HK1227316B - Use of lgg in the manufacture of a medicament for the prevention or treatment of respiratory allergies - Google Patents

Description

Use of LGG in the manufacture of a medicament for preventing or treating respiratory allergies

The present application is a divisional application of an invention patent application filed on March 22, 2006, with application number 200680012467.1 and invention name “Use of LGG in the manufacture of a drug for preventing or treating respiratory allergy”.

Cross-references to related patents and patent applications

This application is a continuation-of and claims priority to U.S. patent application serial number 11/106,792, filed April 15, 2005, which is incorporated herein by reference in its entirety.

Background of the Invention

(1) Field of the Invention

The present invention generally relates to the use of LGG in the manufacture of a medicament for preventing or treating respiratory allergies.

(2) Description of related technologies

Allergy is defined as "an abnormal hypersensitivity reaction to a normally tolerated substance and generally considered harmless." Symptoms of an allergy can range from a runny nose to anaphylactic shock. Nearly 50 million Americans suffer from allergic diseases, and the prevalence of these diseases is rising.

An allergic reaction has two basic phases. The first phase involves the development of the early stages of an immediate hypersensitivity reaction to an allergen. When the allergen first encounters the immune system, no allergic reaction occurs. Instead, the immune system prepares itself for future exposure to the allergen. Macrophages, which act as scavenger cells, surround and break down the invading allergen. The macrophages then display the allergen fragments on their cell walls to T lymphocytes, the master orchestrators of the body's immune system.

This recognition signal plus several non-recognition signals (such as cytokines) activates naive T-cells and instructs T-cells to differentiate into T-cell effector subsets. The key factor in the allergic cascade is the Th-2 phenotype (TH-2) of T-cells. TH-2 type T-cells are characterized by the secretion of several cytokines including interleukin-4 (IL-4), IL-5 and IL-13. The cytokines IL-4 and IL-13 then activate B lymphocytes that produce antibody subclass E (IgE). IgE antibodies directly oppose specific allergens. The interaction of specific IgE antibodies on the surface of effector cells (mast cells and basophils) with allergens triggers the early stages of immediate hypersensitivity reactions.

Mast cell activation usually occurs within a few minutes after the second exposure to the allergen. During the sensitization phase, IgE antibodies on the mast cells that are built up recognize the allergen and bind to this invader. Once the allergen binds to the receptor, the granules in the mast cells release their contents. These contents or mediators are pro-inflammatory substances such as histamine, platelet-activating factor, prostaglandins, cytokines, and leukotrienes. These mediators actually activate the allergic reaction. Histamine stimulates mucus production and causes redness, swelling, and inflammation. Prostaglandins cause airway constriction and vasodilation.

The second stage of the allergic immune response is characterized by inflammatory cell infiltration, such as eosinophil infiltration into the airways after allergen exposure. T-cells represent an important link between sensitization and inflammation, and the mediators they secrete are not only involved in IgE synthesis, but also in the recruitment, activation and survival of eosinophils. Tissue mast cells and adjacent cells produce chemical messengers that signal circulating basophils, eosinophils and other cells to migrate into the tissue and help fight foreign substances. Chemical substances secreted by the eosinophils themselves maintain inflammation, causing tissue damage and even recruiting more immune cells. This stage can occur anywhere from a few hours to a few days after allergen exposure and can last for several hours or even days.

Respiratory allergies are a specific type of allergy that affects the respiratory tract. The lining of the airways from the nose to the lungs is structurally similar and is often affected by similar allergic diseases. Therefore, allergens that affect the nose or sinuses can also affect the lungs.

For example, allergic rhinitis, also known as hay fever, is caused by an allergic reaction in the nasal mucosa and airways to airborne allergens. Symptoms of allergic rhinitis typically include itching in the nose, throat, and eyes, as well as frequent sneezing. This is often followed by nasal congestion or mucus discharge.

Because allergens in one part of the respiratory tract can affect the respiratory tract in another part, rhinitis in the nasal passages can lead to asthma, which is a more serious disease occurring in the lungs. Asthma is characterized by the occurrence of airway hyperresponsiveness, shortness of breath, wheezing when breathing, a dry cough and a feeling of tightness in the chest. Repeated exposure to allergens can maintain an inflammatory immune response in the airways, leading to airway remodeling, commonly known as chronic asthma. Not every allergic rhinitis develops into asthma symptoms, but a significant number of patients, especially those suffering from recurrent untreated allergies, show inflammatory changes in the lungs. About 40% of people suffering from allergic rhinitis actually develop asthma completely.

If the nasal inflammation associated with allergic rhinitis spreads to the sinuses, the result can be an unpleasant infection called sinusitis or rhino-sinusitis, in which the sinuses are unable to clear themselves of bacteria. Symptoms include nasal congestion, mucus discharge, sore throat, fever, headache, fatigue, and cough. They can also include pain in the forehead, behind the cheek, and even pain in the teeth and jaw.

Respiratory allergies are one of the most common diseases in childhood. As in adults, respiratory allergies in children are most likely to present in the form of allergic rhinitis and asthma.

