitis and / or one or more of the above-mentioned symptoms.

[0136] Gastrointestinal autoimmune diseases

[0137] Celiac disease

[0138] Celiac disease (or coeliac disease) is an autoimmune disease in which gluten intake leads to damage to the epithelial cells of the small intestine. It typically occurs in individuals with a genetic predisposition and is associated with type 1 diabetes. Celiac disease and type 1 diabetes may share similar pathogenesis, in which heritable genetic factors, as well as diet and microbial exposure, may play a role, particularly in early life (see, for example, Verdu and Danska, Nature Immunology | VOL19 | JULY 2018 | 685–695).

[0139] When people with celiac disease consume gluten (a protein found in wheat, rye, and barley), their bodies trigger an immune response that attacks the small intestine, damaging the villi (the finger-like projections lining the small intestine). When the villi are damaged, the intestines cannot properly absorb nutrients. Symptoms include abdominal cramps, malnutrition, and osteoporosis.

[0140] Several serological (blood) tests can be used to screen for celiac disease antibodies, but the most commonly used is the tTG-IgA test. For this test to work, the patient must consume gluten. Alternatively, celiac disease can be diagnosed via endoscopic biopsy. A biopsy of the small intestine is then performed, and the tissue can be analyzed to see if there is any damage consistent with celiac disease. A definitive diagnosis can be made when improvement is seen on a gluten-free diet.

[0141] Currently, the only treatment for celiac disease is a strict gluten-free diet. People living a gluten-free lifestyle must avoid foods containing wheat, rye, and barley, such as bread and beer. Even small amounts of gluten can cause damage to the small intestine. The inventors have discovered that, in addition to or as an alternative to the above treatments, the pharmaceutical agents according to the invention can be used to prevent and / or treat celiac disease and / or one or more of the above-mentioned symptoms.

[0142] Inflammatory bowel disease

[0143] Inflammatory bowel disease (IBD) is the term used for two diseases characterized by chronic inflammation of the gastrointestinal tract (GI) (Crohn's disease and ulcerative colitis). IBD is thought to be caused by an dysregulation of the immune response. Symptoms of IBD include persistent diarrhea, abdominal pain, rectal bleeding / bloody stools, weight loss, and fatigue. In IBD, the immune system responds incorrectly to environmental triggers, leading to inflammation of the gastrointestinal tract. There also appears to be a genetic factor—people with a family history of IBD are more likely to develop this inappropriate immune response.

[0144] The diagnosis of IBD combines endoscopy (for Crohn's disease) or colonoscopy (for ulcerative colitis) with imaging studies (such as contrast imaging, magnetic resonance imaging (MRI), or computed tomography (CT) scans).

[0145] Several types of medications are available for treating IBD: aminosalicylates, corticosteroids (such as prednisone), immunomodulators, and recently approved "biologics" for IBD, such as anti-TNFα. Vaccination is recommended for IBD patients to prevent infection. Severe IBD may require surgical removal of damaged portions of the gastrointestinal tract, but advances in drug treatment mean that surgery is not as common as it was decades ago. The inventors have discovered that, in addition to or as an alternative to the treatments described above, agents according to the invention can be used to prevent and / or treat IBD and / or reduce the severity of one or more of its symptoms.

[0146] Nervous system diseases

[0147] Guillain-Barré syndrome

[0148] Guillain–Barré syndrome (GBS) is a rapidly progressing myasthenia gravis (acute polyneuropathy) caused by damage to the peripheral nervous system by the immune system. Initial symptoms are usually altered sensation or pain, along with muscle weakness, starting in the feet and hands and typically spreading to the arms and upper body, affecting both sides. Symptoms can develop over hours to weeks. In the acute phase, the disease can be life-threatening; approximately 15% of people develop respiratory muscle weakness and require mechanical ventilation.

[0149] Although the cause is unknown, the underlying mechanism involves an autoimmune disease in which the body's immune system mistakenly attacks peripheral nerves and destroys their myelin sheath insulation. Sometimes this immune dysfunction is triggered by infection or surgery, and rarely by vaccines. Diagnosis is usually made based on signs and symptoms, after ruling out other causes, and supported by tests such as nerve conduction studies and cerebrospinal fluid examination. Many subtypes exist based on the location of the weakness, the results of nerve conduction studies, and the presence of certain antibodies. Intravenous immunoglobulin or plasma exchange therapy, along with supportive care, will lead to a good recovery for most people. Recovery can take anywhere from weeks to years, and about one-third of people will have some permanent weakness. The inventors have discovered that, in addition to or as an alternative to the above treatments, the agents according to the invention can be used to prevent and / or treat GBS and / or reduce the severity of one or more of its symptoms.

[0150] CDIP

[0151] Chronic inflammatory demyelinating polyneuropathy (CDIP) is an acquired immune-mediated inflammatory disease of the peripheral nervous system. This disease is sometimes referred to as chronic relapsing polyneuropathy (CRP) or chronic inflammatory demyelinating polyradiculopathy (because it involves nerve roots). CIDP is closely associated with Guillain-Barré syndrome and is considered the chronic counterpart of this acute condition. The inventors have discovered that the pharmaceutical agents according to the invention can be used to prevent and / or treat CDIP and / or reduce the severity of one or more of its aforementioned symptoms.

[0152] Multiple sclerosis

[0153] Multiple sclerosis (MS) is a demyelinating disease in which the insulating covering of nerve cells in the brain and spinal cord is damaged. This damage disrupts the ability of parts of the nervous system to transmit signals, leading to a range of signs and symptoms, including physical, mental, and sometimes even psychiatric problems. Specific symptoms may include diplopia, monocular blindness, muscle weakness, and sensory or coordination impairments. MS has multiple forms, with new symptoms either appearing in isolated episodes (relapsing form) or accumulating over time (progressive form). Between episodes, symptoms may disappear completely; however, permanent neurological problems often persist, especially as the disease progresses. While the causes are not fully understood, potential mechanisms are thought to include disruption of the immune system or depletion of myelin-producing cells. Possible causes include genetic and environmental factors, such as those triggered by viral infections. Diagnosis of MS is typically based on the present signs and symptoms, along with the results of auxiliary medical examinations. There is currently no cure for multiple sclerosis. Treatment attempts to improve function after an episode and prevent new episodes. The inventors have discovered that the pharmaceutical agents according to the invention can be used to prevent and / or treat MS and / or reduce the severity of one or more of its aforementioned symptoms.

[0154] Asthma and COPD

[0155] In the context of this invention, given that autoimmune mechanisms may also play a role in asthma, the prevention and / or treatment of asthma are also anticipated.

[0156] Asthma is a common chronic inflammatory disease of the lungs' airways. It is characterized by reversible airflow obstruction and bronchospasm. Symptoms include cough, wheezing, chest tightness, and shortness of breath.

[0157] Currently, there is no definitive diagnostic test for asthma; diagnosis is usually based on symptom patterns and response to treatment over time. Asthma can be diagnosed if there is a history of recurrent wheezing, coughing, or shortness of breath, and these symptoms appear or worsen due to exercise, viral infections, allergens, and / or air pollution; a post-bronchodilator FEV1 test is also performed to investigate the effect on lung function.

[0158] An effective treatment for asthma is to identify the triggers, such as cigarette smoke, pets, or aspirin, and eliminate exposure to these triggers. In addition, bronchodilators are often recommended. For mild but persistent cases, low-dose inhaled corticosteroids or alternatively, leukotriene antagonists or mast cell stabilizers can be used. For severe asthma, i.e., patients with daily attacks, inhaled corticosteroids, i.e., higher doses of corticosteroids, can be used.

[0159] The inventors have discovered that, in addition to or as a substitute for the treatments described above, the pharmaceutical agents according to the present invention can be used to prevent and / or treat asthma and / or one or more of the above-described symptoms.

[0160] The effectiveness of the treatment according to the invention confirms the link between gut microbiome composition and the risk of developing asthma, as hypothesized by Korpela et al. (Nat Commun. 2016 Jan 26; 7:10410).

[0161] (Lung)emphysema is one of the diseases that includes COPD (Chronic Obstructive Lung Disease). Emphysema involves progressive damage to lung tissue, particularly the thinning and destruction of alveoli or air sacs. The agents according to the invention can be used for the prevention and / or treatment of COPD, or specifically (lung)emphysema, and / or one or more of its symptoms as described above.

[0162] Other diseases

[0163] This invention can also be used for the prevention and / or treatment of other autoimmune diseases, particularly including autoimmune hepatitis, type 1a and / or type 1b diabetes, polyglandular autoimmune syndrome, myasthenia gravis, pernicious anemia, primary biliary cirrhosis, sclerosing cholangitis, antiphospholipid antibody syndrome, dermatomyositis, mixed connective tissue disease, polymyalgia rheumatica, polymyositis, scleroderma, and Sjögren's syndrome. However, it is also contemplated that any of the above-mentioned diseases be excluded from this invention.

[0164] Furthermore, the agents according to the invention can be used to prevent and / or treat allergies, also known as allergic diseases, which are symptoms caused by a hypersensitive reaction of the immune system to substances in the environment that are normally harmless. Common allergies include hay fever (plant pollen allergy) and food allergies (related to, for example, milk, soy, eggs, wheat, peanuts, nuts, fish, and / or shellfish).

[0165] This invention may also allow for the prevention and / or treatment of the following diseases, but optionally, these diseases are excluded from the scope of this invention: gastrointestinal diseases, Clostridium difficile infection, Crohn's disease, ulcerative colitis or inflammatory bowel disease (IBD) and / or irritable bowel syndrome (IBS). Optionally and / or additionally, any of the following diseases may be excluded from the scope of this invention: systemic and local (organ-specific) autoimmune diseases, endocrine autoimmune diseases, type 1 diabetes, type 2 diabetes, Hashimoto's disease, Graves' disease or Addison's disease, cutaneous autoimmune diseases, psoriasis or vitiligo, rheumatoid autoimmune diseases, rheumatoid arthritis, Bekhterev disease and gastrointestinal autoimmune diseases, celiac disease, vasculitis, COPD, CIDP, MS, SLE, Guillain-Barré syndrome. Diseases according to the invention need not be inflammation-related.

[0166] Treatment according to the present invention

[0167] Agents used for the prevention or treatment of autoimmune diseases as described herein may be species of the genus *Desulfovibrio*, preferably selected from *Desulfovibrio piger* (ATCC29098), *Desulfovibrio fairfieldensis* (ATCC700045), *Desulfovibrio desulfurization* (Essex 6ATCC 29577), *Desulfovibrio desulfurization* (MB ATCC 27774), *Desulfovibrio indica* (NCIMB 13468), *Desulfovibrio alaskaensis* (NCIMB 13491), *Desulfovibrio vinifera* (DSM 10520), *Desulfovibrio giantii* (DSM 1382), *Desulfovibrio intestinalis* (DSM 11275), *Desulfovibrio longreachensis* (ACM 3958), *Desulfovibrio termitidis* (DSM 5308), and *Desulfovibrio common subspecies*. *Vulgaris subsp. vulgaris* (DSM644) and *Oxamicus* subsp. *desulfovibrio* (DSM 1925). Alternatively or additionally, the agent may be a species of the genus *Bacteroides*, preferably *Bacteroides stercoris* or a relative thereof, such as a relative having at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, or 99.9% sequence identity with the 16S rDNA sequence of the type strain of *Bacteroides stercoris*.

[0168] Most preferably, the *Desulfovibrio* species is *Desulfovibrio piger*, or a relative of *Desulfovibrio piger* having at least 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.5, or 99.9% sequence identity with the 16S rDNA sequence of *Desulfovibrio piger* (e.g., SEQ ID NO: 1). This threshold based on 16S rDNA similarity can define species with similar characteristics and / or functions.

[0169] Preferably, a composition containing, for example, a species of the genus *Desulfovibrio* can be used, containing at least 10 per milliliter or per gram of the composition. 4 10 5 10 6 10 7 10 8 The amount of *Vibrio desulfurans* cells. Optionally or additionally, it may be preferred to use, for example, in a composition containing *Vibrio desulfurans* species, for example, a total of 10 per milliliter or per gram of composition. 4 10 16 Between 10 4 10 15 Between 10 4 10 14 Between 10 4 10 12 Between 10 6 10 12 Between 10, preferably 10 8 10 10 The desulfurized Vibrio cells between each cell.

[0170] Optionally or additionally, the *Desulfovibrio* cells may be live, but it is also conceivable to use (only) attenuated or dead cells, such as those obtained after pasteurization, or those obtained, for example, after incubation at 50-100°C, 60-80°C, 65-75°C, or 70°C, preferably for at least 5, 10, 15, 20, 25, 30, 40, or 50 minutes, or those obtained after exposure to UV or γ radiation, preferably for at least 1, 5, or 60 minutes. The time is 10, 20, or 30 seconds, or 1, 5, 10, 15, 20, 25, 30, 40, or 50 minutes, or obtained after incubation with oxygen (e.g., a gas containing at least 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 99, or 100% by volume oxygen), preferably for at least 1, 5, 10, 20, or 30 seconds or 1, 5, 10, 15, 20, 25, 30, 40, or 50 minutes. Preferably, the *Desulfovibrio* species is the first, second, third, fourth, or fifth most dominant bacterial species in the composition, i.e., having the highest cell count compared to other bacterial species included in the composition, or at least among the top 5.

[0171] Preferably, the fecal matter does not contain any *Desulfovibrio* species according to the invention, or, if *Desulfovibrio* species are included in the fecal matter (e.g., as a substitute for the above-described composition), they are enriched, i.e., the number of *Desulfovibrio* cells is higher than that in fecal matter in the prior art, for example, *Desulfovibrio* cells have been added to the fecal matter, or the fecal matter has been exposed to conditions favorable to the growth of said *Desulfovibrio* species. If the *Desulfovibrio* species according to the invention are included in the fecal matter, preferably, for example, at least 10 per milliliter or per gram of fecal matter. 4 10 5 2×10 5 3×10 5 4×10 5 5×10 5 6×10 5 7×10 5 8×10 5 9×10 5 10 6 2×10 6 3×10 6 4×10 6 5×10 6 6×10 6 7×10 6 8×10 6 9×10 6 10 7 2×10 7 3×10 7 4×107 5×10 7 6×10 7 7×10 7 8×10 7 9×10 7 10 8 10 9 10 10 10 11 10 12 10 13 1,000 desulfovibrio cells. Preferably, the desulfovibrio species is the first, second, third, fourth, or fifth most abundant bacterial species in the feces, that is, it has the highest cell count compared to other bacterial species contained in the feces, or is at least in the top 5.

