METHODS AND COMPOSITIONS FOR TREATING INFECTIOUS, AUTOIMMUNE, AND ALLERGIC DISEASE
20210386796 · 2021-12-16
Assignee
Inventors
- Cathryn R. Nagler (Chicago, IL, US)
- Riyue Bao (Chicago, IL, US)
- Dionysios ANTONOPOULOS (Chicago, IL, US)
- John W. COLSON (Chicago, IL, US)
- Lauren Anders HESSER (Chicago, IL, US)
Cpc classification
A61K31/047
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K31/718
HUMAN NECESSITIES
C12Q1/04
CHEMISTRY; METALLURGY
A61K31/702
HUMAN NECESSITIES
A61K31/718
HUMAN NECESSITIES
A61P1/00
HUMAN NECESSITIES
A23L33/21
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K31/047
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K31/702
HUMAN NECESSITIES
G01N2570/00
PHYSICS
International classification
A23L33/135
HUMAN NECESSITIES
A23L33/21
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
Abstract
The current disclosure provides methods and compositions for treating food allergies, infections, autoimmune conditions, and other allergic conditions. Accordingly, the current disclosure relates to a method for treating an infectious, autoimmune, or allergic disease in a subject comprising administering a composition comprising bacterium Anaerostipes caccae to the subject. Further aspects relate to a method for treating a food allergy or for reducing an allergic response to an allergen or for treating or preventing an anaphylactic response in a subject comprising administering a composition comprising bacterium Anaerostipes caccae and a prebiotic to the subject.
Claims
1. A method for treating a food allergy or for reducing an allergic response to an allergen or for treating or preventing an anaphylactic response in a subject comprising administering a composition comprising bacterium Anaerostipes caccae and a prebiotic to the subject.
2. A method for treating a patient at risk of a food allergy or of an anaphylactic response comprising administering a composition comprising bacterium Anaerostipes caccae and a prebiotic to the subject.
3. A method for treating an atopic disease in a subject in need thereof, the method comprising administering a composition comprising bacterium Anaerostipes caccae and a prebiotic to the subject.
4. The method of claim 3, wherein the atopic disease comprises eczema, atopic dermatitis, asthma, or allergic rhinitis.
5. The method of any one of claims 1-4, wherein the prebiotic comprises one or more of galactooligosacchararide, lactulose, lactitol, erythritol, isomalt, polyglycitol, acetate and lactate.
6. The method of claim 5, wherein the prebiotic comprises one or more of galactooligosacchararide, lactulose, lactate, acetate, and lactitol.
7. The method of any one of claims 1-6, wherein the prebiotic comprises one or more of a digestible and a non-digestible oligosaccharides.
8. The method of any one of claims 1-7, wherein the prebiotic comprises at least 6 grams of non-digestible oligosaccharides.
9. The method of claim 7 or 8, wherein the oligosaccharide comprises a modified oligosaccharide.
10. The method of claim 9, wherein the modified oligosaccharide comprises an A. caccae fermentable butyrate-releasing oligosaccharide.
11. The method of any one of claims 1-10, wherein 1×10.sup.6 to 1×10.sup.15 CFU of A. caccae is administered to the subject.
12. The method of any one of claims 1-11, wherein the method further comprises administration of a butyrate carrying compound.
13. The method of claim 12, wherein the butyrate carrying compound comprises pHPMA-b-pBMA.
14. The method of claim 12 or 13, wherein the butyrate carrying compound is administered orally.
15. The method of any one of claims 1-14, wherein the A. caccae, the prebiotic, and/or the butyrate carrying compound are administered simultaneously.
16. The method of any one of claims 1-11, wherein the A. caccae is administered prior to the prebiotic and/or butyrate carrying compound.
17. The method of any one of claims 1-11, wherein the A. caccae is administered after the prebiotic and/or the butyrate carrying compound.
18. The method of any one of claims 1-17, wherein the A. caccae is administered at least 1 hour before the prebiotic and/or the butyrate carrying compound.
19. The method of any one of claims 1-17 wherein the butyrate carrying compound is administered after the prebiotic.
20. The method of any one of claims 1-19, wherein at least 10 grams of prebiotic is administered to the subject.
21. The method of any one of claims 1-20, wherein the ratio of the colony forming units of A. caccae to grams of prebiotic is 1000:1-10000:1.
22. The method of any one of claims 1-21, wherein the food allergy comprises a cow's milk, egg, peanut allergy, soy allergy, wheat/gluten allergy, shellfish allergy, sesame allergy, or tree nut allergy.
23. The method of any one of claims 1-22, wherein the subject has been diagnosed with a food allergy or with an atopic disease.
24. The method of any one of claims 1-23, wherein the subject has previously been treated for a food allergy or atopic disease.
25. The method of claim 24, wherein the subject has been determined to be resistant to the previous treatment.
26. The method of any one of claims 1-25, wherein the subject is a human.
27. The method of claim 26, wherein the subject is less than one year old, less than five years old, less than twelve years old or less than eighteen years old.
28. The method of any one of claims 1-27, wherein the A. caccae comprises a live bacterial product.
29. The method of any one of claims 1-28, wherein the bacteria are lyophilized or freeze-dried.
30. The method of any one of claims 1-29, wherein the A. caccae and/or prebiotic are administered orally.
31. The method of claim 30, wherein the A. caccae and/or prebiotic are administered in a tablet or capsule.
32. The method of any one of claims 1-31, wherein the method further comprises administration of a lactate-containing formula or food.
33. The method of any one of claims 1-32, wherein the subject is determined to have a ratio of protective/non-protective operational taxonomic units (OTUs) of less than 3.
34. A method for diagnosing a subject with a food allergy, the method comprising determining the ratio of protective/non-protective operational taxonomic units (OTUs); wherein the subject is diagnosed with a food allergy when the ratio is less than 3.
35. The method of claim 34, wherein the method further comprises treating the subject diagnosed with a food allergy with a composition comprising bacterium Anaerostipes caccae and a prebiotic to the subject.
36. The method of claim 35, wherein the prebiotic comprises one or more of galactooligosacchararide, lactulose, lactitol, erythritol, isomalt, polyglycitol, lactate and acetate.
37. The method of claim 36, wherein the prebiotic comprises one or more of galactooligosacchararide, lactulose, lactate, acetate, and lactitol.
38. The method of any one of claims 35-37, wherein the prebiotic comprises one or more of a digestible and a non-digestible oligosaccharides.
39. The method of any one of claims 35-38, wherein the prebiotic comprises at least 6 grams of non-digestible oligosaccharides.
40. The method of claim 38 or 39, wherein the oligosaccharide comprises a modified oligosaccharide.
41. The method of claim 40, wherein the modified oligosaccharide comprises an A. caccae fermentable butyrate-releasing oligosaccharide.
42. The method of any one of claims 35-41, wherein 1×10.sup.6 to 1×10.sup.15 CFU of A. caccae is administered to the subject.
43. The method of any one of claims 35-42, wherein the method further comprises administration of a butyrate carrying compound.
44. The method of claim 43, wherein the butyrate carrying compound comprises pHPMA-b-pBMA.
45. The method of claim 43 or 44, wherein the butyrate carrying compound is administered orally.
46. The method of any one of claims 35-45, wherein the A. caccae, the prebiotic, and/or the butyrate carrying compound are administered simultaneously.
47. The method of any one of claims 35-42, wherein the A. caccae is administered prior to the prebiotic and/or the butyrate carrying compound.
48. The method of any one of claims 35-42, wherein the A. caccae is administered after the prebiotic and/or the butyrate carrying compound.
49. The method of any one of claims 35-48, wherein the A. caccae is administered at least 1 hour before the prebiotic and/or the butyrate carrying compound.
50. The method of any one of claims 43-49 wherein the butyrate carrying compound is administered after the prebiotic.
51. The method of any one of claims 35-50, wherein at least 10 grams of prebiotic is administered to the subject.
52. The method of any one of claims 35-51, wherein the ratio of the colony forming units of A. caccae to grams of prebiotic is 1000:1-10000:1.
53. The method of any one of claims 34-52, wherein the food allergy comprises a cow's milk allergy.
54. The method of any one of claims 34-53, wherein the subject has not been diagnosed with a food allergy and/or has not exhibited symptoms of a food allergy.
55. The method of any one of claims 34-54, wherein the subject is a human.
56. The method of claim 55, wherein the subject is less than one year old, less than five years old, less than twelve years old or less than eighteen years old.
57. The method of any one of claims 35-56, wherein the A. caccae comprises a live bacterial product.
58. The method of any one of claims 35-57, wherein the bacteria are lyophilized or freeze-dried.
59. The method of any one of claims 35-58, wherein the A. caccae and/or prebiotic are administered orally.
60. The method of claim 59, wherein the A. caccae and/or prebiotic are administered in a tablet or capsule.
61. The method of any one of claims 35-60, wherein the method further comprises administration of a lactate-containing formula or food.
62. A composition comprising bacterium Anaerostipes caccae and a prebiotic.
63. The composition of claim 62, wherein the prebiotic comprises one or more of galactooligosacchararide, lactulose, lactitol, erythritol, isomalt, polyglycitol.
64. The composition of claim 63, wherein the prebiotic comprises one or more of galactooligosacchararide, lactulose, and lactitol.
65. The composition of any one of claims 62-64, wherein the composition further comprises a butyrate carrying compound.
66. The composition of claim 65, wherein the butyrate carrying compound comprises pHPMA-b-pBMA.
67. The composition of any one of claims 62-64, wherein the prebiotic comprises one or both of digestible and non-digestible oligosaccharides.
68. The composition of any one of claims 62-67, wherein the prebiotic comprises at least 6 grams of non-digestible oligosaccharides.
69. The composition of claim 67 or 68, wherein the oligosaccharide comprises a modified oligosaccharide.
70. The composition of claim 69, wherein the modified oligosaccharide comprises an A. caccae fermentable butyrate-releasing oligosaccharide.
71. The composition of any one of claims 62-70, wherein the A. caccae is in an amount of 1×10.sup.6 to 1×10.sup.15 CFU dosage.
72. The composition of any one of claims 62-71, wherein the ratio of the colony forming units of A. caccae to grams of prebiotic is 1000:1-10000:1.
73. The composition of any one of claims 62-72, wherein the composition further comprises a pharmaceutical excipient.
74. The composition of any one of claims 62-73, wherein the composition is formulated for oral administration.
75. A tablet, capsule, or powder comprising the composition of any one of claims 62-74.
76. A method for treating an infectious, autoimmune, or allergic disease in a subject comprising administering a composition comprising bacterium Anaerostipes caccae to the subject.
77. The method of claim 76, wherein the disease comprises an atopic disease.
78. The method of claim 77, wherein the atopic disease comprises eczema, atopic dermatitis, asthma, or allergic rhinitis.
79. The method of any one of claims 76-78 wherein 1×10.sup.6 to 1×10.sup.15 CFU of A. caccae is administered to the subject.
80. The method of any one of claims 76-79, wherein the subject has been diagnosed with an infectious, autoimmune, or allergic disease.
81. The method of any one of claims 1-23, wherein the subject has previously been treated for the infectious, autoimmune, or allergic disease.
82. The method of claim 81, wherein the subject has been determined to be resistant to the previous treatment.
83. The method of any one of claims 76-82, wherein the subject is a human.
84. The method of claim 83, wherein the subject is less than one year old, less than five years old, less than twelve years old or less than eighteen years old.
85. The method of any one of claims 76-84, wherein the A. caccae comprises a live bacterial product.
86. The method of any one of claims 76-85, wherein the bacteria are lyophilized or freeze-dried.
87. The method of any one of claims 76-86, wherein the A. caccae is administered orally.
88. The method of claim 87, wherein the A. caccae is administered in a tablet or capsule.
89. The method of any one of claims 76-88, wherein the method further comprises administration of a lactate-containing formula or food.
90. The method of any one of claims 76-89, wherein the subject is determined to have a ratio of protective/non-protective operational taxonomic units (OTUs) of less than 3.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
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DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0058] To better understand the role of the microbiota in regulating allergic disease in humans the inventors colonized germ free (GF) mice with bacteria derived from the feces of healthy or cow's milk allergic (CMA) infants. The inventors show here that colonization of germ-free mice with bacteria from healthy infants protected against sensitization to the cow's milk allergen β-lactoglobulin. Mice colonized with bacteria from CMA infants exhibited anaphylactic responses to BLG challenge and significantly increased serum BLG-specific IgE. Differences in bacterial composition separated the healthy and CMA populations in both the human donors and the colonized mice. RNA-Seq analysis of ileal intestinal epithelial cells revealed differentially expressed genes (DEGs) that distinguished healthy- and CMA-colonized mice across all donors. Correlation of ileal OTUs with DEGs in the ileum of healthy-colonized mice identified a Clostridial species, Anaerostipes caccae, that protected against an allergic response to food. The findings demonstrate that the composition of the intestinal microbiota is critical for regulating allergic responses to dietary antigens and suggest that interventions that modulate bacterial communities may be therapeutically relevant for food allergy.