Prevention of respiratory allergies in infants and young children is particularly important because early allergic sensitization to allergens appears to be associated with a delay in the maturation of normal immune responses. In addition, allergic sensitization is often considered an initial step in the development of atopic disease. Baena-Cagnani, Role of Food Allergy in Asthma in Childhood, Allergy. Clin. Immun. 1(2): 145-149 (2001). Asthma that begins early in life is often associated with atopy, so early allergic sensitization also appears to play an important role in persistent asthma. Martinez, F., Development of Wheezing Disorders and Asthma in Preschool Children, Pediatr. 109: 362-367 (2002).

Allergic sensitization and asthma are not only closely linked, but this link also appears to be age-dependent. Although few children develop allergic sensitization in the first few years of life, most of those sensitized during this period develop asthma-like symptoms later in life. Martinez, F., Viruses and Atopic Sensitization in the First Years of Life, Am. J. Respir. Crit. Care Med., 162: S95-S99 (2000). Therefore, it is important to find methods to prevent early allergic sensitization and respiratory allergies later in life.

There is increasing evidence that many aspects of health and disease are determined not only in infancy but also during pregnancy. When the immune response during childbirth suggests that intrauterine exposure is the primary sensitizing factor, this is even more certain for such allergic diseases. For example, allergen-specific T-cells are already present at childbirth, and early sensitization to food allergens is identified as a predictor of respiratory allergies that develop later. Illi et al., The Natural Course of Atopic Dermatitis from Birth to Age 7 Years and the Association with Asthma, Clin. Exp. Allergy 27: 28-35 (1997). In addition, lung development begins very early after fertilization and continues for at least 2 or 3 years after childbirth. Therefore, airway development before and after birth is important for the pathogenesis of respiratory allergies in infants and children.

It has also been shown that the human fetus develops IgE-producing B cells early in pregnancy and is capable of producing IgE antibodies in response to appropriate antigenic stimulation in a manner similar to the well-established IgM responses observed in various prenatal infections. Weil, G et al., Prenatal Allergic Sensitization to Helminth Antigens in Offspring of Parasite-Infected Mothers, J. Clin. Invest. 71: 1124-1129 (1983). This also demonstrates the importance of preventing prenatal and postnatal allergic sensitization to respiratory allergens.

Conventional medications for respiratory allergies include antihistamines, topical nasal steroids, decongestants, and sodium cromolyn solution. As an alternative to conventional medications, probiotics have shown potential for treating certain types of allergies.

Probiotics are live microorganisms that benefit the host's health. Lactobacillus spp. and Bifidobacterium spp., which normally inhabit the healthy intestine, are common species of probiotics. Unfortunately, little research has been published on the clinical efficacy of probiotic supplementation in infants. Agostoni, C. et al., Probiotic Bacteria in Dietetic Products for Infants: A Commentary by the ESPGHAN Committee on Nutrition, J. Pediatr. Gastro. Nutr. 38:365-74 (2004).

U.S. Patent Application No. 20040208863 to Versalovic et al. relates to a compound secreted by lactobacilli with anti-inflammatory activity. The application describes the use of Lactobacillus GG (LGG) to inhibit proinflammatory cytokines. However, the reference focuses on adults and does not disclose or suggest that administering LGG during pregnancy or infancy would be beneficial.

U.S. Patent Application No. 6,506,380 to Isolauri et al. describes a method for suppressing food-induced hypersensitivity reactions in patients with food allergies by administering probiotics thereto. However, this reference does not disclose the use of probiotics for treating respiratory allergies. Furthermore, while this patent discusses methods for treating infants with probiotics, it does not disclose the importance of both prenatal and postnatal treatment.

Several studies have focused on administering probiotics before and after birth to prevent certain allergic reactions in infants and children. For example, one study concluded that administering probiotics during pregnancy and lactation can protect infants from atopic eczema. Rautava, S et al., Probiotics During Pregnancy and Breast-Feeding Might Confer Immunomodulatory Protection Against Atopic Disease in the Infant, J. Allergy Clin. Immunol. 109: 119-121 (2002). Although the study found that fewer infants developed atopic eczema in the probiotic group than in the placebo group, the study concluded that administering probiotics "is ineffective for conventional treatment targets associated with atopy and atopic diseases (i.e., skin prick test results and serum IgE antibodies). Therefore, since probiotic supplementation does not affect the usual markers of atopy, it must be assumed that administering probiotics is only effective for atopic eczema, not all allergic reactions.

Similarly, a 2001 study concluded that probiotics could prevent atopic eczema, but "the frequency of increases in total and antigen-specific IgE concentrations and the rate of positive skin prick test reactions were very similar between the probiotic and placebo groups." Kalliomaki, M., Probiotics in Primary Prevention of Atopic Disease: a Randomised Placebo-Controlled Trial, Lancet 357:1076-79 (2001). Furthermore, these results suggest that while probiotics may affect atopic eczema, they may not be effective for other types of allergies.

SUMMARY OF THE INVENTION

Briefly, the present invention provides a novel use of LGG for the manufacture of a medicament for preventing or treating respiratory allergies in a subject.

The present invention also provides a new use of LGG for manufacturing a drug for preventing or treating respiratory allergy in a subject.

