Probiotic composition based on the enterococcus strain and used as a treatment means and method for the production thereof



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Bacampicillin, Cloxicillin, Penicillin VK, and the like); with a member of the Fluoroquinolone family of antibiotics (e. g. , Ciprofloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin,

Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, and the like) ; a member of the Macrolide antibiotic family (e. g. , Azithromycin, Erythromycin, and the like); or Metronidazole; and the like.


[00102] Similarly, a therapeutically-effective concentration of an anti-fungal agent may be included in the inactivated probiotic formulation. Such anti-fungal agents include, but are not limited to: Clotrimazole, Fluconazole, Itraconazole, Ketoconazole, Miconazole, Nystatin, Terbinafine, Terconazole, and Tioconazole.
[00103] Inactivated probiotic bacteria can be formulated with one or more agents for treating allergy. Suitable therapeutic agents for the treatment of allergies which can be formulated with inactivated probiotic bacteria include, but are not limited to, antihistamines such as loratadine (Claritin;), fexofenadine (Allegra;), terfenadine; astemizole, cetirizine, hydroxyzine, diphenhydramine; leukotriene synthesis inhibitors zileutron (Zyflo ( ) ; leukotriene receptor antagonists such as zafirlukast (Accolate;), and montelukast ; ss-adrenergic agonists such as

epinephrine, isoproterenol, isoetharine, metaproterenol, albuterol, terbutaline, bitolterol, pirbuterol, and salmeterol; proinflammatory cytokine antagonists; proinflammatory cytokine receptor antagonists; anti-CD23; anti-IgE; anticholinergics such as atropine and ipratropium bromide; immunomodulating drugs; glucocorticosteroids; steroid chemical derivatives ; anti- cyclooxygenase agents; anti-cholinergic agents; methylxanthines, cromones; anti-CD4 reagents; anti-IL-5 reagents; anti-thromboxane reagents; anti-serotonin reagents; ketotiphen; cytoxin; cyclosporin; methotrexate; macrolide antibiotics; heparin; and low molecular weight heparin.


Nutraceutical formulations [00104] The term"nutraceutical formulation"refers to a food or part of a food that offers medical and/or health benefits including prevention or treatment of disease. Nutraceutical products range from isolated nutrients, dietary supplements and diets, to genetically engineered designer foods, functional foods, herbal products and processed foods such as cereal, food bars, soups, and beverages. The term"functional foods, "refers to foods that include"any modified food or food ingredients that may provide a health benefit beyond the traditional nutrients it contains. "Thus, by definition, pharmaceutical compositions comprising an inactivated probiotic bacterium include nutraceuticals. Also by definition, pharmaceutical compositions comprising inactivated probiotic bacteria include compositions comprising inactivated probiotic bacteria and a food-grade component. Inactivated probiotic bacteria may be added to food products to provide a health benefit.
[00105] Nutraceutical formulations of interest include foods for veterinary or human use, including food bars (e. g. cereal bars, breakfast bars, energy bars, nutritional bars); chewing gums; drinks; fortified drinks; drink supplements (e. g. , powders to be added to a drink) ; tablets; and the like. These foods are enhanced by the inclusion of an inactivated probiotic bacterium. For example, in the treatment of an inflammatory bowel disease, the normal diet of a patient may be supplemented by an inactivated probiotic bacterium nutraceutical formulation taken on a regular basis, e. g. , at meal times, before meals, between meals, or after meals. As another example, in the treatment of diarrhea, inactivated probiotic bacteria are included in an electrolyte-containing beverage that the individual consumes periodically throughout the day when the individual is experiencing diarrhea.
[00106] The present invention provides compositions (e. g., nutraceutical compositions) comprising an inactivated probiotic bacterium and a food-grade pharmaceutically acceptable excipient. In many embodiments, subject nutraceutical compositions include one or more components found in food products. Thus, the instant invention provides a food composition

and products comprising an inactivated probiotic bacterium and a food component. Suitable components include, but are not limited to, mono-and disaccharides; carbohydrates; proteins; amino acids; fatty acids; lipids; stabilizers; preservatives; flavoring agents; coloring agents; sweeteners; antioxidants, chelators, and carriers; texturants ; nutrients ; pH adjusters; emulsifiers; stabilizers; milk base solids; edible fibers; and the like. The food component can be isolated from a natural source, or can be synthesized. All components are food-grade components fit for human consumption.


[00107] Examples of suitable monosaccharides include sorbitol, mannitol, erythrose, threose, ribose, arabinose, xylose, ribulose, glucose, galactose, mannose, fructose, and sorbose. Non- limiting examples of suitable disaccharides include sucrose, maltose, lactitol, maltitol, maltulose, and lactose.
[00108] Suitable carbohydrates include oligosaccharides, polysaccharides, and/or carbohydrate derivatives. As used herein, the term"oligosaccharide"refers to a digestible linear molecule having from 3 to 9 monosaccharide units, wherein the units are covalently connected via glycosidic bonds. As used herein, the term"polysaccharide"refers to a digestible (i. e. , capable of metabolism by the human body) macromolecule having greater than 9 monosaccharide units, wherein the units are covalently connected via glycosidic bonds. The polysaccharides may be linear chains or branched. Carbohydrate derivatives, such as a polyhydric alcohol (e. g., glycerol), may also be utilized as a complex carbohydrate herein. As used herein, the term"digestible"in the context of carbohydrates refers to carbohydrate that are capable of metabolism by enzymes produced by the human body. Examples of polysaccharides non- digestible carbohydrates are resistant starches (e. g. , raw corn starches) and retrograded amyloses (e. g. , high amylose corn starches). Non-limiting examples carbohydrates include raffinoses, stachyoses, maltotrioses, maltotetraoses, glycogens, amyloses, amylopectins, polydextroses, and maltodextrins.
[00109] Suitable fats include, but are not limited to, triglycerides, including short-chain (C2-C4) and long-chain triglycerides (C16-C22).
[00110] Suitable texturants (also referred to as soluble fibers) include, but are not limited to, pectin (high ester, low ester); carrageenan; alginate (e. g. , alginic acid, sodium alginate, potassium alginate, calcium alginate); guar gum; locust bean gum; psyllium; xanthan gum; gum arabic ; fructo-oligosaccharides; inulin; agar ; and functional blends of two or more of the foregoing.
[00111] Suitable emulsifiers include, but are not limited to, propylene glycol monostearate (PGMS), sodium stearoyl lactylate (SSL), calcium stearoyl lactylate (CSL), monoglycerides,

diglycerides, monodiglycerides, polyglycerol esters, lactic acid esters, polysorbate, sucrose esters, diacetyl tartaric acid esters of mono-diglycerides (DATEM), citric acid esters of monoglycerides (CITREM) and combinations thereof. Additional suitable emulsifiers include

DIMODAN, including DIMODANTM B 727 and DIMODANTM PV, GRINDSTEDTM

CITREM, GRINDSTEDTM GA, GRINDSTEDTM PS such as GRINDSTEDTM PS 100, GRINDSTEDTMPS 200, GRINDSTEDTMPS 300, GRINDSTEDTMPS 400; RYLOTM (manufactured and distributed by DANISCO CULTOR), including RYLOTM AC, RYLOTM CI, RYLOTM LA, RYLOTMMD, RYLOTMMG, RYLOTM PG, RYLOTMPR, RYLOTM SL, RYLOTMSO, RYLOTM TG; and combinations thereof.


[00112] Edible fibers include polysaccharides, oligosaccharides, lignin and associated plant substances. Suitable edible fibers include, but are not limited to, sugar beet fiber, apple fiber, pea fiber, wheat fiber, oat fiber, barley fiber, rye fiber, rice fiber, potato fiber, tomato fiber, other plant non-starch polysaccharide fiber, and combinations thereof.
[00113] Suitable flavoring agents include natural and synthetic flavors, "brown flavorings" (e. g. , coffee, tea); dairy flavorings; fruit flavors; vanilla flavoring; essences; extracts; oleoresins; juice and drink concentrates; flavor building blocks (e. g. , delta lactones, ketones); and the like; and combinations of such flavors. Examples of botanic flavors include, for example, tea (e. g. , preferably black and green tea), aloe vera, guarana, ginseng, ginkgo, hawthorn, hibiscus, rose hips, chamomile, peppermint, fennel, ginger, licorice, lotus seed, schizandra, saw palmetto, sarsaparilla, safflower, St. John's Wort, curcuma, cardamom, nutmeg, cassia bark, buchu, cinnamon, jasmine, haw, chrysanthemum, water chestnut, sugar cane, lychee, bamboo shoots, vanilla, coffee, and the like.
[00114] Suitable sweeteners include, but are not limited to, alitame; dextrose; fructose; lactilol; polydextrose; xylitol; xylose; aspartame, saccharine, cyclamates, acesulfame K, L-aspartyl-L- phenylalanine lower alkyl ester sweeteners, L-aspartyl-D-alanine amides; L-aspartyl-D-serine amides ; L-aspartyl-hydroxymethyl alkane amide sweeteners; L-aspartyl-l-hydroxyethylalkane amide sweeteners; and the like.
[00115] Suitable anti-oxidants include, but are not limited to, tocopherols (natural, synthetic); ascorbyl palmitate; gallates; butylated hydroxyanisole (BHA); butylated hydroxytoluene (BHT) ; tert-butyl hydroquinone (TBHQ); and the like.
[00116] Suitable nutrients include vitamins and minerals, including, but not limited to, niacin, thiamin, folic acid, pantothenic acid, biotin, vitamin A, vitamin C, vitamin B2, vitamin B 3, vitamin B6, vitamin B12, vitamin D, vitamin E, vitamin K, iron, zinc, copper, calcium, phosphorous, iodine, chromium, molybdenum, and fluoride.

[00117] Suitable coloring agents include, but are not limited to, FD & C dyes (e. g. , yellow ;5, blue ;2, red ;40), FD & C lakes; Riboflavin; (3-carotene ; natural coloring agents, including, for example, fruit, vegetable, and/or plant extracts such as grape, black currant, aronia, carrot, beetroot, red cabbage, and hibiscus.