[0172] The pharmaceutical agents according to the invention may additionally or optionally be amino acids substituted with one or more halogens, preferably one halogen, and more preferably chlorine, fluorine, or bromine-substituted amino acids. Aromatic amino acids are preferred, optionally substituted with one or more halogens, preferably, for example, substituted with one halogen at the 6-position, and more preferably, for example, aromatic amino acids substituted with chlorine, fluorine, or bromine at the 6-position. More preferably, tryptophan, tyrosine, or phenylalanine are optionally substituted with one or more halogens, preferably one halogen (e.g., at the 6-position), preferably substituted with chlorine, fluorine, or bromine at the 6-position, for example, chlorinated tryptophan, fluorinated tryptophan, or bromine-substituted tryptophan. Even more preferred are, for example, halogenated tryptophan at the 6-position, preferably chlorotryptophan, fluorotryptophan, or bromotryptophan. Most preferred is 6-bromotryptophan or any derivative or functional equivalent thereof. For example, in a composition comprising a pharmaceutical agent per milliliter or per gram, the pharmaceutical agent may be used in amounts of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 μg (micrograms). Optionally or additionally, for example, in a composition comprising a pharmaceutical agent per milliliter or per gram, it is preferable to use a total amount of the pharmaceutical agent between 0.1-10, 0.5-15, 1-20, 1-100, 5-100, 1-500, or 50-750 μg (micrograms). Optionally, for example, in a composition comprising the pharmaceutical agent per milliliter or per gram, the pharmaceutical agent may be used in an amount of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 mg. Optionally or additionally, for example, in a composition comprising the pharmaceutical agent per milliliter or per gram, the total amount of the pharmaceutical agent may preferably be between 0.1-10, 0.5-15, 1-20, 1-100, 5-100, 1-500, or 50-750 μg (micrograms). Optionally or additionally, for example, in the composition comprising the agent per milliliter or per gram, a total amount of the agent may preferably be used between 0.1-10, 0.5-15, 1-20, 1-100, 5-100, 1-500, or 50-750 mg. Administration may be oral, subcutaneous, or intravenous. The total amount to be administered may depend on the weight of the individual to be treated and may be determined by a technician. For example, a single dose may contain between 10 μg and 100 g, or between 10 mg and 50 g, or between 50 mg and 10 g, or between 100 mg and 5 g. This dose may be administered periodically as described elsewhere herein.Alternatively or additionally, the agent is preferably not contained in fecal matter, or if it is contained in fecal matter (e.g., as a substitute for the above-described composition), it is enriched, i.e., the amount of the agent is higher than that in fecal matter in the prior art, i.e., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% by weight higher than any fecal matter or fecal microbiota graft without the agent added. According to the invention, the agent can be added to feces. If the agent is contained in fecal matter, for example, at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 ng per milliliter or per gram of fecal matter, or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, The agent comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 μg (micrograms), or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 mg is included in the said fecal matter. Preferably, the agent is the first, second, third, fourth, or fifth most important metabolite in fecal matter, that is, it has the highest weight compared with other metabolites contained in fecal matter, or is at least in the top 10 or top 5.

[0173] Alternatively or alternatively, the agent according to the invention may be mono-fatty acid substituted glycerophosphate choline (GPC) or di-fatty acid substituted glycerophosphate choline (GPC), preferably wherein the fatty acid is (independently) saturated or (mono- or poly-)unsaturated fatty acid.

[0174] Preferred unsaturated fatty acids include myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, trans-oleic acid, vaccenic acid, linoleic acid, trans-linolenic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid. More preferably, substituted glycerophosphocholine (GPC) containing one or more of myristoleic acid and arachidonic acid is preferred. The use of 1-myristoyl-2-arachidonico-glycerophosphocholine (MA-GPC) and 1-arachidonico-glycerophosphocholine (A-GPC), or any derivatives or functional equivalents thereof, is more preferred or has yielded better results. In compositions comprising, for example, a pharmaceutical agent per milliliter or per gram, the pharmaceutical agent may be used in amounts of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 μg (micrograms). Optionally or additionally, for example, in compositions comprising, a pharmaceutical agent per milliliter or per gram, the pharmaceutical agent may preferably be used in amounts between 0.1-10, 0.5-15, 1-20, 1-100, 5-100, 1-500, or 50-750 μg (micrograms). Optionally, for example, in a composition comprising the pharmaceutical agent per milliliter or per gram, the pharmaceutical agent may be used in an amount of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 mg. Optionally or additionally, for example, in a composition comprising the pharmaceutical agent per milliliter or per gram, it is preferable to use an amount of the pharmaceutical agent in a total quantity between 0.1-10, 0.5-15, 1-20, 1-100, 5-100, 1-500, or 50-750 μg (micrograms). Optionally or additionally, for example, in a composition comprising the pharmaceutical agent per milliliter or per gram, a total amount of the pharmaceutical agent between 0.1-10, 0.5-15, 1-20, 1-100, 5-100, 1-500, or 50-750 mg may be used. Administration may be oral, subcutaneous, or intravenous. The total amount to be administered may depend on the weight of the individual to be treated and may be determined by a technician. For example, a single dose may contain between 10 μg and 100 g, or between 10 mg and 50 g, or between 50 mg and 10 g, or between 100 mg and 5 g.Optionally or additionally, administration may result in plasma concentrations in the treated individual preferably between 0.1-100, 0.2-50, 0.5-25, 0.5-20, 0.5-3, 1-15, 2-10, and 2-5 nanomoles / ml or between 0.1-100, 0.2-50, 0.5-3, 0.5-25, 0.5-20, 1-15, 2-10, and 2-5 micromoles / ml, or, in the case of pediatric use, 50% of these concentrations. This dosage may be administered periodically as described elsewhere herein. Alternatively or additionally, the agent is preferably not contained in fecal matter, or, if it is contained in fecal matter (e.g., as a substitute for the above-described composition), it is enriched, i.e., the amount of the agent is higher than that in fecal matter in the prior art, i.e., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% by weight higher than any fecal matter or fecal microbiota graft without the agent added. According to the invention, the agent can be added to fecal matter. If the agent is contained in fecal matter, for example, at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 ng per milliliter or per gram of fecal matter, or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2 The agent is included in the fecal matter in amounts of 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 μg (micrograms), or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 mg. Preferably, the agent is the first, second, third, fourth, or fifth most important metabolite in the fecal matter, i.e., it has the highest weight compared to other metabolites contained in the fecal matter, or is at least among the top 10 or top 5.

[0175] The agents according to the invention can be used in any combination for the prevention or treatment of the autoimmune diseases described herein. For example, *Desulfovibrio* species can be combined with chlorinated, fluorinated, or bromine-substituted tryptophan, such as 6-BT, and / or with mono- or di-fatty acid-substituted glycerophosphocholine (GPC), such as MA-GPC or A-GPC. Alternatively, chlorinated, fluorinated, or bromine-substituted tryptophan, such as 6-BT, can be combined with mono- or di-fatty acid-substituted glycerophosphocholine (GPC), such as with MA-GPC or with A-GPC. Alternatively, MA-GPC can be combined with A-GPC.

[0176] The agent according to the invention can modulate the immune system by resetting B cell clonal function and regulating T cells, which in turn can suppress autoimmune responses.

[0177] Preferably, the agent according to the invention is not contained in or combined with fecal matter, although the agent may be obtained from fecal matter. Additionally or optionally, the agent may be contained in a composition comprising no more than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 bacterial species.

[0178] Preferably, the pharmaceutical agent according to the invention is contained in the composition in amounts of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or 0.1-10, 0.5-15, 1-20, 1-100 mg, 5-50 mg or 1-25 mg per gram or per milliliter of composition or carrier (e.g., particularly in the case of intravenous administration, containing, for example, 0.5-1.5% by weight of NaCl, such as an aqueous solution containing, for example, 0.9% by weight of NaCl).

[0179] Prevention and / or treatment according to the invention may involve oral administration of the agent, or administration to the individual's small intestine, preferably the duodenum. In this regard, fecal matter may be administered enterically, preferably orally, nasally, or rectally, and / or duodenally, for example, through the (nasal)duodenal tube. Intravenous and subcutaneous administration are also anticipated (e.g., via a subcutaneous drug delivery system with a matchstick-sized (4×44 mm) implantable device for delivering an effective and consistent chronic dose for 3-6 months of treatment (followed by replacement)), particularly for substituted amino acids according to any agent of the invention, and especially for GPCs according to the invention, such as MA-GPC and A-GPC.

[0180] The agent according to the invention can be administered to an individual's gastrointestinal tract, preferably to the small intestine, and most preferably to the duodenum. The duodenum is the first part of the small intestine in most higher vertebrates, including mammals. Located before the jejunum and ileum, the duodenum is the shortest part of the small intestine. In humans, the duodenum is a hollow tube 25-38 cm long that connects the stomach to the distal duodenum. It begins at the duodenal bulb and terminates at the suspensory dorsum of the duodenum. Although the agent can also be administered to the colon (or cecum), administration to the colon (or cecum) is preferably not included in this invention.

[0181] The agents according to the invention can be combined with bacteria, i.e., microbial communities or intestinal microbial cells, wherein the phylum can be selected from one (or a combination thereof):

[0182] Firmicutes, including species such as *Eubacterium*, *Enteromonas*, *Faecalibacterium*, *Christensenella*, *Anaerostipes*, *Agathobacter*, *Roseburia*, *Coprococcus*, *Clostridium*, *Subdoligranulum*, *Anaerotruncus*, and *Flavobacterium*. The genera *Vinobacter*, *Ruminococcus*, *Butyricicoccus*, *Butyrovibrio*, *Sporobacter*, *Papilibacter*, *Oscillobacter*, *Oscillospora*, *Veilonella*, *Lactobacillus*, and *Streptococcus*.

[0183] Proteobacteria, such as genera belonging to the genera Escherichia or Enterobacter;

[0184] Actinobacteria, such as genera belonging to the genera Bifidobacterium or Collinsella;

[0185] The phylum Bacteroidetes, such as genera belonging to the genera *Bacteroides*, *Prevotella*, or *Alistipes*; and / or

[0186] Phylum Verrucomicrobia, such as genera belonging to the genus Akkermansia.

[0187] The agent can also be combined with microbial communities or gut microbial cells selected from eukaryotes, archaea, and bacteria, preferably free from other microorganisms. The 1057 species disclosed in de Vos (2014 FEMS Microbiol Rev.38(5):996-1047).

[0188] For example, a total of 10 per milliliter or per milligram of carrier may be preferred. 4 10 16 Between 10 4 10 15 Between 10 4 10 14 Between 10 6 10 12 Between 10, preferably 10 8 10 10 Any of the above-mentioned microbial cells between 1 and 2.

[0189] A medication may be administered in an effective amount, that is, an amount sufficient to achieve the desired therapeutic and / or preventative effect, such as an amount sufficient to treat and / or prevent a given condition. In the case of therapeutic or preventative application, the amount administered to an individual may depend on the type and severity of the disease or condition, as well as the individual's characteristics, such as general health status, age, sex, weight, and tolerance to the medication. It may also depend on the extent, severity, and type of the disease or condition. A skilled technician will be able to determine the appropriate dosage based on these and other factors.

[0190] In a preferred embodiment, the prevention and / or treatment according to the invention involves the individual application of the agent at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times and / or up to 10, 20, 30, 40, 50 times, preferably with an interval of at least 1, 2, 3, 4, 5, 6, 7, 8, 10 weeks and / or up to 10, 20, 30, 40, 50 weeks between said individual applications. Prevention and / or treatment may also involve daily, weekly, or monthly application, for example, once or twice every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days / weeks / months, and / or may be applied over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 weeks (or months or even years).

[0191] The reagent may be contained in a liquid medium and / or preferably (e.g., in a composition containing solids) not combined with solids having a diameter greater than 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 200, 400, 600, 800, or 1000 μm. The liquid medium may be an aqueous solution containing 0.5-1.5% by weight of NaCl, for example, 0.9% by weight of NaCl. The term "solid" refers to discrete particles containing up to 30, 20, 10, 5, or 1% by weight of water.

[0192] Further, it is envisioned that the pharmaceutical agent according to the invention is contained in a composition, preferably a pharmaceutical composition, more preferably a liquid or solid dosage form, and most preferably a capsule, tablet or powder.

[0193] For oral administration, the medication can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. Additionally, a carrier, such as activated charcoal, can be used.

[0194] Pharmaceutical formulations may be used as pharmaceutical products and / or accompanied by physiologically acceptable carriers, which may be any inert carrier. For example, non-limiting examples of suitable physiologically or pharmaceutically acceptable carriers include any well-known physiological or pharmaceutical carriers, buffers, diluents, and excipients. It should be understood that the selection of a suitable physiological carrier will depend on the intended manner of administration (e.g., oral) and the intended form of the composition taught herein (e.g., beverage, yogurt, powder, capsule, etc.). Those skilled in the art know how to select physiologically acceptable carriers suitable for or compatible with compositions used as taught herein.

[0195] Particularly preferred is that the drug is contained in and / or encapsulated in an enteric coating, preferably wherein the coating is insoluble and / or does not disintegrate in the gastric environment of an individual. This coating helps the drug reach its intended site of delivery, such as the duodenum, without being decomposed due to the acidic environment of the stomach. Preferred enteric coatings function by providing a surface that is stable at the high acidity of the stomach but decomposes more rapidly at lower pH levels. For example, it will not dissolve in the gastric acid (pH ~ 3) but will dissolve in the alkaline environment (pH 7-9) present in the small intestine or duodenum.

