Patent Application
20110251156
The present disclosure relates to a vehicle for delivering a compound to a mucous membrane compositions, methods and systems. In particular, the present disclosure relates to vehicles for delivering a compound to a mucous membrane of the digestive tract.
Effective delivery of a desired compound or substance to mucous membrane has been challenging in particular when referred to delivery to the digestive tract, and in particular to human gastrointestinal tract.
The human gastrointestinal tract harbors an extraordinary number of microbes (known as the gut microbiota) that have a profound effect on the development and function of the immune system. Of the innumerable species of bacteria that inhabit the gastrointestinal tract of mammals, Bacteroidetes are the most abundant Gram-negative bacterial phylum.
Identification of immunomodulatory microorganisms and compounds is of particular interest. In particular, Bacteroides fragilis is a human commensal microorganism that has been shown to have immunomodulatory properties. In particular, immunomodulatory properties of Bacteroides fragilis have been associated to Polysaccharide A (PSA) production by the bacterium.
Gram-negative bacteria have evolved intricate mechanisms to export bacterial products; type III secretion systems (T3SS), T4SS and T6SS, which assemble complex surface appendages that translocate bacterial effectors following direct contact with host cells. Another secretion strategy is outer membrane vesicles (OMVs) which are released from the surface of Gram-negative bacteria and deliver a suite of molecular cargo to distant target cells. Although many (if not all) Gram-negative bacteria appear to produce OMVs, genome sequences of human commensal bacteria reveal a universal absence of T3SS, T4SS and T6SS.
However, identification of the mechanism and/or molecular triggers by which B. fragilis (or any commensal bacteria) delivers beneficial microbial molecules to the immune system and performs the immunomodulatory properties has been challenging.
Inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis represent serious disorders whereby uncontrolled intestinal inflammation leads to severe pathology of the digestive tract. IBD afflicts an estimated 1 million people in the United States. In addition to the medical and social burden of IBD, the incidences of disease are alarmingly increasing in ‘Western’ societies and current therapies are largely inadequate. Although the cause(s) for IBD remain enigmatic even after decades of research, defects in immune regulation toward intestinal bacteria appear to play an important role in disease3.
The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.
We reveal herein that PSA is delivered to the host by outer membrane vesicles (OMVs), secretion structures that target bacterial molecules to host cells. OMVs containing PSA are internalized by dendritic cells of the host immune system. Following uptake of OMVs, PSA programs dendritic cells to induce the differentiation of regulatory T cells (Treg) that express Foxp3 and the anti-inflammatory cytokine interleukin-10 (IL-10). Treg development by OMVs requires toll-like receptor 2 (TLR2) expression and IL-10 production by dendritic cells. Remarkably, purified OMVs direct the in vitro differentiation of functional Tregs with potent suppressive activity in a PSA dependent manner. Treatment of animals with OMVs containing PSA prevents experimental colitis and suppresses pro-inflammatory cytokine responses in the gut. These findings reveal that commensal bacteria provide beneficial microbial factors through vesicle secretion, a process that may be engineered into a novel approach for delivery of probiotic therapies for IBD.
In one embodiment, a pharmaceutical composition for delivering a compound to a mucous membrane is provided, comprising an outer membrane vesicle formed by a lipid membrane enclosing an aqueous environment, the vesicle comprising a PSA ligand; and a pharmaceutically appropriate carrier.
In another embodiment, a pharmaceutical composition for delivering a compound to a mucous membrane is provided, comprising an outer membrane vesicle formed by a lipid membrane enclosing an aqueous environment, the vesicle comprising a PSA ligand; and a pharmaceutically appropriate carrier, wherein the lipid membrane is derived from the outer membrane of B. fragilis.
In a more particular embodiment of the composition in paragraph [0012], the PSA ligand is L-PSA.
In yet another embodiment of the composition in paragraph [0012], the pharmaceutical composition further comprises one or more medicaments or treatments known to be useful in the treatment of inflammatory bowel disease.
In one embodiment, a method of treating an inflammatory disease or inflammation in an individual, comprising administering an effective amount of a composition comprising an outer membrane vesicle formed by a lipid membrane enclosing an aqueous environment, the vesicle comprising a PSA ligand.
In a particularized embodiment of the method in paragraph [0016], the inflammatory disease is inflammatory bowel disease.
In yet another embodiment, a vesicle for delivering a compound to a mucous membrane, comprising an outer membrane vesicle formed by a lipid membrane enclosing an aqueous environment, the vesicle comprising a PSA ligand.
In a more particularized embodiment of the vesicle in paragraph [0018], the vesicle further comprises a compound other than PSA.
In another embodiment, a method for directing the differentiation of functional Tregs with potent suppressive activity is provided, comprising an outer membrane vesicle formed by a lipid membrane enclosing an aqueous environment, the vesicle comprising a PSA ligand.
In a more particularized embodiment of the method in paragraph [0020], the Treg cells inhibit T cell proliferation and/or further suppresses immunity by reducing dendritic cell activation.
In another embodiment of the method in paragraph [0020], the method further comprises providing DCs for co-culture, wherein IL-10 is produced and production of IL-10 is dependent upon the presence of TLR2 expression on the DCs, in comparison to PSA activity alone (in the absence of OMVs) on the DC production of IL-10 where TLR2 expression on the DCs is not required.
In another embodiment, a method for identifying compounds that reduce inflammation or improve the symptoms of an inflammatory disease in vitro is provided, comprising providing a candidate compound and incubating the compound with Treg cells; further providing T cells, and then determining whether said Treg cells inhibit T cell activation.
In a more particularized embodiment of the method in paragraph [0023], the method further comprises dendritic cells and/or wherein the Treg cells express Foxp3.
In another embodiment, a bacterial substance for use as a medicament is provided comprising an outer membrane vesicle formed by a lipid membrane enclosing an aqueous environment, the vesicle comprising a PSA ligand.
In a more particularized embodiment of the medicament in paragraph [0025], the bacterial substance further comprises an excipient and/or one or more medicaments known to be useful in the treatment of inflammatory bowel disease.
In another embodiment, a bacterial substance for use in the treatment of inflammatory disease or inflammation is provided comprising an outer membrane vesicle formed by a lipid membrane enclosing an aqueous environment, the vesicle comprising a PSA ligand.
In a more particularized embodiment of the bacterial substance in paragraph [0027], the inflammatory disease is inflammatory bowel diseases.
In another embodiment, a composition for directing the differentiation of functional Treg with potent suppressive activity, comprising an outer membrane vesicle formed by a lipid membrane enclosing an aqueous environment, the vesicle comprising a PSA ligand.
In a more particularized embodiment of the composition in paragraph [0029], the Treg inhibit T cell activation and/or wherein the Treg cells express Foxp3.
Also provided herein, are methods and systems for screening microorganisms and related substances having immunomodulatory and, in particular, anti-inflammatory properties. In particular, provided herein are methods and systems that in several embodiments allow identification of bacterial substances that are able to induce in an individual an immunomodulatory response comparable to the one of Bacteroides fragilis.
According to another embodiment, a method is described for identifying a bacterial substance having immunomodulatory ability. The method comprises contacting a candidate bacterial substance with a T cell alone or in presence of an antigen presenting cell, and detecting expression of at least one of one or more anti inflammatory biomarkers selected from the group consisting of IL-10, Foxp3, TGFβ1, TGFβ2, Perforin and Granzyme B, and one or more inflammatory biomarkers selected from the group consisting of IFNγ, IFNα, IFNβ, IL-1, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-21, IL-22, IL-23, IL-17, or TNFα. The method further comprises determining an anti-inflammatory ability of the candidate bacterial substance through detection of an increase of the expression of the one or more anti-inflammatory biomarkers or a decrease of the expression of the one or more inflammatory biomarkers following the contacting.
According to yet another embodiment, a method is described for identifying a bacterial substance having immunomodulatory ability. The method comprises contacting a candidate bacterial substance with an antigen presenting cell, and incubating the antigen presenting cell with a T cell following the contacting. The method further comprises detecting expression of at least one of one or more anti-inflammatory biomarker selected from the group consisting of IL-10, Foxp3, TGFβ1, TGFβ2, Perforin and Granzyme B, and one or more inflammatory biomarker selected from the group consisting of IFNγ, IFNα, IFNβ, IL-1, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-21, IL-22, IL-23, IL-17 or TNFα. The method further comprises determining an anti inflammatory ability of the candidate bacterial substance through detection of an increase of the expression of the one or more anti-inflammatory biomarkers or a decrease of the expression of the one or more inflammatory biomarkers following the incubating.
According to another aspect, a method is described for identifying a bacterial substance having immunomodulatory ability in animals. The method comprises treating a transgenic marker non-human animal with a candidate bacterial substance, the transgenic marker non-human animal genetically modified to express at least one of one or more labeled anti-inflammatory biomarkers selected from the group consisting of IL-10, Foxp3, TGFβ1, TGFβ2, Perform and Granzyme B, and one or more labeled inflammatory biomarker selected from the group consisting of IFNγ, IFNα, IFNβ, IL-1, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-21, IL-22, IL-23, IL-17 or TNFα. The method further comprises detecting expression in the transgenic marker non-human animal of at least one of the one or more anti-inflammatory biomarkers or at least one of the inflammatory biomarkers following the treating and determining an anti-inflammatory ability of the candidate bacterial substance through detection of an increase of the expression of the one or more anti-inflammatory biomarkers or a decrease of the expression of the one or more inflammatory biomarkers following the treating.