Another aspect of the present invention provides a novel use of LGG for the manufacture of a medicament for preventing or treating the release of one or more pro-inflammatory cytokines in a subject.

In addition, the present invention points out a new use of LGG for manufacturing a drug for preventing or treating the production of serum IgE antibodies in a subject.

The present invention also points out a new use of LGG for manufacturing a drug for increasing the production of serum IgA antibodies in a subject.

Among the several advantages achieved by the present invention, one advantage is that subjects benefit by reducing or preventing allergy-induced inflammation in the lungs or airways, including reducing inflammation of airway tissue and airway lumen. The present invention can also reduce mucus production or enlarge the airways. The present invention can also reduce or inhibit the release of one or more proinflammatory cytokines and serum IgE antibodies, and increase serum IgA production in a subject. These benefits are surprising and unexpected, as similar studies have provided contrary results.

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG1 shows the scheme utilized by the present invention.

Figure 2 shows the comparison of LGG detected in feces of LGG-treated mice and control mice.

FIG3 shows the comparison of fecal bacteria between LGG-treated and control mice.

FIG4 shows the effects of LGG on serum anti-OVA-lgE, serum anti-OVA-lgG1, and serum anti-OVA-lgG2a.

FIG5 shows the effect of LGG on serum IgA antibody levels.

Figure 6 shows the effects of LGG on various proinflammatory cytokines, IFN-γ (Panel A), MCP-1 (Panel B), IL-10 (Panel C), and IL-6 (Panel D) in OVA-sensitized infants.

FIG7 shows the effect of LGG on the distribution of monocytes in allergic airways.

FIG8 shows the inhibition of bovine milk-specific antibody production.

Figure 9 shows the immune effector function of milk-specific antibodies assessed by passive skin sensitization test and mast cell proteinase-1 release assay.

Detailed description of preferred embodiments

What will be mentioned in detail at present is an embodiment of the present invention, and one or more examples of such embodiment are listed below. Each example is provided in a manner to explain the present invention rather than to limit the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the present invention. For example, the features of one embodiment part illustrated or described can be used in another embodiment to produce another embodiment.

Therefore, the present invention is intended to cover such modifications and variations as come within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the present invention are disclosed or become apparent from the following detailed description. Those skilled in the art will appreciate that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the present invention.

definition

The term "probiotic" refers to microorganisms that exert beneficial effects on the health of the host.

As used herein, the term "prebiotic" refers to a non-digestible food ingredient that promotes the growth and/or activity of probiotic bacteria.

The term "subject" refers to any mammal, preferably a human.

As used herein, the term "treat" refers to alleviating, ameliorating or treating the condition, disorder or symptoms of a disease or condition.

The term "prevent" or "preventing" refers to an action to stop or inhibit the condition, disorder or symptoms of a disease or condition.

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

The terms "prenatal administration" or "prenatally administering" refer to any method of administration to the pregnant mother of an unborn subject.

The terms "postnatal administration" or "postnatally administering" refer to any method of administration to a subject from birth to approximately one year after birth.

As used herein, the term "infant formula" refers to a composition that meets the nutritional needs of infants as a substitute for breast milk. In the United States, federal regulations promulgated at 21 C.F.R. Sections 100, 106, and 107 direct the contents of infant formula. These regulations define the levels of macronutrients, vitamins, minerals, and other ingredients that contribute to the nutritional and other properties of human breast milk.

invention

The present invention has discovered a new use of LGG for the manufacture of a medicament for preventing or treating respiratory allergies in a subject. According to the present invention, LGG can be administered to unborn and infant subjects prenatally and/or postnatally to prevent or treat respiratory allergies later in life.

LGG is a probiotic strain isolated from healthy human intestinal flora. It is disclosed in U.S. Patent No. 5,032,399 to Gorbach et al., the entire contents of which are incorporated herein by reference. LGG is resistant to most antibiotics, stable in the presence of acid and bile, and readily binds to human intestinal mucosal cells. It survives for 1-3 days in most individuals and up to 7 days in 30% of subjects. In addition to its colonization ability, LGG also benefits mucosal immune responses. LGG is deposited with the American Type Culture Collection under accession number ATCC 53103.

In the use of the present invention, ^a therapeutically effective amount of LGG may be equivalent to about ^1x104-1x1012 cfu/L/kg/day. In another embodiment, the present invention comprises administering about ^{1x106-1x109 cfu} /L/kg/day of LGG. In yet another embodiment, the present invention comprises administering about ^1x108 cfu/L/kg/day of LGG.

The form in which LGG is administered is not critical for the present invention, as long as a therapeutically effective amount of LGG is administered. For prenatal administration, LGG can be administered to the pregnant mother of an unborn child and thereby delivered to the unborn child. Prenatal administration can be administered to the mother in the form of a supplement. For example, LGG can be administered in the form of a pill, tablet, capsule, caplet, powder, liquid, or gel. In this embodiment of the present invention, the LGG supplement can be administered in combination with other nutritional supplements, such as vitamins.

In another embodiment, the LGG administered prenatally can be encapsulated in a sugar, fat or polysaccharide matrix to further increase the chance of bacterial survival. The composition of the present invention can also be provided in a form suitable for consumption selected from beverages, milk, yogurt, juice, fruit drinks, chewable tablets, cookies, biscuits or combinations thereof.