[0196] In one embodiment, the agent according to the invention may be combined with a mucosal binding agent or included in a composition comprising a mucosal binding agent. As used herein, the terms "mucosal binding agent" or "mucosal binding polypeptide" refer to an agent or polypeptide capable of attaching itself to the surface of the intestinal mucosal barrier of a mammal (e.g., a human). Various mucosal binding polypeptides have been disclosed in the art. Non-limiting examples of mucosal binding polypeptides include bacterial toxin membrane binding subunits, including, for example, the B subunit of cholera toxin, the B subunit of Escherichia coli heat-labile enterotoxin, pertussis toxin subunits S2, S3, S4 and / or S5, the B fragment of diphtheria toxin, and membrane binding subunits of shiga toxin or shiga-like toxin. Other suitable mucosal binding peptides include bacterial fimbriae, such as *Escherichia coli* fimbriae K88, K99, 987P, F41, FAIL, CFAIII ICES1, CS2 and / or CS3, CFAIIV ICS4, CS5 and / or CS6), and P fimbriae. Other non-limiting examples of fimbriae include *Bordetella pertussis* filamentous hemagglutinin, *Vibrio cholerae* toxin co-regulated fimbriae (TCP), mannose-sensitive hemagglutinin (MSHA), and fucose-sensitive hemagglutinin (PSHA). Other mucosal binders include viral attachment proteins, including influenza virus and Sendai virus hemagglutinins and animal agglutinins or agglutinin-like molecules, including immunoglobulin molecules or fragments thereof, calcium-dependent (type C) agglutinins, selectins, agglutinins, or Helixpomatia hemagglutinin. Plant agglutinins with mucosal binding subunits include concanavalin A, wheat germ agglutinin, phytohemagglutinin, abrin, and ricin.

[0197] In one embodiment, a composition comprising an agent for use as taught herein may be in liquid form, such as a stable suspension comprising one or more agents, or in solid form, such as a powder of a lyophilized agent as taught herein. For example, cryoprotectants such as lactose, trehalose, or glycogen may be used.

[0198] Optionally, the pharmaceutical preparations according to the invention may be encapsulated in capsules, such as gelatin capsules, together with inactive ingredients and powder carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talc, magnesium carbonate, etc.

[0199] In one embodiment, the pharmaceutical agent according to the invention may comprise one or more ingredients adapted to, for example, promote the survival and / or viability of the agent and / or maintain its integrity during storage and / or exposure to bile and / or passage through the gastrointestinal tract of mammals (e.g., humans). Non-limiting examples of such ingredients include enteric coatings as previously described, and / or controlled-release agents that allow passage through the stomach. Those skilled in the art know how to select appropriate ingredients to ensure that fecal matter reaches its intended destination and exerts its effect there.

[0200] In one embodiment, a composition comprising a pharmaceutical agent for use as taught herein may further comprise an ingredient selected from prebiotics, probiotics, carbohydrates, peptides, lipids, vitamins, minerals, pharmaceutical agents, preservatives, antibiotics, or any combination thereof.

[0201] In a particularly preferred embodiment, the agent according to the invention is combined with bacteria from the genera *Eubacterium*, *Enteromonas*, *Bifidobacterium*, *Lactobacillus*, and / or *Akkermansia*, preferably selected from *Bifidobacterium animalis* subsp. *lactobacter* or *Bifidobacterium breve*, *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Lactobacillus acidophilus*, *Eubacterium hominis*, *Enteromonas butyrate-producing*, and / or *Akkermansia muciniphila*. A total number of 10... 4 and 10 14 Between, 10 6 and 10 12 Between, preferably in 10 8 and 10 10 The bacterial cells between these (e.g., per milliliter or per milligram). The above combination can provide a synergistic effect. The agent and bacteria can be contained in different compositions, or together in a single composition (e.g., in capsules or other dosage forms described herein).

[0202] The pharmaceutical agents according to the invention can additionally or optionally be combined with hormone supplements (thyroid, hydrocortisone, insulin, etc.) preferably selected from infliximab, adalimumab, cetuzumab, and golimumab, tumor necrosis factor α (TNFα) inhibitors, and / or DMARDs (rheumatoid arthritis). The inventors believe that treatment with a TNFα inhibitor can increase the response to treatment with the agents of the invention, and / or vice versa. Preferably, the TNFα inhibitor is administered in a different or the same composition as agent (a) (e.g., in capsules or other dosage forms as described herein). The TNFα inhibitor can be administered, for example, at doses of 1-10 mg / kg, 2-8 mg / kg, 3-7 mg / kg, 4-6 mg / kg, or 5 mg / kg weekly or daily, and / or intravenously / orally at least (or at most) 1, 2, 3, or 4 times.

[0203] Further envisioning, the agents according to the invention, particularly *Desulfovibrio* species, are present in lyophilized and / or microencapsulated forms, such as capsules containing said substance. Preferably, the agents (e.g., *Desulfovibrio* species) are present in solid, lyophilized, or dried forms (i.e., containing less than 20, 10, 5, 2, or 1% water by weight), for example, in powder or granule form. For example, it can be present in microencapsulated form. Those skilled in the art can lyophilize or microencapsulate agents based on known techniques, where anaerobic conditions can be used to preserve the viability of any bacteria contained in fecal matter.

[0204] Microencapsulation technology is well known in the field of bacterial preservation (e.g., as reviewed in Serna-Cock and Vallejo-Castillo, 2013. Afr J of Microbiol Res, 7(40):4743-4753). For example, any preservation technique and system taught by Serna-Cock and Vallejo-Castillo can be used in this invention.

[0205] Freeze-drying methods include, but are not limited to, slow, gradual freezing to -40°C before drying, rapid freezing at -80°C before drying, or ultra-rapid freezing by dripping cells containing cryoprotectants into liquid nitrogen before drying. Cryoprotectants are commonly used to protect the composition during freeze-drying and extend its shelf life. Without limitation, cryoprotectants selected from sucrose, maltose, maltodextrin, trehalose, mannitol, sorbitol, inulin, glycerol, DMSO, ethylene glycol, propylene glycol, 2-methyl-2,4-pentanediol, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyglycerol, skim milk powder, milk protein, whey protein, UHT milk, betaine, adonitol, sucrose, glucose, lactose, or any combination thereof can be used.

[0206] Prebiotics such as starch and wheat bran can be further added to the pharmaceutical preparations of the present invention, for example, prior to freeze-drying, to enhance their efficacy. Adding antioxidants such as riboflavin, riboflavin phosphate or their physiologically acceptable salts, glutathione, ascorbate, glutathione, and cysteine ​​to the freeze-dried mixture can further enhance the viability of any bacteria contained therein.

[0207] After adding a cryoprotectant (e.g., glycerol) disclosed herein and / or freezing at -80°C, the agent (especially *Desulfovibrio* species) can be stored for extended periods (e.g., at least 10, 20, 40, 52 weeks or at least 1, 2, 3 years). Alternatively, freeze-drying stabilizes the agent during this period. Finally, *Desulfovibrio* species can also be inoculated, as described by de Vos (2013 Microb Biotechnol. 2013 Jul; 6(4):316-25).

[0208] In one embodiment, the pharmaceutical agent used as taught herein may be or can be contained in a food or food supplement composition. Such a food or food supplement composition may include dairy products, more preferably fermented dairy products, and most preferably yogurt or yogurt beverages.

[0209] In one embodiment, the pharmaceutical agent or composition comprising the pharmaceutical agent as taught herein may further comprise one or more ingredients that further enhance the nutritional and / or therapeutic value of the fecal material taught herein. For example, it may be advantageous to add one or more ingredients (e.g., nutritional, veterinary, or pharmaceutical ingredients, etc.) selected from proteins, amino acids, enzymes, mineral salts, vitamins (e.g., HCl thiamine, riboflavin, HCl pyridoxine, niacin, inositol, choline chloride, calcium pantothenate, biotin, folic acid, ascorbic acid, vitamin B12, para-aminobenzoic acid, vitamin A acetate, vitamin K, vitamin D, vitamin E, etc.), sugars and complex carbohydrates (e.g., water-soluble and water-insoluble monosaccharides, disaccharides, and polysaccharides), pharmaceutical compounds (e.g., antibiotics), antioxidants, trace element components (e.g., compounds of cobalt, copper, manganese, iron, zinc, tin, nickel, chromium, molybdenum, iodine, chlorine, silicon, vanadium, selenium, calcium, magnesium, sodium, and potassium, etc.). Those skilled in the art are familiar with methods and ingredients suitable for enhancing nutritional and / or therapeutic / pharmaceutical value.

[0210] The present invention also provides a method for predicting the response of patients with autoimmune diseases to treatment with an agent (or autologous fecal matter) according to the present invention, the method comprising:

[0211] The abundance levels of at least one bacterium selected from Bacteroides caccae and Coprococcus catus in the patient's fecal microbiota were measured.

[0212] Measurements above a reference level indicate a response to therapy in patients with autoimmune diseases. The reference level can be, for example, between 50% and 150% of the abundance of at least one bacterium selected from *Bacteroides coccidioides* and *Coccidioidomyces cerevisiae* in the fecal microbiota of a healthy control individual, preferably between 75% and 125%, more preferably between 90% and 120%, 95% and 110%, and 98% and 105%.

[0213] In the context of this invention, the individuals receiving treatment are preferably animals, more preferably mammals, and most preferably humans. As will be clear, the treatments of this invention are preferably not used as control or placebo treatments and / or conducted in clinical trials, i.e., in studies where participants are assigned to groups receiving one or more interventions / treatments, one or more control or placebo interventions / treatments, or no intervention, so that researchers can assess the impact of the intervention on biomedical or health-related outcomes.

[0214] In this invention document and its claims, the verb "comprising" and its variations are used in a non-limiting sense to mean including the items that follow the word, but not excluding items not specifically mentioned. Furthermore, the indefinite article "a" or "an" in reference to an element does not exclude the possibility that there is more than one element, unless the context explicitly requires exactly one element. Therefore, the indefinite article "a" or "an" generally means "at least one".

[0215] "Sequence identity" can be determined by aligning two peptide sequences or two nucleotide sequences using an alignment algorithm (when performing optimal alignment using default parameters through programs such as GAP or BESTFIT). GAP uses the Needleman and Wunsch global alignment algorithms to align the two sequences across their entire length, maximizing the number of matches and minimizing the number of gaps. Typically, using GAP's default parameters, the gap creation penalty = 50 (nucleotides) / 8 (proteins), and the gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, while for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignment and percentage sequence identity scores can be determined using computer programs such as the GCG Wisconsin Package, version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or EmbossWin version 2.10.0 (using the program “needle”). Alternatively, the similarity or identity percentage can be determined by searching a database using algorithms such as FASTA, BLAST, etc. For illustration, a polynucleotide having a nucleotide sequence that is at least, for example, 95% “identical” to a reference nucleotide sequence encoding a polypeptide of a given sequence means that the polynucleotide sequence is identical to the reference sequence except that the reference polypeptide sequence may contain up to 5 point mutations per 100 nucleotides (which can be (conserved) substitutions, deletions, and / or insertions). In other words, to obtain a polynucleotide with a nucleotide sequence having at least 95% identity with a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted and / or substituted with another nucleotide, and / or up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the 5' or 3' ends of the reference nucleotide sequence, or at any position between these ends, either individually or in one or more consecutive groups within the reference sequence. Similarly, by a polypeptide having an amino acid sequence having at least, for example, 95% “identity” with the reference amino acid sequence of SEQ ID NO:1, it means that the amino acid sequence of the polypeptide is identical to the reference sequence, except that changes may include up to 5 amino acid changes per 100 amino acids of the reference amino acid sequence of SEQ ID NO:1.In other words, to obtain a polypeptide with an amino acid sequence having at least 95% identity with a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence may occur at the amino-terminal or carboxyl-terminal position of the reference amino acid sequence or at any position between these positions, either individually or as one or more consecutive groups within the reference sequence. Sequence identity can be determined over the entire length of the sequence under consideration.

[0216] sequence list

[0217]

[0218] Attached Figure Description

[0219] Figure 1 The top 10 small gut microbiota ranked by relative importance in predicting treatment group allocation (XGBoost predictive modeling algorithm). Percentages are scaled towards the maximum value, which is set to 100%. The top 4 microbiota stand out with higher relative importance.

[0220] Figure 2 A: Top 10 metabolites in the best-predicted treatment group allocation (XGBoost predictive modeling algorithm). Percentages are scaled to the maximum value, set to 100%. The top 3 metabolites showed high relative importance in the analysis. BD: Relative abundance of the top 3 metabolites over time for each treatment group (in each figure, the upper line represents the autologous FMT group; the lower line represents the allogeneic FMT group). Median ± IQR is reported. The Mann-Whitney U test was used to calculate p-values ​​between groups at 12 months. At 12 months, there was a difference in 1-myristoyl-2-arachidonicoyl-GPC between groups, p = 0.020. E: Spearman correlation between changes in fasting C-peptide and changes in 1-myristoyl-2-arachidonicoyl-GPC. F: Abundance of D. piger in feces over time. p-values ​​were calculated using the Mann-Whitney U test. p-value = 0.024 at 6 months and 0.023 at 12 months. G: Fold change in D. piger between groups (the dominant upper limit represents the autologous FMT group). δp-values ​​were calculated using the Mann-Whitney U test for δ between 0 and 12 months in each group; p-value = 0.006. H: Spearman correlation plot of δ (0–12 months) of fecal D. piger and δ (0–12 months) of fasting C-peptide. I: Correlation plot of fecal D. piger and 1-arachidonicyl-GPC. J: Correlation plot of fecal D. piger and Prevotella 1. K: Correlation plot of fecal D. piger and Prevotella 2.

[0221] Figure 3 The output of the fecal microbiota prediction model shows the top 30 differences in changes among treatment groups.

