HK1227299A - Product containing inactivated probiotic for children or infants - Google Patents

Description

Product containing inactivated probiotic for children or infants

The application is a divisional application of PCT application with the international application date of 22/2008, the international application number of PCT/US2008/054717, the application number of 200880006384.0 entering the national stage and the invention name of 'a product containing inactivated probiotics for children or infants'.

Cross reference to related patents and patent applications

This application is a non-provisional patent application but claims priority from U.S. provisional patent application serial No. 60/904,122, filed on day 28, 2/2007, which is incorporated herein by reference in its entirety.

Background

(1) Field of the invention

The present invention relates generally to products containing at least one inactivated probiotic and methods of using at least one inactivated probiotic.

(2) Background of the invention

Inflammatory responses are efforts to restore and maintain homeostasis after the body has been attacked by infectious agents, antigenic attacks, or physical, chemical, or traumatic injuries. Localized inflammation is localized to a specific site and may exhibit different symptoms including redness, swelling, heat, and pain.

While inflammatory responses are generally considered to be a healthful response to the damage, adverse physiological responses may occur if the immune system is improperly regulated. In this case, the body's normal protective immune system causes damage to self-tissue by treating healthy tissue as is the case with infected or abnormal tissue. Alternatively, if damage occurs, the inflammatory response is not proportional to the threat of causing the damage. When this occurs, the inflammatory response may cause much more damage to the body than the factor itself does.

Studies have shown that the inflammatory response is composed in part of increased expression of both pro-inflammatory and anti-inflammatory cytokines. Cytokines are low molecular weight, biologically active proteins involved in the coordination of immune and inflammatory responses, as well as in the communication between specific immune cell populations. During the inflammatory response, various cell types produce cytokines, including neutrophils, monocytes, and lymphocytes.

Cytokines produced at sites of inflammation affect the inflammatory response by a variety of mechanisms that exist. However, if the pro-inflammatory response is not successfully counteracted by the anti-inflammatory cytokines, uncontrolled systemic inflammation may occur.

Systemic inflammation is distributed throughout the body, as compared to local inflammation. This type of inflammation may include localized inflammation at specific sites, but may also be associated with common "flu-like" symptoms, including fever, chills, fatigue or loss of vitality, headache, loss of appetite, and muscle stiffness. Systemic inflammation can lead to protein degradation, catabolism and hypermetabolism. Thus, the structure and function of vital organs (e.g., muscles, heart, immune system and liver) may be impaired and multiple organ failure may result, ultimately leading to death. Jeschke et al, insulin attenuated systemic inflammatory response to burns, mol. Med.8 (8): 443-450(2002). Although significant progress has been made in understanding the mechanisms of systemic inflammation, the high mortality rates resulting from this disease remain unacceptable.

Respiratory infections are extremely common, especially in infants. Infants are susceptible to recurrent respiratory infections during the first year of life, often experiencing 3-6 infections in the first year alone. In the united states alone, about 6% of infants less than 1 year old are hospitalized annually for lower respiratory tract infections.

Respiratory infections and their symptoms can range from mild to severe, depending on the type of virus and the site of infection. Upper respiratory tract infections often manifest themselves as colds, causing inflammation and swelling of the mucosa of the nose, throat and sinuses. Influenza, commonly referred to as flu, is a highly contagious upper respiratory viral infection. Symptoms of influenza include fever, chills, headache, muscle aches, dizziness, cough, sore throat, runny nose, nausea and diarrhea. Another upper respiratory infection, grubbs (croup), causes very severe coughing and varying degrees of dyspnea, mainly when inhaling.

Lower respiratory tract infections are generally considered to be more severe than upper respiratory tract infections. Respiratory Syncytial Virus (RSV) is the most common cause of lower respiratory tract infections in infants and children under 4 years of age. Van woensel, j, et al, viral lowerer respiratory tract infection transmenoninfatand young childrens (viral lower respiratory tract infection of infants and young children), BMJ 327: 36-40(2003). This is a common virus such that virtually all children are infected with RSV by the age of 3. In most infants and children, RSV is a mild respiratory infection that is indistinguishable from the cold. It typically causes nasal obstruction, nasal discharge, and cough.

Protection against RSV includes both T and B cell responses, antibody responses (IgM, IgG, and IgA), and other immune system responses activated by bacterial and viral infections. Studies have shown a correlation between RSV infection in infancy and the subsequent recurrent wheezing, asthma and specific allergies in childhood. Thus, control of RSV infection can prevent serious respiratory complications that extend into childhood.

Bronchitis is an infection of the lower respiratory tract involving the bronchi, and is a narrowing and swelling of the bronchi caused by viral inflammation. Bronchiolitis is similar to bronchitis, but occurs primarily in infants and young children. It is an inflammation of the smaller caliber trachea in the bronchial branching network. Infections cause dyspnea, frequent and intense coughing and wheezing, and may require hospitalization.

The perhaps most severe lower respiratory infection for infants is pneumonia. Pneumonia is caused by infection of the alveoli, filling them with a liquid, often of a viscous, purulent nature, that interferes with the normal exchange of carbon dioxide. The severity of pneumonia will depend on the amount of lung tissue involved.

Most upper and lower respiratory tract infections are caused by viruses, and no particularly effective preventive or therapeutic measures against these viruses have been taken. Some respiratory infections, including influenza, can be prevented by vaccination. However, even if vaccinations were developed for specific respiratory infections, they are expensive and not universally available. As such, the availability of drugs to treat these infections is limited and expensive. Therefore, it would be beneficial to provide a non-pharmaceutical method of treating or preventing respiratory infections in infants and young children.

Frequent respiratory infections are often associated with Acute Otitis Media (AOM), also known as middle ear infections. AOMs are characterized by acute short-term inflammation and fluid in the middle ear. AOM can be associated with rhinitis, cough, fever, sore throat, ear pain, hypoacusis, dysphoria, irritability, anorexia, emesis or diarrhea. Purulent ear discharge via the perforated tympanic membrane is also considered to constitute part of the AOM.

AOM has been initiated at least once by the age of 1 in 50% of children. 80% of children have had at least one episode by the 3 year old birthday. Between the ages of 1 and 3, 35% of children may have repeated attacks of AOM.

AOMs may be caused by viruses or bacteria. The most common bacterial strains causing AOM are streptococcus pneumoniae (streptococcus pneumoniae) (35% of cases), haemophilus influenzae (haemophilus influenzae) (30% of cases) and moraxella catarrhalis (moraxella catarrhalis) (10% of cases). Because bacterial strains often cause infections, AOM is generally treated by administration of antibiotics. In fact, more antibiotics are prescribed by AOM than for any other disease in infancy.

In general, whether the cytokine response is pro-inflammatory or anti-inflammatory depends on the balance of various microorganisms that colonize the intestinal lumen at any particular time. It is well known that the mucosal surfaces of the intestine colonize a very large number of microorganisms, whose composition is extremely complex and constantly changing. The composition of the gut microflora varies with the digestive tract and with different small environments, such as epithelial mucosal layer, crypt deep mucosal layer and mucosal epithelial cell surface. The specific colonization status depends on internal and external factors including the molecules available in the lumen, the nature of the mucosa and the host-microbial interaction and the microbial-microbial interaction. Murch, s.h., tollol along reduction of b biology (probiotics reduce allergy to Toll), Lancet, 357: 1057-1059(2001).

