WO2008031438A2

WO2008031438A2 - Treatment of asthma, eczema and/or allergy using non-pathogenic organisms

Info

Publication number: WO2008031438A2
Application number: PCT/DK2007/050126
Authority: WO
Inventors: Hans Bisgaard
Original assignee: Region Hovedstaden V/Gentofte Hospital
Priority date: 2006-09-13
Filing date: 2007-09-13
Publication date: 2008-03-20
Also published as: WO2008031438A3

Abstract

The present invention relates to a treatment of asthma, eczema and/or allergy in individuals, in particular treatment of infants for reducing the risk of acquiring asthma, eczema and/or allergy at a later stage in life, by administration to the respiratory tract in the individual an effective amount of a probiotic locally to the airways in order to reduce or eradicate pathogenic bacterial growth in the airways thereby inhibiting pathogenic bacteria in stimulating asthma, eczema and/or allergy through various routes. In particular the individual is an infant. Furthermore, the present invention also includes treatment of pregnant women to prevent infants from being colonized with bacteria during birth.

Description

Treatment of asthma, eczema and/or allergy using non-pathogenic organisms

Field of invention

The present invention relates to a treatment of asthma, eczema and/or allergy in individuals, in particular treatment of infants for reducing the risk of acquiring asthma, eczema and/or allergy at a later stage in life.

All patent and non-patent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.

Background of invention

Persistent wheeze represents a major chronic disorder in young children being the main cause of hospitalization, medication use and other healthcare resource utilizations and burdens their quality of life.(19) It reflects a common clinical phenotype with heterogeneous etiologies such as post-bronchiolitic, virus-associated or atopy- related wheeze; as well as proper asthma. Such phenotype has also been separated on a temporal pattern into transient early onset, late onset or persistent pattern. (20) There is a need for a better understanding of different etiologies of persistent wheeze in young children. Biopsies from infants with persistent wheeze and reversible airflow obstruction revealed neither thickening of the reticular basal membrane nor eosinophilic inflammation even in the presence of atopy.(4) Broncho-alveolar-lavage in young children with severe persistent wheeze showed increased lymphocytes and epithelial cells, but in contrast to findings in adult asthmatics, macrophages and neutrophils were up-regulated, while eosinophils and mast-cells were less predominant.(5;6) .

The use of probiotics is known in the art for improving the microbial balance in the intestinal tract of mammals, in order to prevent or treat gastro-enteric infections and other diseases or disorders involving and/or causing changes in or to the intestinal microflora composition, and/or resulting in any change to the microflora composition, maintaining such changes, as well as changes to the microflora composition which actively cause or potentiate such diseases or disorders. WO 01/37865 describes treatment of allergic diseases by administering living probiotics to the gastro-intestinal tract. The reference describes how administration of probiotics, in particular Lactobacillus, to the gastro-intestinal tract lowers IgE levels and thereby preventing allergy towards one or more allergens. The allergen may be present in any part of the body of the individual being treated in that it is the general IgE level that is lowered. Thus, the reference discusses whether stimulating the immune system by giving an active probiotic supplement can prevent or delay the appearance of early signs of asthma, such as wheezing, frequent runny nose, and eczema.

Summary of invention

The present invention is based on the finding of a correlation between bacterial colonization in the airways and later development of asthma, eczema and/or allergy.

Accordingly, the present invention relates to the administration to the respiratory tract for the treatment of asthma, eczema and/or allergy in an individual, in particular prevention or reduction of asthma, eczema and/or allergy, by administering an effective amount of a probiotic locally to the airways in order to reduce or eradicate pathogenic bacterial growth in the airways thereby inhibiting pathogenic bacteria in stimulating asthma, eczema and/or allergy through various routes. In particular the individual is an infant. Furthermore, the present invention also includes treatment of pregnant women to prevent infants from being colonized with bacteria during birth.

Thus, in one aspect the invention relates to the use of a probiotic for the preparation of a composition for the administration to the respiratory tract of an individual for the treatment of asthma, eczema and/or allergy in an infant, or for the preparation of a composition for the administration to a pregnant woman for the treatment of asthma, eczema and/or allergy in her infant to be born.