According to another aspect a system for screening a bacterial substance is described. The system comprises at least two of a T cell, an antigen presenting cell and reagents for detection of at least one of one or more anti-inflammatory biomarkers selected from the group consisting of IL-10, Foxp3, TGFβ1, TGFβ2, Perforin and Granzyme B and one or more labeled inflammatory biomarker selected from the group consisting of IFNγ, IFNα, IFNβ, IL-1, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-21, IL-22, IL-23, IL-17 or TNFα, for simultaneous, combined or sequential use in a method to identify an anti-inflammatory bacterial substance herein described.
The methods and systems herein described can be used in connection with medical, pharmaceutical, veterinary applications as well as fundamental biological studies and various applications, identifiable by a skilled person upon reading of the present disclosure, wherein investigating immunomodulatory ability and in particular anti-inflammatory ability of a substance is desirable.
The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description and example sections, serve to explain the principles and implementations of the disclosure.
Vehicles are herein described that are suitable for delivering a compound to a mucous membrane.
The terms “wild-type OMV”, “PSA containing OMVs”, and “PSA-OMV”, are herein used interchangeably.
The term “vehicle” in the sense of the present disclosure indicates a medium in which a compound is administered, and in which the compound is comprised, expressed, and/or displayed. Exemplary vehicles comprise any of various media acting usually as solvents, carriers, or binders for active ingredients, with particular reference to drugs.
The term “deliver” and delivering” referred to a compound or substance in the sense of the present disclosure indicates passage of the compound or substance from an originating location to a target location, and in particular to a location formed by one or more cells, tissues, or organs of an individual. In particular, a compound is delivered in the sense of the present disclosure when the passage is performed so that at least one activity associated to the delivered compound can be exerted on the one or more cells, tissues or organs of the individual.
The term “compound” as used herein indicates any chemical substance comprised of one or more chemical elements and comprises various substances, molecules or component that include but are not limited to biomolecules and in particular drugs. The term “biomolecule” as used herein indicates a substance compound or component associated to a biological activity including but not limited to sugars, aminoacids, peptides proteins, oligonucleotides, polynucleotides, polypeptides, organic molecules, haptens, epitopes, biological cells, parts of biological cells, vitamins, hormones and the like. The term “drug” as used herein indicates substance that, when absorbed into the body of a living organism, alters normal bodily function. In particular, drugs in the sense of the present disclosure include a chemical substance used in the treatment, cure, prevention, or diagnosis of disease or used to otherwise enhance physical or mental well-being.
If PSA is for instance the above ‘compound’ then additional different compounds could also be delivered, in addition to PSA, by the vesicle.
The term “mucous membrane” in the sense of the present disclosure indicates a lining of mostly endodermal origin, covered in epithelium, which are involved in absorption and secretion. In an individual, mucous membranes line various body cavities that are exposed to the external environment and internal organs. Mucous membranes are at several places continuous with skin: at the nostrils, the mouth, the lips, the eyelids, the ears, the genital area, and the anus.
In the compositions, and related methods and systems herein described, the vehicle is formed by a vesicle comprising one or more compounds to be delivered.
A “vesicle” in the sense of the present disclosure is a supramolecular complex formed by a membrane forming lipid and additional molecules assembled in an aqueous environment. In particular, in vesicles herein described the membrane forming lipids are arranged in a lipid layer enclosing an internal aqueous environment herein also indicated as cytosol.
The term “membrane forming lipid” or “amphipatic lipid” as used herein indicates a lipid possessing both hydrophilic and hydrophobic properties that in an aqueous environment assemble in a lipid layer structure that consists of either one or two opposing layers of amphipatic molecules known as polar lipid. Each polar lipid has a hydrophilic moiety, i.e., a polar group such as, a derivatized phosphate or a saccharide group, and a hydrophobic moiety, i.e., a long hydrocarbon chain. Exemplary polar lipids include phospholipids, sphingolipids, glycolipids, ether lipids, sterols and alkylphosphocholins. Amphipatic lipids include but are not limited to membrane lipids, i.e. amphipatic lipids that are constituents of a biological membrane, such as phospholipids like dimyrisoylphosphatidylcholine (DMPC) or Dioleoylphosphoethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC). In an embodiment, the membrane of the vesicle is formed by a lipid bilayer mimicking a plasma membrane (a biological membrane separating the interior of a cell from the outside environment, and enclose an aqueous environment) and in particular the outer membrane of B. fragilis.
Vesicles herein described also comprise a lipopolysaccharide (LPS) either associated with the membrane of the vesicle or comprised in the aqueous environment of the vesicle. The term “lipopolysaccharide” as used herein indicates large molecules consisting of a lipid and a polysaccharide joined by a covalent bond; they are found in the outer membrane of Gram-negative bacteria, act as endotoxins and elicit strong immune responses in animals. In particular, vesicles herein described comprise one or more LPS of B. fragilis which are identifiable by a skilled person.
Vesicles herein described can also comprise a peptidoglycan either associated with the membrane of the vesicle or comprised in the aqueous environment of the vesicle. The term “peptidoglycan” as used herein indicates a polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of bacteria (Eubacteria, not Archaebacteria), forming the cell wall. In particular, vesicles herein described comprise one or more peptidoglycans of B. fragilis which are identifiable by a skilled person.
Additional compounds that can be comprised in the vesicles, comprise membrane proteins, membrane lipids carbohydrates and nucleic acids, and in particular, membrane proteins, membrane lipids carbohydrates and nucleic acids of B. fragilis.
Exemplary vesicles in the sense of the present disclosure comprise small membrane-enclosed sacs that can store or transport substances. Vesicles can form naturally because of the properties of the membrane forming lipid, or they may be prepared from bacterial membranes. Most vesicles have specialized functions depending on what materials they contain on the membrane and/or the aqueous environment.
In an embodiment, the vesicles herein described are formed by portions of membranes of bacteria. In an embodiment, vesicles are formed by Outer Membrane Vesicles (OMVs) of the bacteria.
In an embodiment, the vesicles herein described are formed by portions of membranes of B. fragilis and in particular from the OMV of B. fragilis as exemplified in the Examples section.
In an embodiment, the delivered compound can be a drug, candidate drug, a supplement (a product taken orally that contains one or more ingredients (as vitamins or amino acids) that are intended to supplement one's diet and are not considered food) or any other compound whose delivery to the mucous membrane is desired. One or more of those compounds can be include in the vesicles herein described in a suitable dosage that can be determined by the skilled person in view of the specific compound to be delivered.
In an embodiment, the compound comprises one or more zwitterionic polysaccharide and in particular Polysaccharide A.
The term “zwitterionic polysaccharide” as used herein indicates synthetic or natural polymers comprising one or more monosaccharides joined together by glicosidic bonds, and including at least one positively charged moiety and at least one negatively charged moiety. Zwitterionic polysaccharides include but are not limited to polymers of any length, from a mono- or di-saccharide polymer to polymers including hundreds or thousands of monosaccharides. In some embodiments, a zwitterionic polysaccharide can include repeating units wherein each repeating unit includes from two to ten monosaccharides, a positively charged moiety (e.g. an free positively charged amino moiety) and a negatively charged moiety (such as sulfonate, sulfate, phosphate and phosphonate). In some embodiment ZPs can have a molecular weight comprised between 500 Da and 2,000,000 Da. In some embodiments, the ZPs can have a molecular weight comprised between 200 and 2500. Exemplary ZPS include but are not limited to PSA and PSB from Bacteroides Fragilis, CP5/CD8 from Staphylococcus aureus, and Sp1/CP1 from Streptococcus pneumonia. Zwitterionic polysaccharides can be isolated from natural sources, and in particular from bacterial sources, e.g. by purification. Zwitterionic polysaccharides can also be produced by chemical or biochemical methods, as well as by recombinant microorganism technologies all identifiable by a skilled person. Thus, those methods and technologies will not be further described herein in detail.
The wording “polysaccharide A” (or PSA, or PSA ligand) as used herein indicates a molecule produced by the PSA locus of Bacteroides fragilis and derivatives thereof which include but are not limited to polymers of the repeating unit {→3) α-d-AAT Galp(1→4)-[β-d-Galf(1→3)] α-d-GalpNAc(1→3)-[4,6-pyruvate]-β-d-Galp(1→}, where AATGal is acetamido-amino-2,4,6-trideoxygalactose, and the galactopyranosyl residue is modified by a pyruvate substituent spanning O-4 and O-6. The term “derivative” as used herein with reference to a first polysaccharide (e.g., PSA), indicates a second polysaccharide that is structurally related to the first polysaccharide and is derivable from the first polysaccharide by a modification that introduces a feature that is not present in the first polysaccharide while retaining functional properties of the first polysaccharide. Accordingly, a derivative polysaccharide of PSA, usually differs from the original polysaccharide by modification of the repeating units or of the saccharidic component of one or more of the repeating units that might or might not be associated with an additional function not present in the original polysaccharide. A derivative polysaccharide of PSA retains however one or more functional activities that are herein described in connection with PSA in association with the anti-inflammatory activity of PSA.