I. Definitions
[0059] The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired responses discussed herein in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the effect desired. The actual dosage amount of a composition of the present embodiments administered to a patient or subject can be determined by physical and physiological factors, such as body weight, the age, health, and sex of the subject, the type of disease being treated, the extent of disease penetration, previous or concurrent therapeutic interventions, idiopathy of the patient, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance. For example, a dose may also comprise from about 1 μg/kg/body weight to about 1000 mg/kg/body weight (this such range includes intervening doses) or more per administration, and any particular dose derivable therein. In non-limiting examples of a range derivable from the numbers listed herein, a range of about 5 μg/kg/body weight to about 100 mg/kg/body weight, about 5 μg/kg/body weight to about 500 mg/kg/body weight, etc., can be administered. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
[0060] “Subject” and “patient” refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human.
[0061] As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” when used in reference to a disease, disorder or medical condition, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a condition is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, and an increased lifespan as compared to that expected in the absence of treatment.
[0062] The term “isolated” encompasses a bacterium or other entity or substance that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man. Isolated bacteria may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, isolated bacteria are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components.
[0063] The terms “purify,” “purifying” and “purified” refer to a bacterium or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production. A bacterium or a bacterial population may be considered purified if it is isolated at or after production, such as from a material or environment containing the bacterium or bacterial population, and a purified bacterium or bacterial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “isolated.” In some embodiments, purified bacteria and bacterial populations are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. In the instance of bacterial compositions provided herein, the one or more bacterial types present in the composition can be independently purified from one or more other bacteria produced and/or present in the material or environment containing the bacterial type. Bacterial compositions and the bacterial components thereof are generally purified from residual habitat products.
[0064] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.
[0065] As used herein the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. With respect to pharmaceutical compositions, the term “consisting essentially of” includes the active ingredients recited, excludes any other active ingredients, but does not exclude any pharmaceutical excipients or other components that are not therapeutically active.
[0066] The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
[0067] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
[0068] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
[0069] The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein, the term “another” may mean at least a second or more.
[0070] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
[0071] The phrase “effective amount” or “therapeutically effective amount” or “sufficient amount” means a dosage of a drug or agent sufficient to produce a desired result.
II. Microbial Compositions
[0072] Embodiments of the present disclosure concern microbial compositions for the treatment of infectious, autoimmune, or allergic disease.
[0073] The present disclosure also provides a pharmaceutical composition comprising one or more microbial cultures as described above. The bacterial species therefore are present in the dose form as live bacteria, whether in dried or lyophilized form. This may be preferably adapted for suitable administration; for example, in tablet or powder form, potentially with an enteric coating, for oral treatment.
[0074] In particular aspects, the composition is formulated for oral administration. Oral administration may be achieved using a chewable formulation, a dissolving formulation, an encapsulated/coated formulation, a multi-layered lozenge (to separate active ingredients and/or active ingredients and excipients), a slow release/timed release formulation, or other suitable formulations known to persons skilled in the art. Although the word “tablet” is used herein, the formulation may take a variety of physical forms that may commonly be referred to by other terms, such as lozenge, pill, capsule, or the like.
[0075] While the compositions of the present disclosure are preferably formulated for oral administration, other routes of administration can be employed, however, including, but not limited to, subcutaneous, intramuscular, intradermal, transdermal, intraocular, intraperitoneal, mucosal, vaginal, rectal, and intravenous.
[0076] The desired dose of the composition of the present disclosure may be presented in multiple (e.g., two, three, four, five, six, or more) sub-doses administered at appropriate intervals throughout the day, week, month or year.
[0077] In one aspect, the disclosed composition may be prepared as a capsule. The capsule (i.e., the carrier) may be a hollow, generally cylindrical capsule formed from various substances, such as gelatin, cellulose, carbohydrate or the like.
[0078] In another aspect, the disclosed composition may be prepared as a suppository. The suppository may include but is not limited to the bacteria and one or more carriers, such as polyethylene glycol, acacia, acetylated monoglycerides, carnuba wax, cellulose acetate phthalate, corn starch, dibutyl phthalate, docusate sodium, gelatin, glycerin, iron oxides, kaolin, lactose, magnesium stearate, methyl paraben, pharmaceutical glaze, povidone, propyl paraben, sodium benzoate, sorbitan monoleate, sucrose talc, titanium dioxide, white wax and coloring agents.
[0079] In some aspects, the disclosed microbial composition may be prepared as a tablet. The tablet may include the bacteria and one or more tableting agents (i.e., carriers), such as dibasic calcium phosphate, stearic acid, croscarmellose, silica, cellulose and cellulose coating. The tablets may be formed using a direct compression process, though those skilled in the art will appreciate that various techniques may be used to form the tablets.
[0080] In other aspects, the disclosed microbial composition may be formed as food or drink or, alternatively, as an additive to food or drink, wherein an appropriate quantity of bacteria is added to the food or drink to render the food or drink the carrier.
[0081] In some embodiments, the microbial composition may further comprise a food or a nutritional supplement effective to stimulate the growth of A. caccae present in the gastrointestinal tract of the subject. In some embodiments, the nutritional supplement is produced by another bacterium associated with a healthy human gut microbiome.
III. Administration of Therapeutic Compositions
[0082] The therapy provided herein comprises administration of a combination of therapeutic agents, such as microbial compositions and prebiotics. The therapy may be administered in any suitable manner known in the art. For example, the microbial composition and the prebiotic may be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, the microbial composition and the prebiotic are in a separate composition. In some embodiments, the microbial composition and the prebiotic are in the same composition.
[0083] Embodiments of the disclosure relate to compositions and methods comprising bacteria and one or more prebiotics. The bacteria and/or prebiotic(s) may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed, for example, a bacterium (or composition comprising bacteria) is “A” and a prebiotic is “B”:
TABLE-US-00001 A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[0084] In some embodiments, the microbial composition is administered prior to the prebiotic. In some embodiments, the microbial composition is administered at least, at most, or about 1, 2, 3, 5, 6, 12, 24 hours or 2, 3, 4, 6, 8, 10, days or 2, 3, 4, 5, 6, 7, or 8 weeks (or any derivable range therein) prior to the prebiotic. In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 doses (or any derivable range therein) of the microbial composition is administered at least, at most, or about 1, 2, 3, 5, 6, 12, 24 hours or 2, 3, 4, 6, 8, 10, days or 2, 3, 4, 5, 6, 7, or 8 weeks (or any derivable range therein) prior to the prebiotic. In some embodiments, the microbial composition is administered after the prebiotic. In some embodiments, the microbial composition is administered at least, at most, or about 1, 2, 3, 5, 6, 12, 24 hours or 2, 3, 4, 6, 8, 10, days or 2, 3, 4, 5, 6, 7, or 8 weeks (or any derivable range therein) after the prebiotic or after at least one of the prebiotics or after at least 2 of the prebiotics. In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 doses (or any derivable range therein) of the microbial composition is administered at least, at most, or about 1, 2, 3, 5, 6, 12, 24 hours or 2, 3, 4, 6, 8, 10, days or 2, 3, 4, 5, 6, 7, or 8 weeks (or any derivable range therein) after the prebiotic or after at least one of the prebiotics or after at least 2 of the prebiotics.
[0085] In some embodiments, the microbial modulator composition is formulated for oral administration. The skilled artisan knows a variety of formulas which can encompass living or killed microorganisms and which can present as food supplements (e.g., pills, tablets and the like) or as functional food such as drinks or fermented yogurts.
[0086] The agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some embodiments, the prebiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the microbial composition is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.
[0087] For example, the therapeutically effective or sufficient amount of each of the at least one isolated or purified population of bacteria or each of the at least two, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, or 15 isolated or purified populations of bacteria of the microbial modulator compositions of the embodiments that is administered to a human will be at least about 1×10.sup.3 colony forming units (CFU) of bacteria or at least about 1×10.sup.4, 1×10.sup.5, 1×10.sup.6, 1×10.sup.7, 1×10.sup.8, 1×10.sup.9, 1×10.sup.10, 1×10.sup.11, 1×10.sup.12, 1×10.sup.13, 1×10.sup.14, 1×10.sup.15 CFU (or any derivable range therein). In some embodiments, a single dose will contain bacteria (such as a specific bacteria or species, genus, or family described herein) present in an amount of least, at most, or about 1×10.sup.4, 1×10.sup.5, 1×10.sup.6, 1×10.sup.7, 1×10.sup.8, 1×10.sup.9, 1×10.sup.10, 1×10.sup.11, 1×10.sup.12, 1×10.sup.13, 1×10.sup.14, 1×10.sup.15 or more CFU (or any derivable range therein). In some embodiments, a single dose will contain at least, at most, or about 1×10.sup.4, 1×10.sup.5, 1×10.sup.6, 1×10.sup.7, 1×10.sup.8, 1×10.sup.9, 1×10.sup.10, 1×10.sup.11, 1×10.sup.12, 1×10.sup.13, 1×10.sup.14, 1×10.sup.15 or greater than 1×10.sup.15 CFU (or any derivable range therein) of total bacteria.
[0088] In some embodiments, the therapeutically effective or sufficient amount of each of the at least one isolated or purified population of bacteria of the microbial compositions of the embodiments that is administered to a human will be at least about 1×10.sup.3 cells of bacteria or at least about 1×10.sup.4, 1×10.sup.5, 1×10.sup.6, 1×10.sup.7, 1×10.sup.8, 1×10.sup.9, 1×10.sup.1°, 1×10.sup.11, 1×10.sup.12, 1×10.sup.13, 1×10.sup.14, 1×10.sup.15 cells (or any derivable range therein). In some embodiments, a single dose will contain bacteria (such as a specific bacteria or species, genus, or family described herein) present in an amount of at least, at most, or about 1×10.sup.4, 1×10.sup.5, 1×10.sup.6, 1×10.sup.7, 1×10.sup.8, 1×10.sup.9, 1×10.sup.10, 1×10.sup.11, 1×10.sup.12, 1×10.sup.13, 1×10.sup.14, 1×10.sup.15 or more cells (or any derivable range therein). In some embodiments, a single dose will contain at least, at most, or about 1×10.sup.4, 1×10.sup.5, 1×10.sup.6, 1×10.sup.7, 1×10.sup.8, 1×10.sup.9, 1×10.sup.10, 1×10.sup.11, 1×10.sup.12, 1×10.sup.13, 1×10.sup.14, 1×10.sup.15 or greater than 1×10.sup.15 cells (or any derivable range therein) of total bacteria.
[0089] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some embodiments, a unit dose comprises a single administrable dose.
[0090] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg/kg to 200 mg/kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg/kg, mg/kg, μg/day, or mg/day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.
[0091] In certain embodiments, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 μM to 150 μM. In another embodiment, the effective dose provides a blood level of about 4 μM to 100 μM.; or about 1 μM to 100 μM; or about 1 μM to 50 μM; or about 1 μM to 40 μM; or about 1 μM to 30 μM; or about 1 μM to 20 μM; or about 1 μM to 10 μM; or about 10 μM to 150 μM; or about 10 μM to 100 μM; or about 10 μM to 50 μM; or about 25 μM to 150 μM; or about 25 μM to 100 μM; or about 25 μM to 50 μM; or about 50 μM to 150 μM; or about 50 μM to 100 μM (or any range derivable therein). In other embodiments, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM or mM or any range derivable therein. In certain embodiments, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.
[0092] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.
[0093] It will be understood by those skilled in the art and made aware that dosage units of μg/kg or mg/kg of body weight can be converted and expressed in comparable concentration units of μg/ml or μM or mM (blood levels), such as 4 μM to 100 μM. It is also understood that uptake is species and organ/tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.
[0094] Prebiotics may be formulated using techniques of pharmaceutical formulation known in the art. They may be formulated using specialized techniques known for delivery to specific regions of the gastrointestinal tract. Two examples known in the art are described in published PCT application WO 2018/195067 A1 and also in U.S. patent application Ser. No. 15/257,673, each of which are incorporated by reference. Other formulations for prebiotics or combinations of prebiotics and A. caccae are known in the art. In some embodiments, the compositions of the disclosure include a butyrate carrying compound, such as those described in WO 2018/195067.