[00118] Exemplary preservatives include sorbate, benzoate, and polyphosphate preservatives.
[00119] Suitable emulsifiers include, but are not limited to, diglycerides ; monoglycerides; acetic acid esters of mono-and diglycerides; diacetyl tartaric acid esters of mono-and diglycerides; citric acid esters of mono-and diglycerides; lactic acid esters of mono-and diglycerides; fatty acids; polyglycerol esters of fatty acids; propylene glycol esters of fatty acids; sorbitan monostearates ; sorbitan tristearates; sodium stearoyl lactylates; calcium stearoyl lactylates ; and the like.
[00120] Suitable agents for pH adjustment include organic as well as inorganic edible acids.
The acids can be present in their undissociated form or, alternatively, as their respective salts, for example, potassium or sodium hydrogen phosphate, potassium or sodium dihydrogen phosphate salts. Exemplary acids are edible organic acids which include citric acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid and mixtures thereof.
[00121] Inactivated probiotic bacteria are present in the food product/nutraceutical formulation in an amount of from about 5% to about 90% by weight or by volume, e. g. , from about 5% to about 7%, from about 7% to about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 40%, from about 40% to about 50%, from about 50% to about 60%, from about 60% to about 70%, from about 70% to about 80%, or from about 80% to about 90% by weight or by volume. In some embodiments, the inactivated probiotic bacteria present in the food product are homogenous, e. g. , substantially all the inactivated probiotic bacteria in the food product are of the same species. In other embodiments, the inactivated probiotic bacteria in the food product comprise inactivated probiotic bacteria of two or more different species.
[00122] Where the food product is a beverage, the food product generally contains, by volume, more than about 50% water, e. g. , from about 50% to about 60%, from about 60% to about 95% water, e. g. , from about 60% to about 70%, from about 70% to about 80%, from about 80% to about 90%, or from about 90% to about 95% water.
[00123] Where the food product is a bar, the food product generally contains, by volume, less than about 15% water, e. g. , from about 2% to about 5%, from about 5% to about 7%, from

about 7% to about 10%, from about 10% to about 12%, or from about 12% to about 15% water.


[00124] In some embodiments, the food product is essentially dry, e. g. , comprises less than about 5%, water.
[00125] Monosaccharides, disaccharides, and complex carbohydrates, if present, are generally present in an amount of from about 0. 1 % to about 15%, e. g. , from about 0. 1 % to about 1 %, from about 1% to about 5%, from about 5% to about 7%, from about 7% to about 10%, or from about 10% to about 15%, by weight each. Soluble fibers, edible fibers, and emulsifiers, if present, are generally present in an amount of from about 0.1% to about 15%, e. g. , from about

0. 1 % to about 1 %, from about 1 % to about 5%, from about 5% to about 7%, from about 7% to about 10%, or from about 10% to about 15%, by weight each.


[00126] Other components discussed above, if present, are present in amounts ranging from about 0.001% to about 5% by weight of the composition.
TREATMENT METHODS [00127] The present invention provides methods of treating a variety of disorders, the methods generally involving administering to the individual suffering from the disorder a subject formulation. As used herein, the term"administration"includes self administration, e. g., ingestion. Disorders amenable to treatment by administration of a subject formulation include any disorder that is amenable to treatment with viable probiotic bacteria. Disorders amenable to treatment by administration of a subject formulation thus include, but are not limited to, gastrointestinal inflammation; microbial infections; diarrheal diseases; allergic disorders; antigen-stimulated inflammation; microbial infections; irritable bowel syndrome; non- alcoholic liver disease; and asthma.
[00128] The present invention provides methods of treating gastrointestinal inflammation. The methods generally involve administering to an individual in need thereof an effective amount of a subject formulation comprising inactivated probiotic bacteria."Gastrointestinal inflammation"encompasses a variety of disorders, including, but not limited to, inflammatory bowel disease (IBD) ; irritable bowel syndrome ; viral, bacterial, fungal, and parasitic colitis; colitis induced by environmental insults (e. g., gastrointestinal inflammation (e. g., colitis) caused by or associated with (e. g., as a side effect) a therapeutic regimen, such as administration of NSAIDS, chemotherapy, radiation therapy, and the like); colitis in conditions such as chronic granulomatous disease, celiac disease, celiac sprue; food allergies, e. g. , lactose intolerance; gastritis; infectious gastritis or enterocolitis (e. g., Helicobacterpylori-infected chronic active gastritis) and other forms of gastrointestinal inflammation caused by an

infectious agent, e. g., Cryptosporidiumparvum infection, rotavirus gastroenteritis, tropical acute watery diarrhea,"traveler's diarrhea,"Clostridium difficile-induced colitis, Salmonella infections, Shigella infections.


[00129] A subject method of treating a gastrointestinal inflammatory disorder generally involves administering to an individual in need thereof a subject formulation in an amount effective to treat the disorder. The subject methods of treating a gastrointestinal inflammatory disorder include methods of treating individuals who have been diagnosed as having a gastrointestinal inflammatory disorder; methods of reducing the incidence of recurrence, or "flare up"of the disorder; methods of reducing the risk of flare up in an individual who has been diagnosed as having a gastrointestinal inflammatory disorder, has been treated for such by conventional therapies, and is in remission; and methods of treating a gastrointestinal inflammatory disorder in an individual who has failed to respond to conventional therapy for treating the disorder.
[00130] In a subject method for treating a gastrointestinal inflammatory disorder, an"effective amount"of a subject formulation is an amount that reduces the severity of a symptom and/or reduces a measurable parameter associated with the disease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more, when compared with the symptom (e. g. , the severity of the symptom), or when compared with the measurable parameter associated with the disease, in the absence of treatment with a subject formulation.
[00131] The present invention provides methods of treating allergic disorders. The term "allergic disorder"generally refers to a disease state or syndrome whereby the body produces an immune response to environmental antigens comprising immunoglobulin E (IgE) antibodies which evoke allergic symptoms such as itching, sneezing, coughing, respiratory congestion, rhinorrhea, skin eruptions and the like, as well as severe reactions, such as asthma attacks and systemic anaphylaxis. Examples of allergic diseases and disorders which can be treated by the methods of this invention include, but are not limited to, drug hypersensitivity, allergic rhinitis, bronchial asthma, ragweed pollen hayfever, anaphylactic syndrome, urticaria, angioedema, atopic dermatitis, erythema nodosum, erythema multiforme, Stevens-Johnson Syndrome, cutaneous necrotizing venulitis, bullous skin diseases, allergy to food substances and insect venom-induced allergic reactions, as well as any other allergic disease or disorder.
[00132] A subject method of treating an allergic disorder generally involves administering a subject formulation to an individual who is sensitized to an antigen (e. g. , an allergen). A

subject formulation is administered in an amount effective to treat the allergic disorder, e. g. , to reduce production of IgE specific for the antigen (e. g. , the allergen); to reduce the severity of a symptom of the allergic disorder; to reduce the amount of a conventional therapeutic agent that is required to treat the disorder ; to reduce the frequency and/or severity of an allergic reaction to the allergen; and the like. Thus, e. g. , an effective amount of a subject formulation is an amount that reduces the severity of a symptom and/or reduces a measurable parameter associated with the allergic disorder by at least about 10%, at least about 20%, at least about

25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more, when compared with the symptom (e. g. , the severity of the symptom), or when compared with the measurable parameter associated with the allergic disorder, in the absence of treatment with a subject formulation.
[00133] In some embodiments, an effective amount of a subject formulation reduces the level of serum IgE in an individual by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more, when compared with the level of serum IgE in the absence of treatment with a subject formulation. In some embodiments, an effective amount of a subject formulation reduces the severity of symptoms (e. g. , reduces the frequency of coughing, sneezing, wheezing, etc. ) by at least about 10%, at least about 20%, at least about

25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more, when compared with the frequency of coughing, sneezing, wheezing, etc. in the absence of treatment with a subject formulation.


[00134] The present invention provides methods of treating a diarrheal disease. The methods generally involve administering to an individual in need thereof an effective amount of a subject formulation. Diarrheal diseases that are amenable to treatment with a subject method include diarrhea caused by a bacterial infection ; diarrhea caused by a viral infection; diarrhea caused by a mixed bacterial and viral infection ; radiation-induced diarrhea; and antibiotic- induced diarrhea. In the treatment of a diarrheal disease, an"effective amount"of a subject formulation is an amount that is effective to reduce the incidence and/or severity of a diarrheal disease., or that is effective to reduce the time to recover from the disease, by at least about

10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or


more, when compared with the incidence, severity, or recovery time in the absence of treatment with a subject formulation.


[00135] In the treatment of a diarrheal disease, a subject formulation can be taken (administered) prophylactically. For example, a subject formulation can be taken immediately before, and/or during travel to a destination where the risk of contracting a diarrheal disease is high, thereby diminishing the risk that the individual will suffer from diarrhea. As another example, a subject formulation can be administered to an individual who is about to undergo radiation therapy for cancer, or who has recently undergone radiation therapy for cancer. For example, a subject formulation is administered to an individual from about 24 hours to about

72 hours before radiation treatment and/or from about 1 hour to about 24 hours following radiation treatment. Administration of a subject formulation can be initiated from about 1 hour to about 24 hours following radiation treatment, and continued for a period of time thereafter, e. g. , for one day to about 2 weeks following radiation treatment. As another example, a subject formulation can be administered to an individual concurrently with a course of antibiotics, or immediately following a course of antibiotics, to reduce the incidence and/or severity of antibiotic-induced diarrhea.


[00136] The present invention provides a method of treating irritable bowel syndrome (IBS) in an individual. The methods generally involve administering to an individual in need thereof an effective amount of a subject formulation. In the treatment of IBS, an"effective amount"of a subject formulation is an amount that is effective to reduce the severity and/or incidence of one or more symptoms associated with IBS by at least about 10%, at least about 20%, at least about

25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more, when compared with the incidence or severity in the absence of treatment with a subject formulation. Symptoms associated with IBS include bloating, gastrointestinal cramping, loose stool, frequent bowel movement, gas, and the like.


[00137] The present invention provides methods of treating non-alcoholic liver disease, including steatosis, non-alcoholic hepatitic steatohepatitis, and the like. The present invention further provides methods of reducing the risk that an individual will develop hepatic fibrosis or cirrhosis as a result of a non-alcoholic liver disease. The methods generally involve administering to an individual in need thereof an effective amount of a subject formulation. In some embodiments, an"effective amount"of a subject formulation is an amount that is effective to reduce the severity and/or incidence of one or more symptoms or parameters associated with non-alcoholic liver disease by at least about 10%, at least about 20%, at least

about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more, when compared with the incidence or severity of the symptom or the parameter in the absence of treatment with a subject formulation. In other embodiments, an"effective amount"of a subject formulation is an amount that is effective to liver function by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more, when compared with liver function in the absence of treatment with a subject formulation.