[0222] Figure 4 A: Shows the number of responders at 6 and 12 months and the number of individuals in each treatment group. Response was defined as a decrease of <10% in C-peptide AUC compared to baseline. All analyses were performed using 12-month responders. B: Shows the individual subject line for C-peptide AUC over time. C and D: Show the abundance of *F. coliformis* and *F. claustrophoblastus* over time, respectively. In both figures, the upper line represents the responders. The Mann-Whitney U test was used to calculate the p-value between groups at each time point. For *F. coliformis* at baseline, the p-value = 0.0099; for *F. claustrophoblastus* at baseline, the p-value = 0.00049. E: Shows the correlation between Δ*F. claustrophoblastus* (0–12 months) and Δ*C-peptide AUC (0–12 months). Spearman's rho(r) is shown, and p-values ​​were calculated using Spearman's rank.

[0223] Figure 5 : Predictive model output showing the top 30 differences in fecal microbiota among treatment groups.

[0224] Figure 6 Abundance of the top 10 fecal microbiota over time (see: Figure 3 At baseline, the strains that best distinguished between responders and non-responders were selected (in A, B, and D, the top line represents responders and the other line represents non-responders; in C and E, the top line represents non-responders and the other line represents responders). Strains with responders and non-responders at baseline or during the study were selected for presentation. The Mann-Whitney U test was used to calculate p-values ​​at each time point. A: *Paraprevotella* spp., p = 0.019; B: *Eubacterium ramulus*, p = 0.043; C: *Collinsella aerofaciens*, p = 0.043; D: *Bacteroides eggerthii*, p = 0.006; E: *Ruminococcus callidus*, p = 0.026. There was no significant difference in *Faecalibacterium prausnitzii* (ranked 10th out of the top 10) at baseline (p = 0.063).

[0225] Figure 7 Effects of 6-bromotryptophan (6-BT), 1-arachidonico-glycerophosphate choline (20:0) (A-GPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphate ethanolamine (16:0-18:2PE), and 1-myristoyl-2-arachidonico-glycerophosphate choline (MA-GPC) on NFκB pathway activation at different doses.

[0226] Figure 8 Effects of 6-BT, MA-GPC, and A-GPC on bone marrow cells: Mouse CD11b+ monocytes were activated with LPS (10 ng / ml) and stimulated with TLR4 for 24 hours.

[0227] Figure 9 Effects of 6-BT, MA-GPC, and A-GPC on bone marrow cells: 24-hour TLR3 stimulation of mouse monocytes CD11b+ activated with the dsRNA analog Poly(I:C).

[0228] Figure 10 Effects of 6-BT on human monocytes stimulated by LPS.

[0229] Figure 11 Effects of 6-BT, A-GPC, and MA-GPC on T lymphocytes: Mouse CD4+ T cells activated with anti-CD3 and anti-CD28 mAbs.

[0230] Figure 12 INS1eβ cells treated with 6-BT for 24 hours: gene expression of β cell differentiation markers.

[0231] Figure 13 INS1eβ cells treated with 6-BT for 24 hours: gene expression of β cell differentiation markers.

[0232] Figure 14 Mouse bone marrow-isolated monocytes (Christ A, Cell 2018) or bone marrow-derived macrophages (Swansen, JEM, 2017) were exposed to specified concentrations of 6-BT (10-100 μM) for 24 hours in the presence or absence of 10 ng / ml LPS, 10 μg / ml P3C, or 10 μg / ml poly(I:C). Using ELISA, the inventors found that 6-BT inhibited the secretion of the pro-inflammatory cytokine TNFα during TLR4 and TLR2 conjugation, and the secretion of IFNβ during TLR3 activation.

[0233] Figure 15 The inventors investigated the effect of 6-BT on mouse dendritic cells (DCs) differentiated from bone marrow cells using GM-CSF (40 ng / ml). Regarding monocytes / macrophages, after activation of TLR4 (100 ng / ml LPS) or TLR3 (10 μg / ml poly(I:C)) respectively, 6-BT inhibited the secretion of pro-inflammatory cytokines TNFα and IFNβ through DCs.

[0234] Figure 16 The inventors further investigated the effects of 6-BT on CD4 T cells. To mimic antigen presentation, mouse CD4 T cells (isolated from the spleen; Uchimura T, Immunity 2019) were activated with monoclonal antibodies against CD3 and CD28 (2.5 and 1 μg / ml, respectively). Consistent with findings on bone marrow cells, 6-BT significantly reduced the production of the Th1 cytokine IFNγ.

[0235] Figure 17 The inventors investigated whether 6-BT could have a direct effect on β cells. Indeed, we see here that 6-BT induces gene expression of transcription factors PDX1 and MAFA in IS1E β cells, which are crucial for β cell maturation and function. Similarly, 6-BT also promotes insulin secretion during homeostasis and glucose-stimulated insulin secretion (data shown as the difference in insulin release under starvation [1 mM glucose] and hyperglycemic [22 mM] conditions) (Paula S, FASEBJ 2015).

[0236] Figure 18 The inventors investigated the effect of 6-BT on NF-κB pathway activation, a central pathway in all inflammatory diseases (except autoimmune diseases). Therefore, we quantified the expression of the phosphorylated p65 subunit, considered a marker of NF-κB activation. Following T cell activation with PMA (50 ng / ml) and iomycin (1 μg / ml), 6-BT inhibited NF-κB signaling at a very early time point (5–10 minutes post-activation). This effect was observed in both mouse and human (Jurkat cell) CD4 T cells.

[0237] Figure 19 Using the RAW264.7 mouse macrophage cell line (GroenewegM, J Lipid Res 2006) stably expressing the NFkB luciferase reporter gene, the inventors disclosed that macrophages exposed to 6-BT (10-200 μm) overnight and stimulated with LPS (10 ng / ml) for 2 hours inhibited the transcriptional activity of the NFkB complex in a dose-dependent manner.

[0238] Figure 20 In mouse CD4 T lymphocytes (isolated from mouse spleen; Uchimura T, Immunity 2019), 6-BT, rather than tryptophan, inhibited IFNγ production during CD3 / CD28 conjugation. This suggests that 6-BT and tryptophan induce different biological activities.

[0239] Figure 21 Monocytes (isolated from mouse bone marrow; Christ A, Cell 2018) exposed to 6-BT or tryptophan showed that the anti-inflammatory effect was specific to 6-bromotryptophan molecules but not to tryptophan.

[0240] Figure 22 The inventors discovered that 6-BT (100 μM) promotes mitochondrial metabolism in mouse and human (Jurkat) CD4 T cells. OCR = oxygen consumption rate, used as a representative of cellular utilization of mitochondrial oxidative phosphorylation. Measured using a hippocampal XF analyzer (OCRUchimura T, Immunity 2019; Chou, Nature 2021).

[0241] Figure 23 6-BT exposure enhances mitochondrial metabolism in pro-inflammatory M1 macrophages (differentiation in the presence of LPS and IFNγ; Cheng N, JCI Insight 2018) without affecting glycolytic flux. Intracellular metabolism was measured using a hippocampal XF analyzer (Uchimura T, Immunity 2019; Chou, Nature 2021).

[0242] Figure 24 The inventors investigated whether 6-BT could affect mitochondrial metabolism in β-cells, which rely on ATP and mitochondrial metabolites for insulin exocytosis. 6-BT increased mitochondrial metabolism in β-cells (INS1E β-cells) under both homeostatic and hyperglycemic (25 mM glucose) conditions. Furthermore, we tested the effects of tryptophan on intracellular metabolism and found that tryptophan played a different role than 6-BT in terms of inflammatory markers. (Intracellular metabolism was measured using a hippocampal XF analyzer (Uchimura T, Immunity 2019; Chou, Nature 2021)).

[0243] Figure 25 : Relative abundance of desulfurized Vibrio in feces between diabetic and non-diabetic individuals.

[0244] Figure 26 Diabetes: The effect of *Desulfovibrio* spp. Odds ratio for diabetes.

[0245] Figure 27 The relationship between plasma 6BT levels and relative abundance of fecal desulfovibrio in (1) non-diabetic individuals (upper limit) and (2) diabetic patients (lower limit).

[0246] Example 1

[0247] Patients with the following conditions receive the following treatments

[0248] 1. Take one empty enteric-coated capsule orally daily for two consecutive years.

[0249] 2. Administer orally for two consecutive years, containing ~1*10 8 Enteric-coated capsules containing cells of *Desulfovibrio piger* (also known as *Desulfovibrio piger*, *Desulfovibrio desulfurization*, or *Desulfovibrio fairyfield*).

[0250] 3. For two consecutive years, administer orally daily an enteric-coated capsule containing 50 mg of chlorinated, fluorinated, or bromine-substituted tryptophan (6-bromotryptophan (6-BT) or 6-fluorotryptophan (6-FT)).

[0251] 4. For two consecutive years, administer orally daily enteric-coated capsules containing 50 mg of mono-fatty acid-substituted glycerophosphate choline (GPC) or di-fatty acid-substituted glycerophosphate choline (GPC) (1-myristoyl-2-arachidonico-glycerophosphate choline (MA-GPC) or 1-arachidonico-glycerophosphate choline (A-GPC)).

[0252] Table 1

[0253]

[0254]

[0255]

[0256]

[0257] Similar results to the hypothetical effects shown in Table 1 above can be expected in a larger patient cohort.

[0258] Example 2

[0259] Patients with recently diagnosed T1D (<6 weeks) were randomly assigned to two groups to receive three autologous or allogeneic (healthy donor) fecal microbiota transplants (FMTs) over a period of 4 months.

[0260] Several (microbiome-derived) plasma metabolites and (small) gut bacterial strains were found to be associated with improved residual β-cell function in type 1 diabetes.

[0261] Materials and methods

[0262] A double-blind, randomized, controlled trial was conducted using computer randomization in newly diagnosed T1D individuals. The study investigated the effects of allogeneic (healthy donor) versus autologous (self) gut microbiota infusion on residual β-cell function and autoimmune T-cell responses, related to changes in the (small) gut microbiota over one year post-treatment.

[0263] Patient recruitment

[0264] We are recruiting newly diagnosed type 1 diabetes patients from outpatient clinics in the Amsterdam area. Inclusion criteria include male / female, age 18-35 years, and normal BMI (18.5-25 kg / m²). 2 Patients diagnosed with T1D within a maximum of 6 weeks prior to enrollment and possessing residual β-cell function (as indicated by plasma C-peptide >0.2 mmol / L and / or >1.2 ng / mL after MMT).

[0265] Exclusion criteria include a diagnosis or symptom of another autoimmune disease (such as hypothyroidism or hyperthyroidism, celiac disease, rheumatoid arthritis, or inflammatory bowel disease), (expected) long-term impaired immunity (due to recent cytotoxic chemotherapy or HIV infection with a CD4 count <240), and use of antibiotics, proton pump inhibitors, or any other type of systemic medicine other than insulin within the past 3 months.

[0266] Donor Recruitment

[0267] Recruiting lean individuals (BMI < 25 kg / m²) 2 Omnivorous, healthy Caucasian men and women were selected as fecal donors. They completed questionnaires regarding their diet and bowel habits, travel history, and comorbidities (including a family history of diabetes and medication use). As previously mentioned, donors are screened for infectious diseases (van Nood et al., 2013). Blood is screened for human immunodeficiency virus (HIV); human T-lymphovirus (HIL); hepatitis A, B, and C; cytomegalovirus (CMV); Epstein-Barr virus (EBV); strongyloides; amebiasis; and syphilis. Although previous and inactive EBV and CMV infections are permissible, the presence of infection leads to exclusion. As previously recommended, donors are also excluded if stool screening shows the presence of pathogenic parasites (such as *Bacillus humanis*, *Entamoeba fragilis*, *Giardia lamblia*), multidrug-resistant bacteria (Shigella, Campylobacter, Yersinia, MRSA, ESBL, Salmonella, enteropathogenic *Escherichia coli*, and *Clostridium difficile*), or viruses (norovirus, rotavirus, astrovirus, adenovirus (40 / 41 / 52), enterovirus, and pasteurellosis).

[0268] Inspection and visit

[0269] Prior to each study visit, participants were asked to complete a one-week online nutrition diary to monitor calorie intake, including dietary carbohydrates, fats, proteins, and fiber. During the study visits, blood pressure, height, weight, and daily insulin dosage were recorded. Fasting blood samples were collected at each visit, centrifuged, and stored at -80°C for later analysis. Whole blood heparin sodium tubes were stored at room temperature and processed within 24 hours for immunological analysis.

[0270] Fecal transplantation using fresh feces was performed three times at 0, 2, and 4 months. Mixed diet trials (for residual β-cell function) and gut microbiota analysis were conducted at 0, 2, 6, 9, and 12 months. Plasma metabolites were measured at 0, 6, and 12 months. Biostatistical measurements and fasting plasma monitoring were performed at all time points to ensure safety parameters.

[0271] Description of each research visit

[0272] All visits were conducted after an overnight fast, and individuals had not taken long-acting insulin the previous night. Blood, stool, and urine samples, as well as biometric measurements, were taken at each visit. Nasoduodenal tube localization was performed at baseline / 0 months. As previously mentioned, a standardized 2-hour mixed diet test was performed when the patient was fully awake after catheter placement. (Moran et al., 2013) to investigate residual β-cell function. At 2, 9, and 12 months, patients underwent a mixed diet trial again to detect residual β-cell C-peptide secretion. Then, a duodenal tube was placed via the CORTRAK enteral route, and fecal transplantation was repeated. A mixed diet trial was performed at 6 months.

[0273] fecal transplant surgery

[0274] Individuals were randomly assigned to receive three autologous or allogeneic fecal transplants. All patients and researchers were unaware of their treatment allocation. Upon admission, a duodenal tube was placed via endoscopy or the CORTRAK enteral access system. Each patient then received 2–4 L of Klean's solution via the duodenal tube. A complete colonic irrigation was performed using polyethylene glycol (PEG) until researchers determined the intestines were adequately irrigated (i.e., no solid waste, but clear fluid) for approximately 3 hours. Then, 200–300 grams of donor feces were treated by diluting it in 500 ml of 0.9% saline solution and filtering it through an unfolded cotton mesh. Klean was then administered for the final time via the duodenum using a 50 cc syringe over approximately 30 minutes. Two hours later, the filtrate was used for transplantation. After a short observation period, the patient was sent home.