If the subject is breastfed during the first year of life, LGG administered postnatally can be administered via breast milk. In this embodiment, the mother can continue to use LGG supplementation while she is breastfeeding, thereby delivering an effective amount of LGG to her offspring via breast milk.

If the subject is formula-fed or breastfed in combination with formula during the first year of life, LGG can be added to infant formula and then given to the subject. This is also a method of administering LGG after birth.

In one embodiment, the infant formula used in the present invention is nutritionally complete and contains the appropriate types and amounts of lipids, carbohydrates, proteins, vitamins, and minerals. The amount of lipid or fat can generally vary between about 3g/100 kcal and about 7g/100 kcal. The amount of protein can generally vary between about 1g/100 kcal and about 5g/100 kcal. The amount of carbohydrates can generally vary between about 8g/100 kcal and about 12g/100 kcal. Any protein source in the art, such as skim milk, whey protein, casein, soy protein, hydrolyzed protein, amino acids, etc., can be used. Any carbohydrate source in the art, such as lactose, glucose, corn syrup solids, maltodextrin, sucrose, starch, rice syrup solids, etc., can be used. Any lipid source in the art, such as vegetable oils such as palm oil, soybean oil, sunflower oil, coconut oil, medium-chain triglyceride oil, high-oleic sunflower oil, high-oleic safflower oil, etc., can be used.

Commercially available infant formulas can be readily used. For example, premature infant formula, containing iron and HYDRATOR® (available from Mead Johnson & Company, Evansville, IN, USA) can be supplemented with appropriate levels of LGG and used in the practice of the present invention.

As an alternative to infant formula, LGG can be administered postnatally as a supplement that is not incorporated into formula feeding. For example, LGG can be administered in the form of pills, tablets, capsules, caplets, powders, liquids, or gels. In this embodiment of the use, LGG supplementation can be administered in combination with other nutritional supplements, such as vitamins.

In another embodiment, the LGG administered prenatally can be encapsulated in a sugar, fat or polysaccharide matrix to further increase the chance of bacterial survival. The composition of the present invention can also be provided in the form of a follow-on formula, beverage, milk, yogurt, juice, fruit drink, chewable tablet, cookie, biscuit or a combination thereof suitable for infant consumption.

In one embodiment of the use of the present invention, LGG is administered for at least 3 months after birth. In another embodiment of the present invention, LGG is administered for at least 6 months after birth. In yet another embodiment of the present invention, LGG is administered for at least 12 months after birth. In a specific embodiment of the present invention, LGG is administered indefinitely after birth.

In one embodiment of the present invention, LGG administered prenatally or postnatally can be combined with one or more additional probiotics. Any probiotic known in the art is acceptable in this embodiment. In a specific embodiment, the probiotic is selected from the genera Lactobacillus and Bifidobacterium.

In another embodiment of the present invention, LGG administered prenatally or postnatally can be combined with one or more prebiotics. Any prebiotic known in the art is acceptable in this embodiment. Prebiotics of the present invention can include lactulose, galacto-oligosaccharides, fructo-oligosaccharides, isomalto-oligosaccharides, soy oligosaccharides, lactosucrose, xylo-oligosaccharides, and gentio-oligosaccharides.

In one use of the invention, LGG is administered prenatally and/or postnatally to prevent or treat the development of allergic rhinitis, asthma or sinusitis.

In another use of the present invention, LGG is administered prenatally and/or postnatally to prevent or treat allergy-induced inflammation in the lungs or trachea of a subject. In particular, the use of the present invention can prevent or treat inflammation of airway tissue, inflammation of air spaces, reduce mucus production, or dilate airways.

In another use of the present invention, LGG is administered prenatally and/or postnatally to reduce or inhibit the release of one or more proinflammatory cytokines. As used herein, "proinflammatory" cytokines include those known in the art that are involved in upregulating inflammatory responses. Examples include, but are not limited to, IFN-γ, MCP-1, IL-6, and IL-10.

In a specific embodiment of the present invention, antenatal and/or postnatal administration of LGG inhibits or reduces serum IgE antibody production in a subject. In another embodiment, antenatal and/or postnatal administration of LGG increases serum IgA antibody production in a subject.

The following examples describe various embodiments of the present invention. Other embodiments within the scope of the presently claimed invention will be apparent to those skilled in the art upon consideration of this specification or practice of the invention disclosed herein. The description together with the examples should be considered as illustrative only, with the scope and spirit of the invention being indicated by the claims following the examples. In the examples, all percentages are by weight unless otherwise indicated.

Example 1

This example describes the experimental materials and methods necessary to demonstrate the effects of prenatal administration of LGG on the development of respiratory allergies and inflammation in the lungs and airways. Female BALB/c mice, 6-8 weeks old, were obtained from Harlaan Hinkelmann (Hannover, Germany). They were maintained on an ovalbumin (OVA)-free diet. All experimental procedures were approved by the Animal Ethics Committee.