These microorganisms constitute the gut microflora and actively participate in the immune response. They interact with the epithelium under conditions where there is a mutual interest in the two parties (mutualism), or under conditions where one is beneficial and the other is not harmful (partiality). Hooper et al, HowHost-microbiological interactions Shapepe Nutrient environmental impact on Mammarian Intestine (how host-microbial interactions form the nutritional environment of the mammalian gut), Annu.Rev.Nutr.22: 283-307(2002). In fact, there is a great deal of evidence for strong interactions or "interactions" between gut microflora and different cell populations in the intestinal mucosa. Bourlioux et al, the interest and Microfloraarreparrnersfor the Protectionnofthest: reporton the dinonesymposium "the intelligentintestine" (gut and its microflora are partners for host protection: darby monograph report "smart gut"), held in paris 6, 14 th month 2002, am.j. clin. nutr.78: 675 (2003); hooper, l.v. and Gordon, j.i., CommensalHost-bacterial relationship between enterosymbiotic hosts, sci.292: 1115 (2001); haller et al, Non-pathogenic bacterial cells in intestinal epithelial cell/leukocyte co-culture inducing different cytokine responses, GUT 47: 79 (2000); walker, w.a., rolleof nutrindsandbacteriol colloids of development of intestinalhostdefense (role of nutrients and bacterial colonization in development of gut host defense), j.pediator.gastroenterol.nutr.30: s2 (2000). In addition, studies have shown that the gut microflora induces specific immune responses at both the local and systemic levels in adults. Isolauri, E, et al, Probiotics: effectsonimmitus (action of probiotics on immunity), am.j.clin.nutr.73: 444S-50S (2001).

It is known that the development degree of intestinal microflora in infants is far less than that of adults. Although the microflora of adults is 10¹³More than one microorganism and up to 500 species of bacteria, but some are harmful and some are beneficial, and the microflora of infants contains only a small fraction of these microorganisms, both in absolute numbers and in species diversity. Infants and young children have a sterile intestinal tract, but the intestinal microflora is obtained from the birth canal, their original environment and the food they ingest. Because gut microflora is very unstable in the early life of a newborn, it is often difficult for the infant's gut to maintain a delicate balance between harmful and beneficial bacteria, thus reducing the ability of the immune system to function properly.

It is particularly difficult for formula-fed infants (formula-fed) to maintain this balance caused by the difference between the intestinal bacterial species of formula-fed infants and breast-fed infants (breast-fed). The feces of breast-fed infants mainly contain bifidobacteria (bifidobacteria), and Streptococcus (Streptococcus) and Lactobacillus (Lactobacillus) as unusual contributors. In contrast, formula-fed infants are more diverse in their microflora, containing bifidobacteria and Bacteroides (Bacteroides) and more pathogenic bacteria-staphylococci (Staphylococcus), escherichia coli (escherichia coli) and Clostridia (clostridium). The feces of breast-fed infants and formula-fed infants also differ in the species of bifidobacterium. Studies have suggested a number of factors responsible for the different fecal flora of breast-fed infants and formula-fed infants, including lower protein content, different protein composition, lower phosphorus content in human milk, high amounts of oligosaccharides in human milk, and various humoral and cellular mediators of immune function in breast milk. Agostoni et al, ProbioticBacteriani Dieticiprocproducts for Infants: amementarybytheneggpachromitteonen nutrition (probiotic bacteria in infant food: opinion of the ESPGHAN committee on nutrition), j.pediatr.gastro.nutr.38: 365-.

The potential for inflammatory disease in formula fed infants is higher because the microflora in formula fed infants is very unstable and the microflora of the gut is involved to a large extent in stimulating intestinal immunity. Many major diseases affecting infants and young children, including chronic lung disease, periventricular leukomalacia, neonatal meningitis, neonatal hepatitis, sepsis and necrotizing enterocolitis, are inflammatory in nature. Depending on the particular disease, concomitant inflammation may occur in a specific organ (e.g., lung, brain, liver, or intestine), or the inflammation may be entirely systemic in nature.

For example, chronic lung disease causes inflammation of the tissues within the lung, while neonatal meningitis includes inflammation of the meninges (lingsoffhebrain) and inflammation of the spinal cord. Periventricular leukomalacia is caused by inflammatory lesions in the periventricular region of the developing brain. Necrotizing enterocolitis causes enteritis which can lead to partial or total intestinal destruction, while neonatal hepatitis includes hepatitis which occurs in early infancy. Sepsis, also known as systemic inflammatory response syndrome, is a serious disease caused by lethal infection of the bloodstream with toxin-producing bacteria. In this disease, pathogens in the bloodstream elicit an inflammatory response throughout the body.

It is also a serious challenge for preterm infants and infants with severe disease in terms of intestinal immune development and prevention of systemic inflammation. Premature or severely ill infants are often placed in a sterile incubator in time so that they remain unexposed to the bacterial population to which healthy, fully pregnant infants may normally be exposed. This may delay or impair the natural colonization process. These infants are also often treated with broad spectrum antibiotics that kill commensal bacteria that attempt to colonize the infant's gut. In addition, these infants are often fed with infant formula rather than breast milk. Each of these factors can lead to inappropriate development of the infant's intestinal microflora, thus causing or accelerating life-threatening systemic inflammation.

In recent years, there have been studies proposing the supplementation of formula-fed infant diets with probiotic bacteria in order to promote intestinal colonization by beneficial microorganisms. Probiotic bacteria are living microorganisms that exert beneficial effects on host health. Fuller, r.probiotics and anandanimals (probiotics in humans and animals), j.appl.bacteriol.66: 365-78(1989).

Although viable probiotic bacteria (viable probiotic bacteria) are effective in normalizing the gut microflora, very few studies have been published to evaluate their safety in preterm infants and immunosuppressed infants. The gut defense barrier of these particular populations is not yet mature, which increases the risk of metastasis of the luminal bacteria (luminal bacteria), leading to an increased likelihood of infection risk. In many cases, viable probiotics (viable probiotics) are generally not recommended for use in immunosuppressed patients, patients after cardiac surgery, patients with pancreatic dysfunction, or patients with fecal blood. It has been reported that at least one death event in immunosuppressed individuals is due to probiotic supplementation. MacGregorg et al, Yoghurtbiotherapy: are related proteinaceous immunological disorders (yoghurt bio-agent therapy: contraindications in immunosuppressed patients)? Postgradmedj.78: 366-367(2002).

Thus, for immunosuppressed patients or preterm infants, it may be beneficial to provide non-viable supplements (non-viable supplements) that can treat or prevent systemic inflammation. Non-viable substitutes (non-viable) for viable (active) or viable (viable) probiotics may have other benefits, such as longer shelf life. Live probiotics are sensitive to heat, humidity and light and ideally should be refrigerated to maintain their viability. Even with these precautions, the shelf life of typical probiotics is relatively short. Non-viable alternatives to live probiotics may avoid the need for refrigeration and may provide products with longer shelf lives. The product can then be distributed to various parts of the world without the need for readily available refrigeration. In addition, non-viable alternatives to probiotics may be less at risk of interacting with other food components, such as fermentation and changes in product taste, texture and freshness. Accordingly, it would be beneficial to provide a method for reducing or preventing systemic inflammation in formula-fed infants, comprising administering inactivated probiotics.