In one embodiment, low molecular weight PSA (L-PSA) is from 70 kDa to 200 kDa and high molecular weight PSA (H-PSA) is above 200 kDa.
In an embodiment, the mucous membrane is a mucous membrane of the digestive tract of an individual, and in particular intestinal mucosa, gastric mucosa, esophageal mucosa, buccal mucosa, oral mucosa and/or buccal mucosa.
In an embodiment, the vesicles herein described can be used in connection with procedures and applications such as the ones described in U.S. Provisional Application Ser. No. 61/321,527 herein enclosed as Exhibit A which forms part of the present disclosure and is incorporated herein by reference in its entirety.
In an embodiment, the vesicles herein described can be used in connection with methods and systems such as the ones described in U.S. patent application Ser. No. 12/766,787 herein enclosed as Exhibit B which forms part of the present disclosure and is incorporated herein by reference in its entirety.
In an embodiment, vesicles herein described can be used in a method to deliver the compound to a mucous membrane of an individual. In particular, the method comprises contacting a vesicle comprising the compound with the mucous membrane for a time and under condition to allow contact of the substance with the membrane. In the method, the vesicle is formed by a lipid membrane enclosing an aqueous environment and the compound to be delivered is comprised in the aqueous environment of the vesicle.
In another embodiment, a composition or pharmaceutical composition comprising of the vesicle or OMV is contacted with the mucous membrane for a time and under condition to allow contact of the substance with the membrane.
The term “contacting” or “incubating” as used herein indicates actions directed to creation of a spatial relationship between two items provided for a time and under condition such that at least one of the reciprocal or non reciprocal action or influence between the two items can be exerted. In particular, incubation can be performed between a vesicle and a mucous membrane and can result in a direct contact and/or interaction between the vesicle and the mucous membrane or can result in a modification of the mucous membrane following an indirect action of the vesicle (e.g. following activation or modification of another substance which directly interacts with the membrane).
Suitable conditions for performing the contacting or incubation are identifiable by a skilled person upon reading of the present disclosure in view of the compound, the mucous membrane, and the experimental design.
In an embodiment, vesicles herein described can be comprised in a composition for delivering a compound to a mucous membrane. The composition comprises one or more vesicles herein described optionally in combination with an additional vehicle acting as a solvent, carrier, binder or diluents for the vesicles.
The term “condition”—when referring to the status of an animal or human—as used herein indicates a usually the physical status of the body of an individual, as a whole or of one or more of its parts, that does not conform to a physical status of the individual, as a whole or of one or more of its parts, that is associated with a state of complete physical, mental and possibly social well-being. Conditions herein described include but are not limited disorders and diseases wherein the term “disorder” indicates a condition of the living individual that is associated to a functional abnormality of the body or of any of its parts, and the term “disease” indicates a condition of the living individual that impairs normal functioning of the body or of any of its parts and is typically manifested by distinguishing signs and symptoms. Exemplary conditions include but are not limited to injuries, disabilities, disorders (including mental and physical disorders), syndromes, infections, deviant behaviors of the individual and atypical variations of structure and functions of the body of an individual or parts thereof.
In particular, in embodiments where the compound is used as an anti-inflammatory compound, the condition can be any condition associated to an inflammation or inflammatory response; or a condition described as an inflammatory disease itself. The wording “associated to” as used herein with reference to two items indicates a relation between the two items such that the occurrence of a first item is accompanied by the occurrence of the second item, which includes but is not limited to a cause-effect relation and sign/symptoms-disease relation.
Conditions associated with an inflammation (or inflammatory diseases) in humans include but are not limited to inflammatory bowel disease, including but not limited to Crohn's disease and ulcerative colitis, asthma, dermatitis, arthritis, myasthenia gravis, Grave's disease, multiple sclerosis (MS), and psoriasis. A person of skill in the art would be able to identify such subjects suffering from the aforementioned diseases using the appropriate clinical criteria.
In one embodiment, compositions, pharmaceutical composition comprising OMVs containing PSA can be used for the prevention of inflammatory diseases, such as, but not limited to, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), asthma, dermatitis, arthritis, myasthenia gravis, Grave's disease, multiple sclerosis, and psoriasis.
The term “treatment” or “treated” as used herein indicates any activity whether part of a medical care, or not, for or deals with a condition medically or surgically in animals or humans. Such treatments can be administered by either medical or non-medical personnel.
In some embodiments, where the composition is to be administered to an individual the composition can be a pharmaceutical composition, and comprise one or more vesicles each comprising PSA. In a more particular embodiment, the pharmaceutical composition can comprise of one or more vesicles each comprising PSA and one or more of another compound, and/or a pharmaceutically acceptable or appropriate carrier/vehicle.
In another embodiment, the above pharmaceutical composition, comprising one or more vesicles each comprising PSA and one or more of another compound, and/or a pharmaceutically acceptable or appropriate carrier/vehicle, wherein an individual/subject with an inflammatory condition or inflammation given this composition shows an improvement.
In some embodiments, the vesicles herein described can be included in pharmaceutical compositions together with an excipient or diluent. In particular, in some embodiments, pharmaceutical compositions contain vesicles herein described, in combination with one or more compatible and pharmaceutically acceptable vehicle, and in particular with pharmaceutically acceptable diluents or excipients.
The term “excipient” as used herein indicates an inactive substance used as a pharmaceutically acceptable or appropriate carrier for the active ingredients of a medication. Suitable excipients for the pharmaceutical compositions herein disclosed include any substance that enhances the ability of the body of an individual to absorb vesicles herein described. Suitable excipients also include any substance that can be used to bulk up formulations with vesicles herein described to allow for convenient and accurate dosage. In addition to their use in the single-dosage quantity, excipients can be used in the manufacturing process to aid in the handling of vesicles herein described. Depending on the route of administration, and form of medication, different excipients may be used. Exemplary excipients include but are not limited to antiadherents, binders, coatings disintegrants, fillers, flavors (such as sweeteners) and colors, glidants, lubricants, preservatives, sorbents.
Pharmaceutically acceptable or appropriate carriers can be, but not limited to, organic or inorganic, solid or liquid excipient which is suitable for the selected mode of application such as oral application or injection, and administered in the form of a conventional pharmaceutical preparation. Such preparation includes solid such as tablets, granules, powders, capsules, and liquid such as solution, emulsion, suspension and the like. Said carrier includes starch, lactose, glucose, sucrose, dextrine, cellulose, paraffin, fatty acid glyceride, water, alcohol, gum arabic and the like. If necessary, auxiliary, stabilizer, emulsifier, lubricant, binder, pH adjustor controller, isotonic agent and other conventional additives may be added.
The pharmaceutically acceptable or appropriate carrier may well include other compounds known to be beneficial to an impaired situation of the gut, (e.g., antioxidants, such as Vitamin C, Vitamin E, Selenium or Zinc); or a food composition. The food composition can be, but is not limited to, milk, yoghurt, curd, cheese, fermented milks, milk based fermented products, ice-creams, fermented cereal based products, milk based powders, infant formulae, tablets, liquid bacterial suspensions, dried oral supplement, or wet oral supplement.
The term “diluent” as used herein indicates a diluting agent which is issued to dilute or carry an active ingredient of a composition. Suitable diluent include any substance that can decrease the viscosity of a medicinal preparation.
In certain embodiments, compositions, compounds, and, in particular, pharmaceutical compositions can be formulated for enteral administration including, but not limited to, i) by mouth (orally) as tablets, capsules, or drops; ii) by gastric feeding tube, duodenal feeding tube, or gastrostomy; and enteral nutrition; and iii) rectally as a suppository.
In some embodiments, vesicles herein described comprising PSA can be used in a method of treating or preventing a condition in an individual.
The method comprises administering to the individual an effective amount of the composition or pharmaceutical composition. The term “individual” as used herein includes a single biological organism wherein inflammation can occur including but not limited to animals and in particular higher animals and in particular vertebrates such as mammals and in particular human beings.
The “effective amount”, “amount effective to,” or “amount of X effective to” refers to an amount of the compound, composition or pharmaceutical composition that is effective to treat, relieve, reduce, or improve to some extent one or more of the symptoms of the disease in need of treatment, or to retard initiation of clinical markers or symptoms of a disease in need of prevention, when the compound is administered. Thus, for example an effective amount refers to an amount of the compound/composition/pharmaceutical ingredient which exhibits the ‘improved’ effects, as noted below.
The “effective amount” may be empirically determined by experimenting with the compounds concerned in known in vivo and in vitro model systems for a disease in need of treatment. An effective amount will vary according to the weight, sex, age and medical history of the individual, as well as the severity of the patient's condition(s), the type of disease(s), mode of administration, and the like. An effective amount may be readily determined using routine experimentation, e.g., by titration (administration of increasing dosages until an effective dosage is found) and/or by reference to amounts that were effective for prior patients. Generally, the effective amount of the present invention will be administered in dosages ranging between about 0.1 mg/kg and about 20 mg/kg of the patient's body-weight.