[0275] Mixed Diet Trial

[0276] Starting the night before each mixed-diet trial, T1D patients discontinued their long-acting insulin injections. After a night of fasting and without taking short-acting morning insulin, the mixed-diet trial was conducted with 6 ml / kg body weight of Boost High Protein (Nestlé Nutrition, Vervey, Switzerland), up to a maximum of 360 ml per person. Blood samples were then taken at -10, 0, 15, 30, 45, 60, 90, and 120 minutes to measure the stimulated C-peptide. The AUC (area under the curve) values ​​were derived using the trapezoidal rule.

[0277] plasma metabolites

[0278] Fasting plasma metabolite measurements were performed by Metabolon (Durham, NC) using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). Raw data were standardized to account for inter-day variability. The levels of each metabolite were then readjusted to set the median for all samples to 1. Missing values ​​were typically due to sample measurements being below the limit of detection and were then estimated using the smallest observed value for the corresponding metabolite.

[0279] Biochemistry

[0280] Glucose and C-reactive protein (CRP, Roche, Switzerland) were measured in fasted plasma samples. C-peptide was measured using radioimmunoassay (Millipore). Total cholesterol, high-density lipoprotein cholesterol (HDLc), and triglycerides (TG) in EDTA-containing plasma were measured using commercially available enzymatic assays (Randox, Antrim, UK, and DiaSys, Germany). All analyses were performed using a Selectra automated analyzer (Sopachem, Netherlands). Low-density lipoprotein cholesterol (LDLc) was calculated using the Friedewald formula. Calcium protectin in feces was measured using a commercially available ELISA (Bühlmann, Switzerland).

[0281] Shotgun sequencing of fecal samples and metagenomic pipeline

[0282] Fecal microbiota analysis was performed on donor fecal samples and fecal samples collected at 0, 6, and 12 months after the start of the study using shotgun sequencing. DNA was extracted from fecal samples for shotgun metagenomics. Subsequently, shotgun metagenomic sequencing was performed (Clinical Microbiomics, Copenhagen, Denmark). Prior to sequencing, the quality of the DNA samples was assessed using agarose gel electrophoresis, NanoDrop 2000 spectrophotometry, and Qubit 2.0 fluorometer quantification. Genomic DNA was randomly spliced ​​into fragments of approximately 350 bp. The fragmented DNA was used for library construction using the NEBNext Ultra LibraryPrep Kit for Illumina (New England Biolabs). The fragment size distribution of the prepared DNA libraries was assessed using Qubit 2.0 fluorometer quantification and an Agilent 2100 bioanalyzer. Quantitative real-time PCR (qPCR) was used to determine the final library concentration prior to sequencing. The libraries were sequenced on the Illumina HiSeq platform, producing 2 × 150 bp paired-end reads. Raw reads were quality filtered using Trimmomatic (v0.38) to remove adaptors, trim the first 5 bp, and then quality-trimmed again using a 4 bp sliding window and a minimum Q score of 15. Reads shorter than 70 bp after trimming were discarded. The surviving paired reads were mapped to the human genome (GRCh37_hg19) using bowtie2 (v2.3.4.3) to remove human reads. Finally, the remaining quality-filtered non-human reads were subsampled to reach 20 million reads per sample and processed using Metaphlan2 (v2.7.7) to infer metagenomic microbial species composition, and gene counts and functional pathways were extracted using Humann2 (v0.11.2). In summary, bowtie2 was used to map microbial pan-genome reads; unmapped reads were translated and mapped to the full Uniref90 protein database using diamond (v0.8.38). Pathway collection was performed using the MetaCyc database.

[0283] Small intestinal microbiota analysis

[0284] Biopsies were added to a bead-beating tube containing 300 μl of Stool Transport and Recovery (STAR) buffer and 0.25 g of sterile zirconia beads (0.1 mm). 6 μl of proteinase K (20 mg / ml; QIAGEN, Venlo, Netherlands) was added and the tube was incubated at 55 °C for 1 h. The biopsies were then homogenized by bead-beating three times (60 sec × 5.5 ms) and incubated at 95 °C for 1000 rpm for 15 min. The sample was then centrifuged at 4 °C and 14,000 g for 5 min, and the supernatant was transferred to a sterile tube. The precipitate was reprocessed with 200 μl of STAR buffer, and the two supernatants were combined. DNA purification was performed using a custom kit (AS1220 Promega) with a 250 μl final supernatant pool. DNA was eluted in 50 μl of DNase- and RNase-free water and analyzed using a DS-11FX+ spectrophotometer / fluorometer (DeNovix Inc., Wilmington, USA) and Qubit. TMThe concentration was measured using a dsDNABR assay kit (Thermo Scientific, Landsmeer, Netherlands). The V5-V6 region of the 16S ribosomal RNA (rRNA) gene was amplified in duplicate PCR reactions of 50 μl per sample. Primary enrichment was achieved using 27F and 1369R primers. The kit included 1 μl of 10 μM primers, 1 μl of a dNTPs mixture, 0.5 μl of Phusion Green Hot Start II high-fidelity DNA polymerase (2 U / μl; Thermo Scientific, Landsmeer, Netherlands), 10 μl of 5× Phusion Green HF buffer, and 36.5 μl of water free of DNAse-RNAse-. The amplification program consisted of an initial denaturation step at 98 °C for 30 seconds, followed by five cycles of denaturation at 98 °C for 30 seconds, annealing at 52 °C for 40 seconds, extension at 72 °C for 90 seconds, and a final extension step at 72 °C for 7 minutes. Nested PCR was performed on the PCR products using a master mix containing 1 μl of unique barcode primers 784F-n and 1064R-n (10 μM per reaction), 1 μl of dNTPs mixture, 0.5 μl of Phusion Green Hot Start II high-fidelity DNA polymerase (2 U / μl; Thermo Scientific, Landsmeer, Netherlands), 10 μl of 5× Phusion Green HF buffer, and 36.5 μl of DNAse- and RNase-free water. The amplification program consisted of an initial denaturation step at 98 °C for 30 sec, followed by five cycles of denaturation at 98 °C for 10 sec, annealing at 42 °C for 10 sec, extension at 72 °C for 10 sec, and a final extension step at 72 °C for 7 min. The PCR products were visualized on a 1% agarose gel (~280 bp) and purified using a CleanPCR kit (CleanNA, Alphen aan den Rijn, Netherlands). The concentration of purified PCR products was measured using the Qubit dsDNA BR detection kit (Invitrogen, California, USA), and 200 ng of microbial DNA from each sample was pooled to create the final amplicon library (150 bp, paired ends) for sequencing on the Illumina HiSeq.2500 platform (GATC Biotech, Constance, Germany).

[0285] Raw reads were demultiplexed using the Je software suite (version 2.0), disallowing barcode mismatches. After removing barcodes, adapters, and primers, human genome-mapped reads were removed using bowtie2 control to eliminate human reads. Surviving microbial forward and reverse reads were pipelined separately using DADA2 (Callahan et al., 2016) (v1.12.1). Amplicon sequence variants (AVS) inferred from reverse reads were reverse complementary and matched with ASVs inferred from forward reads. Only non-chimeric forward read ASVs matched with reverse complementary reverse read ASVs were retained. ASV sample counts were inferred from forward reads. ASV classifications were assigned using the DADA2 and SILVA (v132) databases. The generated ASV table and classification assignments were integrated using the phyloseq R package (v1.28.0) and diluted to 60,000 counts per sample.

[0286] Power Calculation and Statistics

[0287] A sample size of 17 patients per group (34 patients total) was required to provide 80% efficacy for detecting the 50% difference in C-peptide AUC between treatment groups at 12 months (360 mmol / L / min vs. 180 mmol / L / min, standard deviation 170), with a two-sided test at α = 0.05 and 10% dropout. All analyses were based on a pre-specified intention-to-treat cohort (full case analysis) with known measurements; missing values ​​were considered randomized missing. The primary endpoint was the reserve of residual (MMT-stimulated) β-cell function at 6 and 12 months compared to baseline (0 months). Other secondary endpoints were changes in immune markers of autoimmunity in whole blood leukocyte subsets, glycemic control parameters, and fasting plasma metabolites during these 12 months. Finally, changes in small intestinal epithelial genes were measured between baseline and 6 months after FMT initiation. Analyses were performed via intention-to-treat.

[0288] For baseline differences between groups, unpaired Student's t-tests or Mann-Whitney U tests were used, depending on the distribution of the data. Therefore, data are presented as mean ± standard deviation or median of the interquartile range. Postprandial outcomes (e.g., C-peptide) were described as the area under the curve (AUC) at 2-hour follow-up after a meal, calculated using the trapezoidal method. For correlation analysis, Spearman's rank test was used (since all parameters were nonparametric). To compare the primary endpoint, a linear mixed model (LMM) (lme4 package in R) was used, where "assignment" and "time point" were fixed effects, and "number of patient entries" was a random effect. p-values ​​for interactions between "assignment" and "time point" are reported. Furthermore, the Mann-Whitney U test was used to compare intergroup parameters at different time points. A p-value < 0.05 was considered statistically significant. This study was conducted at the Academic Medical Center (Amsterdam) in accordance with the Declaration of Helsinki (2013 update). Written informed consent was provided by all participants, and all research procedures were approved by the Academic Medical Center Ethics Committee (IRB). This study has been prospectively registered with the Dutch Trial Registry (NTR3697).

[0289] Machine learning and subsequent statistical analysis

[0290] Extreme Gradient Boosting (XGBoost) machine learning classification algorithm, combined with a stability selection procedure, was applied to determine which parameters (as baseline values ​​or as relative changes) best predicted treatment groups and responders vs. non-responders. This technique was used for duodenal microbiota composition (16S rRNA sequencing from biopsy), fecal microbiota composition and metabolic pathway abundance, and plasma metabolite levels. To predict treatment groups, we used the relative change (δ(delta)) of each parameter between 0 and 12 months. For duodenal microbiota, we used δ0 versus 6 months. For responder vs. non-responder prediction, we used baseline values, δ0 versus 6 months, and δ0 versus 12 months. Each analysis generated a ranked list of the top 30 most discriminative features. The highest-ranking parameter was selected from each analysis for more closely studied groups assigned to accurate (i.e., ROC AUC > 0.8) or moderate (ROC AUC > 0.7) predictions using arbitrary but reasonable cutoff values. This cutoff value was typically around 30% or higher of relative importance. Then, the changes of the selected parameters over time were visualized (Wilcoxon's signed rank tests), and the differences between study groups at each time point were analyzed (Mann-Whitney U test). Finally, Spearman's rank test was used to correlate these parameters with the primary endpoint and other key parameters identified in this way.

[0291] result

[0292] Patients were randomly assigned to either the donor FMT group (n = 11 individuals) or the autologous FMT group (n = 10 individuals). One participant withdrew their consent after the first study visit. Due to lack of funding, the trial was terminated after 20 individuals enrolled and completed the study. Seven healthy, lean donors (three of whom were used twice) donated allogeneic gut microbiota to 10 newly diagnosed DM1 patients, and the same donor was used for three consecutive FMTs for each DM1 patient. There were no differences between the two groups at baseline and throughout the follow-up period, and no serious adverse events or adverse changes in plasma biochemistry occurred in either treatment group.

[0293] Autologous FMT preserves (stimulated) C-peptide levels better than allogeneic FMT.

[0294] The mean fasting plasma C-peptide at baseline was similar across groups (327 pmol / L + / - 89 in the allogeneic transplant group versus 319 + / - 118 in the autologous transplant group; p = 0.86, Student's t-test), but at 12 months, the allogeneic FMT group was worse than the autologous FMT group (202 + / - 85 vs. 348 pmol / L + / - 115, Student's t-test p = 0.0049; LMM p = 0.00019). Similar effects were observed in the stimulated C-peptide response AUC, which was similar across groups at baseline (361 + / - 154 mmol / L·min in the allogeneic transplant group, compared to 355 + / - 97 in the autologous transplant group; p = 0.92, Student's t-test), but residual β-cell function was significantly better preserved at 12 months after autologous FMT (392 + / - 124 vs. 248 + / - 153 mmol / L·min, Student's t-test p = 0.033, LMM p = 0.000067). As expected, exogenous insulin therapy reduced HbA1c levels in both groups at 12 months. Although the daily exogenous insulin requirement was similar between the allogeneic FMT group (0.45 + / - SD IU / kg / day) and the autologous FMT group (0.47 IU / kg / day), there was no significant improvement in glycemic control between the autologous and allogeneic FMT groups (HbA1c 46 vs. 53.5 mmol / mol, MBU p = 0.19, LMM p = 0.12). Glucose metabolism parameters were measured at 0, 6, and 12 months. Finally, BMI, fecal calprotectin, microalbuminuria, blood lipids, and dietary intake (total calories, fat, saturated fat, protein, carbohydrates, and fiber assessed separately) showed no differences at baseline and throughout the study.

[0295] The success of autologous FMT treatment can be predicted by changes in plasma metabolites and microbiome composition.

[0296] Differences in small gut microbiota among FMT treatment groups

[0297] At baseline, there was no significant difference in α-diversity of the small gut microbiota between treatment groups, but at 6 months, there was a significant difference between autologous and allogeneic FMT groups (p = 0.054), consistent with the significant increase in diversity in the allogeneic FMT group (p = 0.009). When plotted along the axes in redundancy analysis (RDA-plot), the small gut microbiota composition clustered differently between groups at baseline and also varied between treatment groups. FMT treatment group assignments could be reliably predicted by variations in specific small gut bacterial strains (AUC ROC 0.89 ± 0.18), including two Prevotella strains and Streptococcus oralis. Figure 1However, changes at the phylum, family, genus, and species levels indicate no significant alterations in the small intestinal microbiota composition. The relative abundance of all these species decreased after autologous fecal transplantation but increased after allogeneic fecal transplantation. Notably, the relative abundance of *Prevotella* 1 showed baseline differences between groups (p = 0.033). Significant δ differences were observed between the *Prevotella* 2 groups (p = 0.048), but not between the *Prevotella* 1 group (p = 0.069) or the *Streptococcus stomatologica* group. Furthermore, a significant negative correlation was observed between the relative abundance of *Prevotella* 1 and the AUC of stimulated C-peptide (Spearman p = 0.015, rho = -0.55).