Prenatal exposure to Lactobacillus rhamnosus GG (LGG)

Female BALB/c mice were gavaged (i.g.) five times with ¹⁰⁸ colony-forming units (cfu) of lyophilized LGG (reconstituted in phosphate-buffered saline (PBS)) in a 200 μl volume on days 10, 8, 6, 4, and 2 before mating. Post-mating, throughout gestation and lactation, all groups received ¹⁰⁸ cfu of lyophilized LGG by gavage every 2 days. Age-matched, sham-exposed control animals received PBS instead of LGG (control group).

Exposure to OVA during the neonatal period

At 25 days of age and then at 39 days of age, mice were sensitized to OVA by intraperitoneal (ip) injection of 10 μg OVA (grade Vl; Sigma, Deisenhofen, Germany) emulsified in 1.5 mg Al(OH) ₃ (Pierce, Rockford, USA) in a total volume of 200 μl.

To evaluate airway inflammation, mice were placed in Plexiglas chambers and exposed to aerosolized OVA (1% w/v diluted in PBS) for 20 minutes on days 44, 45, 46, and 47. Airway inflammation was evaluated 24 hours after the last allergen-aerosol exposure.

OVA-sensitized mice were evaluated on day 53 for cytokine production and IgE and IgA antibody responses.

LGG-DNA determination in fecal samples

Fecal samples (0.05 g) were aseptically weighed, placed in sterile test tubes, and evenly dispersed in 1.4 ml of lysis buffer. Genomic DNA was extracted according to the manufacturer's instructions (QIAamp DNA Stool Kit, Quiagen). DNA was amplified using a hot-start PCR kit (Quiagen) and LGG-specific primer pairs (LGG sense: gagaagaatggtcggcagag and LGG antisense: catttcaccgctacacatgg).

Offspring: exposed to OVA

Offspring were sensitized to OVA at 25 and then at 39 days of age by intraperitoneal (ip) injection of 10 μg OVA (grade V1; Sigma, Deisenhofen, Germany) emulsified in 1.5 mg Al(OH) ₃ (Pierce, Rockford, USA) in a total volume of 200 μl. Antibody responses of OVA-sensitized mice were evaluated on day 53.

On days 44, 45, 46, and 47, mice were placed in Plexiglas chambers and exposed to aerosolized OVA (1% weight/volume diluted in PBS) for 20 minutes for evaluation of airway inflammation.

Determination of OVA-specific antibody serum levels

OVA-specific IgG1, IgG2a, and IgE antibody titers were determined by ELISA on day 53. 96-well polystyrene flat-bottom microtiter plates (Greiner) were coated with OVA (20 μg/ml) diluted in 0.1 M carbonate coating buffer, pH 8.2 (for IgG1) or PBS (for IgE and IgG2a). The plates were incubated overnight at 4°C, rinsed three times with wash buffer (PBS/0.1% Tween 20), and blocked with blocking solution (3% BSA/PBS) for 2 hours at room temperature. After rinsing the plates three times, the samples (diluted in PBS/0.1% Tween 20) were added and incubated overnight at 4°C. The plates were then incubated with biotin-conjugated anti-mouse IgE, IgG2a, or IgG1 monoclonal antibodies (2.5 μg/ml, all obtained from Pharmingen) for 2 hours at room temperature. The reaction was carried out in the dark at room temperature with streptavidin-peroxidase (diluted 1:1000) and tetramethylbenzidine (Roche) as substrate for 30 minutes. The reaction was stopped with 2N sulfuric acid and the plate was measured at 450/490 nm.

Bronchioalveolar lavage (BAL) and cell differentiation

Lavage fluid was collected 24 hours after the last OVA aerosol exposure. BAL was performed by cannulating the trachea and lavaging twice with 0.8 ml of ice-cold PBS. The recovered BAL volume and total cell count were determined. Cytospins were prepared for each sample by centrifugation of 50 μl BAL fluid/150 μl PBS (100 g, 5 minutes). Cytospins were fixed and stained with Diff Quick (Baxter Dade). Differential cell counts of 100 cells were performed. Cells were classified as neutrophils, eosinophils, macrophages, or lymphocytes using standard morphological criteria.

Example 2

This example illustrates the presence of LGG in the feces of treated mice. The presence of LGG was assessed in fecal samples from LGG-treated mice compared to mice that received only PBS. Following microbial culture, LGG was present in fecal samples from LGG-treated mice, whereas no LGG was detected in fecal samples from PBS-treated mice ( FIG. 2A ). To further confirm that the detected LGG was indeed identical to the LGG supplement, DNA was prepared from fecal samples and amplified using LGG-PCR-specific primers. As shown in FIG. 2B , LGG-specific PCR products were detected in samples from LGG-treated mice, whereas no LGG-specific PCR products were detected in samples from PBS-treated mice ( FIG. 2B ). This result suggests that prenatal and early postnatal treatment of mice results in intestinal colonization, and that LGG was still detectable for at least 3 weeks after the last LGG supplement.

To evaluate whether maternal LGG administration results in long-term (more than 3 weeks after cessation of supplementation) LGG aggregation in mice, DNA was prepared from fecal samples of 53-day-old mice. PCR analysis showed that neither PBS-treated nor LGG-treated mice had LGG aggregation (Figure 2C). This result indicates that maternal LGG delivery does not result in long-term aggregation in offspring after birth.