As used herein, the phrase “prophylactically effective amount” includes the amount of the compound/composition/pharmaceutical composition which is sufficient to result in the prevention or reduction of the development, recurrence or onset of one or more symptoms associated with a disorder (or to enhance or improve the prophylactic effect(s) of another therapy (e.g., another prophylactic agent).
It is further contemplated that the compound/composition/pharmaceutical composition of the present invention can be used with one or more known medicaments known to be useful in the treatment or prevention of inflammatory diseases, either separately or in combination.
For example the compound/composition/pharmaceutical composition of the present invention can be combined with one or a combination of medicaments/treatments known to be useful in the treatment of IBD such as, but not limited to, sulfasalazine (Azulfadine), mesalamine (Asacol, Pentasa), immunosuppressants (Imuran, 6-MP, cyclosporine); methotrexate, TNF-alpha inhibitors (Remicade and Humira); and corticosteroids (Entocort and prednisone). Other treatments (experimental) for ulcerative colitis, include aloe vera, butyrate, boswellia, probiotics, antibiotics, immunosuppressive therapy, and nicotine.
For example the compound/composition/pharmaceutical composition of the present invention can be combined with one or a combination of medicaments/treatments known to be useful in the treatment of MS such as, but not limited to, Avonex®, Betaseron®, and Copaxone®. Reba®; Extavia® Novantrone® (mitoxantrone); Tysabri® (natalizumab), and Gilenya® (fingolimod). Other drugs include intravenous immunoglobulin (IVIg) therapy, methotrexate, azathioprine (Imuran®), and cyclophosphamide (Cytoxan®); corticosteroids; Cytoxan® (cyclophosphamide); Imuran® (azathioprine); methotrexate; plasma exchange; pulse Solu-Medrol® (IV methylprednisolone); prednisone; Decadron® (dexamethasone); Medrol® (oral methylprednisolone); Plasmapheresis (plasma exchange); intravenous immunoglobulin (IVIg) therapy.
In another embodiment, it is possible that these other medicaments could be expressed or delivered within the OMVs themselves.
As used herein, “improved,” “improvement,” and other grammatical variants, includes any beneficial change in the individual/subject resulting from a treatment. A beneficial change is any way in which a patient's condition is better than it would have been in the absence of the treatment. “Improved” includes, but is not limited to, prevention of an undesired condition, slowing the rate at which a condition worsens, delaying the development of an undesired condition, restoration to an essentially normal condition; causing the patient to go into remission more often or remain in remission for longer periods than without treatment (i.e. where the degree of inflammation in the patient is less (or absent); or the person usually is without symptoms); reduction in the number and/or severity of relapses; and reduction in the development of new areas of inflammation as seen on magnetic resonance imaging (MRI) scans.
More particularly, improvement in IBD encompasses a reduction in the severity or duration in any one or more clinical IBD symptoms, such as, but not limited to, Abdominal cramps and pain; Bloody stool; diarrhea; urgency to have a bowel movement; fever; loss of appetite; weight loss; mucus in the stool; ulceration of the large intestine; and anemia (due to blood loss). For example improvement in MS encompasses a reduction in the severity or duration in any one or more clinical MS symptoms, such as, but not limited to fatigue; visual disorders; numbness; dizziness/vertigo; bladder and bowel dysfunction; weakness; tremor; impaired mobility; sexual dysfunction; slurred speech; spasticity (leg stiffness); swallowing disorders; chronic aching pain; depression; mild cognitive and memory difficulties
Moreover, improved,” “improvement,” and other grammatical variants includes any change resulting in the reduction in the severity, duration, and/or risk of developing complications of the inflammatory disease. For IBD, ‘improved’ could mean for example, a reduced risk of the IBD subject developing profuse bleeding from the ulcers; perforation (rupture) of the bowel; strictures and obstruction; fistulae (abnormal passage) and perianal disease; toxic megacolon (acute nonobstructive dilation of the colon); and malignancy (for example, colon cancer).
In an embodiment, at least two of the membrane forming lipid, vesicles, PSA and one or more additional compounds to be delivered can be comprised in a system possibly together with other reagents suitable to be used in the methods herein described.
The systems can be provided in the form of kits of parts. In a kit of parts, the membrane forming lipid, vesicles, PSA, addition compounds, medicaments, and other the reagents can be included in one or more compositions, or each lipid, vesicles, PSA, addition compounds, medicaments compound and reagent can be in a composition together with a suitable vehicle.
Additional components can include labeled molecules and in particular, labeled capture agents specific for an anti-inflammatory or an inflammatory biomarker or a molecule associated to the expression thereof, a microfluidic chip, reference standards, and additional components identifiable by a skilled person upon reading of the present disclosure.
The term “capture agent” as used herein indicates a compound that can specifically bind to a target. The wording “specific” “specifically” or “specificity” as used herein with reference to the binding of a first molecule to second molecule refers to the recognition, contact and formation of a stable complex between the first molecule and the second molecule, together with substantially less to no recognition, contact and formation of a stable complex between each of the first molecule and the second molecule with other molecules that may be present. Exemplary specific bindings are antibody-antigen interaction, cellular receptor-ligand interactions, polynucleotide hybridization, enzyme substrate interactions etc. By “stable complex” is meant a complex that is detectable and does not require any arbitrary level of stability, although greater stability is generally preferred. In some embodiments, the kit can comprise labeled polynucleotides or labeled antibodies.
The components of the kit can be provided, with suitable instructions and other necessary reagents, in order to perform the methods here described. The kit will normally contain the compositions in separate containers. Instructions, for example written or audio instructions, on paper or electronic support such as tapes or CD-ROMs, for carrying out the assay, will usually be included in the kit. The kit can also contain, depending on the particular method used, other packaged reagents and materials (i.e. wash buffers and the like).
The term “immunomodulatory” as used herein indicates the ability to promote a state associated with absence of an inflammatory response. Particular immunomodulatory properties comprise anti-inflammatory properties, wherein the term anti-inflammatory refers to the property of a substance or treatment that prevents or reduces inflammation.
The term “inflammation”, “inflammatory state” or “inflammatory response” as used herein indicate the complex biological response of vascular tissues of an individual to harmful stimuli, such as pathogens, damaged cells, or irritants, and includes secretion of cytokines and more particularly of pro-inflammatory cytokine, i.e. cytokines which are produced predominantly by activated immune cells such as microglia and are involved in the amplification of inflammatory reactions. Exemplary pro-inflammatory cytokines include but are not limited to IL-1, IL-6, TNF-a, IL-17, IL21, and IL23. Exemplary inflammations include acute inflammation and chronic inflammation. The wording “acute inflammation” as used herein indicates a short-term process characterized by the classic signs of inflammation (swelling, redness, pain, heat, and loss of function) due to the infiltration of the tissues by plasma and leukocytes. An acute inflammation typically occurs as long as the injurious stimulus is present and ceases once the stimulus has been removed, broken down, or walled off by scarring (fibrosis). The wording “chronic inflammation” as used herein indicates a condition characterized by concurrent active inflammation, tissue destruction, and attempts at repair. Chronic inflammation is not characterized by the classic signs of acute inflammation listed above. Instead, chronically inflamed tissue is characterized by the infiltration of mononuclear immune cells (monocytes, macrophages, lymphocytes, and plasma cells), tissue destruction, and attempts at healing, which include angiogenesis and fibrosis.
The term “substance” as used herein indicates a matter of particular or definite chemical constitution. The term “bacterial substance” indicates matter of bacterial origin such as, a live bacteria, dead bacteria, and in particular heat-killed bacteria, bacterial extracts, purified molecules from bacteria, a combination of molecules purified from bacteria, or vesicles containing one molecule or a combination of molecules purified from bacteria or purified vesicles from bacteria. In particular, bacterial substance can be formed by, or comprises, the outer membrane vesicles (OMVs) which are vesicles released from the surface of bacteria and deliver a suite of molecular cargo to distant target cells. Even more particularly, the bacterial substance can be formed by, or comprises, the OMVs of B. Fragilis, and more particularly, such OMVs are further comprised of PSA.
Bacterial substance comprises substance derived from a same bacteria and substance derived from two or more different bacteria.
The term “bacteria” as used herein indicates large group of unicellular, prokaryote, microorganisms, typically of a few micrometers in length, and having a wide range of shapes. In particular, bacteria in the sense of the present disclosure comprise bacteria of the human flora, i.e. the assemblage of microorganisms that reside on the surface and in human tissues and bodily fluids such as deep layers of skin, in saliva, oral or vaginal mucosa, and in the gastrointestinal tracts. Bacterial flora comprises gut flora (bacteria detectable in the digestive tract of humans) vaginal flora (bacteria detectable in the fibromuscular tubular tract leading from the uterus to the exterior of the body in female humans) and skin flora (bacteria detectable in human skin). More specifically, bacteria in the sense of the present disclosure can be formed by one or more bacteroidetes of the gut flora and in particular one or more bacteroides, a genus of Gram-negative, non endospore forming anaerobes bacillus bacteria, symbiotic with humans and identifiable by a skilled person. A representative bacteroides is B. fragilis.