[0298] Changes in fasting plasma metabolites after FMT

[0299] Fasting plasma metabolite levels differed between DM1 and donors and changed after FMT. Changes in fasting plasma metabolites between 0 and 12 months reliably predicted treatment group allocation (ROC AUC 0.79 ± 0.23). The relative importance of the ten most predictive metabolites is as follows: Figure 2 As shown in A. At 12 months, the first three metabolites, 1-myristoyl-2-arachidonico-GPC (MA-GPC) (p = 0.02) and 1-arachidonico-GPC (A-GPC) (p = 0.02, Mann-Whitney U test), were different between groups than 1-(1-enyl-palmitoyl)-2-linoleoyl-GPE (EPL-GPE). Figure 2 B- Figure 2 Furthermore, changes in plasma MA-GPC levels were significantly correlated with changes in fasting C-peptide (p = 0.012, Mann-Whitney U test). Figure 2 (E).

[0300] Changes in fecal microbiota after FMT

[0301] At baseline, the fecal microbiota composition differed between Dm1 and healthy donors, and variations also varied between treatment groups. However, no significant differences in alpha diversity were observed between FMT treatment groups or between donors and recipients at baseline, 6 months, or 12 months. Some variations were observed at the phylum, family, genus, and species levels among groups. Group assignment prediction based on changes in fecal microbiota taxa from 0 to 12 months showed a moderate ROC AUC of 0.72 ± 0.24. *Desulfovibrio piger* stood out as the most differentiated bacterial strain among treatment groups. Figure 3Metabolic pathway-based treatment group predictions showed a relatively poor ROC AUC of 0.68 ± 0.27. Interestingly, the changes in D. pigger abundance differed between treatment groups at 6-month (p = 0.024, MWU) and 12-month (p = 0.023) follow-up. Figure 2 G- Figure 2 Furthermore, changes in D. piger were correlated with changes in fasting C-peptide (p = 0.009). Figure 2 Changes in I) and plasma 1-arachidonico-GPC levels (p = 0.004, Figure 2 The relative abundance of D. piger was positively correlated with that of Prevotella 1 (J). Furthermore, changes in the relative abundance of D. piger were correlated with those of Prevotella 1 (J). Figure 2 K) and Prevotella 2 ( Figure 2 The relative abundance of D. piger was negatively correlated with the change in fasting C-peptide (p = 0.009). Furthermore, changes in D. piger were negatively correlated with changes in fasting C-peptide (p = 0.009). Figure 2 The changes in I) and plasma 1-arachidonico-GPC levels (p = 0.004) Figure 2 The relative abundance of *D. piger* was positively correlated with that of *Prevotella* 1 (J). Furthermore, changes in the relative abundance of *D. piger* were correlated with those of *Prevotella* 1 (J). Figure 2 K) and Prevotella 2 ( Figure 2 The relative abundance of L) is negatively correlated with the change.

[0302] Baseline fecal microbiota composition predicts FMT response.

[0303] Because the gut microbiota composition differs between healthy individuals and individuals with T1D in different age groups, the inventors also inferred that FMT itself is an intervention for autoimmune diseases, as FMT introduces fecal matter into the small intestine. Therefore, a post-hoc analysis was conducted on FMT-treated responders and non-responders, regardless of the treatment group. Thus, the inventors investigated at 12-month follow-up whether baseline characteristics in T1D patients could predict response to FMT therapy, and which bacterial strains and plasma metabolites were associated with this response. Clinical response was defined as a decrease in β-cell function of <10% compared to baseline at 12-month follow-up, significantly lower than the expected 20% natural decrease in β-cell function over one year. At 6-month follow-up, i.e., 2 months after the last FMT, 12 out of 20 individuals were responders. At 12-month follow-up, 10 individuals maintained a clinical response, of whom 3 received allogeneic FMT transplantation and 7 received autologous FMT transplantation. Figure 4 A- Figure 4(B). Therefore, the inventors chose to analyze respondents at 12 months because the primary endpoint (C-peptide stimulated by MMT) differed significantly at 12 months (but not at 6 months) and because honeymoon interference was less at the 12-month time point than at the 6-month time point. The inventors then used predictive models to determine which baseline parameters (their baseline values ​​or δ values ​​from 0 to 12 months) were predictors of clinical response to FMT.

[0304] Baseline fecal microbiota composition is the best predictor of clinical response after FMT.

[0305] Baseline fecal microbiota composition accurately predicted clinical response after FMT (AUC ROC 0.93 ± 0.14). In this respect, the levels of gut *Bacteroides faecalis* and *Factococcus faecium* were the most prominent differentiated microbes. Figure 5 These two microorganisms were significantly more abundant in responders than in non-responders at baseline. Figure 4 C- Figure 4 (D). Among the top 10 most differentially expressed gut bacterial strains, *Pleurotus erythrorhizium*, *Collinsella aerogenes*, *Bacteroides edodes*, and *Gastrococcus leprae* also showed significant differences at baseline between responders and non-responders. Figure 6 A- Figure 6 A significant (negative) correlation was observed between changes in the abundance of *F. shrewd* and the AUC of stimulated C-peptide (p = 0.053, r = -0.44). Figure 4 The change in fecal microbiota composition (AUC ROC 0.76 ± 0.23) was less accurate in predicting response than baseline composition, suggesting that gut microbiota composition can predict gut microbiota-based treatment efficacy at the time of T1D diagnosis. The most differentiated species were Bacteroides bacterium ph8, Actinomyces viscosus, Bacteroides thetaoitaomicron, Streptococcus salivarius, Ruminococcus bromii, and Clostridium leptum. Among them, Bacteroides bacterium ph8 (p = 0.015, Mann-Whitney U test) and Ruminococcus bromii (p = 0.013) became less common in responders and non-responders, while Streptococcus salivarius (p = 0.045) became more common in responders and non-responders. Bacteroides thetaoitaomicron was significantly different at baseline and showed a decreasing trend in responders.

[0306] Integration of multi-omics analysis after FMT

[0307] The correlations among parameters found to be significantly affected by FMT were explored. Since responders were found in both treatment groups, correlations were first explored in our pooled dataset (n=20), and then separately within the treatment groups and among clinical responders after FMT. In the pooled dataset, intertwined clusters of significant parameters with both positive and negative correlations to glucose regulation markers (i.e., C-peptide AUC, fasting C-peptide, and HbA1c) were found. On one hand, highly correlated plasma metabolites MA-GPC and A-GPC accurately predicted the retention of insulin secretion and were positively correlated with D. pigger, which in turn was positively correlated with fasting C-peptide. On the other hand, Prevotella 1, Prevotella 2, and Streptococcus oralis were negatively correlated with glucose regulation and the metabolites MC-GPC and A-GPC. Individual analysis of the treatment groups revealed that the preserved β-cell function (high C-peptide) in the autologous group was characterized at baseline by high levels of *F. fecalithococcus*, followed by a decrease in *Ruminococcus brucelli*. In the allogeneic transplant group, preserved β-cell function was characterized by a decrease in fecal *Roseburia intestinalis* (which was positively correlated with *Prevotella 1* and *Prevotella 2*). Finally, in clinical responders, preserved β-cell function was characterized by a decrease in duodenal *Prevotella 1*, *Prevotella 2*, fecal *Femtococcus*, and the metabolite EPL-GPE, while an increase in *D. piger*.

[0308] analyze

[0309] The inventors here report for the first time the effect of fecal microbiota transplantation (FMT) on residual β-cell function in newly diagnosed T1D patients. This is consistent with recent observational studies supporting the role of the gut microbiota in T1D individuals. Contrary to expectations, autologous FMT performed better than healthy donor FMT, and even in the allogeneic transplantation group, the decline in residual β-cell function was less than expected in the untreated group. An attractive explanation is that the beneficial immune effects of FMT are more significant and durable when the FMT donor microbiota better matches the host's immune regulatory tone. This is to the extent that the beneficial effects of healthy donor feces might be attenuated by (immune) incompatibility. Other observations also point to the immunomodulatory effects of specific plasma metabolites derived from diet and transformed by the gut microbiota. While the overall clinical efficacy of FMT is modest and shows wide variability among newly diagnosed T1D individuals, the intervention is safe and has no side effects. We believe that changes in plasma metabolites (primarily fatty acids and tryptophan derivatives) as a result of altered gut microbiota composition can explain the observed beneficial effects of FMT on residual β-cell function in newly diagnosed T1D patients.

[0310] The maintenance of β-cell function is related to changes in specific gut microbiota strains.

[0311] *D. piger* may suppress autoimmunity in T1D via plasma 1-arachidonicyl-GPC. Predictive models showed that baseline fecal microbiota classification and metabolic pathways accurately predicted responses at 12 months. However, identified microbes (e.g., *Bacteroides coccidioides* and *F. coccidioides*) were not correlated with any of our relevant immune parameters, small intestinal genes, or plasma metabolites. This suggests that fecal microbiota composition is a consequence rather than a cause of host immune characteristics associated with the response. The only exception was *D. piger*, a sulfate-reducing strain. Its beneficial effects are likely modulated through its hydrogen sulfide production. Furthermore, we identified *D. piger* as a prominent predictor of fecal microbiota assigned to the FMT-treated group. Interestingly, this small intestinal bacterial strain was also beneficially associated with changes in FMT-stimulated C-peptide responses, and its abundance increased in both the autologous group and overall responders. Interestingly, *D. piger* was positively correlated with plasma 1-arachidonicyl-GPC levels. Figure 2 1-Arachidonicoyl-GPC is a key metabolite and is also associated with increased C-peptide production. In summary, D. piger may be a strong candidate to suppress autoimmunity by producing A-GPC, for example, through uptake into the intestinal lumen by prominent immune cell dendrites. Interestingly, D. piger has recently been cultured from the human gut, enabling the testing of this bacterial strain in human T1D (Chen et al 2019 Letters in Applied Microbiology 68(6)553-561). Among other bacterial species in the duodenum, the most significant differences between treatment groups were two unnamed species of Prevotella spp. and Streptococcus oralis. Our exploratory integration of multi-omics analyses subsequently showed that these Prevotella spp. and Streptococcus oralis were negatively correlated with our key beneficial metabolite MA-GPC (a glycerophospholipid). Femtobacter feces were positively correlated with *D. piger* and A-GPC, and negatively correlated with *Streptococcus oralis*, but not with C-peptides. Finally, changes in *Ruminococcus brucelli* (autologous FMT group) and *Roseidone* (heterogeneous FMT group) were negatively correlated with changes in C-peptides, even though these two strains are generally considered beneficial microorganisms that thrive in fiber-rich diets, produce SCFAs, and promote intestinal integrity.

[0312] in conclusion

[0313] Fecal transplantation of colon-derived microbiota into the host small and large intestines of T1D patients effectively prolonged the function of residual β-cells, thereby extending the honeymoon phase. Furthermore, several novel bacterial strains, including *D. piger* and *Bacteroides feces* in feces, as well as *Prevotella duodenalis* and *Streptococcus stomatologicus*, were identified as having therapeutic potential. Correspondingly, increases in plasma metabolites such as 1-myristoyl-2-arachidonico-GPC, 1-arachidonico-GPC, and 6-bromotryptophan after fecal transplantation (FMT) were associated with beneficial changes.

[0314] Example 3

[0315] In this embodiment, the effects of the following compounds were evaluated in cell-based experiments:

[0316] 6-Bromoxytryptophan (6-BT)

[0317] -1-Arachidonicoyl-glycerophosphatecholine (20:0) (A-GPC)

[0318] -1-Palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate ethanolamine (16:0-18:2PE)

[0319] -1-Myristoyl-2-arachidonico-glycerophosphatecholine (MA-GPC)

[0320] Materials and methods

[0321] Metabolite preparation and cell culture

[0322] Purchase 6-bromotryptophan (6-BT) in powder form (Alichem) and dissolve it in DMSO at 50 mM.

[0323] Purchase 1-arachidonicyl-glycerophosphate choline (20:0) (LysoPC (20:0) (AvantiPolar) in powder form and dissolve it in PBS at 0.9 mM.

[0324] Purchase a 10 mg / ml chloroform solution of 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate ethanolamine (16:0-18:2 PE) (AvantiPolar); transfer 200 μl (equivalent to 2 mg) to a glass tube and evaporate the chloroform under a nitrogen stream to obtain a transparent film, which is then dissolved in PBS at 1 mM.

[0325] Purchase 1-Myristoyl-2-arachidonicoyl-glycerophosphate choline (MA-GPC) in powder form (Syncom, custom synthesis) and dissolve it in DMSO at 10 mM.

[0326] All cell types were cultured at 37°C in a 5% CO2 atmosphere and treated with metabolites for no more than 24 hours. Under control conditions, an appropriate carrier (DMSO or PBS) was added to the culture medium. NF-κB reporter macrophages and luciferase assays were performed.

[0327] NF-κB signaling activation was assessed by luciferase activity assay. RAW264.7 cells stably transfected with the 3×-κB-luc plasmid (a DNA construct containing three NF-κB sites from the Igκ light chain promoter, which is coupled to the gene encoding firefly luciferase) were grown in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U / ml), streptomycin (100 μg / ml), and L-glutamine (2 mM). Cells were cultured at a rate of 0.5 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1 / well in F-bottom 96-well plates and stimulated with LPS (10 / 100 ng / ml) for 2 hours the next day, with / without different concentrations of metabolites (6-BT 0.1-100 μM, LysoPC(20:0) 1-10 μM, 16:0-18:2 PE 1-50 μM, MA-GPC 1-100 μM). Cells were then lysed with 25 μl / well of 1× passive lysis buffer and subjected to... The measurement system (Promega, E1500) is in The activity of luciferase in fireflies was measured using the Promega detection system.