[00138] As used herein, the term"liver function"refers to a normal function of the liver, including, but not limited to, a synthetic function, including, but not limited to, synthesis of proteins such as serum proteins (e. g. , albumin, clotting factors, alkaline phosphatase, aminotransferases (e. g., alanine transaminase, aspartate transaminase), 5'-nucleosidase, y- glutaminyltranspeptidase, etc. ), synthesis of bilirubin, synthesis of cholesterol, and synthesis of bile acids ; a liver metabolic function, including, but not limited to, carbohydrate metabolism, amino acid and ammonia metabolism, hormone metabolism, and lipid metabolism; detoxification of exogenous drugs; a hemodynamic function, including splanchnic and portal hemodynamics ; and the like. As one non-limiting example, levels of serum alanine aminotransferase (ALT) are measured, using standard assays. In general, an ALT level of less than about 45 international units is considered normal. In some embodiments, an effective amount of a subject formulation is an amount effective to reduce ALT levels to less than about

45 U/ml serum.


[00139] Whether a subject method is effective in reducing liver fibrosis can be determined by any of a number of well-established techniques for measuring liver fibrosis and liver function.
Whether liver fibrosis is reduced is determined by analyzing a liver biopsy sample. An analysis of a liver biopsy comprises assessments of two major components: necroinflammation assessed by"grade"as a measure of the severity and ongoing disease activity, and the lesions of fibrosis and parenchymal or vascular remodeling as assessed by"stage"as being reflective of long-term disease progression. See, e. g., Brunt (2000) HepatoL 31: 241-246; and

METAVIR (1994) Hepatology 20: 15-20. Based on analysis of the liver biopsy, a score is assigned. A number of standardized scoring systems exist which provide a quantitative assessment of the degree and severity of fibrosis. These include the METAVIR, Knodell,

Scheuer, Ludwig, and Ishak scoring systems.
[00140] The METAVIR scoring system is based on an analysis of various features of a liver biopsy, including fibrosis (portal fibrosis, centrilobular fibrosis, and cirrhosis); necrosis

(piecemeal and lobular necrosis, acidophilic retraction, and ballooning degeneration); inflammation (portal tract inflammation, portal lymphoid aggregates, and distribution of portal inflammation) ; bile duct changes; and the Knodell index (scores of periportal necrosis, lobular necrosis, portal inflammation, fibrosis, and overall disease activity). The definitions of each stage in the METAVIR system are as follows: score: 0, no fibrosis; score: 1, stellate enlargement of portal tract but without septa formation; score: 2, enlargement of portal tract with rare septa formation ; score: 3, numerous septa without cirrhosis; and score: 4, cirrhosis.


[00141] Knodell's scoring system, also called the Hepatitis Activity Index, classifies specimens based on scores in four categories of histologic features: I. Periportal and/or bridging necrosis; II. Intralobular degeneration and focal necrosis; III. Portal inflammation; and IV. Fibrosis. In the Knodell staging system, scores are as follows: score: 0, no fibrosis; score: 1, mild fibrosis (fibrous portal expansion); score: 2, moderate fibrosis; score: 3, severe fibrosis (bridging fibrosis); and score: 4, cirrhosis. The higher the score, the more severe the liver tissue damage.
Knodell (1981) Hepatol. 1: 431.
[00142] In the Scheuer scoring system scores are as follows: score: 0, no fibrosis; score: 1, enlarged, fibrotic portal tracts; score: 2, periportal or portal-portal septa, but intact architecture; score: 3, fibrosis with architectural distortion, but no obvious cirrhosis; score: 4, probable or definite cirrhosis. Scheuer (1991) J : Hepatol. 13: 372.
[00143] The Ishak scoring system is described in Ishak (1995) J Hepatol. 22: 696-699. Stage 0,

No fibrosis; Stage 1, Fibrous expansion of some portal areas, with or without short fibrous septa; stage 2, Fibrous expansion of most portal areas, with or without short fibrous septa; stage 3, Fibrous expansion of most portal areas with occasional portal to portal (P-P) bridging; stage 4, Fibrous expansion of portal areas with marked bridging (P-P) as well as portal-central (P-C); stage 5, Marked bridging (P-P and/or P-C) with occasional nodules (incomplete cirrhosis) ; stage 6, Cirrhosis, probable or definite.


[00144] The benefit of a subject treatment method can also be measured and assessed by using the Child-Pugh scoring system which comprises a multicomponent point system based upon abnormalities in serum bilirubin level, serum albumin level, prothrombin time, the presence and severity of ascites, and the presence and severity of encephalopathy. Based upon the presence and severity of abnormality of these parameters, patients may be placed in one of three categories of increasing severity of clinical disease: A, B, or C.
[00145] Secondary, or indirect, indices of liver function can also be used to evaluate the efficacy of treatment with the subject method. Morphometric computerized semi-automated assessment of the quantitative degree of liver fibrosis based upon specific staining of collagen

and/or serum markers of liver fibrosis can also be measured as an indication of the efficacy of a subject treatment method. Secondary indices of liver function include, but are not limited to, serum transaminase levels, prothrombin time, bilirubin, platelet count, portal pressure, albumin level, and assessment of the Child-Pugh score.


[00146] Serum markers of liver fibrosis can also be measured as an indication of the efficacy of a subject treatment method. Serum markers of liver fibrosis include, but are not limited to, hyaluronate, N-terminal procollagen III peptide, 7S domain of type IV collagen, C-terminal procollagen I peptide, and laminin. Additional biochemical markers of liver fibrosis include a-

2-macroglobulin, haptoglobin, gamma globulin, apolipoprotein A, and gamma glutamyl transpeptidase.


Dosages [00147] Dosages that provide for a therapeutic effect range from about 1 x 105 to about 1 x 1014, from about 5 x 105 to about 5 x 1013, from about 1 x 106 to about 1 x 1012, from about 5 x 106 to about 5 x 1011, or from about 1 x 107 to about 1 x 101 bacteria per unit dosage form bacteria per dosing unit. A"dosing unit"or"unit dosage form, "which terms are used interchangeably herein, may be in the form of a tablet or capsule; a unit amount of a liquid or gel formulation; or, where the formulation is in the form of a food product or a nutraceutical, a serving size. In some embodiments, multiple doses of from about 1 x 105 to about 1 x 1014, from about 5 x 105 to about 5 x 1013, from about 1 x 106 to about 1 x 1012, from about 5 x 106 to about 5 x 1011, from about 1 x 107 to about 1 x 101 , or from about 1 x 108 to about 1 x 109 bacteria are required to achieve a therapeutic effect. Thus, in some embodiments, a therapeutically effective dose is the amount of bacteria administered in two, three, four, five, six, seven, eight, nine, ten, or more dosing units.
[00148] For example, a therapeutically effective dose of bacteria is 1-5 x 101 inactivated bacteria per packet, tablet, or capsule administered 1 to 4 times per day; 1-5 x 101l inactivated bacteria per packet, tablet, or capsule administered 1 to 4 times per day; 1-5 x 1012 inactivated bacteria per packet, tablet, or capsule administered 1 to 4 times per day; 1 x 1013 inactivated bacteria per packet, tablet, or capsule administered 1 to 4 times per day; 1 x 1014 inactivated bacteria per packet, tablet, or capsule administered 1 to 4 times per day; 1-5 x 101 inactivated bacteria per ml liquid formulation administered 1 to 4 times per day; 1-5 x 1011 inactivated bacteria per ml liquid formulation; 1-5 x 1012 administered 1 to 4 times per day; 1 x 1013 inactivated bacteria per ml liquid formulation administered 1 to 4 times per day; 1 x 1014 inactivated bacteria per per ml liquid formulation administered 1 to 4 times per day.

Routes of administration [00149] Conventional and pharmaceutically acceptable routes of administration for treatment of disorders such as allergy and gastrointestinal inflammation (e. g., chronic gastrointestinal inflammation such as that of IBD), include, but are not necessarily limited to, oral, intragastric, vaginal, rectal (e. g., enema, suppository), intranasal and other routes of effective inhalation routes, e. g. , intrapulmonary. In general, gastrointestinal routes of administration are of particular interest in the present invention for treatment of gastrointestinal inflammation including, but not necessarily limited to oral, intranasal, intragastric, and rectal administration.


Routes of administration of particular interest for the treatment of allergy include oral and inhalational routes of administration. Routes of administration of particular interest for the treatment of diarrheal diseases include oral and rectal routes of administration. Routes of administration for the treatment of microbial infection include oral, rectal, vaginal, and inhalational routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the inactivated probiotic bacteria and/or the desired therapeutic effect. The inactivated probiotic bacteria composition can be administered in a single dose or in multiple doses, and may encompass administration of additional doses, to elicit and/or maintain the desired effect.
[00150] Subject inactivated probiotic bacteria can be administered to a subject using any available conventional methods and routes suitable for delivery of conventional drugs.
Methods and localized routes that further facilitate production of the anti-gastrointestinal inflammatory (e. g., anti-IBD) activity or allergy-reducing activity of the inactivated probiotic bacteria, e. g. , at or near a site of inflammation or allergic reaction is of interest in the invention.
In general, routes of administration contemplated by the invention include, but are not necessarily limited to, gastroenteral, enteral, vaginal, or inhalational. Gastroenteral routes of administration include, but are not necessarily limited to, oral and rectal (e. g., using an enema or a suppository) delivery. For the treatment of allergy, suitable routes of administration include inhalational routes (e. g. , intranasal, oral).
[00151] Inhalational routes of administration (e. g., intranasal, oral, intrapulmonary, and the like) are particularly useful in some embodiments, e. g. , in the treatment of allergy. Such means include inhalation of aerosol suspensions or insufflation of the polynucleotide compositions of the invention. Nebulizer devices, metered dose inhalers, and the like suitable for delivery of inactivated probiotic bacteria to the nasal mucosa, trachea and bronchioli are well-known in the art and will therefore not be described in detail here. For general review in regard to intranasal drug delivery, see, e. g., Chien, Novel Drug Delivery Systems, Ch. 5 (Marcel Dekker, 1992).

* Timing of administration [00152] A subject formulation can be administered to a subject prior to onset of more severe symptoms (e. g. , prior to onset of an acute inflammatory attack, prior to onset of an allergic reaction), or after onset of acute or chronic symptoms (e. g., after onset of an acute inflammatory attack, after onset of an allergic reaction). As such, inactivated probiotic bacteria can be administered at any time, and may be administered at any interval. Thus, in some embodiments, administration is episodic.