The term “transgenic marker non-human animal” as used herein refers to an animal that contains non-native, genetic material that has been transferred naturally or by any of a number of genetic engineering techniques. Such non-native, genetic material (or transgene) may act as a “biomarker” (as defined herein) and/or retain the ability to produce RNA or protein in the non-human animal.
The term “T cell” as used herein indicates a sub-group of lymphocytes (a type of white blood cell or leukocyte) including different cell types identifiable by a skilled person. T-helper cell according to the present disclosure and include effector Th cells (such as Th1, Th2 and Th17)—i.e. Th cells that secrete cytokines, proteins or peptides that stimulate or interact with other leukocytes, including Th cells—and suppressor Th cells (such as Treg) i.e. Th cells that suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens.
The term “antigen presenting cell” as used herein indicates a cell that displays foreign antigen complex with major histocompatibility complex (MHC) on its surface. In particular, antigen presenting cell comprise dendritic cell, macrophage, B cells and additional cells identifiable by a skilled person.
The term “contacting” or “incubating” as used herein indicates actions directed to creation of a spatial relationship between two items provided for a time and under condition such that at least one of the reciprocal or non reciprocal action or influence between the two items can be exerted. In particular, incubation can be performed between a bacterial substance and a cell and can result in a direct contact and/or interaction between the bacterial substance and the cell or can result in a modification of the cell following an indirect action of the bacterial substance (e.g. following activation or modification of another substance which directly interacts with the cell).
The ‘treating’, or ‘treatment’ can be performed by administering the bacterial substance, compound, composition, or pharmaceutical composition by topical or systemic administration. Systemic administration includes enteral administration (e.g. oral administration, administration by gastric feeding tube, administration by duodenal feeding tube, gastrostomy, enteral nutrition, and rectal administration) and parenteral administration (e.g. intravenous administration, intra-arterial administration, intramuscular administration, subcutaneous administration, intradermal, administration, intraperitoneal administration, and intravesical infusion. Topical administration include but is not limited to epicutaneous administration, inhalational administration (e.g. in asthma medications), enema, eye drops (e.g. onto the conjunctiva), ear drops, intranasal route (e.g. decongestant nasal sprays), and vaginal administration.
Similarly, the amount of pharmaceutical composition given to individuals suffering from an inflammatory disease such as, but not limited to, Crohn's disease and ulcerative colitis, asthma, dermatitis, arthritis, myasthenia gravis, Grave's disease, multiple sclerosis, or psoriasis, can be determined by the skilled person experimentally.
Exemplary incubation of an antigen presenting cell with a T cell can be performed in vitro by mixing a cell culture comprising the antigen presenting cell with a cell culture comprising the T cell, by adding a purified antigen presenting cell to a culture of T cells, or by adding a purified T cell to a culture of antigen presenting cells. Additional exemplary incubation between a T cell and an antigen presenting cell in vivo comprise transplanting antigen presenting cell in a into a tissue of an individual, the tissue comprising T cell, or transplanting T cell into a tissue of an individual the tissue comprising antigen presenting cell. In an embodiment, the individual is a transgenic animal other than humans genetically modified to express a labeled inflammatory or an anti inflammatory biomarker.
Additional procedures and techniques suitable for performing contacting between a substance and a T cell or antigen presenting cell and incubation between an antigen presenting cell and a T cell in vitro or in vivo can be identified by a skilled person upon reading of the present disclosure.
In methods and systems herein described, detection of the expression of an inflammatory or an anti-inflammatory biomarker can be performed in vitro and in vivo by techniques identifiable by a skilled person which comprise use of labeled molecules, including labeled biomarkers or labeled molecule specific for the biomarker or molecule associated thereto.
The term “detect” or “detection” as used herein indicates the determination of the existence, presence or fact of an analyte or related signal in a limited portion of space, including but not limited to a sample, a reaction mixture, a molecular complex and a substrate. A detection is “quantitative” when it refers, relates to, or involves the measurement of quantity or amount of the analyte or related signal (also referred as quantitation), which includes but is not limited to any analysis designed to determine the amounts or proportions of the analyte or related signal. A detection is “qualitative” when it refers, relates to, or involves identification of a quality or kind of the analyte or related signal in terms of relative abundance to another analyte or related signal, which is not quantified.
The terms “label” and “labeled molecule” or a used herein as a component of a complex or molecule referring to a molecule capable of detection, including but not limited to radioactive isotopes, fluorophores, chemiluminescent dyes, chromophores, enzymes, enzymes substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, nanoparticles, metal sols, ligands (such as biotin, avidin, streptavidin or haptens) and the like. The term “fluorophore” refers to a substance or a portion thereof which is capable of exhibiting fluorescence in a detectable image. As a consequence, the wording “signal” or “labeling signal” as used herein indicates the signal emitted from the label that allows detection of the label, including but not limited to radioactivity, fluorescence, chemiluminescence, production of a compound in outcome of an enzymatic reaction and the like.
Detection can be performed by detecting levels of expression of the biomarker, a precursor or analog thereof, and/or of an analyte associated thereto. The wording “associated to” as used herein with reference to two items indicates a relation between the two items such that the occurrence of a first item is accompanied by the occurrence of the second item, which includes but is not limited to a cause-effect relation and sign/symptoms-disease relation. In particular, the detection can be performed qualitatively or quantitatively and can involve detection of molecules such as RNA, protein, their precursors, differing types (i.e. mRNA, tRNA, and rRNA) and/or degradation products, and/or detection or measurable properties associated thereof. Techniques and procedures to perform detection are identifiable by a skilled person upon reading of the present disclosure.
Exemplary methods for detection of a biomarker expression comprise methods known to a skilled person including but not limited to ELISA, Q-PCR and intracellular cytokine staining detected by FACS. In some embodiments, expression of a biomarker can be detected via fluorescent based readouts on a cell culture performed using an antibody specific for the biomarker or molecule associated thereto, labeled with fluorophore, which includes, but not exhaustively, small molecular dyes, protein chromophores, quantum dots, and gold nanoparticles. In an embodiment expression of a biomarker can be detected by detecting expression of a label under the transcriptional control of a biomarker promoter in vivo (e.g., in an animal tissue) or in vitro (e.g. in a cell culture). In some of those embodiments the biomarker can be in particular IL-10 or Foxp3. Additional techniques are identifiable by a skilled person upon reading of the present disclosure and will not be further discussed in detail.
In methods and systems herein described an anti-inflammatory ability of the candidate bacterial substance can be determined through detection of an increase of expression of the one or more anti-inflammatory biomarkers or a decrease of the expression of the one or more inflammatory biomarkers following the contacting and/or the incubating. Determining increase and/or decrease of a biomarker expression can be performed by comparing the detected expression of the biomarker following the contacting and/or incubating, with a predetermined detected expression of the same biomarker in absence of contacting and/or incubating. Determining increase and/or decrease of a biomarker expression can be performed by comparing the detected expression marker with the detected expression of the same biomarker in a control cell in absence of contacting and/or incubating.
Reference is made to the Examples section wherein increase in expression of the anti-inflammatory biomarkers IL-10, Foxp3, TGFβ1, TGF-β2, perforin and granzyme B and decrease of expression in inflammatory biomarkers TNF-α r IL-17A are associated with the mechanism of action of Bacteroides fragilis supporting association between the above expression pattern and the biological activities of Bacteroides fragilis, with particular reference to anti-inflammatory abilities. The anti-inflammatory abilities of Bacteroides fragilis are identifiable by a skilled person and described in various publications and patent including but not limited to U.S. Pat. No. 5,679,654, U.S. Pat. No. 7,083,777, US2004092433, WO07092451 and WO2009062132, each of which is herein incorporated by reference in its entirety. A skilled person will understand that the specific anti-inflammatory ability of the candidate bacterial substance will be possibly determined based on further determination of the specific set of cytokines produced and cells activated by the candidate bacterial substance identified by methods herein described.
In an embodiment, detection of an increase in expression of the anti-inflammatory or detection of a decrease in expression of an inflammatory biomarker indicates the ability of a substance to induce anti-inflammatory molecules such as IL-10, Foxp3, TGFβ1 or TGF-β2, perforin and granzyme B or a combination thereof, or suppress inflammatory cytokines such as IFNγ, IFNα, IFNα, IL-1, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-21, IL-22, IL-23, IL-17 or TNFα or a combination thereof.
In an embodiment, the method comprises contacting a candidate bacterial substance in vitro and/or in vivo with a T cell alone or in presence of an antigen presenting cell, and detecting expression of at least one anti inflammatory biomarker selected from the group consisting of IL-10, Foxp3, TGFβ1, TGFβ2, perforin and granzyme B, wherein an increase of the expression of the anti-inflammatory biomarker following the contacting indicates an anti inflammatory ability of the candidate bacterial substance. In particular, in some of those embodiments, expression of IL-10, TGFβ1, TGFβ2, perforin and/or granzyme B can be performed on Foxp3 expressing T cell, and more particularly Foxp3 expressing Treg cell.