To further evaluate whether prenatal and early postnatal LGG supplementation affects the distribution of gut bacterial colonization, several strains (LGG, Enterococci, Escherichia coli, Staphylococcus aureus, and Bacteroides) were evaluated in fecal samples. As shown in Figure 3, LGG supplementation showed a trend toward beneficial shifts in bacterial colonization. Compared to control mice, higher numbers of Enterococci, Escherichia coli, and Bacteroides bacteria were detected in fecal samples from LGG-supplemented mice (Figure 3). Furthermore, compared to PBS mice, there was a trend toward reduced intestinal Staphylococcus aureus colonization. These results suggest that LGG treatment improves normal healthy gut colonization and simultaneously inhibits the proliferation of (pathological) bacteria in the mouse intestine.

Example 3

This example illustrates the effect of LGG on serum allergen-specific antibody production in allergic subjects. We further investigated whether prenatal and early postnatal LGG supplementation inhibits the development of an allergic phenotype later in life. Therefore, mouse offspring were sensitized intraperitoneally with OVA and then exposed to an OVA-allergen aerosol to assess the production of allergen-specific antibodies. As shown in Figures 4A and 4B, prenatal and early postnatal exposure to LGG significantly reduced anti-OVA IgG1 production compared to PBS controls (Figures 4A, B), indicating that maternal LGG supplementation inhibits the development of allergen-specific antibody responses. The murine IgG1 antibody subclass is an effector molecule in the development of allergic manifestations such as positive skin prick tests and airway inflammation. With respect to this allergic effector function, the murine IgG1 antibody subclass is comparable to the human IgE antibody subclass. Significant differences were detected in the levels of anti-OVA IgG2a antibody production (Figure 4C). These results suggest that maternal LGG-supplementation reduces allergic sensitization to respiratory allergens later in life.

Example 4

This example demonstrates the effect of LGG on the elevation of serum IgA antibodies in subjects. Breast milk contains high levels of secretory antibodies of the IgA subclass. It is also well known that this antibody subclass is associated with improved immune tolerance following allergen exposure. Therefore, the results surprisingly showed that offspring from LGG-supplemented mothers produced significantly higher levels of serum IgA antibodies (PBS) compared to offspring from control mothers (PBS) ( Figure 5 ). This result suggests that maternal LGG supplementation not only inhibits the development of allergic immune responses in offspring, as evidenced by a significant reduction in IgG1 antibody production, but also induces the production of an antibody class (IgA) associated with improved immune tolerance.

Example 5

This example illustrates the effect of LGG on the production of proinflammatory cytokines by splenocytes in subjects. To further evaluate whether the beneficial effects of maternal LGG supplementation on offspring antibody production are related to immune responses involved in immune tolerance, mononuclear cells from the spleens of OVA-sensitized and non-sensitized offspring were cultured in the presence of LGG or an allergen (OVA). Cytokine production was assessed after 72 hours. Offspring from LGG-supplemented mouse mothers showed significantly reduced production of IFN-γ, MCP-1, IL-10, and IL-6 (Figures 6A-D). These results indicate that maternal LGG exposure leads to a significant downregulation of proinflammatory cytokines in these mice.

Example 6

This example illustrates the effect of LGG on suppressing airway inflammation in allergic subjects. To examine whether prenatal exposure to LGG affects the development of experimental asthma, OVA-sensitized mice were challenged four times with aerosolized allergen. Airway inflammation was investigated by analyzing bronchoalveolar lavage (BAL) fluid. Compared to OVA-sensitized mice exposed to PBS prenatally, BAL fluid from OVA-sensitized mice maternally exposed to LGG contained significantly lower numbers of granulocytes ( FIG. 7 ). Furthermore, the cellularity of the BAL fluid revealed a significant decrease in eosinophils and macrophages, while lymphocytes showed only a trend toward a decrease in number.

Example 7

This example describes the experimental materials and methods necessary to demonstrate the effects of prenatal LGG administration on the development of respiratory allergies and inflammation in the lungs and airways. Female BALB/c mice were gavage-administered LGG prior to mating and during gestation. Following birth, LGG supplementation continued during lactation until 21 days after delivery. Control mice received PBS instead of LGG.

Female BALB/c mice were gavaged five times with ¹⁰⁸ colony-forming units (cfu) of lyophilized LGG (reconstituted in PBS) in a 200 μl volume on days 0, 2, 4, 6, 8, 10, and 12. A control group received PBS alone. On days 21 and 28, all animals were sensitized with 100 μg of milk protein and 1.5 mg of Al(OH) ₃ adjuvant via intraperitoneal injection. Blood samples were collected on days 0 and 49 to assess serum antibody levels and immune effector function (PCA).

Wistar rats were depilated on their backs and flanks and injected intradermally with 0.1 ml of diluted test serum from 49-day-old mice. Twenty-four hours later, 1 ml of a 1:1 mixture of milk protein (Mead Johnson Nutritionals, Evansville, IN, USA) and Evans blue solution (2% in sterile saline) was injected intravenously. After 20-30 minutes, animals with positive reactions were examined. The diameter of dye extravasation at the serum injection site was measured.