[0328] In vitro stimulation of primary monocytes

[0329] Immature bone marrow mononuclear cells were isolated from BM cells. Bone marrow monocytes (BMs) were removed from the hind legs of three mice, and the surrounding muscle was cleared. The femur and tibia were trimmed at their ends, and the contents of the BM were washed away with cold PBS using a 10 ml syringe and a 25 G needle, and then filtered through a 40 μm filter. Red blood cells (RBCs) were lysed on ice for 5 minutes with 1×RBC lysis buffer (Biolegend). CD11b+ monocytes were further purified by positive selection using a CD11b magnetic bead mixture (cocktail) (Miltenyi Biotec, #130-049-601) and a magnetized MS column (Miltenyi Biote) according to the manufacturer's instructions. Subsequently, the monocytes were quantified at 1×10⁶ cells per well. 5Mononuclear cells were seeded at a density of 10% heat-inactivated fetal bovine serum, penicillin (100 U / ml), streptomycin (100 μg / ml), and L-glutamine (2 mM) in F-bottom 96-well RPMI 1640 plates and activated with 10 μg / ml poly(I:C). The cells were then activated with 10 ng / ml LPS (Sigma-Aldrich) and treated with 6-BT (10 / 100 μM), LysoPC (20:0) (10 / 50 μM), MA-GPC (10 / 50 / 100 μM), or a suitable vector. Cells were stored at a final volume of 200 μl / well for 24 hours, after which the supernatant was harvested and stored at -80°C.

[0330] In vitro stimulation of mouse macrophages / dendritic cells (DCs)

[0331] Bone marrow-derived macrophages (BMDM) and dendritic cells (BMDC) were obtained by differentiating freshly isolated BM cells from the femur and tibia of C57 / Bl6 mice (N=3 per experiment). For BMDM, BM cells were cultured at 3 × 10⁶ cells per 10 cm culture dish. 6 0.5 × 10⁶ cells were seeded and cultured for 7 days in 12 ml RPMI 1640 medium containing 20% ​​fetal bovine serum and 30% L-929 cell conditioned medium as a source of mouse macrophage colony-stimulating factor (M-CSF). For BMDCs, BM cells were seeded at 0.5 × 10⁶ cells per cell line. 6 BMDM / DC cells were seeded at / ml in 10cm culture dishes and cultured for 7 days in 25ml of 5% FBS-RPMI 1640 medium in the presence of GM-CSF. After differentiation, BMDM / DCs were harvested, counted, and cultured at 1×10⁶ cells per well. 5 Macrophages were seeded at a density of 10 μg / ml in F-bottom 96-well plates and allowed to adhere for 20 hours before the experiment. Macrophages were activated for 24 hours with 10 μg / ml polyinosinic-polycytidylic acid (poly(I:C)(InvivoGen)) in the presence or absence of 6-BT (10 / 100 μM), LysoPC (20:0) (10 / 50 μM), or MA-GPC (10 / 50 / 100 μM), with a final volume of 200 μl / well. At the end of the assay, the supernatant was collected and stored at -80°C.

[0332] In vitro CD4+ T cell activation assay

[0333] Primary CD4+ T cells were freshly isolated from the spleens of C57 / Bl6 mice using negative selection (N=3 per experiment). Briefly, the spleens were pulverized in culture dishes and passed through sieves twice (70 μm and 40 μm, respectively) to obtain a single-cell suspension. After lysing red blood cells with 1×RBC lysis buffer (Biolegend) (on ice for 10 min), the cells were counted and stained with a mixture of biotin-conjugated antibodies against CD8a, CD11b, CD11c, CD19, CD45R (B220), CD49b (D×5), CD105, anti-MHC class II, Ter-119, and TCRγ / δ, followed by magnetic labeling with anti-biotin microbeads.

[0334] Negative selection was used (CD4+ T cell isolation kit, Miltenyi Biotec, #130-104-454). Non-CD4 T cells were depleted by retaining them in an LS magnetized column (Miltenyi Biotec).

[0335] The isolated CD4 T cells were cultured in 96-well plates (1×10⁻⁶). 5 In RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin, and 2 mM L-glutamine, the culture medium was cultured in 200 μl / well of complete RPMI 1640 medium. Immediately after inoculation into 200 μl / well of complete RPMI 1640 medium, the culture was treated with metabolite 6-BT (1 / 10 μM), LysoPC (20:0) (10 / 50 μM), or MA-GPC (10 / 50 / 100 μM), followed by treatment with 2.5 μg / ml soluble anti-CD3 antibody (145-2C11, eBioscience) and 1 μg / ml soluble anti-CD28 antibody (37.51, eBioscience). The supernatant was then collected and stored at -80°C.

[0336] In vitro stimulation of human mononuclear cells

[0337] Using Lymphoprep TM (Axis-Shield) isolated monocyte fractions from the blood of healthy volunteers (Sanquin Bloodbank, Amsterdam, Netherlands) by density centrifugation, and used human CD14 magnetic beads according to the manufacturer's instructions. Cell separation was performed using a Miltenyi Biotec column. The isolated primary human mononuclear cells were counted and analyzed at a rate of 1 × 10⁻⁶. 6Cells were seeded at 100 cells / mL in 24-well plates, with 1 mL of medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 2 mM L-glutamine. After seeding, cells were stimulated for 24 hours with 10 ng / mL LPS or 25 mM D-glucose (both Sigma-Aldrich), with / without 100 μM 6-BT. Cells were then lysed using Tripeure separation reagent (Roche) and stored at -80°C until RNA separation.

[0338] In vitro stimulation of pancreatic β cells

[0339] INS1E cells (rat pancreatic β-cell line) were maintained in a complete RPMI 1640 culture supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 5 μM β-mercaptoethanol, 1 mM sodium pyruvate, 10 mM HEPES, 100 units / ml penicillin, and 100 μg / ml streptomycin. INS1E cells were then cultured at 1 × 10⁻⁶ cells / ml. 5 Cells were seeded at a density of 10 cells / well in 48-well plates and incubated for one day. Subsequently, the medium was replaced with 0.5 ml / well of INS1E complete RPMI 1640 medium containing the carrier or metabolite: 6-BT (1 / 10 / 25 μM), LysoPC (20:0) (5 / 10 / 50 μM), or MA-GPC (10 / 50 / 100 μM). After 24 hours, cells and supernatant were collected for further analysis (gene expression and insulin secretion, respectively) and stored at -80°C. Cells were lysed in Tripeure separation reagent (Roche) before storage.

[0340] For glucose-stimulated insulin secretion (GSIS) assays, cells were pre-cultured at 37°C for 30 min in Krebs-Ringer bicarbonate buffer (KRB) [115 mM NaCl, 5 mM KCl, 2.56 mM CaCl2, 1 mM MgCl2, 10 mM NaHCO3, 15 mM HEPES, and 0.3% BSA (pH 7.4)]. Cells were then stimulated with 1 mM glucose in KRB for 1 h (0.5 ml / well) and then again with 22 mM glucose in KRB for 1 h (0.5 ml / well). After treatment with 1 mM glucose (Sigma-Aldrich) and 22 mM glucose, the supernatant was recovered, and the cells were stored at -80°C. β-cells were then subjected to GSIS after treatment with 10 μM 6-BT for 24 hours.

[0341] ELISA

[0342] According to the manufacturer's instructions, the concentrations of TNFα, IFNβ, and IFNγ in the cell supernatants of mouse monocytes, macrophages, and T cells were measured using a specific ELISA (R&D system). According to the manufacturer's instructions, insulin concentrations were determined using a rat insulin ELISA (Mercodia) 24 hours after metabolite treatment or after GSIS. GSIS was calculated by subtracting the insulin concentration at 1 mM glucose from the insulin rate at 22 mM glucose.

[0343] Gene expression analysis

[0344] Total RNA was extracted from cell lysates using the Tripe separation reagent. RNA was converted to cDNA using the iScript kit (BioRad). Quantitative polymerase chain reaction (qPCR) was performed using a SYBR Green-SensiMix (Bioline) system on a CFX384 touch real-time PCR detection system (BioRad). Gene expression was calculated using the δδCt method as a fold change compared to the control (unstimulated condition).

[0345] Statistical data

[0346] Student's t-test was used for comparisons between two groups, and one-way ANOVA and Dunnett's test were used for comparisons among multiple groups for statistical analysis. Data are expressed as mean and standard error of the mean (SEM). P < 0.05 was considered significant.

[0347] result

[0348] The results are as follows Figures 7-13 As shown:

[0349] -6-BT, A-GPC, and MA-GPC can inhibit the activation of the NFκB pathway in macrophages at different doses. Figure 7 ).

[0350] -6-BT, A-GPC, and MA-GPC inhibit the secretion of cytokines by monocytes. Figure 8 );

[0351] -6-BT and MA-GPC impair type 1 IFN secretion ( Figure 9 );

[0352] -6-BT reduces the production of cytokines by human monocytes. Figure 10 );

[0353] -6-BT and A-GPC inhibit Th1 responses in CD4 T cells. Figure 11 );

[0354] -6-BT enhances pancreatic β-cell function ( Figure 12 and Figure 13 );

[0355] Of particular interest is 6-bromotryptophan, which inhibits NFκB pathway activation, hinders immune responses in monocytes / macrophages and CD4 T cells, and improves pancreatic β-cell function. MA-GPC inhibits NFκB pathway activation, hindering immune responses in monocytes / macrophages and CD4 T cells.

[0356] Example 4

[0357] In a cross-sectional cohort study (n=369 individuals), plasma 6-bromotryptophan levels were negatively correlated with type 2 diabetes and glycemic control.

[0358] In a cohort of 369 individuals, evidence suggests that 6-bromotryptophan (6-BT) may prevent the onset and progression of type 2 diabetes. More specifically, plasma 6-bromotryptophan levels were found to be negatively correlated with the presence of type 2 diabetes and glycemic control. This suggests that 6-BT may contribute to the prevention and treatment of type 2 diabetes, thereby improving cardiovascular (microvascular and macrovascular) complications of type 2 diabetes. 6-BT may also help reduce macrovascular disease (i.e., cardiovascular disease) and microvascular complications in individuals without type 2 diabetes.

[0359] Materials and methods

[0360] As previously described [Koh A, Molinaro A, Stahlman M, et al. Microbially Produced Imidazole Propionate Impairs Insulin Signaling through mTORC1. Cell 2018; 175:947-961.e17. doi:10.1016 / j.cell.2018.09.055], fasting plasma targeted metabolites were measured using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS / MS) via Metabolon (Durham, NC).

[0361] result

[0362] The results are shown in Tables 2 and 3.

[0363] Table 2

[0364]

[0365] Table 3 - Correlation between compounds and diabetes, BMI, glucose levels, and HbA1c levels

[0366]

[0367] *The correlation was significant at the 0.05 level (two-tailed).

[0368] 6-Bromotryptophan inhibits the inflammatory response of bone marrow cells.

[0369] 6-BT is a bromoindole derivative of tryptophan, known to be metabolized by the native gut microbiota of the colon and small intestine. Until now, the physiological functions of 6-BT were completely unknown. The inventors' clinical findings suggest a protective function against inflammation and diabetes.

[0370] Through a series of in vitro / in vitro experiments, the inventors elucidated some functions of 6-BT on immune cells and insulin-producing pancreatic β cells.

[0371] 6-BT can inhibit the secretion of the pro-inflammatory cytokine TNFα during TLR4 and TLR2 binding, and the secretion of IFNβ during TLR3 activation.

[0372] Here, mouse bone marrow-derived monocytes (Christ A, Western Diet Triggers NLRP3-Dependent Innate Immune Reprogramming. Cell 2018) or bone marrow-derived macrophages (Swanson KV, Anoncanonical function of cGAMP in inflammasome priming and activation. JEM 2017) were exposed to specified concentrations of 6-BT (10-100 μM) for 24 hours, with or without 10 ng / ml LPS, 10 μg / ml P3C, or 10 μg / ml poly(I:C). Using ELISA assays, the inventors found that 6-BT inhibits the secretion of the pro-inflammatory cytokine TNFα upon TLR4 and TLR2 conjugation and inhibits IFNβ secretion upon TLR3 activation. See also Figure 14 .

[0373] Inhibiting TLR signaling-induced cytokine secretion is particularly important for the treatment of inflammatory and infectious diseases in which damage is caused by excessive inflammatory repose (e.g., sepsis and systemic inflammatory response syndrome, SIRS).

[0374] 6-BT inhibits the secretion of pro-inflammatory cytokines TNFα and IFNβ.

[0375] Since dendritic cells (DCs) are the primary antigen-presenting cells crucial for T cell activation, the inventors next investigated the effects of 6-BT on mouse DCs differentiated from bone marrow cells using GM-CSF (40 ng / ml). Regarding monocytes / macrophages, after activation of TLR4 (with 100 ng / ml LPS) or TLR3 (10 μg / ml poly(I:C)), respectively, 6-BT inhibited the secretion of pro-inflammatory cytokines TNFα and IFNβ via DCs. See also Figure 15 .

[0376] 6-BT significantly reduces the production of the Th1 cytokine IFNγ.

[0377] Especially in the context of autoimmune diabetes, T cell activity drives disease onset and progression. Therefore, the inventors further investigated the effects of 6-BT on CD4 T cells. To mimic antigen presentation, mouse CD4 T cells (isolated from the spleen; Uchimura T, The Innate Immune Sensor NLRC3 Acts as a Rheostat that Fine-Tunes T Cell Responses in Infection and Autoimmunity. Immunity 2019) were activated with monoclonal antibodies against CD3 and CD28 (2.5 and 1 μg / ml, respectively). Consistent with findings on bone marrow cells, 6-BT significantly reduced the production of the Th1 cytokine IFNγ. See also Figure 16 .

[0378] 6-BT stimulates β-cell differentiation and insulin production

[0379] Given the positive correlation between plasma 6-BT levels and C-peptide concentrations (found in clinical studies), the inventors then questioned whether 6-BT could have a direct effect on β cells. Indeed, the inventors found that 6-BT induces gene expression of transcription factors PDX1 and MAFA in IS1E β cells, which are important for β cell maturation and function. Consistently, 6-BT also promotes insulin secretion during homeostasis and glucose-stimulated insulin secretion (data shown as the difference in insulin release between starvation [1 mM glucose] and hyperglycemia [22 mM]) (Paula S, Exercise increases pancreatic β-cell viability in a model of type 1 diabetes through IL-6 signaling. FASEB J 2015). See also Figure 17 .