In an embodiment, the method comprises contacting a candidate bacterial substance with an antigen presenting cell, and incubating the antigen presenting cell with a T cell following the contacting. The method further comprises detecting the expression of at least one anti-inflammatory biomarker selected from the group consisting of Foxp3, IL-10, TGFβ1, TGFβ2, TGFβ2, Perforin and Granzyme B, wherein detection of an increase of the biomarker expression following the incubating indicates an anti-inflammatory ability of the candidate substance. In particular, in some of those embodiments, expression of IL-10, perforin and/or granzyme B can be performed on Foxp3 expressing T cell, and more particularly Foxp3 expressing Treg cell.
In an embodiment, the method comprises contacting a candidate bacterial substance with a T cell alone or in presence of an antigen presenting cell, and detecting expression of at least one inflammatory biomarker selected from the group consisting of IL-17 and TNFα, wherein a decrease of the expression of the inflammatory biomarker following the contacting indicates an anti-inflammatory ability of the candidate bacterial substance.
In an embodiment, the method comprises contacting a candidate bacterial substance with an antigen presenting cell, and incubating the antigen presenting cell with a T cell following the contacting. The method further comprises detecting the expression of at least one inflammatory biomarker selected from the group consisting of IL-17 and TNFα, wherein a decrease of the expression of the inflammatory biomarker following the contacting indicates an anti-inflammatory ability of the candidate bacterial substance.
In an embodiment, the candidate bacterial substance comprises bacteria in a pure pool. In other embodiments, the candidate bacterial substance comprises bacteria in a mixed pool. The bacteria can be live bacteria, dead bacteria, extracts or products isolated from bacteria, or combinations of each. The bacteria may also be from laboratory strains, isolates from repositories such as ATCC, isolates from animals, isolates from humans, or combinations of each.
In an embodiment, the isolate from animals is isolated from feces. The isolate can also come from intestinal contents or combinations of feces and other intestinal contents. In one embodiment, the isolate from humans is isolated from feces. The isolate can also come from intestinal contents or combinations of feces and other intestinal contents.
In an embodiment, the method comprises treating a transgenic marker non-human animal and in particular a transgenic marker mammal such as a mouse or a rat, with a candidate substance, the transgenic non-human marker animal genetically modified to express a labeled anti-inflammatory or inflammatory biomarker selected from the group consisting of IL-10 and Foxp3 but also perforin and granzyme B, IL17 and TNFα; and detecting biomarker expression in the transgenic marker animal following the treating.
In particular, in one embodiment the aforementioned transgenic non-human animal models can be treated either orally or intravenously with the substance, or permanently colonized with live bacteria by oral gavage and the amount of biomarker expression Foxp3 or IL-10 expression will be monitored in the various compartments of the mouse including the spleen, mesenteric lymph nodes, small and large intestine, lungs, pancreas, and bone marrow as a measure of the immunomodulatory ability of the substance.
In one embodiment dendritic cells, T cells, T regulatory, B cells or macrophages can be purified or differentiated from mice where expression of IL-10 (or Foxp3 in the case of T cell analysis) is marked by the fluorophore Green fluorescent protein (GFP). In those embodiments, the candidate bacterial substance is contacted with one of the aforementioned cell types and the amount of GFP expression will be determined indicating the immunomodulatory ability of the substance being tested. Alternatively, dendritic cells or other antigen presenting cells can be incubated with the substance, the substance can be washed off and the dendritic cells can be subsequently incubated with T cells to determine the ability of the dendritic cells to elicit immunomodulatory activity from the T cell. GFP expression can be determined using fluorescent activated cell sorting (FACS) or a microplate reader.
In an embodiment, a T cell, antigen presenting cell, and reagents for detection of at least one of one or more anti-inflammatory biomarkers selected from the group consisting of IL-10, Foxp3, TGFβ2, Perforin and Granzyme B and one or more labeled inflammatory biomarker selected from the group consisting of IL-17 and TNFα, an be comprised in a system for identifying a bacterial substance having immunomodulatory ability according to methods herein described.
In an embodiment, the system comprises: dendritic cell, T cell, T regulatory, B cells and/or macrophages that, in some embodiments, can be purified or differentiated from mice where expression of IL-10 (or Foxp3 in the case of T cell analysis) is marked by the fluorophore Green fluorescent protein (GFP).
The systems can be provided in the form of kits of parts. In a kit of parts, the multi-ligand capture agent and other reagents to perform the method can be comprised in the kit independently. The antigen presenting cell, the T cell and the reagents can be included in one or more compositions, and each cell and reagent can be in a composition together with a suitable vehicle.
Additional components can include labeled molecules and in particular, labeled capture agents specific for an anti-inflammatory or an inflammatory biomarker or a molecule associated to the expression thereof, a microfluidic chip, reference standards, and additional components identifiable by a skilled person upon reading of the present disclosure.
Further details concerning the identification of the suitable carrier agent or auxiliary agent of the compositions, and generally manufacturing and packaging of the kit, can be identified by the person skilled in the art upon reading of the present disclosure.
The vesicles, compositions and related methods and systems herein described are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.
In particular, the following examples illustrate exemplary cell cultures, methods for contacting OMVs comprising PSA (or PSA ligand) with cells and detecting expression of a biomarker. A person skilled in the art will appreciate the applicability of the features described in detail for OMVs and PSA and Bacteroides fragilis for additional vesicles and delivery of substances additional to PSA in OMVs or other vesicles according to the present disclosure.
The following material and methods were used for all the methods and systems for detection of immunomodulatory substances exemplified herein.
Bacterial strains and culture conditions and mice B. fragilis strain NCTC 9343 was obtained from the American Type Culture Collection, its isogenic PSA deletion mutant and mpi44 mutant (produces only PSA but not other polysaccharides) has been described (M. J. Coyne, A. O. Tzianabos, B. C. Mallory, V. J. Carey, D. L. Kasper and L. E. Comstock, (2001) Polysaccharide biosynthesis locus required for virulence of Bacteroides fragilis, Infect. Immun. 69: 4342-4350.) Bacteria were grown either in a rich medium containing 37 g BHI (BD #237200), 0.5 μg/ml Hemin (Sigma H5533), and 0.5 μg/ml Vitamin K (Sigma V3501) in 1 L ddH2O or a customized minimum medium (MM), which contained 8 g Glucose, 1% FBS, 0.5 μg/ml Hemin, and 0.5 μg/ml Vitamin K in 1 L of RPMI (Invitrogen SKU#11835-030). SPF mice of strains C57BL/6 and Balb/c were purchased from Taconic Farms (Germantown, N.Y.). TLR2 knockout mice and IL-10 knockout mice were purchased from Jackson laboratories IL-10GFP mice were procured from the laboratory of Christopher Karp from Cinncinati Children's medical hospital, and, Foxp3GFP mice were a given by the laboratory of Talal Chatila from the University of California Los Angeles.
Isolation of EDL-enriched bacterial population. Percoll (GE Healthcare #17-0891-01) discontinuous density-gradient centrifugation was used for EDL isolation of both wild-type B. fragilis and B. fragilis ΔPSA (Patrick S, Reid J H. (1983) Separation of capsulate and non-capsulate Bacteriodes fragilis on a discontinuous density gradient. J Med. Microbiol. 16(2): 239-41.) Briefly, a 20%, 40%, 60%, 80% Percoll gradient (diluted with PBS) was created in a 14 ml test tube (2 ml for each layer). Then B. fragilis culture resuspended in PBS was carefully added on top of the 20% Percoll layer. Subsequently, the gradient was centrifuged at 800 g for 20 min at RT. EDL-enriched bacteria can be recovered from the 40%-60% interface of the gradient after the separation.
OMV purification and labeling. This method is adapted from a previously described protocol for the preparation of OMVs from E. coli (Amanda L. Horstman and Meta J. Kuehn. (2000) Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles. J Biol. Chem. 275: 12489-12496.) Briefly, EDL-enriched B. fragilis was grown in customized MM. OMVs were recovered from the bacteria-free supernatant of the culture by centrifugation at 40,000 g for 2 hrs at 4 C and further washed twice with PBS and filtered through 0.45 μm spin columns (Millipore #20-218). Total protein concentration of the purified OMVs was determined by Bradford assay (Biorad #500-0205). FITC-labeled OMVs were prepared as previously described (Nicole C. Kesty and Meta J. Keuhn. (2004) Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles. J Biol. Chem. 279: 2069-2076). Briefly, OMVs were incubated in the staining buffer (1 mg/ml FITC (Thermo Scientific #46424), 100 mM NaCl, 50 mM Na2CO3, pH 9.2) for 1 hr at RT. Labeled OMVs were collected by centrifugation at 40,000 g for 30 min at 4 C and washed twice with PBS+200 mM NaCl.
Electron microscopy of bacterial ultrathin section. Ultrathin sections of EDL-enriched B. fragilis were prepared as previously described (Patrick S, McKenna J P, O'Hagan S, Dermott E. (1996) A comparison of the haemagglutinating and enzymic activities of Bacteroides fragilis whole cells and outer membrane vesicles. Microb Pathog. 20(4):191-202.) Briefly, samples were fixed in 2.5% (v/v) glutaraldehyde (Sigma, G5882) in cacodylate buffer overnight at 4° C., followed by further fixation in osmium tetroxide (1%, w/v) for 3 hrs at RT in the dark. Ruthenium Red (1 mg/ml, Sigma R2751) was included in both of the fixation processes. Then fixed samples were embedded in epoxy resin after dehydration in a graded series of alcohols. Ultrathin sections (100-200 nm) were cut and negatively stained with 2% uranyl acetate and lead citrate on formvar/carbon coated copper grids (EMS #FCF200-Cu) before visualization by TEM.