IV. Methods of Treatment
[0095] The methods of the disclosure relate to the treatment of infectious, autoimmune, or allergic disease. In some embodiments, the method is for the treatment of a food allergy. In some embodiments, the food allergy comprises a milk allergy. In some embodiments, the milk allergy comprises cow's milk allergy. In some embodiments, the milk allergy comprises cow, goat, or sheep milk allergy. In some embodiments, the method is for treating allergies, asthma, diabetes (e.g. type 1 diabetes), graft rejection, arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, and systemic juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, atopy including atopic diseases such as hay fever and Job's syndrome, dermatitis including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, x-linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS, primary progressive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (for example, Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis such as ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease), bowel inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage as in meningitis, herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune condition, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen's encephalitis and limbic and/or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or acute glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN, proliferative nephritis, autoimmune polyglandular endocrine failure, balanitis including balanitis circumscripta plasmacellularis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, eythema multiform, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reaction, eczema including allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular palmoplantar eczema, asthma such as asthma bronchiale, bronchial asthma, and auto-immune asthma, conditions involving infiltration of T cells and chronic inflammatory responses, immune reactions against foreign antigens such as fetal A-B-O blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus, juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), and adult onset diabetes mellitus (Type II diabetes) and autoimmune diabetes. Also contemplated are immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, including vasculitis, large-vessel vasculitis (including polymyalgia rheumatica and gianT cell (Takayasu's) arteritis), medium-vessel vasculitis (including Kawasaki's disease and polyarteritis nodosa/periarteritis nodosa), microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated small-vessel vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), Addison's disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Behcet's disease/syndrome, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus), autoimmune polyendocrinopathies, Reiter's disease or syndrome, thermal injury, preeclampsia, an immune complex disorder such as immune complex nephritis, antibody-mediated nephritis, polyneuropathies, chronic neuropathy such as IgM polyneuropathies or IgM-mediated neuropathy, autoimmune or immune-mediated thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, scleritis such as idiopathic cerato-scleritis, episcleritis, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), experimental autoimmune encephalomyelitis, myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, gianT cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsed or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan's syndrome/nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's disease/syndrome, rosacea autoimmune, zoster-associated pain, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler's syndrome, alopecia greata, alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), and telangiectasia), male and female autoimmune infertility, e.g., due to anti-spermatozoan antibodies, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, gianT cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway/pulmonary disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, asperniogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyreoiditis, acquired spenic atrophy, non-malignant thymoma, vitiligo, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis, autoimmune disorders associated with collagen disease, rheumatism, neurological disease, lymphadenitis, reduction in blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, ischemic re-perfusion disorder, reperfusion injury of myocardial or other tissues, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndromes, cytokine-induced toxicity, narcolepsy, acute serious inflammation, chronic intractable inflammation, pyelitis, endarterial hyperplasia, peptic ulcer, valvulitis, graft versus host disease, contact hypersensitivity, asthmatic airway hyperreaction, and endometriosis.
V. Kits
[0096] Certain aspects of the disclosure also encompass kits for performing the methods of the disclosure, such as kits comprising the compositions described herein. Kits may comprise a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container may hold a composition which includes a probe that is useful for prognostic or non-prognostic applications, such as described above. The label on the container may indicate that the composition is used for a specific prognostic or non-prognostic application, and may also indicate directions for either in vivo or in vitro use, such as those described above. The kit may comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
VI. EXAMPLES
[0097] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1—Healthy Infants Harbor Intestinal Bacteria that Protect Against Food Allergy
[0098] B. Results
[0099] Work from the inventors' laboratory and others demonstrated that the fecal microbial communities of infants with CMA are markedly different from those of their healthy counterparts (5, 6). Based on these results, as well as evidence that members of the microbiota can be allergy protective (7), the inventors used a gnotobiotic mouse model to investigate whether commensal bacteria play a causal role in protection against an allergic response to the cow's milk allergen β-lactoglobulin (BLG). Germ free (GF) mice were colonized with human feces from four healthy and four IgE-mediated cow's milk allergic (CMA) infant donors who were matched for age, gender and mode of birth (8, 9) (Supplementary Table 1). It has previously been reported that diet is important for the stable colonization of germ-free mice with human feces (ref. 10). To support the growth of human bacteria in the murine hosts, mice received feces from formula-fed healthy or CMA infants and were fed the same formulas consumed by their human infant donors in addition to plant-based mouse chow. The CMA infant donors received an extensively hydrolyzed casein formula (EHCF) to manage ongoing allergic symptoms while the healthy donors received a standard cow's milk-based formula (5). Initial transfer recipients were used as living repositories for subsequent experiments (see Methods).
[0100] Groups of GF mice and mice colonized with either the healthy or CMA infant microbiota were sensitized with BLG and the mucosal adjuvant cholera toxin (CT). GF mice, devoid of any bacterial colonization, were highly susceptible to anaphylactic responses to food as evidenced by a drop in core body temperature (
[0101] Analysis of fecal samples from the eight formula-fed human infant donors (Supplementary Table 1) identified 58 operational taxonomic units (OTUs) that were differentially abundant between healthy and CMA infants (
[0102] Tolerance to dietary antigens begins with their absorption in the small intestine (4, 12). Most commensal bacteria reside in the colon; in the small intestine, bacteria are most numerous in the ileum (13). The interaction of these bacteria with IECs is central to regulation of immunity at the host-microbe interface (13, 14). Ileal IECs were isolated from groups of mice colonized by each of the eight infant donors and quantified gene expression by RNASeq (
[0103] To determine whether the fecal OTU signatures identified in
[0104] It was next examined whether A. caccae can mimic the changes in gene expression and protection against anaphylaxis associated with the healthy microbiota by monocolonizing GF mice (see Methods). Some of the genes significantly upregulated in healthy-colonized mice (Fbp1, Tgfbr3) were also significantly upregulated in A. caccae monocolonized mice (
[0105] The inventors have shown that anaerobic, mucosa-associated bacteria in the Clostridia class have attracted considerable interest because of their reported roles in the maintenance of intestinal homeostasis through induction of regulatory T cells (28, 29), production of immunomodulatory metabolites (30, 31), and regulation of colonization resistance (32). The inventors have shown that such immunomodulatory bacteria are present in the ileum, at the site of food absorption and have demonstrated their role in protection against an anaphylactic response to food. Mechanistic analysis of the Clostridia-associated changes in ileal gene expression described herein is likely to reveal additional pathways critical to the maintenance of tolerance to dietary antigens. The model described in this report does not address whether the allergic state drives dysbiosis (33) or dysbiosis precedes allergy. Indeed, many factors are likely to contribute to the development of food allergies. This data demonstrate that the commensal bacteria play an important role in preventing allergic responses to food and provides proof of concept for the development of microbiome-modulating strategies to prevent or treat this disease.
[0106] C. Methods
[0107] Gnotobiotic Mouse Husbandry. All mice were bred and housed in the Gnotobiotic Research Animal Facility (GRAF). Mice were maintained in Trexler style flexible film isolator housing units (Class Biologically Clean) with Ancare polycarbonate mouse cages (catalog #N10HT) and Teklad Pine Shavings (7088; sterilized by autoclave) on a 12-hour light/dark cycle at a room temperature of 20-24° C. Mice were provided with autoclaved sterile water, USP grade, at pH 5.2 ad libitum. Bedding was changed weekly; cages of formula fed mice required near daily bedding changes due to leakage of formula from the bottles. All mice were fed Purina Lab Diet® 5K67, stored in a temperature-controlled environment in accordance with The Guide for the Care and Use of Laboratory Animals (8th Edition, 2013). The diet was sterilized by autoclaving at 121° C.×30 minutes. The sterility of the isolators was checked weekly by both cultivation and 16S rRNA analysis of fecal samples by qPCR. Cultivation was in BHI, Nutrient and Sabbaroud Broth at 37° C. aerobic and anaerobic and 42° C. aerobic for 96 hours. All mice are initially screened upon rederivation or receipt for all internal and external parasites, full serology profile and/or PCR, bacteriology, and gross and histologic analysis of major organs through either IDEXX Radil or Charles River Lab using an Axenic Profile Screen. Germ free (GF) C3H/HeN mice were transferred within the facility from T. Golovkina (University of Chicago).
[0108] Preparation of human fecal samples. Healthy (non-allergic) fecal samples were obtained from participants in a vaccination program. These subjects were not at risk for atopic disorders and their clinical history was negative for any allergic condition. Infants with CMA were diagnosed at a tertiary pediatric allergy center (Pediatric Allergy Program at the Department of Translational Medical Science of the University of Naples ‘Federico II’); for complete patient information see Supplementary Tables 1 and 2. All aspects of this study were conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University of Naples ‘Federico II’. Written informed consent was obtained from the parents/guardians of all children involved in the research. Fresh fecal samples were collected in the clinic in sterile tubes, weighed, mixed with 2 mL LB broth+30% glycerol per 100-500 mg, aliquoted into sterile cryovials and immediately stored at −80° C. Samples were shipped to the University of Chicago on dry ice where they were stored at −80° C. until homogenization. To colonize mice, frozen fecal samples were introduced into an anaerobic chamber and thawed. Thawed feces were mixed with 3 mm borosilicate glass beads in a sterile 50 mL tube with 2.5 mL pre-reduced PBS+0.05% cysteine and vortexed gently to dissociate. The resulting homogenate was filtered through a 100 μm filter. This homogenization and filtration process was repeated three more times and the final filtrate was mixed with an equal volume of 30% glycerol+0.05% cysteine. This solution was aliquoted into Balch tubes with rubber stoppers for transport and introduction into the gnotobiotic isolator. The remaining fecal solution was frozen in aliquots at −80° C.
[0109] Colonization of germ free mice. All mice were weaned at 3 weeks of age onto a plant-based mouse chow (Purina Lab Diet® 5K67) and colonized at weaning. GF mice received autoclaved sterile water. Both male and female mice were used for all experiments. Each experiment was littermate controlled. All mice were identified by unique 5 digit ear tags. All work was performed in accordance with the Institutional Biosafety and Animal Care and Use Committees. Each human infant donor transfer was maintained in its own flexible film isolator to avoid cross contamination. In all experiments, repository mice were created from human fecal donors by intragastric gavage of GF mice with 500 μL of freshly prepared infant fecal homogenate. These repositories were then used to colonize subsequent experimental mice via mouse to mouse transfer by intragastric gavage of mouse feces. Fecal samples from both repository and experimental mice were examined regularly by 16S rRNA analysis which demonstrated that mouse to mouse transfer from repository to experimental mice by gavage was highly reproducible and stable over time. For colonization of experimental mice, a freshly voided fecal pellet from a repository mouse was homogenized in 1 mL of sterile PBS and 250 μL of this homogenate was used to gavage one recipient mouse. For mice fed infant formula, the drinking water was replaced by formula four hours prior to colonization. Mice colonized with healthy infant feces were given Enfamil Infant (Mead Johnson Nutrition, Evansville, Ind.) and CMA-colonized mice were given extensively hydrolyzed casein formula (EHCF), Nutramigen I (Mead Johnson Nutrition) ad libitum. Both dry and liquid forms of the formulas were utilized. Dry formula was mixed with autoclaved sterile water, USP grade, according to the manufacturer's instructions. All formulas were refreshed daily.
[0110] For Anaerosfipes caccae monocolonized mice, A. caccae (DSM-14662, DSMZ) was cultured in an anaerobic chamber (Coy, Model B) in reduced Schaedler's Broth (Remel) overnight at 37° C. to an optical density (OD600) of 1.08. 250 μL (approximately 2.5×10.sup.8 CFU) was gavaged to GF mice. These mice were monitored for colonization by qPCR with species-specific primers (Supplementary Table 6) and were maintained as living repositories. For colonization of experimental mice, Enfamil Infant formula (liquid) was added to the drinking water four hours prior to colonization. A freshly voided fecal pellet from a repository mouse was then homogenized in 1 mL of sterile PBS and 2504, of this homogenate was used to gavage one recipient mouse. Monocolonization with A. caccae was confirmed by 16S rRNA-targeted sequencing of fecal samples collected at sacrifice for all experimental mice.