[00153] In other embodiments, administration is at regular intervals. In one embodiment, inactivated probiotic bacteria are administered about 5 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12 hours, about 24 hours, about 2 days, about 4 days, about 8 days, about 16 days, about 30 days or 1 month, about 2 months, about 4 months, about 8 months, or about 1 year after initial onset of symptoms (e. g. , gastrointestinal inflammation-associated symptoms) and/or after diagnosis of a disorder (e. g. , gastrointestinal inflammation, irritable bowel syndrome, etc. ) in the subject, or after initial onset of an allergic reaction. As described in more detail below, the invention also provides for administration of subsequent doses of inactivated probiotic bacteria.
[00154] When multiple doses are administered, subsequent doses are administered within about

16 weeks, about 12 weeks, about 8 weeks, about 6 weeks, about 4 weeks, about 2 weeks, about

1 week, about 5 days, about 72 hours, about 48 hours, about 24 hours, about 12 hours, about 8 hours, about 4 hours, or about 2 hours or less of the previous dose. In one embodiment, inactivated probiotic bacteria are administered at intervals ranging from at least every two weeks to every four weeks (e. g., monthly intervals) in order to maintain the maximal desired therapeutic effect (e. g., to provide for maintenance of relief from symptoms). In another embodiment, inactivated probiotic bacteria are administered at intervals ranging from once per week, to twice per week, to three times per week, to once per day, to twice per day, or to three times per day.
[00155] In view of the teaching provided by this disclosure, those of ordinary skill in the clinical arts will be familiar with, or can readily ascertain, suitable parameters for administration of inactivated probiotic bacteria according to the invention.
Determining therapeutic efficacy [00156] Where the disorder is gastrointestinal inflammation, the effectiveness of therapy can be monitored by monitoring the reduction of disease activity in the subject. Reduction in disease activity can be monitored by, for example, monitoring reduction of incidence of diarrhea or volume of stool, reduction of rectal bleeding, reduction of weight loss, reduction of size or

number of colon lesions, reduction or opening of strictures, reduction or closure of fistulae, and the like. Therapeutic effectiveness can also be measured by for example, a decrease in C- reactive protein (CRP) level, a decrease in anti-neutrophil cytoplasmic antibodies (ANCA) in a biological sample, a decrease in erythrocyte sedimentation rate (ESR), a decrease in colonic myelo-peroxidase (MPO) activity, reduction of anemia (as detected by, for example, hemoglobin levels, and the like), or other conventional indicator of gastrointestinal inflammation. Many of these methods for assessing therapeutic efficacy can be accomplished through endoscopy or through blood tests. Methods for monitoring gastrointestinal inflammation are well known in the art and well within the skill and knowledge of the ordinarily skilled artisan. Indicators of efficacy of the treatment can include a reduction in severity and/or absence of symptoms, an increase in the number of symptom-free days per time period (e. g. , per week, per month) and/or a reduction in the need for conventional medications.


[00157] Where the disorder is allergy, the efficacy of the treatment can be monitored according to clinical protocols well known in the art for monitoring the treatment of allergic disorders.
For example, such clinical parameters as allergy symptoms (itching, sneezing, coughing, respiratory congestion, rhinorrhea, skin eruption, etc. ), assays and skin prick tests (wheal and flare response) to known allergens and serum levels of IgE and allergy-associated cytokines (e. g., interleukin-4, interleukin-5) can be monitored for determining efficacy.
Indicators of efficacy of the treatment can include a reduction in severity and/or absence of symptoms, an increase in the number of symptom-free days per time period (e. g. , per month) and/or a reduction in the need for conventional medications such as decongestants, anti- histamines, mast cell stabilizers and corticosteroids.
[00158] If the treatment of this invention is carried out in conjunction with immunotherapy, efficacy can be evaluated by observing an increase in tolerated dose of a given allergen (s).
These parameters can be monitored weekly or monthly, as well as at greater time intervals (e. g. , every 3-6 months). In a particular example, clinical parameters that can be monitored for asthma can include the number and severity of attacks as determined by symptoms of wheezing, shortness of breath and coughing. The measurement of airway resistance by the use of respiratory spirometry, the extent of disability and the dependence on immunosuppressive medications or bronchodilators can also be determined.
[00159] The efficacy of treatment for preventing an allergic disorder in a subject not known to have an allergic disorder, but known to be at risk of developing an allergic disorder, can be determined by evaluating clinical parameters such as allergy symptoms (itching, sneezing, coughing, respiratory congestion, rhinorrhea, skin eruption, etc. ), assays and skin prick tests

(wheal and flare response) to known allergens and serum levels of IgE and allergy-associated cytokines (e. g. , interleukin-4, interleukin-5), over time following administration of the nucleic acid or fusion protein of this invention. This time interval can be very short (i. e, minutes/hours) or very long (i. e., years/decades). The determination of who would be at risk for the development of an allergic disorder would be made based on current knowledge of the known risk factors for a particular allergic disorder as would be familiar to clinicians and researchers in this field, such as a particularly strong family history of an allergic disorder or exposure to or acquisition of factors or conditions (i. e. , environmental factors or conditions) which are likely to lead to development of an allergic disorder.


[00160] Where the disorder is diarrhea, the efficacy of a particular treatment is determined by monitoring symptoms reported by the individual or observed by a clinician. Efficacy can be assessed by determining the number of bacteria and/or virus in the stool of an individual who has diarrhea.
Reduction of Risk of Subsequent Disease [00161] The methods of the invention can also provide for reduced risk of other conditions for which gastrointestinal inflammation is a risk factor. For example, ulcerative colitis is a risk factor for colonic carcinoma. Thus, treatment of ulcerative colitis (e. g., by reduction of inflammation) according to the methods of the invention also reduces the risk of colonic cancer (e. g., colonic carcinoma, colonic adenoma, and the like). The methods of the invention can thus be applied as prophylactic measure to prevent or reduce the risk of onset of colonic carcinoma, particularly in those patients that are high risk of colon cancer.
[00162] Established risk factors for colon cancer in those patients having ulcerative colitis include long duration of the disease, large extent of the disease, low activity of the disease, young age at onset, presence of complicating primary sclerosing cholangitis or stenotic disease and possibly lack of adequate surveillance, inadequate pharmacological therapy, folate deficiency and smoking. Crohn disease is associated with an increased risk of colorectal carcinoma in patients with long-standing disease, strictures and fistulae under the condition that the colon is involved, tumors of the small intestine may occur occasionally. Thus treating using inactivated probiotic bacteria according to the invention can be of particular benefit in these patients.
Combination therapy [00163] Inactivated probiotic bacteria can be administered in combination therapy with additional therapeutic agents. For example, in some embodiments, the methods provide for treatment of a gastrointestinal inflammatory disorder, a diarrheal disease, a microbial infection,

an allergic disorder, etc. , involving administering inactivated probiotic bacteria, and a second therapeutic agent.


4] Inactivated probiotic bacteria can be administered in combination therapy with conventional agents used for treatment of gastrointestinal inflammation, where appropriate.
Exemplary agents used in conventional gastrointestinal inflammation therapy, such as those used in therapy for chronic gastrointestinal inflammation such as in IBD, include, but are not necessarily limited to, 5-aminosalicylate (5-ASA), sulfasalazine, corticosteroids, azathioprine, cyclosporine, and methotrexate, as well as tumor necrosis factor-a (TNF-a) antagonists (including antibodies specific for TNF-a ; soluble TNF receptor; and the like), cytokines such as IL-10, or other drug useful in the treatment of chronic gastrointestinal inflammation. Such additional agents can be administered separately or included in the inactivated probiotic bacteria formulation. In addition inactivated probiotic bacteria can be administered in combination therapy with other anti-inflammatory agents, with the proviso that such agents do not substantially interfere with the efficacy of inactivated probiotic bacteria. Exemplary agents include, but are not necessarily limited to, antacids, H2 blockers, proton pump inhibitors, and the like (e. g., famotidine, ranitidine hydrochloride, omeprazole, and the like).
5] Suitable H2 blockers (histamine type 2 receptor antagonists) include, but are not limited to, Cimetidine (e. g. , Tagamet, Peptol, Nu-cimet, apo-cimetidine, non-cimetidine);

Ranitidine (e. g. , Zantac, Nu-ranit, Novo-randine, and apo-ranitidine); and Famotidine (Pepcid,

Apo-Famotidine, and Novo-Famotidine).
6] Subject inactivated probiotic bacteria can be administered in combination therapy with an immunosuppressive agent. Suitable immunosuppressive agents include, but are not limited to, a steroidal immunosuppressive agent, azathioprine, 6-mercaptopurine, methotrexate, cyclosporine, tacrolimus, mycophenolate mofetil, thalidomide, and the like.
71 Suitable TNF-a antagonists that can be administered in combination therapy with a subject inactivated probiotic formulation include soluble TNF-a receptors, chimeric TNF-a receptors, antibodies to TNF-a, etc. Suitable TNF-a antagonists include, but are not limited to, ENBRELO (a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kilodalton (p75) TNFR linked to the Fc portion of human IgGl ; Smith et al.
(1990) Science 248: 1019-1023; Mohler et al. (1993) J Immunol. 151: 1548-1561; U. S. Pat. No.
5, 395, 760; and U. S. Pat. No. 5,605, 690); Infliximab (REMICADE (g) ; a chimeric monoclonal anti-TNF-a antibody that includes about 25% mouse amino acid sequence and about 75% human amino acid sequence; Elliott et al. (1993) Arthritis Rheum. 36: 1681-1690; Elliott et al.
(1994) Lancet 344: 1105-1110; Baert et al. (1999) Gastroenterology 116: 22-28) ; and

Adalimumab (HUMIRA ; a human, full-length IgGl monoclonal antibody that was identified using phage display technology. Piascik (2003) J. Am. Pharm. Assoc. 43: 327-328); and the like.