Immunogold labeling of purified OMVs. This method was adapted from a previously described protocol (Patrick S, McKenna J P, O'Hagan S, Dermott E. (1996) A comparison of the haemagglutinating and enzymic activities of Bacteroides fragilis whole cells and outer membrane vesicles. Microb Pathog. 20(4):191-202.) Briefly, a tiny drop of purified OMV was applied to formvar/carbon coated gold grids (EMS #FF200-Au) and air-dried. Immunogold labeling was performed at RT by floating these grids with “OMVs”-side down on a series of small drops of antibody and wash solutions. Particularly, samples were blocked in 10% FBS for 10 min after 5 min incubation in 0.12% glycine. After blocking, samples were further incubated with antibody against PSA diluted in 10% FBS for 20 min, followed by 5 washes×3 min with PBS. Subsequently, secondary antibody-IgG conjugated with 5 nm gold (kind gift from Dr. Paul Webster, House Ear Institute, Los Angeles) was applied to the samples for 20 min, followed again by 5 washes×4 min with PBS. After labeling, samples were fixed in 1% glutaraldehyde for 5 min and washed extensively by transferring grids to drops of PBS 4×1 min and H2O 4×1 min Contrast staining was performed by placing the grids on drops of 3-5% uranyl acetate in 2% methylcellulose for 10 min on ice. Finally, grids were removed from the staining solution by a wire loop and air-dried. Samples covered by a thin film of methylcellulose were removed from loop and used for visualization by transmission electron microscopy (TEM).
Glycoprotein assay. PSA purified from whole cell extracts or OMVs (from B. fragilis mutant mpi44) were subjected to SDS-PAGE and the gel was stained subsequently by glycoprotein staining kit (G bioscience #786-254) to show the presence of PSA.
Chemical (TNBS)-induced experimental colitis. This protocol is adapted from a previously described method (Scheiffele and Fuss. (2001) Induction of TNBS colitis in mice. Current Protocols in Immunology. 15.19.1-15.19.14.) Briefly, wild-type (Balb/c) male mice were orally treated with PBS, WT-OMV (5 μg) or ΔPSA-OMV (5 μg) every other day for a week before TNBS administration. The treated mice were anesthetized with isofluorene and rectal administration of 2% TNBS (in 50% EtOH, Sigma P2297) was applied through a 3.5 F catheter (Instech Solomon; SIL-C35). Oral treatment continued for another two times after TNBS administration and mice were analyzed 1-2 days after the last treatment.
In a prophetic example, the same mice may be treated with the TNBS—as indicated above—prior to orally treating the mice with PBS, WT-OMV (5 μg) or ΔPSA-OMV (5 μg) every other day for a week before TNBS administration. The treated mice would then be anesthetized with isofluorene and rectally administered with 2% TNBS (in 50% EtOH, Sigma P2297) as applied through a 3.5 F catheter (Instech Solomon; SIL-C35). Oral treatment would be continued for another two times after TNBS administration and then mice were analyzed 1-2 days after the last treatment. We expect that using this protocol, the degree of colitis in these mice would be improved, reduced or cured. This expectation is based on the fact that our previous studies using PSA alone (Round et al. 2010) both prior to and after TNBS treatment have both prevented and reduced colitis, respectively; and the fact that some of our present results show that our OMV effect on these mice is PSA dose dependent.
Tissue pathology analysis Mouse colons were fixed in neutral buffered 10% formalin (ScyTek Laboratories CAS#50-00-0) and processed by Pacific Pathology for H & E staining. Colitis scores for each colon section were evaluated in a blinded fashion by a pathologist (Dr. Gregory Lawson, David Geffen School of Medicine, UCLA, Los Angeles). Histology images were taken using light microscopy (Zeiss) at 20× magnification.
Quantitative real-time PCR RNA was collected either from mouse tissues using Trizol (Invitrogen #15596-018) or from purified cells using RNeasy Mini Kit (Qiagen #74104). iSCRIPT cDNA synthesis kit (BioRad #170-8890) was used for conversion of cDNA and IQ SYBR Green supermix (BioRad #172-8882) was used for real-time PCR. Primers used in this study are: TNF (F-5′ACG GCA TGG ATC TCA AAG AC 3′ (SEQ ID NO:1); R-5′ GTG GGT GAG GAG CAC GTA GT 3′) (SEQ ID NO 2); IL-17 (F-5′ TTA AGG TTC TCT CCT CTG AA 3′(SEQ ID NO:3); R-5′ TAG GGA GCT AAA TTA TCC AA 3′) (SEQ ID NO: 4) IL-10 (F-5′ GGT TGC CAA GCC TTA TCG GA 3′ (SEQ ID NO:5); R-5′ ACC TGC TCC ACT GCT TGC T 3′) (SEQ ID NO: 6) Foxp3 (F-5′ GCA ATA GTT CCT TCC CAG AGT TCT 3′(SEQ ID NO: 7); R-5′ GGA TGG CCC ATC GGA TAA G 3′ (SEQ ID NO: 8)) actin (F-5′ TTC GTT GCC GGT CCA CA 3′(SEQ ID NO:9); R-5′ ACC AGC GCA GCG ATA TCG-3′ (SEQ ID NO: 10)).
Fluorescent microscopy In vitro differentiated BMDCs were plated into Lab-Tek II 8-well chamber slide (Nunc #154534) at 50,000 cells/well. FITC labeled OMVs were added into the cell culture at 10 μg/ml. After 2 hrs incubation, cells were fixed in 4% PFA for 20 min at RT. After 3 washes×5 min with PBS, cell membrane was stained with 1 μg/ml tetramethylrhodamine conjugate of WGA (Invitrogen W849) for 1 hr at 4 C. And ProLong Gold anti-fade reagent (P36930) was applied to the sample after extensive washes following membrane staining. Fluorescent images were taken using LSM 510 microscope and Plan-Neofluar 63×/1.25 oil objective.
Flow cytometry and staining BMDCs from OMV uptake assay or activation assay were collected and blocked in 5% mouse serum for 30 min on ice. After blocking, cells were stained with anti-CD11c-APC, anti-MHCII-FITC or anti-CD86-PE (ebioscience) for 30 min on ice and washed 2× with FACS buffer (HBSS (no Ca2+/Mg2+), 1% FBS, 2 mM EDTA, 10 mM HEPES) at 4° C. before flow cytometry analysis. Similarly, cells from in vitro BMDC-T cell co-culture were blocked, and stained with anti-CD4-APC/anti-CD25-PE the same way except that the cells were re-stimulated using PMA/Ionomycin for 4-4.5 hrs before collecting. All flow cytometry was done with BD FACSCalibur and results were analyzed using FlowJo.
In vitro BMDC-T cell co-culture. Bone marrow was collected from different stains of mice and differentiated in vitro in the presence of 20 ng/ml GM-CSF (Miltenyi Biotec #9517571) for 8 days as described previously (Mazmanian, Liu, Tzianabos, and Kasper (2005) An Immunomodulatory Molecule of Symbiotic Bacteria Directs Maturation of the Host Immune System. Cell 122:1 107-118.). (Cell purity was >90%.) CD4+ splenic T cells were isolated by magnetic microbead purification (Miltenyi Biotec #130-090-860) (Cell Purity is >95%.) OMV-pulsed BMDCs (10 μl/ml OMVs, 100,000 cells/ml, 12 hrs-24 hrs) were washed with HBSS and then incubated with CD4+ T cells (1,000,000 cells/ml) in a round bottom 96 well plate with addition of 0.01 μl/ml anti-CD3 (day 0,
In vitro suppression assay: CD4+CD25+ cells purified from BMDC (pulsed with WT-OMV or ΔPSA-OMV)-T cell co-culture were used as a source of Tregs (Miltenyi Biotec, #130-091-041). CD4 depleted mouse splenocytes treated with Mitomycin C (Sigma M4278) were used as APCs (100,000 cells/ml). CD4+CD25− T cells directly purified from mouse spleen were pulsed with CFSE for 10 min at 37° C., followed by first wash with PBS and a second wash with culture media, and used immediately (500,000 cells/ml) as responder cells (Teff). This assay was conducted in a round bottom 96 well plate with an addition of 5 μg/ml of anti-CD3 (ebioscience #16-0031-86) in 200 μl volume. Teff:Treg ratio was titrated and cells were collected after 2-3 days of culture for FACS analysis.
Statistical Analyses: Student T test and one-way ANOVA were applied for pair-wise comparisons and comparisons among >2 groups, respectively. Significant differences among groups detected by ANOVA were analyzed using Newman-Keuls test as the post-hoc test to identify groups exhibiting statistically significant differences. All error bars indicate SEM. NS: not significant; * p<0.05; *** p<0.01; *** p<0.001.
Ultrathin sections of EDL-enriched B. fragilis were prepared as described in materials and methods and imaged by transmission electron microscopy.