Milk protein-specific IgG1, IgG2a, and IgE antibody titers were determined on day 49 by ELISA. 96-well flat-bottom microplates (NUNC, Germany) were coated with milk protein (5 μg/ml) diluted in 0.1 M carbonate coating buffer, pH 9.6, and then blocked with PBS/1% BSA/0.02 Tween 20 for 1 hour at 37°C. A standard curve was generated using positive pooled serum (day 49 control) and measured on each plate (starting dilutions: 1:10 for IgE, 1:100 for IgG2a, and 1:6400 for IgG1). 1000 arbitrary units/ml (AU/ml) was set as the specific antigen-specific antibody level. For detection of IgE, IgG1, and IgG2a antibodies, PO-conjugated antibodies were added (37°C for 1 hour). 3,3',5,5'-Tetramethylbenzidine (TMB; Sigma, Germany, 100 μl/well; 6 mg/ml DMSO) was then used for color development, the reaction was stopped with 100 μl/well of 2N H ₂ SO ₄ solution, and the absorbance was measured at 450 nm. The titer of milk protein-specific antibodies was expressed as arbitrary units (AU) compared with standard serum.

On day 56, blood samples were collected per animal before and 1 hour after oral challenge with 100 mg of bovine milk protein, and serum levels of mmcp-1 were determined using an ELISA kit (Moredun, Scotland). ELISA was performed according to the manufacturer's instructions. Data were analyzed using GraphPad Prism software, version 3.02. Differences between groups were determined using the Mann Whitney-U test. A value of p < 0.05 was considered statistically significant.

Example 8

This example illustrates the presence of LGG in the feces of mice treated with LGG before and after birth. The presence of LGG in fecal samples of LGG-treated mice was evaluated compared to mice that received only PBS. After microbial culture, LGG was present in fecal samples of LGG-treated mice, while no LGG was detected in PBS-treated mice ( FIG. 2A ). To further confirm that the LGG detected was indeed the same as the LGG supplement, DNA was prepared from fecal samples and amplified using LGG-PCR-specific primers. As shown in FIG. 2B , LGG-specific PCR products were detected in samples from LGG-treated mice, while no LGG-specific PCR products were detected in samples from PBS-treated mice. This result indicates that prenatal and early postnatal treatment of mice leads to intestinal colonization, and LGG can still be detected at least 3 weeks after the last LGG supplement.

To evaluate whether maternal LGG administration leads to LGG accumulation in newborn mice, DNA was prepared from fecal samples of 53-day-old newborn mice. PCR analysis showed that neither PBS-treated nor LGG-treated mice had LGG accumulation. This result suggests that maternally delivered LGG is not transmitted to newborn mice during the prenatal or postnatal period.

To further evaluate whether prenatal and early postnatal LGG supplementation affects the distribution of gut bacterial flora (bacterial gut colonization), several strains (LGG, Enterococci, Escherichia coli, Staphylococcus aureus, Bacteroides) were evaluated in fecal samples. LGG supplementation showed a trend toward a beneficial shift in bacterial flora. Higher numbers of Enterococci, Escherichia coli, and Bacteroides bacteria were detected in fecal samples from LGG-supplemented mice compared to control mice. Furthermore, there was a trend toward reduced Staphylococcus aureus colonization in the gut of LGG-treated mice compared to PBS-treated mice. These results suggest that LGG treatment improves normal healthy gut flora and simultaneously inhibits the proliferation of (pathological) bacteria in the mouse gut.

Example 9

This example illustrates the effects of prenatal and early postnatal LGG supplementation on serum allergen-specific antibody production in allergic neonatal mice and control mice. Allergen-specific antibody production following intraperitoneal OVA sensitization and exposure to OVA-allergen aerosol was evaluated. Maternal LGG supplementation inhibited allergen-specific antibody responses, as demonstrated by a significant decrease in anti-OVA IgG1 production in prenatal and early postnatal LGG-treated mice compared to PBS controls. The murine IgG1 antibody subclass is equivalent to the human IgE antibody subclass. The murine IgG1 antibody subclass is an effector molecule in the development of allergic manifestations such as positive skin prick tests and airway inflammation. Anti-OVA IgG2a antibody production levels differed significantly between LGG-treated and PBS-treated mice. These results suggest that maternal LGG supplementation reduces allergic sensitization to respiratory allergens later in life.

Example 10

This example illustrates the effects of prenatal and early postnatal LGG supplementation on serum IgA antibody production in both allergic neonatal mice and control mice. Breast milk contains high levels of secretory antibodies of the IgA subclass. It is also well known that this antibody subclass is associated with improved immune tolerance following allergen exposure. Therefore, the results surprisingly showed a significant increase in serum IgA antibody production in neonatal mice from LGG-supplemented mothers compared to neonatal mice from control mothers (PBS). This result clearly demonstrates that maternal LGG supplementation not only inhibits the development of allergic immune responses in neonatal mice, as evidenced by a significant reduction in IgG1 antibody production, but also induces the production of an antibody class (IgA) associated with improved immune tolerance.