In another aspect the present invention also relates to a non-pathogenic organism formulation for administration to the respiratory tract, said non-pathogenic organism formulation comprising at least one non-pathogenic organism. In yet another aspect the invention also relates to a non-pathogenic organism formulation for administration to the genital tract, said non-pathogenic organism formulation comprising at least one non-pathogenic organism.

Description of Drawings

Figure 1 Flow chart of subjects included into the analyses of the primary outcome wheeze.

Figure 2 First wheezy episode as observed during the first 4 years of life. The cumulated risk is indicated for neonates with (•) and without (— ) early colonization with S. pneumoniae, M. catarrhalis and H. influenzae.

Figure 3 Persistent wheeze. Development during the first 4 years of life. The cumulated risk is indicated for neonates with (•) and without (— ) early colonization with S. pneumoniae, M. catarrhalis and H. influenzae.

Figure 4 Acute severe exacerbation of wheeze. Development during the first 4 years of life. The cumulated risk is indicated for neonates with (•) and without (— ) early colonization with S. pneumoniae, M. catarrhalis and H. influenzae.

Figure 5 Hospitalization for acute severe exacerbation of wheeze during the first 4 years of life. The cumulated risk is indicated for neonates with (•) and without (— ) early colonization with S. pneumoniae, M. catarrhalis and H. influenzae.

Figure 6 Eosinophils in blood: relative mean increase (95%CI) during the first 4 years of life for neonates with and without early colonization with S. pneumoniae, M. catarrhalis and H. influenzae.

Detailed description of the invention

Definitions Allergy: In the present context the term allergy refers to any type of disease related to allergy, such as the most common allergic conditions that include hay fever (allergic rhinitis), asthma, allergic eyes (allergic conjunctivitis), allergic eczema, hives (urticaria), and allergic shock (also called anaphylaxis and anaphylactic shock).

Asthma: Asthma is a chronic inflammatory pulmonary disorder that is characterized by reversible obstruction of the airways resulting in chest tightness, coughing and wheezing.

Atopic dermatitis: Atopic dermatitis, commonly referred to as eczema, is a chronic skin disorder categorized by scaly and itching rashes. People with eczema often have a family history of allergic conditions like asthma, hay fever, or eczema.

Delay of asthma, eczema and/or allergy: By the term is meant that onset of asthma, eczema and/or allergy is delayed to a later stage in life. The typical onset of asthma in untreated population is an age of 2-3 years. When the onset is delayed it is normally delayed by at least Vz year, more preferably at least 1 year.

Eczema: Eczema is an itching dermatitis often with vesicles. Atopic dermatitis is the most common form of eczema.

Infant: A child in the earliest period of life, preferably before 1 year, more preferably before 6 months, more preferably before 3 months.

Inhibition of asthma, eczema and/or allergy: By the term is meant that the infant does not acquire asthma, eczema and/or allergy at a later stage in life.

Non-pathogenic organisms: Non-pathogenic organisms are defined as "live microorganisms administered in adequate amounts which confer a beneficial health effect on the host". Most non-pathogenic organism products contain bacteria from the genera Lactobacillus or Bifidobacterium, as well as other genera, including Escherichia, Enterococcus, Bacillus and Saccharomyces (a yeast).

Pathogenic bacteria: Bacteria causing disease or capable of causing disease. Prevention of asthma, eczema and/or allergy: By the term is meant that the infant has a reduced risk of acquiring asthma, eczema and/or allergy at a later stage in life.

Probiotics: Probiotics are defined as "live microorganisms administered in adequate amounts which confer a beneficial health effect on the host". Most probiotic products contain bacteria from the genera Lactobacillus or Bifidobacterium, as well as other genera, including Escherichia, Enterococcus, Bacillus and Saccharomyces (a yeast).

Reduction of asthma, eczema and/or allergy: By the term is meant that the infant has a reduced risk of acquiring asthma, eczema and/or allergy at a later stage in life.