Disclosure of Invention

Briefly, therefore, the present invention is directed to a new product comprising at least one inactivated probiotic, wherein the probiotic is non-viable, but the cellular components of the inactivated probiotic retain the same or similar biological response profile as the viable or non-inactivated cells of the probiotic.

In other embodiments, the invention relates to methods of using one or more inactivated strains of probiotics that have the same or similar biological response benefits as viable probiotics or live probiotics.

In other embodiments, the present invention relates to methods for treating, preventing or reducing systemic inflammation and/or respiratory inflammation in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one inactivated probiotic, wherein the probiotic in its viable form is useful for treating, preventing or reducing such systemic inflammation and/or respiratory inflammation in the subject.

In other embodiments, the present invention relates to methods for treating, preventing or reducing respiratory inflammation in a subject, the methods comprising administering to the subject a therapeutically effective amount of at least one inactivated probiotic, wherein the probiotic in its viable form is useful for such treating, preventing or reducing respiratory inflammation in the subject.

In other embodiments, the present invention relates to a method for reducing or preventing the systemic release of one or more pro-inflammatory cytokines or chemokines in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one inactivated probiotic.

In a particular embodiment, the present invention relates to a method for treating, preventing or reducing systemic inflammation in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one inactivated probiotic in combination with at least one long chain polyunsaturated fatty acid (LCPUFA) and/or at least one viable probiotic. In particular embodiments, the LCPUFA may be docosahexaenoic acid (DHA) or arachidonic acid (ARA).

One of the several advantages obtained by the present invention is that systemic inflammation or respiratory inflammation can be reduced or prevented. The present invention may also reduce inflammation of the liver, plasma, lungs and intestines. In addition, the present invention reduces or prevents the release and growth-related oncogene (GRO/KC) levels of various proinflammatory cytokines and chemokines, including interleukin-1 beta (IL-1 beta), IL-8, CINC-1. Since the present invention can be used to improve inflammatory conditions, it can also prevent the occurrence of infections (deleteriousinfection) and diseases that cause injury.

Drawings

For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawing.

FIG. 1 shows the effect of active probiotic and inactivated probiotic on cytokine-induced neutrophil chemoattractant-1 (CINC-1) peptide production in the liver as determined by enzyme-linked immunosorbent assay (ELISA). Inactivated lactobacillus rhamnosus gg (lactobacillus rhamnosus gg) (LGG), an exemplary inactivated probiotic, is labeled "heat-LGG".

FIG. 2 shows the effect of active probiotic and inactivated probiotic on CINC-1 peptide production in plasma as determined by ELISA. Inactivated LGG was labeled "thermo-LGG".

FIG. 3 shows the effect of active probiotic and inactivated probiotic on CINC-1 peptide production in the lung as determined by ELISA. Inactivated LGG was labeled "thermo-LGG".

FIG. 4 shows the effect of activated probiotic and inactivated probiotic on the production of growth-related oncogenes (GRO/KC) in the liver as determined by a cytokine multiplex assay. Inactivated LGG was labeled "thermo-LGG".

FIG. 5 shows the effect of active probiotic and inactivated probiotic on GRO/KC production in the lung as determined by a cytokine multiplex assay. Inactivated LGG was labeled "thermo-LGG".

FIG. 6 shows the effect of active probiotic and inactivated probiotic on interleukin-1 β (IL-1 β) levels in the liver as determined by a cytokine multiplex assay. Inactivated LGG was labeled "thermo-LGG".

Detailed Description

The details of an embodiment of the invention, one or more examples of which are set forth below are provided for reference. The examples are provided for the purpose of illustrating the invention and are not to be construed as limiting the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

The following abbreviations are used herein: LGG, lactobacillus rhamnosus GG; LCPUFA, long chain polyunsaturated fatty acids; LPS, lipopolysaccharide; IL, interleukin; CINC-1, cytokine-induced neutrophil chemoattractant-1; GRO/KC, growth-related oncogene; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription-polymerase chain reaction; ANOVA, analysis of variance; SD, standard deviation; RMS, rat milk replacer, TLR, Toll-like receptor; nuclear factor κ B, NF- κ B; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; ARA, arachidonic acid.

TLRs are a family of vertebrate recognition receptors. They evolve into key molecules in innate and adaptive immunity. They play a crucial role in the recognition of conserved microbial components. Cell wall components, DNA and double stranded RNA of an organism are apparently recognized by different TLRs. These bacterially derived components (whether LPC, peptidoglycan, or CpGDNA) are natural TLR ligands that retain strong immunomodulatory properties in the absence of pathogenic consequences (diarrhea, tissue destruction, systemic inflammation, barrier permeability) that can normally result from normal bacterial proliferation. The bacterial component generally contributes to the adaptive immune response, while the bacteria themselves are perceived by the innate immune response.

The term "probiotic" refers to a living, active or viable microorganism that exerts beneficial effects on the health of the host.

The term "prebiotic" refers to a non-digestible food ingredient that stimulates the growth and/or activity of probiotics.

The term "treating" as used herein refers to ameliorating, improving or treating a disease, disorder or symptoms of a disease or disorder.

The term "mitigate" refers to a decrease in range, amount, or degree.

The term "preventing" refers to the termination or retardation of a disease, disorder, or symptom of a disease or disorder by some means.

The term "systemic" as used herein refers to the association with or involvement of the entire body.

The term "respiratory infection" or "respiratory disease" refers to a disease or infection that affects the group of organs responsible for the transport of oxygen from the air into the blood stream and for the removal of carbon dioxide.

The term "inactivated probiotic" or "inactivated LGG" means that the internal metabolic activity or reproductive capacity of the probiotic or LGG organism is reduced or destroyed. It is generally believed that "inactivated probiotics" or "inactivated LGG" still retain at least part of its native TLR ligand at the cellular level, which in turn retains at least part of the immunomodulatory properties. The term "inactivated" is used herein synonymously with "non-viable".

The term "therapeutically effective amount" refers to an amount that results in the treatment or amelioration of a disease, disorder, or symptom of a disease or disorder.

The term "preterm infant" refers to an infant born before the end of week 37 of gestation.

The term "infant" refers to a person less than 1 year of age after birth.

The term "child" refers to a person between the ages of about 1 year and about 12 years. In certain embodiments, the child is between the ages of about 1 year and about 6 years. In other embodiments, the child is between the ages of about 7 and about 12 years old.

The term "infant formula (infantformula)" as used herein refers to a composition that satisfies the nutritional needs of infants by replacing human milk.

The present invention invented a new product and method of using probiotics. The products and methods comprise administering a therapeutically effective amount of at least one inactivated probiotic and administering it to a subject. In some embodiments, the subject is an infant.

Previous attempts to effectively administer inactivated probiotics have met with significant obstacles. For example, Kirjavainen, p. et al reported that nearly 40% of children supplemented with inactivated LGG experienced severe diarrhea in a comparison of viable LGG and heat-inactivated LGG. ProbioticBacteriainthomanagementof AtomicDisase: underworcolingthe immunopotentiation of viatility (probiotic bacteria in atopic disease control: emphasis on the importance of viability), j.ped. gastro.36: 223-227(2003). No adverse reactions were reported in either the placebo or viable LGG groups (supra, P225). Since diarrhea is largely associated with inflammation, studies by Kirjavainen indicate that inactivated LGG may actually cause gastrointestinal inflammation. Indeed, this study indicated that "heat inactivation methods can cause surface peptide denaturation and heat shock protein expression, thus altering the immunostimulatory properties of LGG in such a way that a heat-inactivated form can induce an inflammatory response and thereby increase intestinal permeability" (supra, P226). In contrast, the inventors of the present invention developed a novel method for treating or preventing inflammation by administering at least one inactivated probiotic by ingestion of a product containing such inactivated probiotic.