[0111] 16S rRNA-targeted sequencing. Bacterial DNA was extracted using the Power Soil DNA Isolation Kit (MoBio). 16S rRNA gene amplicon sequencing was performed on an Illumina MiSeq at the Environmental Sample Preparation and Sequencing Facility at Argonne National Laboratory. Procedures described in reference 34 were used to generate 151 bp paired-end reads from the fecal samples with 12 bp barcodes. The V4 region of the 16S rRNA gene was PCR amplified with region-specific primers (515F-806R) that include sequencer adapter sequences used in the Illumina flowcell. The microbiota signature cohort consisting of infant donor fecal samples, and gnotobiotic mouse fecal and ileal samples (n=99) was analyzed by Quantitative Insights into Microbial Ecology (QIIME) (version 1.9) (35). Raw reads were trimmed to remove low quality bases; paired-end 3′ overlapping sequences were merged using SeqPrep (found on the world wide web at github.com/jstjohn/SeqPrep). The open reference OTU picking protocol was used at 97% sequence identity against the Greengenes database (August 2013 release) (36). Sequences were aligned with PyNAST (37). Taxonomic assignments were made with the uclust consensus taxonomy assigner (38); predicted chimeric sequences were removed using ChimeraSlayer (v20110519) (found on the world wide web at microbiomeutil.sourceforge.net). Data were rarefied to an even depth of 3,160 reads for the donor and colonized mouse cohort (n=99, consisting of donor fecal samples, mouse fecal samples at 2- and 3-weeks post-colonization, and mouse ileal samples), and 10,050 reads for the mouse cohort shown in
where H′ is Shannon index and S as the maximum number of OTUs. Discrete False-Discovery Rate (DS-FDR) (40) was used to identify differentially abundant bacterial taxa between fecal communities of the CMA and healthy groups with parameters “transform type=normdata, method=meandiff, alpha=0.10, numperm=1000, fdr_method=dsfdr” (accessed 02262018) (https://github.com/biocore/dsFDR). Compared to the Benjamin-Hochberg-FDR (BH-FDR) method, the DS-FDR method has increased power with limited sample size and is robust to sparse data structure (low proportion of non-zero values in microbe abundance table), therefore is uniquely suited for data analysis of microbe communities (40). The DS-FDR algorithm does not compute adjusted P-values; instead, it estimates the false-discovery rate from a permutation test (default 1000 permutations), which controls the FDR at the desired level (0.10). As such, it computes the raw P-values, test statistics and rejected hypotheses in the output (Supplementary Table 3 and Supplementary Table 5). In each comparison, OTUs present in less than 4 samples were removed prior to applying the DS-FDR test. Linear discriminant analysis effect size (LEfSe) was used to identify genera significantly enriched in CMA or healthy groups compared to the other, using the per-sample normalization value of 1,000,000 and default values for other parameters (41). In LEfSe analysis, linear discriminant analysis (LDA) score was computed for taxa differentially abundant between the two groups. A taxon at P<0.05 (Kruskal-Wallis test) and log 10(LDA)≥2.0 (or ≤−2.0) was considered significant. For
[0112] Food allergen sensitization and challenge. Protocols were adapted from reference7. All mice were weaned onto a plant-based mouse chow (Purina Lab Diet® 5K67) at 3 weeks of age. GF mice received autoclaved sterile water. For mice colonized with feces from infant donors, or monocolonized with A. caccae, the drinking water was replaced by formula four hours prior to colonization. Mice colonized with healthy feces or A. caccae received Enfamil; CMA colonized mice received Nutramigen (both from Mead Johnson). On day 0, one week post weaning (GF) or colonization (healthy/A. caccae/CMA), all mice were fasted for 4 hours and then given a gavage of 200 mM sodium bicarbonate. 30 minutes later, mice were given 20 mg BLG (Sigma) plus 10 μg CT (List Biologicals). This protocol was repeated weekly for 5 weeks. For formula-fed mice, formula was replaced by sterile water for the week after the last sensitization. Prior to challenge on day 42, mice were fasted for 4 hours and given sodium bicarbonate by gavage. Two doses of 100 mg BLG each were then administered via intragastric gavage 30 minutes apart. Core body temperature was measured in a blinded fashion prior to allergen challenge and every 5 minutes after the first challenge until at least 30 minutes after the second challenge using a rectal probe (PhysiTemp). Serum was collected 1 hour after the second challenge to measure mMCPT-1 levels. Serum was collected 24 hours after challenge for antibody measurements.
[0113] ELISAs. mMCPT-1 was quantified in serum collected 1 hour after the second challenge according to the manufacturer's protocol (eBioscience). BLG-specific ELISAs were performed using protocols modified from reference7. Briefly, plates were coated overnight at 4° C. with 100 μg/mL BLG in 100 mM carbonate-bicarbonate buffer (pH 9.6). Plates were blocked for 2 hours at room temperature with 3% BSA. Samples were added in 1% BSA and incubated overnight at 4° C. Assays were standardized with BLG-specific antibodies (IgE or IgG1) purified on a CNBr-Sepharose affinity column from mice immunized with BLG+alum (42). BLG-specific antibodies were detected with goat anti-mouse IgE-UNLB (Southern Biotech) and rabbit anti-goat IgG-AP (ThermoFisher) then developed with p-NPP (KPL Labs) or IgG1-HRP (Southern Biotech) and developed with TMB (Sigma).
[0114] For cytokine analysis spleens were harvested 24 h post challenge from A. caccae or CMA colonized mice sensitized with BLG+CT for 5 weeks. Splenocytes were stimulated at a concentration of 2×10.sup.6 cells/ml at 37° C., 10% CO.sub.2 with 10 mg/ml BLG (Sigma) in cDMEM (with 4% FCS (HyClone), 10 mM HEPES (Gibco), 100 U/ml Penicillin/Streptomycin (Gibco) and 55 μM 2-mercaptoethanol (Gibco). Cytokine concentrations in 72 hr culture supernatants were determined by ELISA for IL-13 and IL-4 (both from Invitrogen).
[0115] Epithelial cell isolation. As in sensitization experiments, mice were weaned at 3 weeks of age and placed on infant formula prior to colonization. Seven days after colonization, mice were euthanized and ileum was removed. For IEC isolation, tissues were cleaned and inverted as described in ref. (43). IECs were collected by inflating inverted tissue in Cell Recovery Solution (Corning) every 5 minutes for 30 minutes. IEC samples were lysed in TRIZol (ThermoFisher) and RNA was extracted with PureLink RNA Mini Kit (Ambion) plus on-column DNAse treatment (PureLink DNAse Set, Ambion).
[0116] RNASeq. RNA libraries were prepared using TruSeq Stranded Total Library Preparation Kit with Ribo-Zero human/mouse/rat (Illumina). Samples were sequenced at the University of Chicago Functional Genomics Core, using 50 bp single reads chemistry in a HiSeq2500 instrument, with sequencing replicates in two lanes. The quality of raw reads was assessed by FastQC (v0.11.5) (44). The QC30 score across 39 RNAseq samples was 96.81%±0.06% (mean±s.e.m), which represents the percentage of bases with quality score ≥Q30. Alignment to the mouse reference transcriptome was performed with Gencode gene annotation (vM16, GRCm38) by Kallisto (v0.43.1) with the strand-specific mode (45). This mode implements a kmer-based pseudoalignment algorithm to accurately quantify transcripts from RNASeq data while robustly detecting errors in the reads. The average mapping rate was 62.77%±1.10% (mean±S.E.M) based on the Kallisto pseudoalignments to the reference transcriptome. On average, 35 million raw sequencing reads were generated per sample, and 22 million were mapped to transcriptome using Kallisto. Transcript-level abundance was quantified specifying strand-specific protocol, summarized into gene level using tximport (v1.4.0) (46), normalized by trimmed mean of M values (TMM) method, and log 2-transformed. Genes expressed in at least 3 samples (counts per million of reads (CPM)>3) were kept for further analysis. Genes differentially expressed between groups were identified using limma voom algorithm with precision weights (v3.34.5) (47). duplicateCorrelation function was used to estimate the correlation among mouse samples with Donor (1 to 8) as the blocking factor. 1mFit function was used to fit all mouse samples (n=39, 18 CMA-colonized, 18 healthy-colonized, and 3 GF) into one linear model incorporating the correlation structure computed from above. Contrasts were set as CMA versus healthy, CMA versus GF and healthy versus GF to identify DEGs in each comparison. Genes that are significantly differentially expressed between CMA and healthy, and also different from GF mice were identified using a two-step procedure: (1) Genes were detected as different in CMA vs healthy comparison with fold change ≥1.5 or ≤−1.5 at false-discovery rate (FDR) corrected p-value smaller than 0.10; (2) Genes from step (1) were further filtered by fold change ≥1.5 or ≤−1.5 in CMA vs GF or healthy vs GF comparison at FDR 0.05. A more stringent FDR threshold (0.05) was applied in step (2) to prioritize potentially true positives when compared to the negative control (GF). Multiple testing correction was performed using Benjamini-Hochberg FDR (BH-FDR) method (48). A total of 32 DEGs passed these thresholds, which represent four types of gene expression changes in colonized mice: (1) Up in healthy: genes that are up-regulated in healthy mice relative to both CMA and GF; (2) Up in CMA: genes that are up-regulated in CMA mice relative to both healthy and GF; (3) Down in healthy: genes that are down-regulated in healthy mice relative to both CMA and GF; and (4) Down in CMA: genes that are down-regulated in CMA mice relative to healthy and GF. The four groups of DEGs are shown in
[0117] qPCR. Gene expression was measured by qPCR as described in reference 7. In brief, cDNA was prepared from RNA using the iScript cDNA Synthesis kit (BioRad). Gene expression was measured with PowerUp SYBR green master mix (Applied Biosystems) according to the manufacturer's instructions. Primers are listed in Supplementary Table 7 (20, 50-54). Expression of genes of interest was normalized to Hprt. Relative expression was measured using ΔΔCt centered around the geometric mean; GF mice were used as a reference.
[0118] The presence of A. caccae in fecal and ileal samples was confirmed using qPCR as described in ref (25). Bacterial DNA was extracted using the Power Soil DNA Isolation Kit (MoBio) and qPCR was performed using PowerUp SYBR green master mix (Applied Biosystems) using 4 μl of each primer at 10 μM working dilution and 41 of bacterial DNA. Primers are listed in Supplementary Table 6. The cycling conditions for the reaction consisted of an activation cycle of 50° C. for 2 min followed by one cycle of 95° C. for 10 min and 40 cycles at 94° C. for 20 sec, 55° C. for 20 sec and 72° C. for 50 sec. The fluorescent probe was detected in the last step of this cycle. A melt curve was performed at the end of the PCR to confirm specificity of PCR product. Relative abundance is expressed as 2.sup.−Ct normalized to total 16S rRNA copies per g fecal material and multiplied by a constant (1×10.sup.25) to bring all values above 1.
[0119] Histopathologic analysis. For histological analysis 3 mm pieces of mid-colon and mid-ileum tissue were fixed in either 10% formalin for H&E staining or Carnoy's fixative for Periodic-acid Schiff (PAS) staining. Sectioning and staining were performed by the Human Tissue Resource Center at the University of Chicago. All sections were reviewed by a GI pathologist in a blinded fashion.