[00168] Subject inactivated probiotic bacteria are in some embodiments administered in combination therapy with a nutritional beverage, e. g. , peptide-based liquid preparations; beverages comprising nutrients that are easily absorbed by the gut epithelium, e. g. , peptides, fatty acids, electrolytes, monosaccharides, disaccharides, and the like; nutritional beverages such as Ensure, Sustacal, etc.; and the like.
[00169] Inactivated probiotic bacteria can be administered in combination therapy with conventional agents that treat diarrhea, e. g. , loperamide (Imodium (g), Imodium (g) A-D); bismuth subsalicylate; diphenyloxylate/atropine (Lomotil (g)) ; attapulgite (Kaopectateg) ; and the like.
[00170] Inactivated probiotic bacteria can be administered in combination therapy with one or more antibiotics, e. g. , for the treatment of Cryptosporidium parvum infection, Shigella infection, or Salmonella infections. Antibiotics include, but are not limited to, Gentamicin;

Vancomycin; Oxacillin ; Tetracyclines; Nitroflurantoin; Chloramphenicol ; Clindamycin;

Trimethoprim-sulfamethoxasole; a member of the Cephlosporin antibiotic family (e. g.,

Cefaclor, Cefadroxil, Cefixime, Cefprozil, Ceftriaxone, Cefuroxime, Cephalexin, Loracarbef, and the like); a member of the Penicillin family of antibiotics (e. g. , Ampicillin,

Amoxicillin/Clavulanate, Bacampicillin, Cloxicillin, Penicillin VK, and the like); with a member of the Fluoroquinolone family of antibiotics (e. g. , Ciprofloxacin, Grepafloxacin,

Levofloxacin, Lomefloxacin, Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, and the like); a member of the Macrolide antibiotic family (e. g. , Azithromycin, Erythromycin, and the like); or metronidazol.


[00171] Similarly, a therapeutically-effective concentration of an anti-fungal agent may be administered in combination therapy with a subject inactivated probiotic formulation. Such anti-fungal agents include, but are not limited to: Clotrimazole, Fluconazole, Itraconazole,

Ketoconazole, Miconazole, Nystatin, Terbinafine, Terconazole, and Tioconazole.


[00172] Inactivated probiotic bacteria can be administered in combination therapy with a second therapeutic agent for the treatment of allergy. Therapeutic agents for the treatment of allergy include, but are not limited to, a steroid, an anti-histamine, an anti-inflammatory agent, a leukotriene synthesis inhibitor, an immunosuppressant, a bronchodilator, a vasoconstrictor, a decongestant, a leukotriene inhibitor, and the like.

[00173] Suitable therapeutic agents for the treatment of allergies which can be used in combination therapies with an agent of the instant invention include, but are not limited to, antihistamines such as loratadine (Claritin;), fexofenadine (Allegra), terfenadine; astemizole, cetirizine, hydroxyzine, diphenhydramine; leukotriene synthesis inhibitors zileutron (Zyflo (g)) ; leukotriene receptor antagonists such as zafirlukast (Accolate ( ), and montelukast; p-adrenergic agonists such as epinephrine, isoproterenol, isoetharine, metaproterenol, albuterol, terbutaline, bitolterol, pirbuterol, and salmeterol; proinflammatory cytokine antagonists; proinflammatory cytokine receptor antagonists; anti-CD23; anti-

IgE; anticholinergics such as atropine and ipratropium bromide; immunomodulating drugs; glucocorticosteroids; steroid chemical derivatives; anti-cyclooxygenase agents; anti- cholinergic agents; methylxanthines, cromones; anti-CD4 reagents; anti-IL-5 reagents; anti- thromboxane reagents; anti-serotonin reagents ; ketotiphen ; cytoxin; cyclosporin; methotrexate; macrolide antibiotics; heparin; and low molecular weight heparin.
[00174] Inactivated probiotic bacteria and an additional therapeutic agent may be administered in the same formulation or in separate formulations. Where the inactivated probiotic bacteria and the additional therapeutic agents are administered in separate formulations, they may be administered substantially simultaneously, or within about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 16 hours, about 24 hours, about 36 hours, about 72 hours, about 4 days, about 7 days, or about 2 weeks of one another.
SUBJECTS SUITABLE FOR TREATMENT [00175] Subjects suitable for treatment with the formulations and methods of the instant invention include any individual who has been diagnosed as having a gastrointestinal inflammatory disorder. Also suitable are individuals who failed treatment with one or more standard therapies for treating a gastrointestinal inflammatory disorder. Also suitable are individuals who have been treated for a gastrointestinal inflammatory disorder, and are in remission. Suitable individuals include immunocompetent as well as immunocompromised individuals.
[00176] Subjects suitable for treatment with the formulations and methods of the instant invention include any individual who has been diagnosed as having an allergy. Subjects amenable to treatment using the methods and agents described herein include individuals who are known to have allergic hypersensitivity to one or more allergens. Subjects amenable to treatment include those who have any of the above-mentioned allergic disorders. Also amenable to treatment are subjects that are at risk of having an allergic reaction to one or more

allergens. Also suitable are individuals who failed treatment with one or more standard therapies for treating an allergic disorder.


[00177] Subjects suitable for treatment with a subject formulation and method include individuals suffering from IBS.
[00178] Subjects suitable for treatment with a subject formulation and method include individuals having a microbial infection. In some embodiments, the individual is immunocompromised. Immunocompromised individuals include CD4+ T cell deficient individuals; individuals who are immunocompromised following a course of cancer chemotherapy; individuals having an inherited immunodeficiency; individuals who are immunocompromised following a course of radiation therapy; and the like.
[00179] In some embodiments, an immunocompromised individual is a CD4+-deficient individuals, e. g. , individuals who have lower than normal numbers of functional CD4+ T lymphocytes. As used herein, the term"immunocompetent"refers to an individual having CD4+ T lymphocyte levels and function (s) within the normal range in the population, for humans, typically 600 to 1500 CD4+ T lymphocytes per mm3 blood. CD4+-deficient individuals who have an acquired immunodeficiency, or a primary immunodeficiency. An acquired immunodeficiency may be a temporary CD4+ deficiency, such as one caused by radiation therapy, or chemotherapy. In some embodiments, an immunocompromised individual suitable for treatment has a bacterial infection, a viral infection, or a helminth infection (e. g. , a Cryptosporidium parvuni infection).
[00180] Subjects suitable for treatment with a subject formulation and method include individuals having diarrhea. Such individuals include those infected with a virus, bacteria, or combination of virus and bacteria, who have diarrhea as a result of the infection; individuals who are being treated with antibiotics and who have diarrhea as a result; individuals who have been treated for cancer with radiation and who have diarrhea as a result. Subjects suitable for treatment with a subject formulation and method include individuals at risk of developing diarrhea. Individuals at risk of developing diarrhea include individuals traveling in an area where drinking water that is contaminated with viruses and/or bacteria that cause diarrhea is prevalent; individuals who are about to be treated with a course of antibiotics or who are undergoing treatment with a course of antibiotics; and individuals who are undergoing radiation therapy for cancer.
[00181] Subjects suitable for treatment with a subject formulation and method include individuals who have been diagnosed with non-alcoholic liver disease. Such subjects include individuals in whom non-alcoholic liver disease has given rise to fibrosis or cirrhosis.

EXAMPLES [00182] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed.


Efforts have been made to ensure accuracy with respect to numbers used (e. g. amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for.
Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.
Standard abbreviations may be used, e. g. , bp, base pair (s); kb, kilobase (s); pl, picoliter (s); s, second (s) ; min, minute (s); hr, hour (s); i. g. , intragastric; i. r. , intrarectal/intrarectally; cfu, colony forming units; and the like.
Example 1: Treatment of DSS-induced colitis

Materials and Methods

Animals [00183] Balb/c, C57B1/6 (B6), 129XB6F2, congenic mice bearing the C3H-Tlr4LPs~dmutation (i. e. , resistant to LPS) on the Balb/c background, and IL-10 deficient mice were purchased from The Jackson Laboratory (Bar Harbor, ME). MyD88 (B6), TLR2 (B6) and TLR9 (129xB6F2) deficient mice were used as described and are currently bred in the UCSD vivarium. Takeda et al. (2003) Annu Rev Immunol 21: 335-376; and Hemmi et al. (2000)

Nature 408: 740-745.


Probiotic preparations [00184] Probiotic bacteria (VSL-3) were purchased from VSL Pharmaceutical Inc.
(Gaithersburg, MA). Each packet contains viable lyophilized gram+ bacteria of four strains of lactobacilli (L. casei, L. plantarum, L. acidophilus, and L. delbruechii subsp bulgaricus), three strains of bifidobacteria (B. Iongum, B. breve, and B. inJrantis), and one strain of Streptococcus salivarius subsp. T17erti7ophilus. Original packets (450 x 109 CFU per packet) were irradiated with 1.2 M rad using a 137Cs source at a rate of 8 Gy/min overnight. Heat-killed VSL were prepared by resuspending viable probiotics in PBS at 28 x 108 CFU/ml followed by incubation for 30 min at 100 C (heat block), centrifuged at 8,000 RPM for 5 min, washed in PBS and resuspended in fresh PBS prior to their administration. All VSL preparations were resuspended in phosphate-buffered saline (PBS) at a final concentration of 28 x 108 CFU/ml and then cultured as described. Madsen et al. (2001) Gastroenterology 121: 580-591. The resulting

viability was determined by plating the cells on MRS-agar plates (Difco Laboratories, Detroit,

MI) under anaerobic conditions for 16 hours at 37 C. No colonies were detected in the irradiated or heat-killed VSL while 22. Ix 10 6. 1 CFU/ml were recovered for viable (untreated) VSL (28 x 108 as specified by the manufacturer).
Genomic DNA and oligodeoxvnucleotide preparations [00185] Genomic DNA was isolated from VSL-3 packets (VSL Pharmaceutical) and from E. coli (DH5a, Invitrogen, Carlsbad, CA) using DNA Isolation Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The purity of DNA was confirmed by measuring the UV 260/280-absorbance ratio ( < 1.8). LPS levels in the DNA preparations were detected by limulus amebocytes lystate (BioWhittaker Inc. , Wakersville MD) and were < 0.2 EU per u, g of

DNA.
[00186] Cytosine methylation of CpG dinucleotides in isolated probiotic DNA was performed by Sss I methylase (CpG methylase) (New England BioLabs, Beverly, MA) according to the manufacturer's instructions. Methylated DNA was extracted with phenol/chloroform for deproteination. Methylation of DNA was confirmed by digestion with restriction endonuclease

BstUl followed by agarose gel electrophoresis.
[00187] Calf thymus DNA was purchased from Sigma (St Louis MO). Immunostimulatory oligodeoxynucleotide (ISS-ODN) (5'-TGACTGTGAACGTTCGAGATGA-3' ; SEQ ID

NO : 01) and the control ODN (5'-TGACTGTG AAGGTTAGAGATGA-3; SEQ ID NO : 02) on a phosphothioate backbone and were purchased from Tri-Link (San Diego, CA).