Respiratory tract: The term respiratory tract is used in it's conventional meaning, i.e. mouth and nose, airways and lungs.

Detailed description of the invention

The present invention relates to the use of a probiotic for the preparation of a composition for the administration to the respiratory tract for the treatment of asthma, eczema and/or allergy in an individual, preferably an infant. The treatment is preferably prevention of asthma, eczema and/or allergy or reduction of asthma, eczema and/or allergy. In another embodiment the treatment is delay of asthma, eczema and/or allergy symptoms.

The present inventors have shown a correlation between airway colonization with pathogenic bacteria in asymptomatic neonates and later development of severe persistent lung symptoms and asthma.

Furthermore, a correlation between airway colonization and blood eosinophil concentration has been found. Also a correlation between airway colonization and total immunoglobulin E (total IgE) has been found. These correlations point to a correlation between airway colonization and later development of asthma, eczema and allergy.

Reduction or eradication of the pathogenic bacteria by administration of probiotics reduces the risk of acquiring asthma, eczema and/or allergy at a later stage in life, thereby preventing asthma, eczema and/or allergy or reducing asthma, eczema and/or allergy.

Furthermore, the invention relates to treatment of older children as well as adults in order to prevent symptoms or reduce symptoms or asthma, eczema and/or allergy.

The pathogenic bacteria colonizing the airways may be any pathogenic bacteria, in particular pathogenic bacteria normally infecting the airways. The pathogenic bacteria typically found to colonize the airways of the infants are selected from the group consisting of Streptococcus pneumoniae (S. p.), Haemophilus influenze (H. L), Moraxella catarrhalis (M.c), and Staphylococcus aureus.

Thus, selected probiotics according to the present invention preferably have the ability to inhibit or reduce the growth of the pathogenic bacteria colonizing the airways, whereby the probiotics can be used in infants to protect against asthma, eczema and/or allergy onset in children or later in life.

The probiotics are administered locally to the airways as described more detailed below.

Without being bound by theory it is believed that the infants are colonized with bacteria received from the mother, in particular bacteria colonizing the genital tract of the mother. Thus, in another aspect the invention relates to treatment of the mother during pregnancy in order to reduce the bacteria load in the mother. Therefore, the present invention also relates to the treatment of asthma, eczema and/or allergy in infants by administering to a pregnant woman a sufficient amount of a probiotic.

Non-pathogenic organisms - Probiotics

In the present context the term probiotics is used synonymously with the term non- pathogenic organisms.

The probiotics used according to the present invention may be any probiotics or yeast useful for reducing or eradicating pathogenic bacterial growth in the airways. Without being bound by theory the probiotics may lead to probiotic-induced suppression of pathogen growth and/or invasion. This may be accomplished by probiotic secretion or production of acids, hydrogen peroxide, antimicrobial substances, and/or bacteriocins (proteins elaborated by probiotic organisms that have specific antimicrobial effects against sensitive species) antagonistic to pathogen growth; competition for nutrients required for growth of pathogens; or competitive inhibition of adhesion of pathogens.

Each of the types of probiotics or yeast occupies its own ecological niche, each having particular conditions for optimal survival and multiplication rate.

Pathogenic bacteria, which may cause various diseases or disorders, also occupy their own particular environmental niches or habitats. Competition between pathogenic and probiotics may occur under various conditions, but maximal competitive effect occurs when the conditions for optimal survival and multiplication rate of both pathogenic and probiotics are similar. Under such conditions, survival depends upon more stringent competition for nutrients or growth factors, a well as upon synergistic nutrient utilization and competition for receptor sites. Factors such as production of substances, intensity of multiplication, and creation of restrictive environment, including induction of immunological processes and stimulation of epithelial cell turnover also have great significance under such conditions.

According to the present invention the composition may comprise one or more types and/or species of probiotics, such probiotics preferably being selected from the group consisting of Bifidobacterium, Lactobacillus, Lactococcus, Saccharomyces and Streptococcus.

In the following the various bacteria and yeasts used as probiotics according to the present invention are described in more details.