[0120] Statistical analysis. Prism 7.0 (GraphPad) was used to perform one-way (
[0121] D. Tables
TABLE-US-00002 Supplementary TABLE 1 Patient data for formula-fed infant donors used in this study Healthy CMA Donor ID # 1 2 3 4 5 6 7 8 Sex Male Male Male Male Male Male Male Male Mode of Cesarean Cesarean Cesarean Cesarean Cesarean Cesarean Cesarean Cesarean Delivery section section section section section section section section Gestational At At At At At At At At Age term term term term term term term term Breast- <14 days <14 days <14 days <14 days <14 days <14 days <14 days <14 days feeding Age at 6 months 6 months 6 months 6 months 6 months 6 months 6 months 6 months stool collection Age at CMA — — — — 2 months 2 months 6 months 5 months symptoms 12 days 20 days onset Symptoms at — — — — Urticaria Urticaria Eczema, Eczema, CMA and and urticaria, vomiting diagnosis vomiting vomiting vomiting IgE-mediated — — — — Yes Yes Yes Yes mechanism Skin prick test value at diagnosis (mm) Alphalacto- — — — — 5 12 10 4 albumin Betalacto- — — — — 3 3 1 10 globulin Casein — — — — 3 5 4 8 Whole milk — — — — 7 6 5 7 Total serum — — — — 340 127 267 358 IgE at diagnosis (kU/L)
TABLE-US-00003 Supplementary Table 2 Patient data for breast-fed infant donors in Supplementary Figure 4 Description Healthy infant Infant with cow's milk allergy Donor ID # 9 10 Sex Female Female Mode of Delivery Spontaneous Spontaneous Gestational Age At term At term Breastfeeding Yes Yes (mother consuming (mother consuming cow's milk protein) cow's milk protein) Age at stool collection 6 months 6 months Age at CMA 5 months symptoms onset Symptoms at CMA Eczema, irritability, vomiting, diagnosis failure to thrive, diarrhea IgE-mediated Yes mechanism Skin prick test value at diagnosis (mm) Alphalactoalbumin — 15 Betalactoglobulin — 1 Casein — 7 Whole milk — 15 Total serum IgE at 334 diagnosis (kU/L)
TABLE-US-00004 Supplementary Table 3 58 OTUs differentially abundant between CMA- and healthy-donor fecal samples, shown in FIG. 2a. Change DS-FDR In DS-FDR DS-FDR Raw OTU Direction Reject Test Statistic Pvalues Taxonomy 1111294 UP in TRUE −0.184530795 0.015984016 k_Bacteria; Healthy p_Proteobacteria: c_Gammaproteobacteria; o_Enterobacteriales; f_Enterobacteriaceae; g_; s.sub.— 360015 UP in TRUE −0.068956969 0.001998002 k_Bacteria; p_Firmicutes; Healthy c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_[Ruminococcus]; s_gnavus New.Reference UP in TRUE −0.039674728 0.000999001 k_Bacteria; OTU147 Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_; s.sub.— 628226 UP in TRUE −0.034540622 0.000999001 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_; s.sub.— 349024 UP in TRUE −0.01594088 0.013986014 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s.sub.— 712677 UP in TRUE −0.012704055 0.027972028 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales 780650 UP in TRUE −0.010383613 0.012987013 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_; s.sub.— 843459 UP in TRUE −0.009899159 0.002997003 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_; s.sub.— 262095 UP in TRUE −0.007552857 0.01998002 k_Bacteria; Healthy p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_; s.sub.— New.Reference UP in TRUE −0.005328787 0.006993007 k_Bacteria; OTU166 Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae 579851 UP in TRUE −0.005308631 0.022977023 k_Bacteria; Healthy p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_[Eubacterium]; s_dolichum 551822 UP in TRUE −0.005216137 0.026973027 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae 298247 UP in TRUE −0.004133646 0.000999001 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Epulopiscium; s 345540 UP in TRUE −0.003450891 0.000999001 k_Bacteria; Healthy p_Protcobactcria: c_Gammaproteobacteria; o_Enterobacteriale s; f_Enterobacteriaceae; g_; s.sub.— 582691 UP in TRUE −0.002271895 0.004995005 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_; s.sub.— 259772 UP in TRUE −0.002219727 0.016983017 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Coprococcus; s.sub.— 325419 UP in TRUE −0.001426612 0.000999001 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_; s.sub.— 797229 UP in TRUE −0.001367202 0.000999001 k_Bacteria; Healthy p_Protcobactcria: c_Gammaproteobacteria; o_Enterobacteriale s; f_Enterobacteriaceae; g _; s.sub.— 557627 UP in TRUE −0.000748282 0.000999001 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_; s.sub.— 828483 UP in TRUE −0.000737522 0.020979021 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_Clostridium 299267 UP in TRUE −0.000617653 0.026973027 k_Bacteria; Healthy p_Protcobactcria: c_Gammaproteobacteria; o_Enterobacteriale s; f_Enterobacteriaceae; g_; s.sub.— 4448331 UP in TRUE −0.000531974 0.030969031 k_Bacteria; Healthy p_Proteobacteria; c_Gammaproteobacteria; o_Enterobacteriale s; f_Enterobacteriaceae; g_; s.sub.— 3376513 UP in TRUE −0.000490385 0.024975025 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_[Ruminococcus]; s_gnavus 813217 UP in TRUE −0.000418414 0.004995005 k_Bacteria; Healthy p_Protcobactcria: c_Gammaproteobacteria; o_Enterobacteriale s_; f_Enterobacteriaceae; g_; s.sub.— New.CleanUp.Reference UP in TRUE −0.000411459 0.001998002 k_Bacteria; OTU56927 Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_[Ruminococcus]; s_gnavus 335701 UP in TRUE −0.000408096 0.032967033 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_[Ruminococcus]; s_gnavus 191999 UP in TRUE −0.000393548 0.008991009 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae 183865 UP in TRUE −0.00036463 0.014985015 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_ [Ruminococcus]; s_gnavus 231787 UP in TRUE −0.000362215 0.003996004 k_Bacteria; Healthy p_Protcobactcria: c_Gammaproteobacteria; o_Enterobacteriale s_; f_Enterobacteriaceae; g_; s .sub.— 342397 UP in TRUE −0.000334965 0.006993007 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_[Ruminococcus]; s_gnavus 581782 UP in TRUE −0.000327138 0.012987013 k_Bacteria; Healthy p_Protcobactcria: c_Gammaproteobacteria; o_Enterobacteriales; f_Enterobacteriaceae; g_; s.sub.— 304641 UP in TRUE −0.000299547 0.014985015 k_Bacteria; Healthy p_Protcobactcria: c_Gammaproteobacteria; o_Enterobacteriale s; f_Enterobacteriaceae; g_; s.sub.— 4389289 UP in TRUE −0.000274745 0.032967033 k_Bacteria; Healthy p_Protcobactcria: c_Gammaproteobacteria; o_Enterobacteriale s; f_Enterobacteriaceae; g_; s.sub.— 541119 UP in TRUE −0.000220503 0.032967033 k_Bacteria; Healthy p_Protcobactcria: c_Gammaproteobacteria; _Enterobacteriale s; f_Enterobacteriaceae; g_; s.sub.— 195258 UP in TRUE 0.000463044 0.000999001 k_Bacteria; CMA p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; g _Bacteroides; s_ovatus 315846 UP in TRUE 0.0009126 0.012987013 k_Bacteria; CMA p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_[Bamesiellaceael; g_; s.sub.— 551902 UP in TRUE 0.001063128 0.002997003 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; g_; s.sub.— 318190 UP in TRUE 0.001133533 0.002997003 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; g_; s.sub.— 591635 UP in TRUE 0.001212852 0.004995005 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; g_Ruminococcus; s.sub.— 585227 UP in TRUE 0.001492734 0.022977023 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; g_Oscillospira; s.sub.— 370225 UP in TRUE 0.001699313 0.008991009 k_Bacteria; CMA p_Actinobactcria: c_Actinobacteria; o_Bifidobacteriales; f_Bifidobacteriaceae; g_Bifidobacterium; s_adolescentis 199354 UP in TRUE 0.003134517 0.012987013 k_Bacteria; CMA p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_ [Bamesiellaceael; g_; s.sub.— 198866 UP in TRUE 0.003516506 0.002997003 k_Bacteria; CMA p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Porphyromonadaceae; g_Parabacteroides; s.sub.— 583398 UP in TRUE 0.003665419 0.000999001 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_ [Ruminococcusl; s.sub.— 583656 UP in TRUE 0.003887232 0.000999001 k_Bacteria; CMA p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; g_Bacteroides; s.sub.— 535375 UP in TRUE 0.004373572 0.000999001 k_Bacteria; CMA p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; g_Bacteroides; s.sub.— 1111191 UP in TRUE 0.004749497 0.021978022 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_[Ruminococcus]; s_gnavus 580629 UP in TRUE 0.005200626 0.017982018 k_Bacteria; CMA p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; g_Bacteroides; s.sub.— 365181 UP in TRUE 0.00614329 0.000999001 k_Bacteria; CMA p_Actinobactcria: c_Coriobacteriia; o_Coriobacteriales; f_Coriobacteriaceae; g_Collinsella; s_aerofaciens 359809 UP in TRUE 0.007206031 0.006993007 k_Bacteria; CMA p_Protcobactcria: c_Betaproteobacteria; o_Burkholderiales; f_Alcaligcnaccac: g_Sutterella; s.sub.— 585914 UP in TRUE 0.007841977 0.001998002 k_Bacteria; CMA p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Porphyromonadaceae; g_Parabacteroides; s_distasonis 365385 UP in TRUE 0.009299774 0.014985015 k_Bacteria; CMA p_Actinobactcria: c_Actinobactcria: o_Bifidobacteriales; f_Bifidobacteriaceae; g_Bifidobacterium; s.sub.— 583117 UP in TRUE 0.009878272 0.000999001 k_Bacteria; CMA p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; g_Bacteroides; s.sub.— 180082 UP in TRUE 0.011856764 0.002997003 k_Bacteria; CMA p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Porphyromonadaceae; g_Parabacteroides; s.sub.— 589071 UP in TRUE 0.016312063 0.002997003 k_Bacteria; CMA p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; g_Bacteroides; s_uniformis 514272 UP in TRUE 0.029745704 0.011988012 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_; s.sub.— 484304 UP in TRUE 0.030173538 0.004995005 k_Bacteria; CMA p_Actinobactcria: c_Actinobactcria: o_Bifidobacteriales; f_Bifidobacteriaceae; g_Bifidobacterium; s.sub.— 589277 UP in TRUE 0.052225784 0.001998002 k_Bacteria; CMA p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; g_Bacteroides; s.sub.—
TABLE-US-00005 Supplementary TABLE 4 32 genes differentially expressed in CMA- and healthy-colonized mouse ileal RNAseq samples, and different from negative control (GF mice), shown in FIG. 3a. RNAseq CMA versus Healthy CMA versus GF Average DEG FDR-adjusted Fold Direction Gene EnsemblGeneID Expression Group P value Change of Change Slc22a13 ENSMUSG00000074028 2.778111515 UP in 0.029753823 −2.401525743 NE Healthy Fbp1 ENSMUSG00000069805 5.555254563 UP in 8.17E−05 −2.063132823 ND Healthy Apcdd1 ENSMUSG00000071847 1.494018834 UP in 0.049270498 −1.609221514 NE Healthy Npl ENSMUSG00000042684 5.041978196 UP in 0.085368856 −1.559896139 NE Healthy Abcc2 ENSMUSG00000025194 9.209485113 UP in 0.010848769 1.505881058 UP in CMA CMA Bhmt ENSMUSG00000074768 1.082085196 UP in 0.071649815 1.509332678 UP in CMA CMA Mfsd2b ENSMUSG00000037336 1.777883345 UP in 0.061865715 1.537225758 UP in CMA CMA Cyp3a59 ENSMUSG00000061292 2.136791204 UP in 0.027389658 1.549562876 UP in CMA CMA Lrrn1 ENSMUSG00000034648 1.595303389 UP in 0.013681852 1.581394892 UP in CMA CMA Slc9b2 ENSMUSG00000037994 1.102430337 UP in 0.085368856 1.605005893 UP in CMA CMA Mroh7 ENSMUSG00000047502 3.89754756 UP in 0.021327414 1.617161783 UP in CMA CMA Grin2a ENSMUSG00000059003 2.458692024 UP in 0.085653544 1.617995585 UP in CMA CMA Cntn1 ENSMUSG00000055022 1.148935328 UP in 0.014593455 1.651618302 UP in CMA CMA Letm2 ENSMUSG00000037363 1.650075654 UP in 0.018797255 1.66583385 UP in CMA CMA Ptafr ENSMUSG00000056529 1.301260118 UP in 0.073471735 1.694815563 UP in CMA CMA Me1 ENSMUSG00000032418 4.3922554 UP in 0.016384547 1.703263564 UP in CMA CMA Smco1 ENSMUSG00000046345 1.23463105 UP in 0.090599686 1.755629553 UP in CMA CMA Cyp2b10 ENSMUSG00000030483 7.581089071 UP in 0.029753823 1.757005744 UP in CMA CMA Cyp2j13 ENSMUSG00000028571 4.136663388 UP in 0.053930421 1.800872438 UP in CMA CMA Acot12 ENSMUSG00000021620 5.494716849 UP in 5.48E−05 2.01501538 UP in CMA CMA Cyp2c29 ENSMUSG00000003053 2.680142327 UP in 8.17E−05 4.735853239 UP in CMA CMA Akr1c19 ENSMUSG00000071551 6.653987069 DOWN in 0.007181957 1.539241268 NE Healthy Gstm1 ENSMUSG00000058135 3.552920591 DOWN in 0.010848769 1.592387077 NE Healthy Ces1f ENSMUSG00000031725 6.194279856 DOWN in 0.027389658 1.893583214 NE Healthy Ces1g ENSMUSG00000057074 2.742386148 DOWN in 0.090559257 1.907760384 ND Healthy Tgfbr3 ENSMUSG00000029287 1.883866476 DOWN in 0.000408545 −2.74659932 DOWN in CMA CMA Acta1 ENSMUSG00000031972 1.201617812 DOWN in 0.027389658 −2.50720332 DOWN in CMA CMA Hist1h2bj ENSMUSG00000069300 1.79373615 DOWN in 0.072249982 −1.935238841 DOWN in CMA CMA Gp2 ENSMUSG00000030954 0.806917328 DOWN in 0.070122494 −1.870775326 DOWN in CMA CMA Ddit4 ENSMUSG00000020108 3.745786556 DOWN in 0.070340591 −1.576061582 DOWN in CMA CMA Hist1h2bn ENSMUSG00000095217 3.070162335 DOWN in 0.08015986 −1.565406563 DOWN in CMA CMA Ror2 ENSMUSG00000021464 2.322930094 DOWN in 0.03422688 −1.545471585 DOWN in CMA CMA CMA versus GF Healthy versus GF FDR-adjusted Fold Direction FDR-adjusted Fold Gene P value Change of Change P value Change Slc22a13 0.063113496 3.40496861 UP in 0.005425032 8.17711977 Healthy Fbp1 0.000581517 2.373595096 UP in 2.38E−07 4.897041951 Healthy Apcdd1 0.435864462 1.265635652 UP in 0.025785895 2.03668812 Healthy Npl 0.293803914 1.388291017 UP in 0.024969934 2.165589797 Healthy Abcc2 2.04E−05 2.175407835 ND 0.042313216 1.444608007 Bhmt 0.010731326 1.975422585 NE 0.410644089 1.308805284 Mfsd2b 0.000867035 2.82533517 NE 0.075749268 1.837944202 Cyp3a59 3.05E−05 3.39446921 ND 0.009662128 2.190597918 Lrrn1 0.007383553 1.714726833 NE 0.777501394 1.084312869 Slc9b2 0.031991401 2.02570437 NE 0.591859824 1.262116469 Mroh7 2.72E−03 2.033697925 NE 0.42241223 1.257572339 Grin2a 0.044336212 1.964067568 NE 0.676602939 1.213889325 Cntn1 0.005875115 1.875544221 NE 0.68375077 1.135579703 Letm2 0.001104306 2.371462071 NE 0.242181977 1.423588595 Ptafr 0.021173615 2.232120867 NE 0.540574153 1.317028776 Me1 0.03823817 1.603254667 NE 0.863312513 −1.062378672 Smco1 0.026988488 2.508688181 NE 0.501298796 1.428939367 Cyp2b10 0.009227326 2.023601722 NE 0.699603833 1.151733129 Cyp2j13 0.001587354 3.68957984 NE 0.120415488 2.048773562 Acot12 5.70E−05 2.402984772 NE 0.462442117 1.192539171 Cyp2c29 6.17E−03 3.403244621 NE 0.593296835 −1.391570036 Akr1c19 0.08837154 −1.323738676 DOWN in 0.000475667 −2.037553198 Healthy Gstm1 0.988990596 −1.002868547 DOWN in 0.020964575 −1.596954914 Healthy Ces1f 0.186446914 −1.487066804 DOWN in 0.00373614 −2.815884739 Healthy Ces1g 0.030963104 −1.97871865 DOWN in 0.001377 −3.774921051 Healthy Tgfbr3 0.007704498 −2.236046082 NE 0.553319278 1.228328585 Acta1 0.006712918 −2.803646543 NE 0.822471304 −1.118236611 Hist1h2bj 0.000113721 −3.822281003 ND 0.039087358 −1.97509523 Gp2 0.001158966 −2.871474818 NE 0.208403405 −1.534911637 Ddit4 0.037955029 −1.679764946 NE 0.861431628 −1.065799056 Hist1h2bn 0.000151693 −2.672909018 ND 0.037500512 −1.707485507 Ror2 7.86E−05 −2.277636991 NE 0.05497608 −1.473748863 NE: Not Evaluable; when DEG does not meet the filtering criteria in either comparison “CMA vs GF” or “Healthy vs GF”, Change of Direction is not evaluable. ND: Not Determined; when DEG meets the filtering criteria in both comparison “CMA vs GF” and “Healthy vs GF”, the one with higher P value is reset to not determined to resolve conflicts.