Rachmilewitz et al. (2002) Gastroenterology 122: 1428-1441.
[00188] To generate DNA-free probiotics, bacteria (VSL-3) were suspended in saline and disrupted by sonication. Bacterial lysates were incubated with DNase I (Roche, Indianapolis,

IN) (10 U/ml) in the presence of 1 mM MgCl2 on ice for 2 hrs. Elimination of DNA was confirmed by ethidium bromide staining on a 1% TAE agarose-gel.


Colitis models [00139] To induce dextran sodium sulfate (DSS) colitis, DSS (Sigma) was given in the drinking water for 7 days. Preliminary studies were performed to identify the concentration of DSS in the drinking water required to elicit a similar disease activity score in different mouse strains.
3. 5% of DSS in Balb/c mice was equivalent to 1.5% DSS in B6 mice and to 1.75% DSS in

129/B6 mice.


[00190] Trinitrobenzenesulfonic acid (TNBS) colitis was induced in 8 week old, Balb/c mice by rectal instillation of 0.5 mg/mouse of 2, 4,6-trinitrobenzene sulfonic acid (Sigma) dissolved in

O. lml of 50% ethanol as described. Rachmilewitz et al. (2002), supra.

[00191] Mice were sacrificed 7 days after the induction of colitis. All studies were performed in a blind fashion.
Probiotics and various DNAs treatment protocols [00192] Probiotics, including live probiotics, irradiated probiotics, and heat killed probiotics, were intragastrically (i. g. ) given starting 10 days prior to the induction of colitis and for 7 days thereafter. In preliminary studies, mice were treated daily by i. g. administration of 0.28 x 108,

2.8 x 108 or 28 x 108 CFU of irradiated probiotics per mouse per day. The administration of 2.8 x 108 CFU/mouse/day was sufficient to inhibit colitis in Balb/c mice, whereas the administration of 28 x 108 CFU/mouse/day was required to inhibit colitis in the other mouse strains. In some experiments chloroquine (10 mg/kg) (Sigma) was injected s. c. daily after the i. g. administration of viable or irradiated probiotics (2.8 x 1 O8 CFU/mouse/day).


[00193] Various DNA preparations (ISS-ODN and control-ODN, 30 pg/mouse ; probiotic DNA, methylated probiotic DNA, E. coli DNA and calf thymus DNA, 50 llg/mouse) and DNase treated probiotics (i. e. , the amount of microorganisms that yielded 50 u. g of probiotic DNA) were s,. c. injected 2 hrs prior to the administration of DSS or TNBS. In another experiment 50 u, g of these DNA preparations were administered i. g. or intrarectally (i. r. ) 2 hrs prior to DSS administration. In the IL-10 deficient colitis model, ten-week old mice were treated s. c. once a week with the various DNA preparations (see above), and this treatment continued for 4 weeks as described (Rachmilewitz et al. (2002) supra). The disease-activity score, histological score, and colonic myelo-peroxidase (MPO) activity were determined as described (Rachmilewitz et al. (2002) supra).
Effect of probiotics on chronic DSS induced colitis: [00194] To evaluate whether probiotics are effective not only in the prevention but also in the treatment of colitis the following experiment was performed: Mice were treated for 7 days with

DSS 3. 5% added to the drinking water. From the 8th day until sacrifice on day 15, the concentration of DSS in the drinking water was reduced to 1. 75%. During the 15 days of the experiment, 2 groups of mice were treated daily i. g. with viable or with irradiated probiotics

2. 8x108 CFU. A third group was treated s. c. on day 8 with ISS-ODN (10 p. g) and a fourth control group was treated i. g. daily with 0.2 ml of saline. Mice were observed for rectal bleeding, weighed and sacrificed on day 15. The colon was isolated, weighed, sections were taken for histology and mucosal samples were obtained for MPO determination.

Effect of chloroquine on normal flora and on probiotic bacterial strains [00195] To test whether chloroquine has antimicrobial activity on probiotics and on the commensal flora, mice were treated s. c. daily for 7 days with 10 mg/kg of chloroquine (Sigma). Control group was treated s. c. daily with 0.2 ml of saline. After 7 days, stool samples were collected, homogenized and cultured on blood, Maconkey, phenylethanol, chocolate,

M. R. S. , and anaerobic agars. In another experiment, all strains of fecal flora and all probiotic strains were tested for susceptibility to chloroquine by the agar dilution method.
Concentrations tested ranged from 0.3-250 , g/ml of choloroquine. No significant differences were observed as to the identity or quantity of bacterial strains grown from the stool of chloroquine and saline treated mice. The flora that grew included: Bacillus spp. ; Enterococcus sp. ; Escherichia coli; Diphtheroid sp.; Lactobacillus sp.; and Bacteroides sp. All strains, inclusive of the probiotic strains, grew on all plates including those containing 250, ug/ml. The

MIC of all bacterial strains is therefore > 250, ug/ml.


Activation of bone marrow derived macrophages (BMDM) by DNAs [00196] Bone marrow derived macrophages were prepared from Balb/c mice as described (Rachmilewitz et al. (2002) supra). BMDM (1 x 106) were incubated for 48 hrs with 0.1-10 pg/ml of the various DNA preparations. The levels of IL-6 and IL-12 in the supernatants were determined by enzyme linked immunosorbent assay (ELISA; BD-Pharmingen, San Diego, CA)

24 hours post-stimulation.


Detection of absorbed DNAs in mice [00197] For the detection of plasmid DNA (pDNA), one mg of pBudCE4 (Invitrogen) was administered i. g. or i. r. to Balb/c mice. Mice were sacrificed at various time points after pDNA administration and DNA was extracted from liver and spleen using DNeasy Tissue Kit (Qiagen). For the detection of probiotic DNA, 28 x 108 CFU of irradiated probiotics was delivered i. g. for 10 days before DSS administration and for 7 days thereafter as described above. Ten ug each of isolated DNA was run on 1% TAE-agarose gel, transferred onto Hybond-NF membrane (Amersham, Piscataway, NJ), and hybridized to 32P-labeled pDNA or

VSL DNA using hybridization solution (Clontech, Palo Alto, CA). The hybridized membrane was exposed to X-ray film (Kodak, Rochester, NY) at-80 C overnight.


Signaling assays [00198] Translocation of nuclear factor-B (NF-KB) was detected by EMSA as described (Lee et al. (2000) JLeukoc Biol 68 : 909-915). For JNK and IKK kinase assays, lysates of cells or tissues were prepared, and JNK1 or IKK were immunoprecipitated using anti-JNKl or anti-

IKK antibodies (Santa Cruz Biotech, Santa Cruz, CA). The kinase activities were determined


by an in vitro kinase assay using GST-cJun for JNK or GST-IKBa for IKK as a substrate, respectively (Lee et al. (2000) supra).


Statistical analysis [00199] Data are expressed as SEM. Statistical analyses for significant differences were performed according to parametric, Student t test (MPO activity), non-parametric, Mann- Whitney test (disease activity score and histological score). In some assays Chi square test was applied.
Viability assays [00200] The viability of the bacteria was determined by plating on MRS-agar plates (MRS:

DeMan, Rogosa, Sharpe; Difco laboratories). The VSL suspensions of various treatments were serially diluted (1: 10) and a 200 u. l aliquot of each dilution was plated on MRS-agar plates. The plates were incubated anaerobically for 16 hours at 37 C. The numbers of colonies on the plates were counted and multiplied by the dilution factor. No colonies were detected in the suspension of y-irradiated or heat-treated bacteria, while 21. 1 x 108 i 7.1 cfu/ml was recovered from non-treated VSL (28 x 108 as specified by the manufacturer).


DSS induced colitis [00201] A mouse model of colitis was used and assessed, as described. Rachmilewitz et al.
(2002) Gastroenterology 122: 1428-1441. Colitis was induced by adding dextran sodium sulfate (DSS, Sigma), 3.5% to the drinking water, and allowing them to drink ad libitum.
Seven days after induction of colitis, mice were weighed and inspected for diarrhea and rectal bleeding. The mice were sacrificed, and the entire colon was dissected and its length measured and weighed. Scores were again defmed as follows: Changes in body weight: No loss-0; 5 to

10%-1; 10 to 25%, -2 ; 15 to 20%, -3 ; > 20%-4. Hemoccult: No blood,-0 ; positive, -2 ; gross blood,-4. Mucosal samples were processed for determination of MPO activity according to: Bradley (1982) JInvest Dermatol 78: 206-9.


Histological score [00202] When indicated, sections from the distal colon were fixed in buffered formalin and routine 5 um sections were prepared and stained with hematoxylin and eosin. Stained sections were examined blindly and scored. The scoring system took into account the depth of the ulcer, the extent of the ulcer, presence of inflammation, extent of inflammation, and location of fibrosis. Minimal score was 0 and maximal score was 20. Scoring was as follows for depth of the ulcer: 0 = no ulcer; 1-mucosal involvement; 2-mucosal + submucosal involvement; 3 = penetration of muscularis propria; 4 = full thickness involvement. Scoring was as follows for extent of the ulcer: 0 = no ulcer; 1 = punctate; 2 = minimal; 3 = moderate; 4 = widespread.

Scoring was as follows for presence of inflammation: 0 = none; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe. Scoring was as follows for extent of inflammation: 0 = none; 1 = mucosal; 2 = mucosal + submucosal involvement; 3 = mucosal + submucosal + muscle penetration; 4 = full thickness involvement. Scoring was as follows for location of fibrosis : 0 = none; 1 = mucosa only ; 2 = mucosa + submucosa ; 3 = including muscle layer; 4 = full thickness fibrosis.


Determination of MPO activity [00203] Fifty mg colonic mucosal scrapings were homogenized with a polytron (Kinematica

GmbH, Krienz-Luzern, Switzerland) in ice-cold hexadecyltrimethyl ammonium bromide (0.5%) in 50 mM phosphate buffer, pH 6.0. The homogenate was sonicated for 10 seconds, freeze-thawed three times, and centrifuged for 15 minutes. An aliquot of the supernatant was taken for determination of myeloperoxidase (MPO) enzyme activity, as described (Rachmilewitz et al. (2002) supra).


RESULTS

Probiotic and E. coli DNA have immunostimulatory activities [00204] In order to evaluate the immunostimulatory properties of probiotic DNA, we assessed the ability of probiotic DNA to activate NF-kB and JNK, two major signaling pathways involved in TLR activation. Probiotic DNA, but not methylated probiotic DNA or calf thymus

DNA, activated NF-kB (EMSA), as did ISS-ODN but not control-ODN (Fig. 1A). Similar results were obtained for JNK activation (Fig. 1B). The activation of these signaling pathways resulted in the induction of IL-12 (p40) and IL-6, which was mediated via TLR9 as both probiotic DNA and ISS-ODN did not induce the secretion of p40 or IL-6 in TLR9 null macrophages (Fig. 1 C). Similar immunostimulatory profile was observed with E. coli genomic

DNA.
[00205] Figures 1A-C : Probiotic DNA has immunostimulatory activities that depend on TLR9.