[0380] Mechanism of action of 6-bromotryptophan

[0381] To investigate the molecular mechanism of 6-BT's action, the inventors first examined the effect of 6-BT on the activation of the NF-κB pathway, a central pathway in all inflammatory diseases (except autoimmune diseases). Therefore, the inventors quantified the expression of the phosphorylated p65 subunit as a marker of NF-κB activation. After T cells were activated with PMA (50 ng / ml) and iomycin (1 μg / ml), 6-BT inhibited NF-κB signaling at a very early time point (5-10 minutes after activation). This effect was observed in both mouse and human (Jurkat cell) CD4 T cells. See also Figure 18 .

[0382] 6-BT inhibits NF-κB activation in macrophages

[0383] Similar to what the inventors observed in lymphocytes, 6-BT inhibits NF-κB activation in macrophages. Using the RAW264.7 mouse macrophage cell line stably expressing the NF-κB luciferase reporter gene (Groeneweg M, Lipopolysaccharide-induced gene expression in murine macrophages is enhanced by prior exposure to oxLDL. J Lipid Res 2006.), the inventors disclosed that overnight exposure of macrophages to 6-BT (10-200 μM) followed by 2 hours of stimulation with LPS (10 ng / ml) inhibited the transcriptional activity of the NF-κB complex in a dose-dependent manner. See also Figure 19 .

[0384] 6-BT and tryptophan trigger different biological activities

[0385] Next, the inventors questioned whether the effect of 6-BT was specific or could also be exerted by tryptophan. In mouse CD4 T lymphocytes (isolated from mouse spleen; Uchimura T, The Innate Immune Sensor NLRC3 Acts as a Rheostat that Fine-Tunes T Cell Responses in Infection and Autoimmunity. Immunity 2019), 6-BT, but not tryptophan, inhibited IFNγ production after CD3 / CD28 binding. This indicates that 6-BT and tryptophan induce different biological activities. See also Figure 20 .

[0386] Consistent with the results from DC, studies on exposure to 6-BT or tryptophan in monocytes (isolated from mouse bone marrow; Christ A, Western Diet Triggers NLRP3-Dependent Innate Immune Reprogramming. Cell 2018) showed that the anti-inflammatory effect was specific to 6-bromotryptophan but not to tryptophan. See also Figure 21 .

[0387] 6-BT affects intracellular metabolism

[0388] Finally, the inventors discovered that 6-BT also affects intracellular metabolism. Specifically, they found that 6-BT (100 μM) promotes mitochondrial metabolism in mouse and human (Jurkat) CD4 T cells. See also Figure 22 .

[0389] OCR (Oxygen Consumption Rate) is used as an indicator of cellular utilization of mitochondrial oxidative phosphorylation. OCR was measured using a SeahorseXF analyzer (Uchimura T, The Innate Immune Sensor NLRC3 Acts as a Rheostat that Fine-Tunes T Cell Responses in Infection and Autoimmunity. Immunity 2019; Chou, AIM2 in regulatory T cells restrains autoimmune diseases, Nature 2021).

[0390] 6-BT enhances mitochondrial metabolism

[0391] Similarly, 6-BT exposure enhanced mitochondrial metabolism in pro-inflammatory M1 macrophages (differentiation in the presence of LPS and IFNγ; Cheng et al JCI Insight. 2018; 3(22):e120638) without affecting glycolytic flux. Intracellular metabolism was measured using a Seahorse XF analyzer (Uchimura T, The Innate Immune Sensor NLRC3 Acts as a Rheostat that Fine-Tunes T Cell Responses in Infection and Autoimmunity. Immunity 2019; Chou, AIM2 in regulatory T cells restrains autoimmune diseases, Nature 2021). See also Figure 23 .

[0392] 6-BT can rescue β-cell dysfunction in type 1 and type 2 diabetes.

[0393] Finally, the inventors investigated whether 6-BT could affect mitochondrial metabolism in β-cells, which rely on ATP and mitochondrial metabolites for insulin exocytosis. 6-BT increased mitochondrial metabolism in β-cells (INS1E β-cells) under both homeostatic and hyperglycemic (25 mM glucose) conditions. Furthermore, the inventors tested the effect of tryptophan on intracellular metabolism and found that it played a different role than 6-BT in terms of inflammatory markers. Intracellular metabolism was measured using a Seahorse XF analyzer (Uchimura T, The Innate Immune Sensor NLRC3 Acts as a Rheostat that Fine-Tunes T Cell Responses in Infection and Autoimmunity. Immunity 2019; Chou, AIM2 inregulatory T cells restrains autoimmune diseases, Nature 2021).

[0394] Importantly, defects in mitochondrial and oxidative metabolism have been reported in T2D (Haythorne, Nature Communications volume 10, Article number: 2474 (2019)), suggesting that 6-BT can rescue β-cell dysfunction in type 1 and type 2 diabetes. Furthermore, increased mitochondrial metabolic utilization can counteract ectopic intracellular lipid accumulation, thereby mitigating obesity.

[0395] In summary:

[0396] ·6-BT exerts pleiotropic effects on multiple cell types

[0397] It has anti-inflammatory effects on bone marrow cells and lymphocytes.

[0398] It promotes insulin secretion from β cells.

[0399] Mechanistically, its biological function differs from that of tryptophan.

[0400] 6-BT does not function by activating AhR, but it inhibits NF-κB activation and enhances mitochondrial metabolism. The latter is typically used by cells with an anti-inflammatory phenotype.

[0401] application

[0402] Due to its broad effects on multiple cell types, its inhibitory effect on NF-κB signaling, and its promotion of mitochondrial metabolism and adaptation, 6-BT could be a novel therapy not only in type 1 and type 2 diabetes, but also in many other inflammation-related diseases such as sepsis, systemic inflammatory response syndrome (SIRS), and cardiovascular disease.

[0403] Example 5

[0404] In a cross-sectional cohort study (n=369 individuals), *Desulfovibrio* levels were negatively correlated with type 2 diabetes and glycemic control.

[0405] In the same cohort of 369 individuals discussed earlier in this paper, evidence was found that bacteria belonging to the genus *Desulfovibrio* (e.g., *Desulfovibrio piger*) can prevent the onset and development of type 2 diabetes. More specifically, the relative abundance of fecal bacteria belonging to the genus *Desulfovibrio* was found to be negatively correlated with the presence of type 2 diabetes and glycemic control. This suggests that administration of *Desulfovibrio* may contribute to the prevention and treatment of type 2 diabetes, thereby improving cardiovascular complications (microvascular and macrovascular) associated with type 2 diabetes. *Desulfovibrio* may also help reduce macrovascular disease (i.e., cardiovascular disease) and microvascular complications in individuals without type 2 diabetes.

[0406] Materials and methods

[0407] As previously described [Koh A, Molinaro A, Stahlman M, et al. Microbially Produced Imidazole Propionate Impairs Insulin Signaling through mTORC1. Cell 2018; 175:947-961.e17. doi:10.1016 / j.cell.2018.09.055], fasting plasma targeted metabolites were measured using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS / MS) via Metabolon (Durham, NC).

[0408] result

[0409] The results are shown in Table 4 and Figure 25 and Figure 26 middle.

[0410] Table 4 – Correlation between relative abundance of *Desulfovibrio* spp. in feces and diabetes, BMI, glucose levels, and HbA1c levels.

[0411] diabetes BMI glucose HbA1c Desulfuric Vibrio -0.03* -0.03* 0.02 -0.02

[0412] *The correlation was significant at the 0.05 level (two-tailed).

[0413] Furthermore, a significant relationship was observed between the relative abundance of fecal desulfovibrio and plasma 6BT levels in patients with type 2 diabetes. See also Figure 27 . <110> Central Nervous System Institute (Academisch Medisch Centrum), Wageningen University (WageningenUniversiteit) <120> Intervention strategies for the prevention or treatment of diabetes, autoimmune diseases, inflammatory diseases, or cardiovascular diseases. <130> KHP222111025.6 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 1542 <212> DNA <213> Desulfovibrio piger <400> 1 agagtttgat cctggctcag attgaacgct ggcggcgtgc ttaacacatg caagtcgtac 60 gcgaaaggga cttcggtccc gagtaaagtg gcgcacgggt gagtaacacg tggataatct 120 gcctctatga tggggataac agttggaaac gactgctaat accgaatacg ctcatgatga 180 actttgtgag gaaaggtggc ctctgcttgc aagctatcgc atagagatga gtccgcgtcc 240 cattagctag ttggtggggt aacggcctac caaggcaacg atgggtagcc gatctgagag 300 gatgatcggc cacactggaa ctgaaacacg gtccagactc ctacgggagg cagcagtggg 360 gaatattgg caatgggcga aagcctgacg cagcgacgcc gcgtgaggga tgaaggtctt 420 cggatcgtaa acctctgtca gaagggaaga aactagggtg ttctaatcat catcctactg 480 acggtacctt caaaggaagc accggctaac tccgtgccag cagccgcggt aatacggagg 540 gtgcaagcgt taatcggaat cactgggcgt aaagcgcacg taggctgtta tgtaagtcag 600 gggtgaaagc ccacggctca accgtggaac tgcccttgat actgcacgac tcgaatccgg 660 gagagggtgg cggaattcca ggtgtaggag tgaaatccgt agatatctgg aggaacatca 720 gtggcgaagg cggccacctg gaccggtatt gacgctgagg tgcgaagcg tggggagca 780 acaggattag ataccctggt agtccacgcc gtaaacgatg gatgctagat gtcgggatgt 840 atgtctcggt gtcgtagtta acgcgttaag catcccgcct ggggagtacg gtcgcaaggc 900 tgaaactcaa agaattgac gggggcccgc acagcggtg gagtatgtgg ttatattcga 960 tgcaacgcga agaacttac ctaggtttga catctgggga accctcccga aaatgagggg 1020 tgcccttcgg ggagccccaa gandaggtgct gcatggctgt cgtcagctcg tgtcgtgaga 1080 tgttgggtta agtcccgcaa cgagcgcaac ccctatgcat agttgccagc aagtaagtt 1140 gggcactcta tgcagactgc ccgggttaac cgggaggaag gtggggacga cgtcaagtca 1200 tcatggccct tacacctagg gctacacacg tactacaatg gcacgcacaa agggcagcga 1260 taccgtgagg tggagccaat cccaaaaaac gtgtcccagt ccggattgca gtctgcaact 1320 cgactgcatg aagtcggaat cgctagtaat tcgaggtcag catactcggg tgaatgcgtt 1380 cccggggcctt gtacaccg cccgtcacac cacgaagtc gttttacc gaagccggtg 1440 agccaactag caatagaggc agccgtctac ggtagggccg atgattgggg tgaagtcgta 1500 acaaggtagc cgtaggggaa cctgcggctg gatcacctcc tt 1542

Claims

1. Use of 6-bromotryptophan in the preparation of a medicament for the prevention or treatment of diabetes or inflammatory diseases in an individual, wherein said inflammatory diseases are selected from gastrointestinal inflammation, hyperthyroidism, hypothyroidism, and liver inflammation, and If the drug is used to prevent or treat diabetes, the fecal matter does not contain the 6-bromotryptophan.

2. The use according to claim 1, wherein the diabetes is selected from type 1 diabetes and type 2 diabetes.

3. The use according to claim 1, wherein the gastrointestinal inflammation is selected from gastritis, gastroenteritis, enteritis, colitis, enterocolitis, appendicitis, and proctitis.

4. The use according to claim 1, wherein the enteritis is selected from duodenitis and ileitis.

5. The use according to any one of claims 1-3, wherein the 6-bromotryptophan is combined with a tumor necrosis factor α (TNFα) inhibitor.

6. The use according to claim 5, wherein the tumor necrosis factor α inhibitor is selected from infliximab, adalimumab, sertozumab, and golimumab.

7. The use according to any one of claims 1-3, wherein the 6-bromotryptophan is derived from the genus *Eubacterium* (…). Eubacterium Enteromonas (Intestinimonas Bifidobacterium spp. Bifidobacteria Lactobacillus () Lactobacillales ) and / or Akkermania spp. Akkermansia ) bacterial combination.

8. The use according to claim 7, wherein the bacteria is selected from Bifidobacterium animalis subsp. lactis (… Bifidobacterium animal is sub lactis ) or Bifidobacterium breve ( Bifidobacterium breve Lactobacillus plantarum ( Lactobacillus plantarum Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Lactobacillus acidophilus ( Lactobacillus acidophilus ), Hodgkin's Eubacterium ( Eubacterium hallii Enterobacter butyrate-producing bacteria ( Intestinimonas butyriciproducens ) and / or Akkermansia myxophilus ( Akkermansia muciniphila ).

9. The use according to any one of claims 1-3, wherein the drug is administered enterically or nasally, or subcutaneously, intravenously, rectally, and / or via the nasoduodenal tube.

10. The use according to claim 9, wherein the drug is administered orally.

11. The use according to any one of claims 1-3, wherein the drug is administered into the small intestine.

12. The use according to claim 11, wherein the drug is administered into the duodenum.

13. The use according to any one of claims 1-3, wherein the medicament is a pharmaceutical composition.

14. The use according to claim 13, wherein the drug is a liquid dosage form or a solid dosage form.

15. The use according to claim 13, wherein the drug is a capsule, tablet or powder.

16. The use according to claim 13, wherein the 6-bromotryptophan is contained in the pharmaceutical composition in an amount of at least 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, or 100 mg.

17. The use according to any one of claims 1-3, wherein the 6-bromotryptophan is contained in and / or encapsulated in an enteric coating.

18. The use according to claim 17, wherein the enteric coating does not dissolve and / or disintegrate in the gastric environment.

19. The use according to any one of claims 1-3, wherein the use relates to administering the drug alone at least once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times.

20. The use according to claim 19, wherein the interval between the individual applications is at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks.

21. The use according to any one of claims 1-3, wherein the individual is a mammal.

22. The use according to claim 21, wherein the individual is a person.

Images