The results illustrated in
To determine if PSA is associated with OMVs of B. fragilis, purified vesicles from wild-type and ΔPSA bacteria were subjected to immunoblot analysis as described in the materials and methods section.
The results illustrated in
The results from immunoblot analysis were confirmed by experiments of immunogold labeling performed as described in the materials and methods section. The results of immunogold labeling of purified vesicles illustrated in
PSA is a heterogeneous polymer of repeating subunits. Size separation of PSA recovered from whole cell extracts by chromatography was performed as well as an immunoblot analysis with anti-PSA of capsular polysaccharide preparations from whole cells and purified OMVs as indicated in material and methods.
The relevant results illustrated in
Together, the above results reveal that the immunomodulatory capsular polysaccharide PSA is actively sorted into OMVs of B. fragilis.
To investigate if OMVs can ameliorate clinical symptoms of disease, mice were treated by gavage with OMVs during the induction of TNBS (2,4,6-trinitrobenzene sulfonic acid) colitis.
The results illustrated in
Our efforts to detect intact vesicles in the colon after intra-gastric gavage were confounded by observations of host-derived vesicles, even in germ-free (sterile) mice as previously reported (data not shown).
Since reduction in colon length is a hallmark of TNBS colitis detection of colon length was performed in unmanipulated colons immediately following resection from cecum to rectum and quantification of length (graph) from vehicle treated (EtOH) and TNBS groups (n=4 animals/group). In particular, measurement were performed at time of sacrifice (day 4 following disease induction).
The results illustrated in
Experimental colitis results in severe pathological alterations in intestinal architecture. Accordingly, hematoxylin and eosin (H & E) stained colon sections representative of each treatment group were provided and are shown in
The results of
The above results establish that PSA is required for the disease protective activity of OMVs.
The production of canonical pro- and anti-inflammatory cytokines associated with colitis was measured in mice treated as exemplified in Example 2. In particular, cytokine transcript analysis was performed by qRT-PCR from RNA recovered from whole colons or purified CD4+ T cell from mesenteric lymph nodes.
The relevant results representative of 3 independent trials are reported in
Dendritic cells (DCs) extend protrusions into the gut lumen and sample intestinal particles, and subsequently migrate to MLNs in order to initiate T cell reactions. Indeed, PSA administered orally to animals is associated with CD11c+DCs in the MLN. Accordingly, Applicants sought to test whether OMVs containing PSA can also be taken up by DCs.
The results illustrated in
PSA mediated induction in the DCs of T cell activation markers was also investigated. The results illustrated in
In light of PSA containing OMVs protective role in colitis shown with experiments exemplified in Examples 1 to 3, the biological effects of OMVs on the induction of suppressive T cell responses were tested. In particular, as various regulatory T cell (Treg) populations are known to inhibit colitis, OMVs' ability to promote Treg development was examined.
The results illustrated in
The results illustrated in
Microbial ligands are sensed by several classes of pattern recognition receptors, and PSA has been shown to signal through toll-like receptor 2 (TLR2) to elicit Th1 cytokine production. A series of experiments were therefore performed to test whether TLR2 is required for induction of IL-10 by wild-type OMVs (containing PSA), as recent reports have shown that Treg function and IL-10 expression are influenced by TLR2.
The results illustrated in
To determine the source of IL-10 induced by PSA on OMVs, CD4+CD25+ and CD4+CD25− T cells were purified following co-culture with DCs and the expression of IL-10 and Foxp3 were measured by qRT-PCR.
The results illustrated in
Co-cultures were set up as for experiments illustrated in
The results representative of 2 independent trials are illustrate in
The use of Tregs as cellular therapies has been proposed for IBD, autoimmunity and allergies. IL-10 expression by CD4+ T cell subsets following 4 day co-culture with DCs treated with OMVs was investigated. Splenic CD4+ T cell were purified from IL-10-GFP mice, stained with anti-CD4 and anti-CD25 following co-culture, and IL-10 expression measured by GFP expression.
The results of the relevant OMV treatment of purified DCs & T cells illustrate in
Based on this finding, the ability of PSA was investigated to promote the suppressive capacity of Tregs ex vivo. in vitro suppression of naïve responder cells by purified CD4+CD25+ T cells following co-culture with DCs treated with media (control), WT-OMVs and ΔPSA-OMVs. CD4+CD25− responder cells (effector cells; Teff) were purified from spleens of wild-type mice, pulsed with intracellular dye CFSE (Carboxyfluorescein succinimidyl ester), incubated with Tregs and stimulated with anti-CD3 for 3 days. Cell proliferation was measured by FC of CFSE dilution.
The results illustrated in
Therefore, the specific absence of PSA from OMVs that otherwise contain numerous microbial ligands (LPS, lipoproteins, peptidoglycan, etc) abrogates the ability of B. fragilis vesicles to induce functional Tregs.
Foxp3-GFP mice were orally treated with purified PSA every other day for 6 days. MLNs were extracted and CD4+Foxp3+ or the CD4+Foxp3− T cells were purified by FACS based on ±GFP expression (purity>99%). RNA was extracted and used for q-PCR.
The results illustrated in
The above results provide a molecular mechanism for the probiotic activity of B. fragilis, and reveal a seminal example for a microbial ligand that links innate immune receptor signaling to regulatory T cell development. In particular the above results indicate that PSA is delivered to the host by outer membrane vesicles (OMVs), secretion structures that target bacterial molecules to host cells. OMVs containing PSA are internalized by dendritic cells of the host immune system. Following uptake of OMVs, PSA programs dendritic cells to induce the differentiation of regulatory T cells (Treg) that express Foxp3 and the anti-inflammatory cytokine interleukin-10 (IL-10). Treg development by OMVs requires toll-like receptor 2 (TLR2) expression and IL-10 production by dendritic cells. Remarkably, purified OMVs direct the in vitro differentiation of functional Tregs with potent suppressive activity in a PSA dependent manner. Treatment of animals with OMVs containing PSA prevents experimental colitis and suppresses pro-inflammatory cytokine responses in the gut. These findings reveal that commensal bacteria provide beneficial microbial factors through vesicle secretion, a process that can be engineered into a novel approach for delivery of probiotic therapies for IBD.
In particular, the above results show that Tregs induced by OMVs containing PSA are functionally suppressive and inhibit T cell activation in culture. This is the first demonstration of in vitro Treg development by a microbial ligand, and provides proof-of-principle to the widely-speculated notion that innate immune receptor signaling modulates Treg function. As current therapies for IBD are either ineffective or have severe side effects, probiotics represent promising new treatment options by harnessing well-evolved evolutionary mechanisms for immunmodulation. Given that Tregs suppress immune responses in multiple tissues, our seminal demonstration of in vitro Treg development by PSA suggests the exciting possibility of novel cellular therapies for numerous inflammatory disorders including autoimmunity, asthma and allergies.
To identify molecules made by microorganisms that mediate immunomodulation, fractions of bacterial products can be purified and the same assays performed as above until a pure compound is found which mimics that outcome when whole bacteria are used. Also, the approaches above could be used to screen a mutant library of a microorganism of interest to identify immunomodulatory molecules that have been deleted in a respective clone of a strain which possesses this activity.
Our previous studies have shown that PSA alone can both treat and prevent colitis in mice (Round et al. 2010). Accordingly, given our present results that show wildtype OMV, containing PSA, can prevent colitis in mice, and that this effect with the OMV seems to be PSA dependent we now propose that wildtype OMV will be effective in treating colitis in the same TNBS mice model, and have a beneficial or improved affect on humans with IBD.
It should be noted at the present time that although predictive markers for the onset of inflammatory diseases (such as IBD) are not known, once identified these predictive markers will mean that subjects/humans at risk of developing the disease can be similarly treated with the substances/compositions/OMVs as disclosed herein, to prevent the onset of the disease.
In another embodiment, OMVs (containing PSA) could be altered so that they either do not express or have reduced expression of certain enzymes (for instance, haemagglutinating and enzymic activities (hydrolyzing enzymes such as alkaline and acid phosphatases, esterase lipase. Phosphohydrolyase, glucosaminidase etc.) using molecular or physical methods known to those of skill in the art. Such modified OMVs could then be used to treat or prevent inflammation or inflammatory diseases.
The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the vesicles, systems and methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. Modifications of the above-described modes for carrying out the disclosure that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.
The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background, Summary, Detailed Description, and Examples is hereby incorporated herein by reference.
It is to be understood that the disclosures are not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The term “plurality” includes two or more referents unless the content clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.
Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the specific examples of appropriate materials and methods are described herein.
A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.
Although the present invention has been described in connection with the preferred embodiments, it is to be understood that modifications and variations may be utilized without departing from the principles and scope of the invention, as those skilled in the art will readily understand. Accordingly, such modifications may be practiced within the scope of the following claims.
This application claims the benefit of Provisional Patent Application No. 61/321,527 filed on Apr. 7, 2010, and Provisional Patent Application No. 61/345,039 filed on May 14, 2010, all of which are incorporated by reference herein.
The U.S. Government has certain rights in this invention pursuant to Grant No. DK078938 awarded by the National Institutes of Health.
| Number | Date | Country | |
|---|---|---|---|
| 61321527 | Apr 2010 | US | |
| 61345039 | May 2010 | US |