The inventors of the present invention have found that inactivated probiotics may be used in order to obtain the same or similar beneficial effect to a person ingesting said inactivated probiotics as may be obtained by a person ingesting the same live probiotics or viable probiotic bacteria. In addition to reproductive and other active properties closely related to living organisms, the inactivated probiotics of the present invention retain cellular and molecular properties and induce the same or similar bioresponse responses in the host ingesting the inactivated probiotics. Thus, the inactivated probiotic of the present invention may be any probiotic or combination of probiotics known in the art.

In other embodiments, the inactivated probiotic may be a member of the genus Lactobacillus. For example, the inactivated probiotic bacteria may be lactobacillus acidophilus (l.acidiphilus), lactobacillus amylovorus (l.amylovorus), lactobacillus bulgaricus (l.bulgaricus), lactobacillus crispatus (l.crispatus), lactobacillus delbrueckii (l.delbrueckii), lactobacillus rhamnosus (l.rhamnous), lactobacillus casei (l.casei), lactobacillus helveticus (l.gallinarum), lactobacillus fermentum (l.fermentum), lactobacillus gasseri (l.gasseri), lactobacillus helveticus (l.helveticus), lactobacillus about gulf-shaped (l.juruti), lactobacillus johnsonii (l.johnsonii), lactobacillus hilgarrisii (l.leichmannii), lactobacillus plantarum (l.plan-shaped) (l.tarum), lactobacillus reuteri (l.reuteri) or lactobacillus salivarius (l.salivariaus). In certain embodiments, the inactivated probiotic may be lactobacillus acidophilus (lLactobacillus acidophilus NCFM, Lactobacillus acidophilus AS-1, Lactobacillus acidophilus DDS-1, Lactobacillus acidophilus HP10, Lactobacillus acidophilus HP100, Lactobacillus acidophilus HP101, Lactobacillus acidophilus HP102, Lactobacillus acidophilus HP103, Lactobacillus acidophilus HP104, Lactobacillus acidophilus HP15, Lactobacillus acidophilus PIM703, Lactobacillus acidophilus SBT2062, Lactobacillus casei DN-114001, Lactobacillus casei LC10, Lactobacillus casei PIM61, Lactobacillus casei(CRL431), Lactobacillus casei F19, Lactobacillus casei Shirota, Lactobacillus casei immunatass, Lactobacillus crispatus BG2FO4, Lactobacillus delbrueckii subspecies bulgaricus (L.delbrueckii sp. bulgaricus), Lactobacillus delbrueckii subspecies bulgaricus 2038, Lactobacillus delbrueckii subspecies bulgaricus MR120, Lactobacillus delbrueckii subspecies bulgaricus PIM695, Lactobacillus plantarum 299V, Lactobacillus reuteri 1063-S, Lactobacillus reuteri 11284, Lactobacillus reuteri SD2112, Lactobacillus reuteri T-1, Lactobacillus reuteri ATTC55730, Lactobacillus reuteri SD2112, Lactobacillus reuteri 1063-SLactobacillus rhamnosus GG (LGG) ATCC53013, Lactobacillus rhamnosusLactobacillus rhamnosus LB21, Lactobacillus rhamnosus R-011, Lactobacillus rhamnosus R-049, Lactobacillus rhamnosus MX1, Lactobacillus gasseri ADH, Lactobacillus helveticus MR220, Lactobacillus helveticus NCK388, Lactobacillus johnsonii 11088(NCK088), Lactobacillus johnsonii La-1, Lactobacillus salivarius UCC500, Lactobacillus salivarius UCC118 or Lactobacillus delbrueckii subsp.

As noted above, in one embodiment of the invention, the inactivated probiotic may be LGG. LGG is a probiotic strain isolated from the intestinal microflora of healthy humans. It is disclosed in U.S. Pat. No. 5,032,399 to Gorbach et al, which is incorporated herein by reference in its entirety. LGG is resistant to most antibiotics, stable in the presence of acids and bile, and has affinity for attachment to human intestinal mucosal cells. Survival was 1-3 days in most individuals and up to 7 days in 30% of subjects. In addition to its colonization ability, LGG beneficially affects mucosal immune responses. LGG is deposited with the American type culture Collection (American TypeCultureCollection) under the accession number ATCC 53103.

In yet other embodiments, the inactivated probiotic may be a member of the genus Bifidobacterium (Bifidobacterium). For example, the inactivated probiotic may be bifidobacterium animalis (b.animalis), bifidobacterium breve (b.breve), bifidobacterium infantis (Binfantis), bifidobacterium lactis (b.lactis), bifidobacterium suis (b.suis) or bifidobacterium longum (b.longum). In certain embodiments, the inactivated probiotic may be bifidobacterium animalis subspecies animalis (bifidobacterium animalisis), bifidobacterium animalis DN-173010, bifidobacterium animalis subspecies lactis (b.animalisis)Bifidobacterium breve Yakult, Bifidobacterium breve R-070, Bifidobacterium infantis BBI, Bifidobacterium infantis 35624, Bifidobacterium lactis HN019(DR10), Bifidobacterium longum BB46, Bifidobacterium longum BBL or Bifidobacterium longum BB 536.

As noted, the inactivated probiotic may be Bifidobacterium animalis subspAvailable from chr, hansen biosystems, located in Milwaukee, WI.Is a gram-positive anaerobic rod-shaped bacterium that can be found in the large intestine of most mammals, including humans.

In yet other embodiments, the inactivated probiotic may be escherichia coli, enterococcus faecalis (enterococcus faecalis), saccharomyces cerevisiae (saccharomyces cerevisiae), lactococcus lactis (lactococcus lactis), bacillus coagulans (bacillus coagulans), pediococcus pentosaceus (pediococcus pentosaceus), pediococcus acidilactici (pediococcus acidilactici), streptococcus sanguis (streptococcus sanguinis), or streptococcus thermophilus (streptococcus thermophilus). In a specific embodiment, the inactivated probiotic may be E.coli Nissle 1917. In another embodiment, the inactivated probiotic may be Saccharomyces cerevisiae (boularii) lyo. In yet another embodiment, the inactivated probiotic may be lactococcus lactis L1A. In yet another embodiment, the inactivated probiotic may be Streptococcus thermophilus TH-4^TM。

In one embodiment of the invention, more than one inactivated probiotic may be used. Any combination of inactivated probiotics is included in this embodiment, so long as the combination achieves the desired effect. In a particular embodiment, the combination may comprise one or more members of the genus bifidobacterium and one or more members of the genus lactobacillus, e.g. one may useAnd LGG. In separate embodiments, may be usedAndcombinations of (a) and (b).

In the methods of the invention, a therapeutically effective amount of a killed probiotic is an amount sufficient to reduce or prevent systemic inflammation in a subject. This amount corresponds to about 1x10⁴Individual cell equivalents/kg body weight/day and 1x10¹²Individual cell equivalents/kg body weight/day. In another embodiment, the invention comprises administering from about 1x10⁶Cell equivalents/kg body weight/day to 1x10⁹Individual cell equivalents/kg body weight/day. In another embodimentIn one embodiment, the invention comprises administering about 1x10⁹Individual cell equivalents/kg body weight/day. In yet another embodiment, the invention comprises administering about 1x10¹⁰Individual cell equivalents/kg body weight/day.