Example 11

This example illustrates the effects of LGG supplementation on the production of proinflammatory cytokines in cells of allergic newborn mice and control mice in both prenatal and postnatal stages. Monoclonal cells derived from the spleens of OVA-sensitized and non-sensitized newborn mice were cultured in the presence of LGG or an allergen (OVA). Cytokine production was evaluated after 72 hours. Newborn mice from mother mice supplemented with LGG showed a significant decrease in the production of IFN-γ, MCP-1, IL-10, and IL-6. These results indicate that maternal LGG exposure results in a significant reduction in the production of proinflammatory cytokines in these newborn mice.

Example 12

This example illustrates the effects of prenatal and early postnatal LGG supplementation on the suppression of airway inflammation in both allergic neonatal mice and control mice. OVA-sensitized mice were challenged four times with aerosol allergen. Airway inflammation was investigated by analyzing bronchoalveolar lavage (BAL) fluid. Compared to OVA-sensitized mice exposed to PBS prenatally, BAL fluid from OVA-sensitized mice maternally exposed to LGG contained significantly fewer granulocytes (p < 0.05). In addition, the cell composition of the BAL fluid showed a significant decrease in eosinophils and macrophages (p < 0.05), while lymphocytes showed only a trend toward a decrease in number.

Example 13

This example illustrates the effect of postnatal LGG supplementation on the development of an allergic phenotype later in life. To evaluate the effect of postnatal LGG exposure on the development of an allergic phenotype later in life, female BALB/c mice were given LGG or PBS saline for 2 weeks (14 days). On days 21 and 28, mice were given two injections of cow's milk protein. On days 49 and 56, the production of cow's milk-specific antibodies (Figure 8) and immune effector functions, such as passive skin anaphylaxis (Figure 9A) and serum mast cell proteinase-1 release, were evaluated following oral challenge.

As shown in Figure 8, postnatal LGG supplementation inhibited the development of allergen-specific antibody responses, as demonstrated by a significant reduction in the production of bovine milk-specific IgE antibodies. This reduction in allergen-specific IgE antibody production was associated with a significant reduction in wheal and flare reactions after allergen challenge (Figure 9A). This reduction in allergen-specific IgE antibody production was also associated with a significant reduction in mast cell proteinase-1 release after allergen challenge (Figure 9B). These results suggest that LGG supplementation reduces the development of allergen-specific immune responses and also reduces allergic symptoms after allergen exposure. Thus, LGG exposure prior to allergic sensitization reduces the development of the allergic phenotype later in life.

All references cited in this specification, including but not limited to all documents, publications, patents, patent applications, instructions, texts, reports, manuals, brochures, books, internet postings, journal articles, periodicals, etc., are incorporated herein by reference in their entirety. The discussion of the references herein is intended solely to summarize the assertions of their authors and does not constitute an admission that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.

These and other modifications and variations of the present invention may be practiced by those skilled in the art without departing from the spirit and scope of the present invention, as more particularly described in the appended claims. Furthermore, it should be understood that aspects of the various embodiments are interchangeable in whole or in part. Furthermore, those skilled in the art will appreciate that the foregoing examples are merely illustrative and not intended to limit the present invention, and are therefore further described in the appended claims. Therefore, the spirit and scope of the appended claims should not be limited to the preferred embodiments described herein.

Claims (12)

1. Use of Lactobacillus rhamnosus GG ATCC 53103 in the manufacture of a composition for preventing or treating the production of serum IgE antibodies in a subject and increasing the production of serum IgA antibodies in a subject, characterized in that the composition is administered to the subject before the subject is allergic sensitized. 2. The use of claim 1, wherein the composition is administered to the subject after birth, prior to allergic sensitization. 3. The use of claim 2, wherein postnatal administration comprises incorporating the composition into an infant formula and consuming the formula by the subject, wherein the infant formula comprises fat in an amount of 3 g/100 kcal to 7 g/100 kcal, protein in an amount of 1 g/100 kcal to 5 g/100 kcal, and carbohydrate in an amount of 8 g/100 kcal to 12 g/100 kcal. The use according to claim 1 , wherein the Lactobacillus rhamnosus GG is administered to the subject in an amount of 1×10 ⁴ -1×10 ¹⁰ cfu/L/kg/day. 5. The use of claim 1, wherein the composition is further for prenatal administration of the composition prior to allergic sensitization of a subject, which comprises ingestion of the composition by the pregnant mother of an unborn subject. 6. The use of claim 2, wherein administering the composition to the subject after birth prior to allergic sensitization comprises administering the composition to the subject after birth via breast milk. 7. The use of claim 2, wherein the composition is administered for at least 3 months after birth. 8. The use of claim 2, wherein the composition is administered for at least 6 months after birth. 9. The use of claim 2, wherein the composition is administered for at least 1 year after birth. 10. The use of claim 1, wherein the amount of Lactobacillus rhamnosus GG administered is about 1 x ¹⁰⁶ to 1 x ¹⁰⁹ cfu/L/kg/day. 11. The use of claim 1, wherein the amount of Lactobacillus rhamnosus GG administered is about 1 x ¹⁰⁸ cfu/L/kg/day. 12. The use of claim 1, wherein the composition additionally comprises at least one other probiotic.