TABLE-US-00006 Supplementary Table 5 108 OTUs differentially abundant between CMA and healthy mouse ileal samples. Change DS- DS-FDR DS-FDR In FDR Test Raw OTU Direction Reject Statistic Pvalues Taxonomy 349024 UP in TRUE −0.22217917 0.000999001 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s.sub.— 259772 UP in TRUE 0.044282455 0.000999001 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Coprococcus; s.sub.— 828483 UP in TRUE 0.017799339 0.021978022 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_Clostridium 262095 UP in TRUE 0.015344578 0.001998002 k_Bacteria; Healthy p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_; s.sub.— 332718 UP in TRUE 0.012123767 0.001998002 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s.sub.— 360238 UP in TRUE 0.007986609 0.011988012 k_Bacteria; Healthy p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; _Erysipelotrichaceae; g_; s.sub.— 238205 UP in TRUE 0.004499814 0.000999001 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_Clostridium; s_butyricum 359750 UP in TRUE 0.004188868 0.021978022 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_Clostridium; s_perfringens New.ReferenceOTU16 UP in TRUE 0.004114455 0.000999001 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s_luteciae 309696 UP in TRUE 0.003306804 0.003996004 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s.sub.— 345540 UP in TRUE 0.003062398 0.000999001 k_Bacteria; Healthy p_Proteobacteria; c_Gammaproteobacteria; o_Enterobacteriales; f_Enterobacteriaceae; g_; s.sub.— 586271 UP in TRUE 0.002603169 0.01998002 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_[Ruminococcusl; s.sub.— 356760 UP in TRUE 0.002063474 0.000999001 k_Bacteria; Healthy p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_; s.sub.— 303809 UP in TRUE 0.001887759 0.000999001 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_Clostridium; s_butyricum 327851 UP in TRUE −0.00174762 0.001998002 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s.sub.— New.Reference UP in TRUE 0.001578146 0.000999001 k_Bacteria; OTU18 Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Coprococcus; s.sub.— 291195 UP in TRUE 0.001382348 0.000999001 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s.sub.— 577710 UP in TRUE 0.000950458 0.010989011 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Blautia; s_producta 292820 UP in TRUE 0.000949102 0.000999001 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s.sub.— 573157 UP in TRUE 0.000929379 0.011988012 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales 529116 UP in TRUE 0.000921743 0.001998002 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_Clostridium 579608 UP in TRUE 0.000882353 0.001998002 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s.sub.— 289925 UP in TRUE 0.000678999 0.000999001 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s.sub.— 177986 UP in TRUE 0.000602909 0.028971029 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Coprococcus; s.sub.— New.CleanUp.Reference UP in TRUE 0.000594549 0.003996004 k_Bacteria; OTU64312 Healthy p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_; s.sub.— 967427 UP in TRUE 0.000546212 0.001998002 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s.sub.— 237444 UP in TRUE 0.000487754 0.000999001 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s.sub.— 328283 UP in TRUE 0.000459212 0.003996004 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s.sub.— 145801 UP in TRUE −0.00045917 0.000999001 k_Bacteria; Healthy p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_; s.sub.— New.ReferenceOTUI9 UP in TRUE 0.000449108 0.026973027 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s.sub.— 592866 UP in TRUE 0.000408332 0.017982018 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_[Ruminococcusl; s.sub.— 4313711 UP in TRUE −0.00035444 0.000999001 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s.sub.— New.ReferenceOTU26 UP in TRUE 0.000337736 0.004995005 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Coprococcus; s.sub.— 351020 UP in TRUE 0.000324044 0.014985015 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_Clostridium; s_neonatale 833731 UP in TRUE 0.000280741 0.000999001 k_Bacteria; Healthy p_Proteobacteria; c_Gammaproteobacteria; o_Enterobacteriales; f_Enterobacteriaceae; g_; s.sub.— 392887 UP in TRUE 0.000260077 0.025974026 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_; s.sub.— 199301 UP in TRUE 0.000256085 0.01998002 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_; s.sub.— New.CleanUp.ReferenceOTU98242 UP in TRUE 0.000237095 0.002997003 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_; s.sub.— 255359 UP in TRUE −0.00020765 0.007992008 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s.sub.— New.CleanUp.ReferenceOTU22852 UP in TRUE 0.000197185 0.010989011 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Coprococcus; s.sub.— New.ReferenceOTU72 UP in TRUE −0.00018357 0.021978022 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Coprococcus; s.sub.— 132041 UP in TRUE 0.000168657 0.017982018 k_Bacteria; Healthy p_Actinobactcria: c_Actinobactcria: o_Bifidobactcrialcs: f_Bifidobacteriaceae; g_Bifidobacterium; s.sub.— New.CleanUp.ReferenceOTUI02634 UP in TRUE 0.000163884 0.022977023 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s.sub.— New.CleanUp.ReferenceOTU48619 UP in TRUE −7.42886E−05 0.014985015 k_Bacteria; Healthy p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Coprococcus; s.sub.— New.CleanUp.ReferenceOTU92891 UP in TRUE −7.35692E−05 0.022977023 k_Bacteria; Healthy p_Firmicutes; c_Bacilli; o_Lactobacillales; f_Streptococcaceae; g_Streptococcus; s.sub.— 77514 UP in TRUE 0.000105274 0.028971029 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Blautia; s.sub.— 560336 UP in TRUE 0.000118911 0.028971029 k_Bacteria; CMA p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; g_Bacteroides; s.sub.— 566434 UP in TRUE 0.000132797 0.016983017 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_;g_; s.sub.— 4352657 UP in TRUE 0.000145962 0.022977023 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Blautia; s.sub.— 367790 UP in TRUE 0.000158719 0.028971029 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Blautia; s.sub.— 3768338 UP in TRUE 0.000171334 0.028971029 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Blautia; s.sub.— 311587 UP in TRUE 0.000184786 0.007992008 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_; s.sub.— 273232 UP in TRUE 0.000198366 0.022977023 k_Bacteria; CMA p_Vcrrucomicrobia: c_Verrucomicrobiae; o_Verrucomicrobiales; f_Verrucomicrobiaceae; g_Akkermansia; s_muciniphila 804526 UP in TRUE 0.000198943 0.021978022 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Coprococcus; s.sub.— 331850 UP in TRUE 0.000211798 0.022977023 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g Blautia; s.sub.— 4434334 UP in TRUE 0.000236036 0.003996004 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_; s.sub.— 4422039 UP in TRUE 0.000259764 0.002997003 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae 343047 UP in TRUE 0.000280211 0.00999001 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Dorea; s.sub.— 295085 UP in TRUE 0.000318338 0.021978022 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Blautia; s.sub.— 195061 UP in TRUE 0.000318762 0.011988012 k_Bacteria; CMA p_Actinobactcria: c_Coriobacteriia; o_Coriobacteriales; f_Coriobacteriaceae; g_Adlercreutzia; s.sub.— 362539 UP in TRUE 0.000376855 0.015984016 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_; s.sub.— New.CleanUp.ReferenceOTU35999 UP in TRUE 0.000385001 0.026973027 k_Bacteria; CMA p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_[Eubacterium]; s_dolichum 587933 UP in TRUE 0.000403677 0.000999001 k_Bacteria; CMA p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_Coprobacillus; s.sub.— 3002161 UP in TRUE 0.000470934 0.01998002 k_Bacteria; CMA p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_[Eubacterium]; s_dolichum 3715618 UP in TRUE 0.000529033 0.008991009 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_[Ruminococcus]; s_gnavus 591635 UP in TRUE 0.000561008 0.000999001 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; g_Ruminococcus; s.sub.— 249142 UP in TRUE 0.000585252 0.027972028 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_[Ruminococcusl; s.sub.— 634449 UP in TRUE 0.000585747 0.018981019 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_; s.sub.— 318190 UP in TRUE 0.000620436 0.00999001 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; g_; s.sub.— 548587 UP in TRUE 0.000660208 0.014985015 k_Bacteria; CMA p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_[Eubacterium]; s_dolichum New.ReferenceOTU4 UP in TRUE 0.000693503 0.013986014 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_[Ruminococcus]; s_gnavus New.CleanUp.ReferenceOTUI1241 UP in TRUE 0.000784602 0.002997003 k_Bacteria; CMA p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_[Eubacterium]; s_dolichum 535955 UP in TRUE 0.000789934 0.002997003 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Peptostreptococcaceae; g_; s.sub.— 551902 UP in TRUE 0.000807109 0.002997003 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; g_; s.sub.— 535601 UP in TRUE 0.000857422 0.002997003 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_; s.sub.— 269611 UP in TRUE 0.000862436 0.016983017 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; g_; s.sub.— 175508 UP in TRUE 0.000942059 0.005994006 k_Bacteria; CMA p_Actinobactcria: c_Coriobacteriia; o_Coriobacteriales; f_Coriobacteriaceae; g_; s.sub.— 585914 UP in TRUE 0.001135819 0.014985015 k_Bacteria; CMA p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Porphyromonadaceae; g_Parabacteroides; s_distasonis 587530 UP in TRUE 0.001147768 0.004995005 k_Bacteria; CMA p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_[Eubacterium]; s_dolichum 659361 UP in TRUE 0.001476101 0.002997003 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Dorea; s.sub.— New.ReferenceOTU85 UP in TRUE 0.001499115 0.010989011 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Dorea; s.sub.— 193744 UP in TRUE 0.001689778 0.017982018 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Blautia; s.sub.— 583656 UP in TRUE 0.001702377 0.014985015 k_Bacteria; CMA p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; g_Bacteroides; s 631764 UP in TRUE 0.001846097 0.002997003 k_Bacteria; CMA p_Actinobactcria: c_Coriobacteriia; o_Coriobacteriales; f_Coriobacteriaceae; g_Adlercreutzia; s.sub.— 564188 UP in TRUE 0.001945213 0.001998002 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Dorea; s.sub.— 531675 UP in TRUE 0.001946525 0.022977023 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Blautia; s.sub.— New.ReferenceOTU35 UP in TRUE 0.002345204 0.024975025 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_; s.sub.— 572860 UP in TRUE 0.002838657 0.001998002 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_[Ruminococcusl; s_gnavus 362342 UP in TRUE 0.00291165 0.02997003 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; g_Ruminococcus; s.sub.— 929836 UP in TRUE 0.003147941 0.000999001 k_Bacteria; CMA p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_Coprobacillus; s.sub.— 1111191 UP in TRUE 0.003451219 0.000999001 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_[Ruminococcus]; s_gnavus 535375 UP in TRUE 0.003453613 0.008991009 k_Bacteria; CMA p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; g_Bacteroides; s.sub.— 551822 UP in TRUE 0.004090919 0.005994006 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae 368486 UP in TRUE 0.004218387 0.003996004 k_Bacteria; CMA p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_[Eubacterium]; s_dolichum 583398 UP in TRUE 0.00561987 0.000999001 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_[Ruminococcus]; s.sub.— New.ReferenceOTUI66 UP in TRUE 0.006055017 0.024975025 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae 367213 UP in TRUE 0.006097534 0.022977023 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; g_; s.sub.— 546876 UP in TRUE 0.009753471 0.022977023 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Blautia; s.sub.— 369763 UP in TRUE 0.010722879 0.00999001 k_Bacteria; CMA p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_Coprobacillus; s.sub.— 829337 UP in TRUE 0.011237604 0.011988012 k_Bacteria; CMA p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_Coprobacillus; s.sub.— 592616 UP in TRUE 0.013715698 0.000999001 k_Bacteria; CMA p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_; s.sub.— 582691 UP in TRUE 0.01557264 0.002997003 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_; s.sub.— 628226 UP in TRUE 0.024223372 0.004995005 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; g_; s.sub.— 1078587 UP in TRUE 0.032792882 0.015984016 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Blautia; s.sub.— 370183 UP in TRUE 0.044102787 0.01998002 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_Blautia; s.sub.— 363731 UP in TRUE 0.055865924 0.025974026 k_Bacteria; CMA p_Verrucomicrobia: c_Verrucomicrobiae; o_Verrucomicrobiales; f_Verrucomicrobiaceae; g_Akkermansia; s_muciniphila 360015 UP in TRUE 0.07274586 0.022977023 k_Bacteria; CMA p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; g_[Ruminococcusl; s_gnavus 579851 UP in TRUE 0.082564668 0.000999001 k_Bacteria; CMA p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; f_Erysipelotrichaceae; g_[Eubacteriuml; s_dolichum