In the present invention, the use of at least one probiotic that has been inactivated, may be inactivated by any method currently known or yet to be developed in the art. For example, inactivation may be achieved by heat treatment, lyophilization, ultraviolet light, gamma irradiation, pressure, chemical decomposition, or mechanical disruption. For example, the probiotic bacteria may be inactivated by heat treatment when stored at between 80 ℃ and 100 ℃ for 10 minutes. The probiotic bacteria may also be inactivated by UV light by irradiation with a 30 watt UVC lamp at a distance of 5cm for 5 minutes. Alternatively, the probiotic may be inactivated by gamma irradiation using a cobalt-60 source at a distance of 20cm by irradiation with 2kg-Gray (kGy).

In the methods of the invention, the mode of administration of the inactivated probiotic is not critical, so long as a therapeutically effective amount is administered. In some embodiments, the at least one inactivated probiotic is administered to the subject by a tablet, pill, capsule (encapsulation), caplet, soft capsule, oil drop, or sachet. In another embodiment, the inactivated probiotic is encapsulated with a sugar, fat, or polysaccharide. In yet another embodiment, the inactivated probiotic is added to a food or beverage product for consumption. The food or drink may be a children's nutrition, such as modified-formula (folow-onformula), growing-up milk (growing-up milk), a beverage, milk, yogurt, fruit juice-based beverage, chewable tablet, biscuit, cracker (cracker) or milk powder. In other embodiments, the product may be an infant nutrition, such as an infant formula or a human milk fortifier (humanmilk fortifier).

If at least one inactivated probiotic is administered by the infant formula, the infant formula is nutritionally complete and contains the appropriate types and amounts of lipids, sugars, proteins, vitamins and minerals. The amount of lipid or fat may generally vary from about 3g/100kcal to about 7g/100 kcal. The lipid source can be any known or used in the art, for example, a vegetable oil, such as palm oil, soybean oil, palm olein, coconut oil, medium chain triglyceride oil, high oleic sunflower oil, high oleic safflower oil, and the like. The protein content may generally vary from about 1g/100kcal to about 5g/100 kcal. The protein source may be any known or used in the art, such as skim milk, whey protein, casein, soy protein, hydrolyzed protein, amino acids, and the like. The sugar content may generally vary from about 8g/100kcal to about 12g/100 kcal. The sugar source may be any known or used in the art, such as lactose, glucose, corn syrup solids (cornsyrupylid), maltodextrin, sucrose, starch, rice syrup solids (processuprylid), and the like.

Commercially available infant formula may be suitably used. For exampleFormula milk powder and ferrum for premature infantAnd(available from Mead Johnson&Company, Evansville, IN, u.s.a.) may be supplemented with appropriate levels of inactivated probiotics and used to practice the methods of the present invention.

In one embodiment of the present invention, at least one inactivated probiotic may be combined with one or more viable probiotic to treat or prevent systemic inflammation in formula fed infants. In this embodiment, any viable probiotic known in the art may be acceptable, provided that it achieves the desired effect. In a particular embodiment, the viable probiotic may be selected from any genus or species of probiotic described herein.

If viable probiotic bacteria are used in combination with the inactivated probiotic bacteria, the amount of viable probiotic bacteria may correspond to about 1x10⁴Colony Forming units (cfu)/kg body weight/day and 1X10¹²Colony forming units (cfu)/kg body weight/day. In another embodiment, viable probiotic bacteria may comprise from about cfu/kg body weight/day 1x10⁶To 1x10⁹cfu/kg body weight/day. In yet another embodiment, viable probiotic bacteria may comprise about 1x10⁹cfu/kg body weight/day. In yet another embodiment, the viable probiotic may comprise about 1x10¹⁰cfu/kg body weight/day.

In another embodiment of the present invention, at least one inactivated probiotic may be combined with one or more prebiotics to treat or prevent systemic inflammation or respiratory inflammation in formula fed infants. In this embodiment, any prebiotic known in the art may be acceptable, provided it achieves the desired effect. Prebiotics useful in the present invention include lactulose, galacto-oligosaccharides (galcto-oligosaccharides), inulin (inulin), polydextrose, galacto-oligosaccharides, fructo-oligosaccharides, isomalto-oligosaccharides, soy oligosaccharides, lactosucrose (lactosucrose), xylo-oligosaccharides and gentiooligosaccharides (gentio-oligosaccharides).

In yet another embodiment of the present invention, infant formulas may contain other active agents, such as LCPUFAs suitable LCPUFAs including, but not limited to, α -linoleic acid, gamma-linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), ARA, and/or DHAIs commercially available and can be supplemented with at least oneThe inactivated probiotic bacteria are used in the present invention.

In one embodiment, DHA and ARA are simultaneously combined with at least one inactivated probiotic to treat systemic inflammation in the infant. In this embodiment, the weight ratio of ARA to DHA is generally from about 1: 3 to about 9: 1. In one embodiment of the invention, the ratio is from about 1: 2 to about 4: 1. In yet another embodiment, the ratio is from about 2: 3 to about 2: 1. In one embodiment, the ratio is about 2: 1. In another embodiment of the present invention, the ratio is about 1: 1.5. In other embodiments, the ratio is about 1: 1.3. In still other embodiments, the ratio is about 1: 1.9. In a specific embodiment, the ratio is about 1.5: 1. In yet another embodiment, the ratio is about 1.47: 1.

In certain embodiments of the invention, the level of DHA is between about 0.0% and 1.00% by weight of fatty acids.

The level of DHA may be about 0.32 wt%. In some embodiments, the level of DHA may be about 0.33 wt%. In another embodiment, the level of DHA may be about 0.64 wt%. In another embodiment, the level of DHA may be about 0.67 wt%. In yet another embodiment, the level of DHA may be about 0.96 wt%. In yet another embodiment, the level of DHA may be about 1.00 wt%.

In an embodiment of the invention, the level of ARA is between 0.0% and 0.67% by weight of fatty acids, and in another embodiment the level of ARA may be about 0.67% by weight. In another embodiment, the level of ARA may be about 0.5% by weight. In yet another embodiment, the level of DHA may be between about 0.47% and 0.48% (by weight).

In embodiments of the invention where DHA is included, the effective amount of DHA is typically from about 3mg/kg body weight/day to about 150mg/kg body weight/day. In one embodiment of the invention, the amount is from about 6mg/kg body weight/day to about 100mg/kg body weight/day. In another embodiment, the amount is from about 10mg/kg body weight/day to about 60mg/kg body weight/day. In yet another embodiment, the amount is from about 15mg/kg body weight/day to about 30mg/kg body weight/day.

In embodiments of the invention where ARA is included, the effective amount of ARA will generally vary from about 5mg/kg body weight/day to about 150mg/kg body weight/day. In one embodiment of the invention, the amount varies from about 10mg/kg body weight/day to about 120mg/kg body weight/day. In another embodiment, the amount varies from about 15mg/kg body weight/day to about 90mg/kg body weight/day. In yet another embodiment, the amount varies from about 20mg/kg body weight/day to about 60mg/kg body weight/day.