BMDM were unstimulated (Unst) or stimulated with ISS-ODN, control (Cont)-ODN (5 p, g/lul), probiotic (prob) DNA, methylated (m) probiotic DNA, or calf thymus (ct) DNA (20 , u g/ml) for 2 hours. A) The activation of NF-kB was determined by electrophoretic mobility shift assay (EMSA). B) JNK. activation (kinase assay). C) Cytokine levels in the supernatants were measured 24 hours post-stimulation, using an ELISA. Results are mean SEM.
TLR signaling is required for anti-inflammatory effects of irradiated probiotics [00206] The administration of non-viable irradiated, or viable probiotics attenuated the severity of DSS induced colitis as reflected in the disease-activity score, histological score, and colonic myeloperoxidase (MPO) activity. In contrast, the administration of heat-killed probiotics had

no effect on the severity of DSS induced colitis (Table 1). Recently, chloroquine has been shown to inhibit the activation of TLR9 induced by its natural ligand, bacterial DNA (Macfarlane and Manzel (1998) JImmunol 160 : 1122-1131). Indeed, when mice were treated with chloroquine, it completely abolished the protective effect of both viable and irradiated probiotics on experimental colitis (Table 1).


[00207] Table 1. Balb/c mice were intragastrically treated daily with 2.8 x 108 CFU of viable, irradiated or heat-killed probiotics 10 days prior to the addition of DSS (3.5%) to the drinking water and for 7 days thereafter. Three groups were also subcutaneously treated with chloroquine (10 mg/kg) dissolved in 0.1 ml of saline once daily (see Materials and Methods).
Disease activity score, colonic MPO activity and histological score were determined after 7 days of DSS administration as described. Results are mean SEM and represent 1 of 3 experiments. The following statistical analyses were employed; for MPO activity-Student t test, for disease activity score as well as for histological score-Mann-Whitney test.
*Significantly different from no treatment or chloroquine treatment (P < 0. 05).
Table 1: Effect of Probiotics and Chloroquine on DSS-Induced Colitis

Treatment N Disease Activity MPO Histological

Score (U/gr) Score

NONE 8 8. 00. 9 1. 90i0. 09 7. 50. 9

Viable probiotics 8 2. 71. 1* 0. 78i0. 20* 2. 80. 8*

Irradiated probiotics 8 0. lO. l* 1. 10=0. 10* 2. 50. 3*

Heat-killed probiotics 9 7. 00. 8 1. 600. 10 5. 41. 0

Chloroquine 5 6. 20. 4 1. 540. 16 8. 00. 8

Irradiated probiotics+chloroquine 5 6. 60. 8 1. 600. 10 7. 70. 6

Viable probiotics+chloroquine 5 6. 8-4-0. 1 2. 300. 48 6. 80. 7 [002081 Histologically, the extensive superficial ulceration with mucosal inflammatory reaction induced by DSS was totally abolished in mice treated with irradiated probiotics whereas in mice co-treated with viable probiotics only minimal superficial ulceration with minimal inflammatory reaction was observed.


[00209] Histological evaluation of a colonic segment of naive Balb/c mice showed normal colonic mucosa, submucosa, and muscularis propria. Histological evaluation of a colonic segment of TLR9 null mice treated with DSS (1.75%) and irradiated probiotic bacteria showed superficial ulceration with severe acute inflammation involving mucosa, submucosa, muscularis propria, and mesenteric fat tissue. Histological evaluation of a colonic segment of
Balb/c mice treated with DSS (3.5%) and viable probiotic bacteria showed minimal superficial ulceration over a lymphoid nodule along with minimal inflammatory reaction involving the mucosa only. Histological evaluation of a colonic segment of Balb/c mice treated with DSS (3.5%) and irradiated probiotic bacteria showed normal colonic mucosa, submucosa, and muscularis propria. Histological evaluation of a colonic segment of Balb/c mice following 7 days of DSS (3.5%) administration showed extensive superficial ulceration with mucosal inflammatory reaction.
[00210] Irradiated and viable probiotics as well as ISS-ODN were also found to equally attenuate the severity of a chronic model of DSS induced colitis (Table 2). In this model the probiotic preparations and the ISS-ODN were administered with or after induction of colitis, respectively, indicating their therapeutic capacity.
[00211] Table 2. Balb/c mice were treated for 7 days with DSS (3.5%) added to the drinking water and for an additional 7 days with DSS (1.75%). One group was treated on day 8 s. c. with

ISS-ODN (10zig) and two other groups were treated daily i. g. with viable or irradiated probiotics 2. 8x108 CFU. Mice were sacrificed on day 15. Results are mean SEM and represent 1 of 3 experiments. For MPO activity, Student t test was employed. For disease activity score and for histological score, Mann-Whitney test was employed * Significantly different from no treatment (P < 0. 05).


Table 2: Effect of Probiotics on Chronic DSS-Induced Colitis

Treatment N Disease Activity MPO Histological

Score (U/gr) Score

NONE 9 5. 40. 6 1. 600. 10 8. 00. 5

Viable probiotics 7 0. 90. 5* 0. 970. 10* 6. 50. 7

Irradiated probiotics 8 1. 60. 5* 1. 180. 10* 5. 90. 1*

ISS-ODN 8 1. 10. 4* 1. 250. 19* 6. 90. 8 ProMotie amd. E. c DNA inhibit DSS-indueed colitis [00212] To evaluate the anti-inflammatory role of probiotic DNA in experimental colitis, probiotic DNA was delivered i. g. , i. r. (Table 3) or s. c. (Table 4) once, two hrs prior to DSS administration. Intragastric and s. c administration of probiotic DNA or ISS-ODN inhibited the severity of DSS-induced colitis whereas i. r. administration of these compounds had no effect on the outcome of colitis. The i. g. administration of methylated probiotic DNA (i. e. , with CpG methylase), calf thymus DNA, or DNase treated probiotics (i. e. , the amount of microorganisms that yielded 50 llg of probiotic DNA) also did not affect the course or the severity of colitis

(Table 3). Intragastric or s. c. administration of E. coli DNA also inhibited the severity of DSS- induced colitis (Table 5). Taken together, these data outline the anti-inflammatory role of certain microbial DNA and the required i. g. or s. c. route of administration for the attenuation of experimental colitis.


[00213] Table 3. Balb/c mice were intragastrically or intrarectally treated with various DNA preparations 2 hours before induction of colitis with DSS. Results are mean SEM and represent 1 of 3 experiments. For MPO activity, Student t test was employed. For disease activity score and for histological score, Mann-Whitney test was employed. * Significantly different from no treatment or treatment with calf thymus DNA (P < 0. 05). ** Significantly different from DNase treated probiotics (P < 0.05).
Table 3: Effect of Intragastric or Intrarectal Administration of Various Probiotic DNA (s) on DSS-Induced Colitis

Treatment N Disease Activity MPO Histological

Score (U/gr) Score

NONE 19 7. 30. 5 1. 90~0. 09 7. 50. 9

Probiotic DNA (i. g. ) 8 3. 2~0. 7*;** 1. 300. 10* ** 3. 00. 4*

Probiotic DNA (i. r. ) 10 5. 9~1. 2 1. 900. 10 6. 51. 1

Methylated Probiotic DNA (i. g. ) 4 7. 80. 5 1. 930. 35 5. 7~0. 7

DNase treated Probiotics (i. g. ) 6 7. 81. 2 1. 600. 20 7. 41. 4 Calfthymus DNA (i. g. ) 8 5. 71. 0 1. 98~0. 20 6. 31. 4

ISS-ODN (i. g. ) 8 3. 6~0. 7* 1. 090. 10* 2. 8~0. 6*

ISS-ODN (i. r. ) 10 6. 30. 7 1. 90~0. 30 5. 9~0. 7

Control-ODN (i. g. ) 10 6. 7i0. 8 1. 900. 20 5. 70. 3 [00214] Table 4. Balb/c mice were subcutaneously injected with various DNA preparations 2 hours before induction of colitis (see Materials and Methods). Results are mean SEM and represent 1 of 3 experiments. For MPO activity, Student t test was employed. For disease activity score and for histological score, Mann-Whitney test was employed. * Significantly different from no treatment (P < 0. 05).'55 Significantly different from treatment with probiotic

DNA (P < 0.05).

Table 4: Effect of Subcutaneous Administration of Various DNA (s) on DSS-

Induced Colitis

Treatment N Disease Activity MPO Histological

Score (U/gr) Score

NONE 10 5. 70. 5 3. 6~0. 3 10. 7~0. 8

Probiotic DNA 8 1. 1~0.3* 0. 8~0. 1* 0. 30. 3*

Methylated Probiotic 8 3. 40. 4** 1. 70. 2** 4. 3~1.3**

DNA


DNase treated 4 5. 0~1. 1** 1. 50. 2** 4. 31. 2**

Probiotics

Calf thymus DNA 4 5. 51. 5 3. 30. 5 6. 0~1. 3

ISS-ODN 4 0. 4i0. 4* 0. 9~0. 1* 0i0*

Control-ODN 4 3. 80. 7 4. 2~0. 8 8. 00. 9 [00215] Table 5. Balb/c mice were subcutaneously or intragastrically treated with E. coli DNA

2 hours before induction of colitis DSS (see Materials and Methods). Results are mean SEM and represent 1 experiment. For MPO activity, Student t test was employed. For disease activity score and for histological score, Mann-Whitney test was employed. * Significantly different from no treatment (P < 0. 01) ; ** Significantly different from no treatment (P < 0.03)