TABLE-US-00007 Supplementary TABLE 6 Primer sequence used for qPCR analysis of Anaerostipes caccae Anaerostipes F GTTTTCGGATGG Ref 24. caccae ATTTCCTATAT (SEQ ID NO: 1) R CTTTTCACACTGA ATCATGCGATT (SEQ ID NO: 2)
TABLE-US-00008 SUPPLEMENTARY TABLE 7 Primer sequences used for qPCR analysis in FIG. 4f Hprt F TGAAGAGCTACT Ref. 50. GTAATGATCAGT CAAC (SEQ ID NO: 3) R AGCAAGCTTGCA ACCTTAACCA (SEQ ID NO: 4) Fbp1 F CCATCATAATCG Ref. 51. AACCTGAG (SEQ ID NO: 5) R CTTCTCAGAAGG CTCATCAG (SEQ ID NO: 6) Tgfbr3 F CCGGTGTGAACT Modified GTCACCGATC from Ref. (SEQ ID 52 NO: 7) R CCTGATGAAAAC TGGACCACAG (SEQ ID NO: 8) Ror2 F ATCGACACCTTG Ref. 20. GGACAACC (SEQ ID NO: 9) R AGTGCAGGATTG CCGTCTG (SEQ ID NO: 10) Acot12 F CCGTGGCACTAA Ref. 53. GGTCAGTT (SEQ ID NO: 11) R ACGTTACGGTGC ACGAATTG (SEQ ID NO: 12) Mel F AGTATCCATGAAC Ref. 54. AAAGGGCAC (SEQ ID NO: 13) R ATCCCATTACAGC CAAGGTC (SEQ ID NO: 14)
Example 2: Isolation and Characterization, In Vitro Fermentation, Initial In Vivo Experimental Design
[0122] A. A. caccae Isolation and Characterization
[0123] A. caccae is isolated from healthy infant feces frozen in 30% glycerol stock. First, undiluted fecal glycerol stock from healthy donor #2 is plated on brain heart infusion (BHI) agar supplemented with Vitamin K, hemin, and antibiotics. The antibiotics used are 16 μg/ml ciprofloxacin or a mixture of 16 μg/ml ciprofloxacin, 6 μg/ml gentamicin, 5 μg/ml aztreonam, and 10 μg/ml colistin (58, 60). This donor was chosen because he had the highest relative abundance of A. caccae of the four healthy donors from previous sequencing data. Plates are incubated in anaerobic chambers at 37° C. for six days. The plates are then scraped and suspended in 50% glycerol solution. This suspension is then aliquoted and one aliquot is used for qPCR and the other is frozen at −80° C. to be used for future cultures. A. caccae abundance is quantified using qPCR with species-specific primers against the 16S rRNA gene (24). The frozen aliquot of the suspension from the plate(s) shown to have the highest abundance of A. caccae are diluted and plated on BHI agar and incubated in anaerobic chambers at 37° C. for six days. Single colonies are then selected from the 10.sup.−5 dilution and inoculated into pre-reduced chopped meat and glucose broth (CMG) and incubated in an anaerobic chamber overnight. An aliquot of this broth culture is then then taken for A. caccae specific PCR and universal PCR 16S rRNA amplification for Sanger sequencing. Another aliquot is taken and diluted 1:1 with 50% glycerol solution and frozen at −80° C. in cryovials. Colonies that are positive for A. caccae by both PCR and Sanger sequencing are then streaked onto BHI agar from the frozen cryovial to increase purity. A single colony from this plate is then inoculated into chopped meat+glucose broth and grown overnight at 37° C. in the anaerobic chamber. Aliquots of this stock culture are diluted 1:1 in 50% glycerol and stored in cryovials at −80° C. for future studies. This isolation is shown in
[0124] Further confirmation of A. caccae isolation is analyzed by CosmosID® whole genome analysis. The method described in
[0125] Antibiotic susceptibility of A. caccae_lah was characterized by growth inhibition around antibiotic discs. An ice-chip of the frozen A. caccae_lah stock is grown in CMG broth overnight and 100 μl of broth is spread on BHI agar. Antibiotic discs (10 μg streptomycin, 30 μg kanamycin, 30 μg tetracycline and 10 μg ampicillin) are then placed on top of the agar to measure zone of clearance which demonstrates antibiotic susceptibility. Plates are incubated at 37° C. in anaerobic conditions for six days. A. caccae_lah is highly susceptible to ampicillin as shown by the large radius of clearance (
[0126] B. In Vitro Fermentation
[0127] A. caccae_lah is not able to ferment complex carbohydrates in monoculture but can ferment simple sugars like those in infant formula, including lactose. An ice-chip of the A. caccae_lah stock was grown for 24 h in CMG broth at 37° C. in anaerobic conditions. This method of preculture is used for all future in vitro fermentation experiments. Then 10 μl of preculture was transferred into 7 mL minimal peptone yeast (PY) broth alone or supplemented with 10 mg/ml of glucose, sucrose, lactose, cellobiose, or potato starch. All of these variations of PY were pre-reduced anaerobically prepared by Anaerobe Systems. Growth and butyrate production were measured after 48 h. A. caccae_lah grew abundantly when PY broth was supplemented with sucrose, glucose, and lactose compared to cellobiose or potato starch as measured by OD600. Butyrate in solution is quantified by HPLC-UV-Vis as described in reference 61 and 62. Similarly to growth, A. caccae_lah produced the most butyrate when grown in sucrose, glucose, or lactose. These simple sugars are similar to those found in infant formula. To test the abundance of A. caccae_lah and its butyrate production in infant formula, A. caccae_lah was grown in PY broth supplemented with Nutramigen® (10 mg/ml carbohydrates), an iron-fortified, hypoallergenic infant formula designed for infants with CMA, or Enfamil® (10 mg/ml carbohydrates), standard infant formula containing cow's milk. These are the same formulas consumed by the healthy (Enfamil) and CMA (Nutramigen) infant donors and are also consumed by all mice colonized with these respective microbiomes. A. caccae_lah abundance was similar when grown with Enfamil or Nutramigen as PY broth, proving that A. caccae_lah does not depend on consuming cow's milk for growth. However, butyrate production was highest in Nutramigen compared to PY broth alone. The majority sugar in Nutramigen is corn syrup which contains fructose and sucrose, which mirrors the high butyrate production by A. caccae_lah when supplemented with sucrose alone. All groups were analyzed by one way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 versus PY. These results are shown in
[0128] A. caccae_lah is able to use lactate and acetate together to produce butyrate. A. caccae and other Clostridia are generally considered as fiber fermenting species, however as seen in previous data A. caccae_lah alone is not able to consume complex fibers for growth or butyrate production. Other groups have shown that primary degraders, namely Bacteroides species, break down complex fibers and produce metabolites lactate and acetate which Clostridia species can then consume to produce butyrate (59). To examine whether this complex cross-feeding could be mimicked in vitro, PY broth is supplemented with only these metabolites to stimulate butyrate production by A. caccae_lah. A. caccae_lah preculture was grown from frozen glycerol stock as described above, then 10 μl was transferred into minimal PY broth supplemented with 33 mM acetate and/or 40 mM lactate (57). Growth and butyrate production were measured after 48 h. As shown in
[0129] As shown in
[0130] The inventors determined if the bacterial mix from the feces of CMA-colonized repository mice would behave similarly to the human CMA mix, because the feces of these repository mice will be used to colonize all future CMA mice. Fecal pellets from repository mice previously colonized from CMA donor 6 were collected, homogenized in sterile PBS, then diluted 1:1 in 50% glycerol solution and frozen at −80° C. in cryovials. Precultures of A. caccae_lah, human CMA microbiota (hCMA) and mouse CMA microbiota (msCMA) were prepared as described above. Precultures were then inoculated into PY broth with or without supplements alone (10 ul), together (5 ul A. caccae+5 ul CMA), or A. caccae_lah and supplements were added 24 h later. The supplements (Nutramigen, lactate/acetate) were added in the same concentration as previously described, and approximately 10 mg/ml wheat bran was used. Growth and butyrate production were measured at t=48 h or 72 h, to allow full 48 h growth of A. caccae in the group in which it was added at the later time point. Neither CMA mix had measurable A. caccae, as expected. The bacterial mix from the CMA repository mouse feces produces more butyrate at baseline than freshly thawed human CMA feces, but addition of A. caccae_lah with lactate and acetate still results in a notable increase in butyrate concentration (
[0131] A. caccae_lah is able to grow when inoculated in lower abundance in vitro. In this series of experiments, the inventors determined if A. caccae_lah would still be able to grow in co-culture when inoculated with lower abundance than the CMA mix, as A. caccae will be in much lower relative abundance when introduced into a CMA-colonized mouse. This experiment was performed to reflect the reality of introducing A. caccae_lah into a CMA-colonized mouse more accurately than a simultaneous, 1:1 inoculation. A. caccae_lah or the CMA pre-cultures were performed as described herein, then 10 μl was transferred into minimal PY broth supplemented with 10 mg/ml carbohydrates (10 mg/ml carbohydrates Nutramigen, Nutramigen plus 10 mg/ml scFOS (short chain fructo-oligosaccharide, a clinical prebiotic), or Nutramigen plus LA (40 mM lactate+33 mM acetate)). A. caccae_lah and CMA precultures were transferred in to supplemented PY broth or CMG broth in various ratios: 10 ul A. caccae_lah:0 ul CMA; 5 ul A. caccae_lah: 5 ul CMA; 3 ul A. caccae_lah:7 ul CMA; 1 ul A. caccae_lah:9 ul CMA. As shown by species-specific qPCR, the volume of A. caccae_lah didn't notably affect its expansion or butyrate production. Addition of the scFOS decreased total butyrate concentration compared to media with Nutramigen alone. The addition of lactate and acetate results in the greatest butyrate concentration. The amount of butyrate in this media is greater in co-culture than in A. caccae_lah alone, and in the A. caccae_lah monoculture there is no detectable lactate or acetate in the media at 48 h. This suggests that the CMA species in culture are contributing additional lactate and acetate to the system, which may allow continued butyrate production by A. caccae_lah after the supplemented metabolites are depleted.
[0132] Before beginning in vivo colonization experiments, the presence of A. caccae in the healthy repository mice and absence in CMA repository mice was confirmed and measured by qPCR. Repository mice colonized with feces from healthy infant donor 2 show measurable A. caccae abundance by qPCR, while repository mice colonized with feces from CMA donor 6 do not. The inventors predict that providing A. caccae_lah to CMA colonized mice with the prebiotic supplements will allow the species to grow and establish a niche in the host. Ideally, the inventors would be able to administer A. caccae_lah such that the abundance of A. caccae_lah in CMA colonized mice reaches a level similar to that detected in healthy colonized mice.