If an infant formula is used, the amount of DHA in the infant formula may vary from about 5mg/100kcal to about 80mg/100 kcal. In one embodiment of the invention, DHA varies from about 10mg/100kcal to about 50mg/100 kcal; in another embodiment, from about 15mg/100kcal to about 20mg/100 kcal. In a particular embodiment of the invention, the amount of DHA is about 17mg/100 kcal.

If an infant formula is used, the amount of ARA in the infant formula may vary from about 10mg/100kcal to about 100mg/100 kcal. In one embodiment of the invention, the amount of ARA varies from about 15mg/100kcal to about 70mg/100 kcal. In another embodiment, the amount of ARA varies from about 20mg/100kcal to about 40mg/100 kcal. In a particular embodiment of the invention, the amount of ARA is about 34mg/100 kcal.

If an infant formula is used, the infant formula may be supplemented with oils containing DHA and ARA using standard techniques known in the art. For example, DHA and ARA may be added to a milk formula by replacing an equivalent amount of oil normally present in the formula (e.g. high oleic sunflower oil). As another example, an oil containing DHA and ARA may be added to a formula by replacing an equal amount of the remaining total fat mixture normally present in a formula without DHA and ARA.

If a source of DHA and a source of ARA are used, then the source of DHA and the source of ARThe a source may be any source known in the art, such as marine oils, fish oils, single cell oils, egg yolk lipids, brain lipids, and the like. In some embodiments, the DHA and ARA are derived from single cell Martek oil,or a modified oil thereof. DHA and ARA may be in natural form as long as the remaining components of the LCPUFA source do not have any significant detrimental effect on the infant. Alternatively, refined forms of DHA and ARA may be used.

In one embodiment of the invention, the source of DHA and the source of ARA are single cell oils, see U.S. patent nos. 5,374,567, 5,550,156, and 5,397,591, the disclosures of which are incorporated herein by reference in their entirety. However, the present invention is not limited to only such oils.

In one embodiment, a LCPUFA source comprising EPA is used in combination with at least one inactivated probiotic. In another embodiment, a source of LCPUFA that is substantially free of EPA is used in combination with at least one inactivated probiotic. For example, in one embodiment of the present invention, an infant formula containing no more than about 16mg EPA/100kcal is supplemented with at least one inactivated probiotic and used in the methods of the present invention. In another embodiment, an infant formula containing no more than about 10mg EPA/100kcal is supplemented with at least one inactivated probiotic and used in the methods of the invention. In yet another embodiment, an infant formula containing no more than about 5mg EPA/100kcal is supplemented with at least one inactivated probiotic and used in the methods of the invention. Another embodiment of the present invention comprises an infant formula supplemented with at least one inactivated probiotic and absent even in trace amounts of EPA.

It is believed that providing at least one inactivated probiotic in combination with DHA and/or ARA provides a complementary effect (complementary effect) or a synergistic effect on the anti-inflammatory properties of formulations containing these ingredients. Without intending to be bound by this theory or any other theory, we believe that the inactivated probiotic anti-inflammatory effect is conferred in part by preventing ubiquitination of the inhibitory-kb (ikb). In normal cells, IkB binds to the nuclear factor kb (nfkb) in the cytoplasm. When ubiquitination of IkB occurs, NFkB is released, enters the nucleus of the cell, and activates genes responsible for the inflammatory response. It is this specific interaction and the resulting alteration in gene expression that we believe is involved in the regulation of inflammation. We believe that the inactivation of probiotics prevents ubiquitination of IkB, thereby preventing NFkB release and reducing or preventing inflammation.

In contrast, omega-3 fatty acids (e.g., DHA) are believed to confer an anti-inflammatory effect by altering the production of fatty acid-derived pro-inflammatory mediators broadly referred to as eicosanoids. Omega-6 fatty acids (e.g., ARA) in the phospholipid layer of the cell membrane (phospholipidol) are released during the inflammatory reaction and release a layer of free ARA. This layer ARA then acts through two classes of enzymes called lipoxygenases and cyclooxygenases, which produce various eicosanoids, including the 2-series prostanoids (prostanoids), such as prostaglandins, thromboxanes and leukotrienes.

These eicosanoids are known to have a surplus of pro-inflammatory actions in many cell types and organs. Diets rich in omega-3 fatty acids (e.g., EPA and DHA) are known to be competitors for omega-6 fatty acids at several steps in the process, thus reducing the pro-inflammatory effects of ARA. For example, omega-3 fatty acids regulate elongation of omega-6 fatty acids to ARA, ARA incorporation into cell membrane phospholipid layers, and production of pro-inflammatory eicosanoids from ARA. Thus, the combination of DHA and ARA provides a unique and complementary effect in modulating inflammatory responses in multiple tissues.

Additionally, in some embodiments of the invention, a viable probiotic and a killed probiotic are used in combination with each other. With respect to the anti-inflammatory properties of formulations containing these ingredients, it is believed that the combination of viable probiotic bacteria and inactivated probiotic bacteria provides a complementary or synergistic effect. Without wishing to be bound by this theory or any other theory, we believe that the viable probiotic moiety confers an anti-inflammatory effect by interacting with specific receptors on the surface of specific immune cells, known as Toll-like receptors (TLRs). The direct or indirect interaction between the viable probiotic and these receptors initiates intracellular signaling cascades that result in altered gene expression in these target cells. It is this specific interaction and the changes induced in gene expression and other cellular effects that we believe are involved in the regulation of inflammation. It is believed that the combination of these components may provide a complementary or synergistic anti-inflammatory effect, as we believe that viable probiotics and inactivated probiotics act through different mechanisms.

Additionally, in some embodiments of the invention, at least one viable probiotic, at least one inactivated probiotic, is used in combination with at least one LCPUFA. Since viable probiotics, inactivated probiotics and LCPUFA each act by different mechanisms, we believe that the combination of these components provides a complementary or synergistic effect on the anti-inflammatory properties of the formulations containing these ingredients.

In some embodiments of the invention, the subject is in need of treatment, reduction, or prevention of systemic inflammation. The subject may be at risk for systemic inflammation due to genetic constitution, diet, lifestyle, disease, disorder, and the like. For example, premature infants or immunosuppressed infants may be at risk for systemic inflammation and such treatment, reduction or prevention may be desirable.

In certain embodiments, the inactivated probiotic may be administered to an infant or child to prevent, treat or reduce systemic inflammation. In one embodiment, the infant may be less than 1 year old. In another embodiment, the child may be between 1 and 6 years of age. In yet another embodiment, the child may be between the ages of 7 and 12.

In one embodiment of the invention, the subject is a formula-fed infant. In one embodiment, the infant is fed with formula from birth. In another embodiment, the infant is breastfed from birth until less than 1 year of age, and then fed with formula milk during which time supplementation with inactivated probiotics begins.

In a particular embodiment of the invention, the method comprises treating or preventing systemic inflammation in formula fed preterm infants. In this method, the inactivated probiotic may be administered to the preterm infant in an infant formula, a human milk fortifier, or any other suitable form. In addition, if desired, the inactivated probiotic bacteria are administered to the preterm infant in combination with DHA, ARA and/or one or more viable probiotic bacteria to produce a potentially synergistic anti-inflammatory effect.