Table 5: Effects of Administration of E. coli DNA on DSS-Induced Colitis

Treatment N Disease Activity MPO Histological

Score (U/gr) Score

NONE 7 3. 00. 8 1. 40i0. 20 6. 700. 7

E. coli DNA (s. c. ) 7 0. 3+-0. 2* 0. 55~0. 10* 3. 100. 6**

E. coli DNA (i.g.) 7 0.6~0. 3* 0. 700. 19** 6. 40i0. 9

Probiotic DNA attenuates different models of experimental colitis [00216] We further evaluated whether the protective effect of probiotic DNA can be reproduced by its s. c. injection in TNBS-induced colitis as well as in spontaneous colitis observed in IL-10 deficient mice (Strober et al. (2002) Annu Rev Immunol 20:495-549). A single s. c. injection of probiotic DNA, but not of methylated probiotic DNA or of calf thymus DNA, decreased the disease-activity score, histological score, and colonic MPO activity of TNBS-induced colitis (Table 6) and attenuated the course and the severity of the colitis that had developed in IL-10 deficient mice over time (Table 7).
[00217] Table 6. Balb/c mice were subcutaneously injected with various DNA preparations 2 hours before induction of colitis (see Materials and Methods). Results are mean SEM and

represent 1 of 3 experiments. For MPO activity, Student t test was employed. For disease activity score and for histological score, Mann-Whitney test was employed. * Significantly different from no treatment or treatment with calf thymus DNA (P < 0. 05). ** Significantly different from treatment with probiotic DNA (P < 0.05)

Table 6: Effect of Various DNAs on TNBS-Induced Colitis

Treatment N Disease Activity MPO Histological

Score (U/gr) Score

NONE 10 1. 80. 7 1. 50~0. 01 9. 0~1. 8

Probiotic DNA 8 0* 0. 880. 08* 1. 7~0. 9*

Methylated Probiotic DNA 8 1. 5~0. 3** 1. 5~0. 09** 5. 00. 9**

DNase treated Probiotics 8 1. 0+0** 1. 600. 06** 5. 50. 9**

Calf thymus DNA 8 3. 4~0. 5 1. 20+0. 07 7. 02. 0

ISS-ODN 8 0. 4i0. 3* 0. 8A0. 10* 00* Control-ODN 4 1. 3~0. 2 2. 00~0. 30 4. 8~1. 8 [00218] Table 7. IL-10 KO mice (B6) were subcutaneously injected once a week with various

DNAs (see Materials and Methods). The following statistical analyses were employed; for rectal prolapse-Chi square, for MPO activity-Student t test and for histological score-Mann-

Whitney test. Results are mean SEM. * Significantly different from no treatment (P < 0.05).
Table 7: Effect of Various DNAs on Spontaneous Colitis in IL-10 KO Mice

Treatment N Rectal Prolapse MPO Histological (N) (U/gr) Score

NONE 13 11 1. 0~0. 10 8. 10. 9

Probiotic DNA 10 2* 0. 2~0. 04* 3. 0~0. 4*

Calf Thymus 8 5 0. 7~0. 10 6. 3~1. 4

DNA


ISS-ODN 6 1* 0. 80. 10 1. 81. 2* Absorption of bacterial DNA from the gastrointestinai tract [00219] As i. g. or s. c. but not i. r. administration of probiotic DNA ameliorates experimental colitis, we reasoned that the probiotic DNA might be absorbed from the upper gastrointestinal tract as was described for phage DNA (Schubbert et al. (1997) Proc Natl Acad Sci U S A

94: 961-966) and act in systemic sites. To explore this possibility, a purified form of bacterial

DNA was delivered, i. e. , plasmid DNA (pDNA), i. g. once to wt mice and the presence of this bacterial DNA in their liver and spleen was evaluated. Indeed, we identified the pDNA and its

fragments in these organs (southern blot) within 2-6 hrs post-i. g. but not post-i. r. administration (Fig. 2A). Interestingly, the efficacy of bacterial DNA absorption was by far lower when the pDNA was delivered i. r rather than i. g. (Fig. 2A). The localization of this bacterial DNA in these organs coincided with its immunostimulatory activities i. e. , the activation of JNK and NF-kB (Fig. 2B), the major signaling pathways initiated by the engagement of TLR9 with its ligand, bacterial DNA. We also identified the probiotic DNA in the liver and spleen after daily i. g. administration of irradiated probiotics which was initiated

10 days prior to induction of colitis with DSS, and for 7 days thereafter (Fig. 2C).
[00220] Figures 2A-C: Detection of bacterial DNA at systemic sites. A) Plasmid DNA is detected after its oral administration (1 mg/mouse) in the liver (L) and spleen (S) but is not detected after rectal administration (southern blot). The uptake of pDNA in these organs after s. c. injection (100 llg/mouse) is shown as control. B) The localization of orally administered pDNA coincides with the activation of IKK and JNK1 in these organs. C) VSL DNA is detected in the liver (L) and the spleen (S) after 17 days of oral administration of irradiated probiotic bacteria (southern blot).
[00221] Taken together, these data indicate that most of the probiotic DNA is absorbed from the upper gastrointestinal tract and most probably acts systemically as occurs with s. c. injection of other types of immunostimulatory DNA (e. g. , ISS-ODN).
Example 2: Administration of Pasteurized Probiotics Ameliorates DSS Induced Colitis [00222] Live bacteria from the VSL-3 preparation were heated at different temperatures for various time periods as indicated. Following the heating, the bacteria were cultured for 48 hours and counted. 2. 8x108 heated bacteria were administered i. g. to Balb/c mice for 10 days prior to the additions of DSS 5% to their drinking water and for 7 days thereafter. Seven days after the addition of DSS, mice were sacrificed disease activity (DAI; % decrease in body weight and rectal bleeding) and MPO activity were determined and sections were obtained for histological analysis (HS ; histological score). The results are shown in Table 8, below. DAI = disease activity score (index).
Table 8
Treatment Time Culture N DAI MPO

63 C 30 min. 106 9 3. 3+0. 7 1. 10. 20

70 C 5 min. 106 10 3. 1+0. 8 1. 500. 20

80 C 10 min. sterile 15 4. 7+0. 5 1. 950. 20

100 C 30 min. sterile 10 7. 3+0. 5 1.98~0. 20

[00223] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.Claims:

CLAIMS

What is claimed is:



1. An enteral formulation comprising inactivated probiotic bacteria; and a pharmaceutically acceptable excipient, wherein the bacteria are inactivated by a process other than heating to 100 C for 30 minutes.
2. The method of claim 1, wherein the bacteria are inactivated by a process selected from gamma irradiation, ultraviolet irradiation, and pasteurization.
3. The formulation of claim 1, wherein the formulation is a liquid or gel formulation comprising an agent selected from the group of a flavoring agent and a coloring agent.
4. The formulation of claim 1, wherein the formulation is a solid formulation comprising a solid-based dry material.
5. The formulation of claim 4, wherein the solid-based dry material is selected from a starch, gelatin, sucrose, dextrose, trehalose, and malto-dextrin.
6. The formulation of claim 1, wherein said formulation is in the form of a capsule, tablet, a liquid, or a gel.
7. The formulation of claim 1, wherein said pharmaceutically acceptable excipient is a food-grade carrier.
8. The formulation of claim 7, wherein the food-grade carrier is selected from an edible oil, an emulsifier, a soluble fiber, a flavoring agent, a coloring agent, an edible fiber, and a sweetener.
9. The formulation of claim 1, wherein the inactivated bacteria are present at a concentration of from about 1 x 105 bacteria per gram to about 1 x 1014 bacteria per gram.

10. The formulation of claim 1, wherein the inactivated bacteria are present in a concentration of least about 5% by weight.


11. The formulation of claim 1, wherein the formulation is a liquid formulation, and the inactivated bacteria are present at a concentration of from about 1 x 105 bacteria per milliliter to about 1 x 1014 bacteria per milliliter.
12. The formulation of claim 1, further comprising an immunosuppressive agent.
13. The formulation of claim 12, wherein the immunosuppressive agent is selected from a steroidal agent, azathioprine, 6-mercaptopurine, methotrexate, cyclosporine, tacrolimus, mycophenolate mofetil, and thalidomide.
14. The formulation of claim 1, further comprising an antibiotic.
15. The formulation of claim 14, wherein the antibiotic is selected from metonidazole and ciprofloxacin.
16. The formulation of claim 1, further comprising sulfasalazine.
17. The formulation of claim 1, further comprising 5-aminosalicylic acid.
18. The formulation of claim 1, further comprising a nutritional beverage comprising nutrients that are readily absorbed by gut epithelium.
19. A food product comprising inactivated probiotic bacteria, wherein the bacteria are inactivated by a process other than heating to 100 C for 30 minutes, and wherein the inactivated bacteria are present in the food product at a concentration of from about 1 x 105 bacteria per gram to about 1 x 1014 bacteria per gram or from about 1 x 105 bacteria per milliliter to about 1 x 1014 bacteria per milliliter.
20. The food product of claim 19, wherein the food product is a milk-based food product.

21. The food product of claim 19, wherein the milk-based food product is ed from milk, cheese, yogurt, butter, ice cream, frozen yogurt, whipped toppings, ., custard, pudding, nutritional drinks, infant formula, and milk chocolate.


22. The food product of claim 19, wherein the food product is a soy-based food ct.
23. A method of treating a disorder that is amenable to treatment with viable Dtic bacteria, the method comprising administering to an individual in need thereof an ive amount of a formulation of claim 1.
24. The method of claim 23, wherein the formulation is administered orally.
25. The method of claim 23, wherein the disorder is gastrointestinal imation.
26. The method of claim 25, wherein the gastrointestinal inflammation is acute intestinal inflammation.
27. The method of claim 25, wherein the gastrointestinal inflammation is chronic intestinal inflammation.
28. The method of claim 27, wherein the chronic gastrointestinal inflammation is d by inflammatory bowel disease.
29. The method of claim 28, wherein the inflammatory bowel disease is five colitis.
30. The method of claim 28, wherein the inflammatory bowel disease is Crohn 3e.
31. The method of claim 23, wherein from about 1 x 105 bacteria per gram to bacteria per unit dosage form are administered.

32. The method of claim 23, wherein the disorder is an allergic disorder.


33. The method of claim 32, further comprising administering an additional therapeutic agent for treating the allergic disorder.
34. The method of claim 32, wherein the allergic disorder is allergic asthma.
35. The method of claim 32, wherein the allergic disorder is an allergic reaction to a plant allergen, a food allergen, an animal allergen, or a drug allergen.
36. The method of claim 32, wherein the allergic disorder is selected from atopic dermatitis, a food allergy, allergic gastroenteritis, and allergic rhinitis.
37. The method of claim 23, wherein the disorder is a diarrheal disease.
38. The method of claim 37, wherein the diarrheal disease is caused by a bacterial infection, a viral infection, a mixed viral and bacterial infection, radiation treatment, or antibiotic treatment.
39. The method of claim 23, wherein the disorder is irritable bowel syndrome.
40. The method of claim 23, wherein the disorder is non-alcoholic liver disease.

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