[0133] C. Experimental Plan for Initial In Vivo Studies (
[0134] In this experiment, the inventors aim to validate colonization of A. caccae_lah into CMA-colonized mice and its dependence on prebiotic supplements. Germ free (GF) C3H/HeN germ free mice are weaned and fed with Nutramigen. At the same time, mice are given a fecal slurry from CMA repository mouse (human donor 6) by intragastric gavage (I.G.) to establish the CMA-microenvironment in the mouse model. The CMA microbes are given 7 days to colonize. The mice receive a continuous supply of Nutramigen throughout the experiment. The ad libitum feeding of the mice with Nutramigen, along with additional lactate/acetate or carbohydrate supplements may provide enough continuous substrate to help A. caccae colonize. Beginning on day 7 after weaning, mice receive I.G. gavage of both A. caccae_lah and prebiotic or control. Cohorts of mice receive 100 ul of PBS (control) or one prebiotic supplement (10 mg/ml lactate plus 10 mg/ml acetate or 10 mg/ml potato starch) just after fecal collection. Thirty minutes later, mice receive 250 ul of live biotherapeutic product (LBP, i.e. approximately 1×10.sup.6 CFU A. caccae_lah), or the same volume of sterile CMG broth in glycerol as the negative control. After the first week, gavage of the LBP is stopped, but the prebiotic (or control) continues to be administered for another week. This is to determine whether administration of the prebiotic is sufficient to maintain the LBP (A. caccae_lah) population in the mouse without introducing more bacteria. Fecals are collected daily during this two week period to analyze abundance of A. caccae_lah by qPCR and fecal butyrate concentration by HPLC UV-Vis. Mice are sacrificed at day 42 or at the first timepoint that A. caccae abundance is not detected in the feces. After sacrifice, cecal butyrate is measured and expression of the genes Ror2, Fbp1, Tgfbr3, Acot1, and Me1 are analyzed in ileal intestinal epithelial cells (iIECs) by qPCR. Cecal butyrate is normally a more sensitive measure than fecal butyrate, and may be a better measure of butyrate production by A. caccae_lah in vivo because most butyrate produced in the colon is immediately consumed by coloncytes. These specific genes are chosen for analysis in iIECs because they are immunologically relevant and were shown to be differentially expressed between healthy-, CMA-, and A. caccae-colonized mice (See
[0135] As an additional experiment, the inventors may aim to enhance the colonization by A. caccae_lah by additionally delivering a butyrate carrying compound to the mice by I.G. gavage. Examples of butyrate carrying compounds are described in published PCT application WO 2018/195067 A1, Hubbell et al. A solution of 80 mg/mL of the butyrate delivering polymer pHPMA-b-pBMA is prepared and diluted to 53.3 mg/mL as described in Hubbell et al. In the experiment described directly above [paragraph 129], the cohorts of mice receiving the prebiotic supplement also receive 125 uL of this diluted butyrate delivering polymer solution by I.G. gavage immediately after receiving the prebiotic supplement. The remainder of the experiment is carried out as described above.
[0136] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
REFERENCES
[0137] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. [0138] 1. Sicherer, S. H., et al. Critical Issues in Food Allergy: A National Academies Consensus Report. Pediatrics (2017). [0139] 2. Iweala, O. I. & Burks, A. W. Food Allergy: Our Evolving Understanding of Its Pathogenesis, Prevention, and Treatment. Curr Allergy Asthma Rep 16, 37 (2016). [0140] 3. Wesemann, D. R. & Nagler, C. R. The Microbiome, Timing, and Barrier Function in the Context of Allergic Disease. Immunity 44, 728-738 (2016). [0141] 4. Plunkett, C. H. & Nagler, C. R. The Influence of the Microbiome on Allergic Sensitization to Food. J Immunol 198, 581-589 (2017). [0142] 5. Berni Canani, R., et al. Lactobacillus rhamnosus GG-supplemented formula expands butyrate-producing bacterial strains in food allergic infants. Isme j 10, 742-750 (2016). [0143] 6. Bunyavanich, S., et al. Early-life gut microbiome composition and milk allergy resolution. J Allergy Clin Immunol 138, 1122-1130 (2016). [0144] 7. Stefka, A. T., et al. Commensal bacteria protect against food allergen sensitization. Proc Natl Acad Sci USA 111, 13145-13150 (2014). [0145] 8. Dominguez-Bello, M. G., et al. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci USA 107, 11971-11975 (2010). [0146] 9. Mueller, N. T., Bakacs, E., Combellick, J., Grigoryan, Z. & Dominguez-Bello, M. G. The infant microbiome development: mom matters. Trends Mol Med 21, 109-117 (2015). [0147] 10. Blanton, L. V., et al. Gut bacteria that prevent growth impairments transmitted by microbiota from malnourished children. Science 351(2016). [0148] 11. Cahenzli, J., Koller, Y., Wyss, M., Geuking, M. B. & McCoy, K. D. Intestinal microbial diversity during early-life colonization shapes long-term IgE levels. Cell Host Microbe 14, 559-570 (2013). [0149] 12. Pabst, O. & Mowat, A. M. Oral tolerance to food protein. Mucosal Immunol 5, 232-239 (2012). [0150] 13. Honda, K. & Littman, D. R. The microbiota in adaptive immune homeostasis and disease. Nature 535, 75-84 (2016). [0151] 14. Thaiss, C. A., Zmora, N., Levy, M. & Elinav, E. The microbiome and innate immunity. Nature 535, 65-74 (2016). [0152] 15. Yanez, A. J., et al. Broad expression of fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase provide evidence for gluconeogenesis in human tissues other than liver and kidney. J Cell Physiol 197, 189-197 (2003). [0153] 16. Ostroukhova, M., et al. The role of low-level lactate production in airway inflammation in asthma. Am J Physiol Lung Cell Mot Physiol 302, L300-307 (2012). [0154] 17. Zhu, Y., et al. NPM1 activates metabolic changes by inhibiting FBP1 while promoting the tumorigenicity of pancreatic cancer cells. Oncotarget 6, 21443-21451 (2015). [0155] 18. Berger, C. N., et al. Citrobacter rodentium Subverts ATP Flux and Cholesterol Homeostasis in Intestinal Epithelial Cells In Vivo. Cell Metab (2017). [0156] 19. Zhang, M., Zola, H., Read, L. & Penttila, I. Identification of soluble transforming growth factor-beta receptor III (sTbetaIII) in rat milk. Immunol Cell Biol 79, 291-297 (2001). [0157] 20. Miyoshi, H., Ajima, R., Luo, C. T., Yamaguchi, T. P. & Stappenbeck, T. S. Wnt5a potentiates TGF-beta signaling to promote colonic crypt regeneration after tissue injury. Science 338, 108-113 (2012). [0158] 21. Planer, J. D., et al. Development of the gut microbiota and mucosal IgA responses in twins and gnotobiotic mice. Nature 534, 263-266 (2016). [0159] 22. Schwiertz, A., et al. Anaerostipes caccae gen. nov., sp. nov., a new saccharolytic, acetate-utilising, butyrate-producing bacterium from human faeces. Systematic and applied microbiology 25, 46-51 (2002). [0160] 23. Duncan, S. H., Louis, P. & Flint, H. J. Lactate-utilizing bacteria, isolated from human feces, that produce butyrate as a major fermentation product. Appl Environ Microbiol 70, 5810-5817 (2004). [0161] 24. Kurakawa, T., et al. Diversity of Intestinal Clostridium coccoides Group in the Japanese Population, as Demonstrated by Reverse Transcription-Quantitative PCR. PLoS One 10, e0126226 (2015). [0162] 25. Donohoe, D. R., et al. The microbiome and butyrate regulate energy metabolism and autophagy in the mammalian colon. Cell Metab 13, 517-526 (2011). [0163] 26. Byndloss, M. X., et al. Microbiota-activated PPAR-gamma signaling inhibits dysbiotic Enterobacteriaceae expansion. Science 357, 570-575 (2017). [0164] 27. Donohoe, D. R., Wali, A., Brylawski, B. P. & Bultman, S. J. Microbial regulation of glucose metabolism and cell-cycle progression in mammalian colonocytes. PLoS One 7, e46589 (2012). [0165] 28. Atarashi, K., et al. Induction of colonic regulatory T cells by indigenous Clostridium species. Science 331, 337-341 (2011). [0166] 29. Atarashi, K., et al. Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota. Nature 500, 232-236 (2013). [0167] 30. Furusawa, Y., et al. Commensal microbe-derived butyrate induces differentiation of colonic regulatory T cells. Nature 504, 446-450 (2013). [0168] 31. Yano, J. M., et al. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell 161, 264-276 (2015). [0169] 32. Kim, Y. G., et al. Neonatal acquisition of Clostridia species protects against colonization by bacterial pathogens. Science 356, 315-319 (2017). [0170] 33. Noval Rivas, M., et al. A microbiota signature associated with experimental food allergy promotes allergic sensitization and anaphylaxis. J Allergy Clin Immunol 131, 201-212 (2013). [0171] 34. Caporaso, J. G., et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6, 1621-1624 (2012). [0172] 35. Caporaso, J. G., et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7, 335-336 (2010). [0173] 36. DeSantis, T. Z., et al. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 72, 5069-5072 (2006). [0174] 37. Caporaso, J. G., et al. PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics 26, 266-267 (2010). [0175] 38. Edgar, R. C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460-2461 (2010). [0176] 39. Oksanen, J., et al. vegan: Community Ecology Package. R package version 2.4.5. (2017). [0177] 40. Jiang, L., et al. Discrete False-Discovery Rate Improves Identification of Differentially Abundant Microbes. mSystems 2(2017). [0178] 41. Segata, N., et al. Metagenomic biomarker discovery and explanation. Genome biology 12, R60 (2011). [0179] 42. Bashir, M. E., Louie, S., Shi, H. N. & Nagler-Anderson, C. Toll-like receptor 4 signaling by intestinal microbes influences susceptibility to food allergy. J Immunol 172, 6978-6987 (2004). [0180] 43. Nik, A. M. & Carlsson, P. Separation of intact intestinal epithelium from mesenchyme. Biotechniques 55, 42-44 (2013). [0181] 44. Andrew, S. FastQC: A quality control application for high throughput sequence data. Babraham Institute Project page: on the world wide web at wwwbioinformaticsbabrahamacuk/projects/fastqc (2016). [0182] 45. Bray, N. L., Pimentel, H., Melsted, P. & Pachter, L. Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol 34, 525-527 (2016). [0183] 46. Soneson, C., Love, M. I. & Robinson, M. D. Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Res 4, 1521 (2015). [0184] 47. Law, C. W., Chen, Y., Shi, W. & Smyth, G. K. voom: Precision weights unlock linear model analysis tools for RNA-seq read counts. Genome biology 15, R29 (2014). [0185] 48. Benjamini, Y. & Hochberg, Y. Controlling the False Discovery Rate: a Practical and Powerful Approach to Multiple Testing. J. R. Statist. Soc. B 57, 289-300 (1995). [0186] 49. Yu, G., Wang, L. G., Han, Y. & He, Q. Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284-287 (2012). [0187] 50. Upadhyay, V., et al. Lymphotoxin regulates commensal responses to enable diet-induced obesity. Nat Immunol 13, 947-953 (2012). [0188] 51. Liu, X., et al. Warburg effect revisited: an epigenetic link between glycolysis and gastric carcinogenesis. Oncogene 29, 442-450 (2010). [0189] 52. Roelen, B. A., Lin, H. Y., Knezevic, V., Freund, E. & Mummery, C. L. Expression of TGF-beta s and their receptors during implantation and organogenesis of the mouse embryo. Dev Biol 166, 716-728 (1994). [0190] 53. Ellis, J. M., Bowman, C. E. & Wolfgang, M. J. Metabolic and tissue-specific regulation of acyl-CoA metabolism. PLoS One 10, e0116587 (2015). [0191] 54. Al-Dwairi, A., Pabona, J. M., Simmen, R. C. & Simmen, F. A. Cytosolic malic enzyme 1 (MEI) mediates high fat diet-induced adiposity, endocrine profile, and gastrointestinal tract proliferation-associated biomarkers in male mice. PLoS One 7, e46716 (2012). [0192] 55. Pinheiro, J. C. & Bates, D. M. Mixed-Effects in Models S and S-Plus, (Springer, New York, 2000). [0193] 56. Kuznetsova, A., Brockhoff, P. B., Rune, H. & Christensen, B. 1merTest Package: Tests in Linear Mixed Effects Models. Journal of Statistical Software 82, 1-26 (2017). [0194] 57. Moens, F., et al. Lactate- and acetate-based cross-feeding interactions between selected strains of lactobacilli, bifidobacteria and colon bacteria in the presence of inulin-type fructans. Int J Food Microbiol 241: 225-236 (2017). [0195] 58. Rettedal, E. A., et al. Cultivation-based multiplex phenotyping of human gut microbiota allows targeted recovery of previously uncultured bacteria. Nat Commun 5: 4714 (2014). [0196] 59. Riviere, A., et al. Bifidobacteria and Butyrate-Producing Colon Bacteria: Importance and Strategies for Their Stimulation in the Human Gut. Front Microbiol 7: 979 (2016). [0197] 60. Surana, N. K. and D. L. Kasper Moving beyond microbiome-wide associations to causal microbe identification. Nature 552(7684): 244-247 (2017). [0198] 61. Torii, T., et al. Measurement of short-chain fatty acids in human faeces using high-performance liquid chromatography: specimen stability. Ann Clin Biochem 47(5): 447-452 (2010). [0199] 62. Tyagi, A. M., et al. The Microbial Metabolite Butyrate Stimulates Bone Formation via T Regulatory Cell-Mediated Regulation of WNT10B Expression. Immunity 49(6): 1116-1131 e1117 (2018).