In one embodiment of the invention, the inactivated probiotic reduces or prevents the systemic release of one or more pro-inflammatory cytokines or chemokines. As used herein, a "proinflammatory" cytokine or chemokine includes a proinflammatory cytokine or chemokine known in the art to be involved in upregulating an inflammatory response. Examples include, but are not limited to TNF- α, IL-1 β, IL-6, IL-8, IL-18, and GRO/KC.

Chemokines are a group of cytokines that enable leukocytes to migrate from the blood to tissues at sites of inflammation. When chemokines are produced in excess, damage to healthy tissue can result. Growth-related oncogenes (GRO/KC) are chemokines that recruit immune cells to sites of inflammation. It is the human counterpart of the rat cytokine-induced neutrophil chemoattractant (CINC-1), and is functionally related to the interleukin-8 family.

In yet another embodiment of the invention, the inactivated probiotic bacteria are shown to inhibit the transport of nuclear factor kb (nfkb). NFkB is a major transcription factor present in all cell types and is believed to play an important role in the development of inflammation. In most cells, NF-kB exists in the cytoplasm as a latent inactive inhibitory kB (ikb) binding complex. When cells receive one of a number of extracellular signals, e.g., from cytokines, bacterial antigens or free radicals, NF- κ B rapidly enters the nucleus and activates the genes responsible for the inflammatory response. Studies have shown that inhibition of NF κ B at the onset of inflammation results in a decrease in inflammatory response. Lawrence et al, missiblenewrolleformnf κ binthemateresolution of inflammation (a possible new role for NF κ B in eliminating inflammation), national med.7: 1291(2001). Thus, in the present invention, inhibition of NF κ B by supplementation with inactivated probiotics helps to reduce or prevent systemic inflammation.

As shown in the examples, inactivated probiotics were shown to reduce systemic inflammation in formula fed infants. In formula fed young rats, CINC-1 and various cytokines levels were reduced to levels similar to those of breast-fed young rats when supplemented with inactivated probiotics.

As shown in the examples, inactivated probiotics have also been shown to significantly reduce IL-8 production, decrease NF- κ B transport and increase IkB production in the intestinal epithelium. In addition, the present inventors have unexpectedly discovered that inactivated probiotics prevent ubiquitination of IkB, whereas viable probiotics do not.

The following examples describe various embodiments of the invention. Other embodiments within the scope of the following claims will be apparent to those skilled in the art from consideration of the specification or practice of the invention disclosed herein.

It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the following claims. In the examples, all percentages given are percentages by weight, unless otherwise indicated.

Example 1

This example illustrates the effect of supplemental inactivated probiotic on systemic inflammation in newborn rat pups fed formula. In this example, LGG was used as a probiotic.

Materials and methods

In two independent experiments, Sprague-Dawley (Taconic, Germantown, N.Y.) pups were randomly assigned to the following 4 gastrostomy feeding groups, 5 rats per group: control group (no LPS or LGG), LPS group, LPS plus viable LGG group and LPS plus inactivated LGG group. Rats were fed with the same age of female mice as controls. The young rats were fed gastrostomically starting on day 7 of birth using the "pup-in-the-cup" model. A gastrostomy feeding tube was placed on a 24cm section of a polyethylene tube inserted into the stomach of a young mouse. Gastrostomy placement was performed under isoflurane anesthesia. A timer-controlled syringe pump was connected to the feeding tube and set to feed the rats at a body weight-dependent flow rate for the first 20 minutes per hour.

During the 2 day acclimation period, the gastrostoma-fed pups were fed with rat milk Replacer (RMS). After the acclimation period, one of the RMS feeding groups was given a supplement of 1x10⁸Individual cell equivalents per kg body weight per day of inactivated LGG. LGG is inactivated by lethal heat treatment. Giving a second set of supplements 1x10⁸cfu/L/kg body weight/day of viable LGG. The third group was fed RMS unsupplemented with any type of LGG. These feeding regimens were continued for 6 days. All gastrostomy feeding groups received the same amount of fat and sugar, with the protein fraction approximating that required for normal growth. Rats were fed with the same age of female mice as controls.

Obtained from e.coli 0127: lipopolysaccharide (LPS) of B8 (LPS; Sigma, St. Louis, Mo.) was dissolved in water at a concentration of 2mg/ml by vortexing. 2 days after the start of artificial feeding, 0.25 mg/kg/day to 0.5 mg/kg/day LPS was started to be administered to the gastrostomy rats through the gastrostomy feeding tube. Young mice were given LPS supplementation for 6 days. This dose was determined in a preliminary study that resulted in occasional briquets, piloerection, and slow weight gain, but was not associated with a significant increase in mortality over a 6 day period.

At the end of the 6 day treatment period, the young rats were euthanized with an excess of sodium pentobarbital. Small intestine was harvested and divided into three fractions: ileum, jejunum and duodenum, stored at-80 ℃ for enzyme assays and ELISA, or fixed in 10% neutral buffered formalin for intestinal morphology studies. Lungs, liver and plasma were stored at-80 ℃ for enzyme assay and ELISA.

The results of the ELISA and cytokine/chemokine multiplex assays for body weight, CINC-1 were analyzed using Sigmastat statistical software (SPSS, Chicago, IL). All data are reported as mean ± Standard Deviation (SD). Analysis of one-way variance between groups (ANOVA) was used to determine if there was a significant difference in all treatment groups. When ANOVA was significant (p < 0.05), two-by-two comparisons were performed using the Holm-Sidak method.

Results and discussion

Growth of

This example illustrates the effect of probiotic administration on growth in young mice after gastrostomy feeding. The young rats were weighed daily after the gastrostomy feeding and compared with the control animals fed by the mother rats. The growth of the female mice fed animals was faster than that of LPS-treated gastrostomed fed young mice. Treatment of viable probiotic bacteria or inactivated probiotic bacteria in young mice with LPS fed via gastrostomy failed to improve body weight.

CINC-1

In the present invention, both viable and inactivated probiotics reduce CINC-1 levels. CINC-1 levels were determined by TiterZyme enzyme immunoassay kit for rat growth-related oncogene/CINC-1 (AssayDesigns, AnnArbor, MI). Tissue samples were isolated from cell extracts of whole tissues of liver, intestine, plasma and lung, absorbance was measured at 450nm, and concentration was calculated using a formula derived from a linear standard curve.

As shown in fig. 1 to 3, the ELISA results showed that LPS increased CINC-1 levels in liver, lung and plasma. Both viable and inactivated probiotics reduced LPS-induced CINC-1 production (p < 005) in both liver (fig. 1) and plasma (fig. 2), and also showed this trend in lung (fig. 3) (p ═ 0.09).

Figure 1 shows that supplementation with viable probiotic bacteria reduced the level of CINC-1 in the liver by about 50% when compared to the LPS group. However, when compared to the LPS group, inactivated probiotics reduced the CINC-1 levels in the liver by about 75%. Thus, the reduction of hepatic CINC-1 levels by inactivated probiotic bacteria is significantly greater than that by viable probiotic bacteria, indicating a stronger anti-inflammatory effect. Likewise, fig. 2 shows that plasma CINC-1 levels were lower in the inactivated probiotic group than in the viable probiotic group. In the lung, both viable and inactivated probiotics reduced CINC-1 levels to the same extent (fig. 3).

GRO/KC

As shown in fig. 4 and 5, the cytokine multiplex assay showed that the GRO/KC levels in the liver and lung were also reduced. In the liver, inactivated probiotics reduce the GRO/KC levels to a greater extent than viable probiotics. This indicates a strong anti-inflammatory effect (figure 4). In the lung, both viable and inactivated probiotics reduced GRO/KC levels to the same extent (fig. 5).

In this experiment, the observed decrease in CINC-1 and GRO/KC levels in the lungs indicates that the anti-inflammatory effects of the inactivated probiotic are extended to distant organs. Thus, the anti-inflammatory effects of the inactivated probiotic are substantially entirely systemic.

In the liver, supplementation with inactivated probiotics reduced CINC-1 levels to levels that were substantially lower than those of breast-fed young rats. In the lungs and plasma, inactivated probiotics reduced CINC-1 levels to levels very similar to those of breast-fed young rats. These results indicate that the inactivated probiotics have the ability to reduce the level of systemic inflammation in formula-fed pups to levels similar to, and in some cases lower than, those of breast-fed pups.

Cytokines and chemokines

Viable probiotics and inactivated probiotics also reduce cytokine and chemokine levels. Multiplex bead kits (multiplex xbead kits) were purchased from LINCOResearch, Inc. Cytokines/chemokines analyzed by the kit include Granulocyte Macrophage Colony Stimulating Factor (GMCSF), interferon- λ (IFN- λ), interleukin-1 α (IL-1 α), IL-1 β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-18, monocyte chemoattractant protein-1 (MCP-1), GRO/KC (rat CINC-1), and TNF- α. Multiple assays were performed according to the manufacturer's instructions. Standard curves for each cytokine/chemokine were plotted against the reference concentrations supplied by the manufacturer. The raw data (mean fluorescence intensity) were analyzed by MasterPlex quantitative software (MiraiBio, inc., Alameda, CA, USA) to give concentration values.

As shown in fig. 6, the level of IL-1 β was significantly higher in the livers of gastrostomically fed LPS-treated pups compared to control pups. Both viable and inactivated probiotics significantly blunted LPS-induced elevations of IL-1 β. In fact, inactivated probiotics reduce IL-1 β levels to a greater extent than do live supplemented probiotics. Inactivated probiotics reduced IL-1 β expression to levels similar to control pups. Thus, this part of the experiment also demonstrates systemic anti-inflammatory activity of the inactivated probiotic.

Taken together, these results indicate that supplementation with inactivated probiotics reduces systemic inflammation. Furthermore, the results show that inactivated probiotics reduce systemic inflammation in formula fed pups to levels similar to breast fed pups. This is illustrated in the results of the comparison of the probiotic-inactivated treated group and the breast-milk-only group described herein. In some cases, the inflammatory response resulting from administration of inactivated probiotics is very similar to that of the breast-fed group.

Example 2

This example further demonstrates the effect of supplementation with inactivated probiotic bacteria on inflammation in newborn rat pups fed formula. In this example, LGG was used as the probiotic.

Intestinal epithelial cells were treated with viable LGG or UV-inactivated LGG at 1X10⁸cfu/L pre-treatment followed by stimulation with flagellin 500 ng/mL. IL-8 production was determined by ELISA. The expression of IkB and ubiquitination-IkB (UbQ-IkB) was determined by Western blotting and immunoprecipitation. NF κ B localization was evaluated by immunofluorescence staining.

Flagellin induced a significant increase in cellular IL-8 production during the experiment (p < 0.05). Cells pretreated with viable LGG or UV-inactivated LGG, then stimulated by flagellin, showed significant changes in IL-8, NF κ B nuclear translocation, IkB and UbQ-IkB (p < 0.05). The results are shown in Table 1. The upward pointing arrow indicates a parameter increase, while the downward pointing arrow indicates a parameter decrease.

TABLE 1 expression changes due to supplementation with viable or inactivated probiotics

	IL-8	NF kappa B transport	IkB	UbQ-IkB
					Flagellin only	↑	↑	↓	↑
Active LGG	↓	↓	↑	↑
					Inactivated LGG	↓	↓	↑	↓

As shown in Table 1, flagellin induced a significant increase in IL-8 production by intestinal epithelial cells (p < 0.05). Production of IL-8 was significantly down-regulated in the presence of both viable and inactivated LGG. In addition, cells stimulated by flagellin exhibit nuclear transport of NF κ B, which is prevented by both viable and inactivated LGG. Flagellin reduced IkB production, but pretreatment with viable and inactivated LGG reversed this effect (p < 0.05). Flagellin and viable LGG increased UbQ-IkB (p < 0.05), while inactivated LGG decreased UbQ-IkB.

This example demonstrates that viable probiotic and inactivated probiotic bacteria effectively reduce the production of the pro-inflammatory cytokine IL-8 and therefore have anti-inflammatory effects. Because flagellin and viable probiotics increase UbQ-IkB, but inactivated probiotics decrease UbQ-IkB, inactivated probiotics are likely to act by mechanisms that prevent ubiquitination of IkB, while viable probiotics are likely not. Thus, this example further demonstrates that viable probiotics and inactivated probiotics are likely to act by different mechanisms and may have a synergistic effect when administered simultaneously.

The present invention demonstrates a reduction in inflammation in the liver, plasma and lungs. Because the present invention can be used to ameliorate inflammatory conditions, it also prevents the occurrence of infections and diseases that cause injury.

All references cited in this specification, including but not limited to all papers, publications, patents, patent applications, presentations, texts, reports, manuscripts, brochures, books, internet uploaded documents, journal articles, periodicals, and the like, are hereby incorporated by reference in their entirety. The discussion of the references is intended merely to summarize the assertions made by their authors and no admission is made that all references constitute prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.

These and other modifications and variations to the present invention may be practiced by those of ordinary skill in the art, without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. Additionally, it should be understood that aspects of the various embodiments may be interchanged both in whole or in part. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention so further described in such appended claims. Therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred versions contained therein.

Claims (9)

1. Heat inactivated lactobacillus rhamnosus (a)Lactobacillusrhamnosus) Use of GG in the manufacture of a children or infant nutritional product for the treatment, prevention or reduction of systemic inflammation.

2. Use of inactivated probiotic bacteria in the manufacture of a child or infant nutritional product for the treatment, prevention or reduction of systemic inflammation in an infant or child, wherein the at least one inactivated probiotic bacteria comprises one or more members of the genus lactobacillus and bifidobacterium (b: (b) (b))Bifidobacterium) One or more ofA combination of members.

3. The use of claim 1 or claim 2, wherein the nutritional composition is effective to provide 1x10⁴To 1x10¹⁰The inactivated lactobacillus rhamnosus GG per kg body weight per day of individual cell equivalents is administered to the child or infant.

4. The use according to claim 1 or claim 2, wherein the nutritional product further comprises at least one viable probiotic.

5. Use according to claim 4, wherein the viable probiotic bacteria are selected from Lactobacillus rhamnosus GG, Bifidobacterium animalis subspB.animalisssp.lactis) And combinations thereof.

6. Use according to claim 1 or claim 2, wherein the product further comprises at least one long chain polyunsaturated fatty acid.

7. The use of claim 6, wherein the long chain polyunsaturated fatty acid is selected from the group consisting of docosahexaenoic acid, arachidonic acid, and combinations thereof.

8. The use of claim 1 or claim 2, wherein the nutritional composition further comprises at least one prebiotic.

9. The use of claim 3, wherein the nutritional composition is effective to provide 1x10⁶To 1x10⁹The amount of individual cell equivalents/kg body weight/day.