Bifidobacterium

Bifidobacteria are normal inhabitants of the human and animal colon. Bifidobacteria are gram-positive anaerobes. They are non-motile, non-spore forming and catalase- negative. They have various shapes, including short, curved rods, club-shaped rods and bifurcated Y-shaped rods. Their name is derived from the observation that they often exist in a Y-shaped or bifid form. The guanine and cytosine content of their DNA is between 54 mol% and 67mol%. They are saccharolytic organisms that produce acetic and lactic acids without generation of CO₂, except during degradation of gluconate. They are also classified as lactic acid bacteria (LAB). To date, 30 species of bifidobacteria have been isolated.

In one embodiment the Bifidobacteria used as probiotics according to the present invention include Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium thermophilum, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium lactis. Specific strains of bifidobacteria used as probiotics include Bifidobacterium breve strain Yakult, Bifidobacterium breve RO7O, Bifidobacterium lactis Bb12, Bifidobacterium longum RO23, Bifidobacterium bifidum RO71, Bifidobacterium infantis RO33, Bifidobacterium longum BB536 and Bifidobacterium longum SBT-2928.

Lactobacillus

Lactobacilli are normal inhabitants of the human intestine and vagina. Lactobacilli are gram-positive facultative anaerobes. They are non-spore forming and non-flagellated rod or coccobacilli. The guanine and cytosine content of their DNA is between 32 mol% and 51 mol%. They are either aerotolerant or anaerobic and strictly fermentative. In the homofermentative case, glucose is fermented predominantly to lactic acid. Lactobacilli are also classified as lactic acid bacteria (LAB). To date, 56 species of the genus Lactobacillus have been identified.

In one embodiment the Lactobacilli used as probiotics according to the present invention include Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus GG (Lactobacillus rhamnosus or Lactobacillus casei subspecies rhamnosus), Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum and Lactobacillus salivarus. Lactobacillus plantarum 299v strain originates from sour dough. Lactobacillus plantarum itself is of human origin. Other probiotic strains of Lactobacillus are Lactobacillus acidophilus BG2FO4, Lactobacillus acidophilus INT-9, Lactobacillus plantarum ST31 , Lactobacillus reuteri, Lactobacillus johnsonii LA1 , Lactobacillus acidophilus NCFB 1748, Lactobacillus casei Shirota, Lactobacillus acidophilus NCFM, Lactobacillus acidophilus DDS-1 , Lactobacillus delbrueckii subspecies delbrueckii, Lactobacillus delbrueckii subspecies bulgaricus type 2038, Lactobacillus acidophilus SBT-2062, Lactobacillus brevis, Lactobacillus salivaήus UCC 1 18 and Lactobacillus paracasei subsp paracasei F19.

Lactococcus

Lactococci are gram-positive facultative anaerobes. They are also classified as lactic acid bacteria (LAB). Lactococcus lactis (formerly known as Streptococcus lactis) is found in dairy products and is commonly responsible for the souring of milk.

In one embodiment the Lactococci that are used as probiotics according to the present invention include Lactococcus lactis, Lactococcus lactis subspecies cremoris (Streptococcus cremoris), Lactococcus lactis subspecies lactis NCDO 712, Lactococcus lactis subspecies lactis NIAI 527, Lactococcus lactis subspecies lactis NIAI 1061 , Lactococcus lactis subspecies lactis biovar diacetylactis NIAI 8 W and Lactococcus lactis subspecies lactis biovar diacetylactis ATCC 13675.

Saccharomyces

Saccharomyces belongs to the yeast family. The principal probiotic yeast is Saccharomyces boulardii. Saccharomyces boulardii is also known as Saccharomyces cerevisiae Hansen CBS 5296 and S. boulardii. S. boulardii \s normally a nonpathogenic yeast. S. boulardii has previously been used to treat diarrhea associated with antibiotic use.

In one embodiment the saccharomyces used as probiotics according to the present invention include Saccharomyces cerevisiae Hansen CBS 5296.

Streptococcus

Streptococcus thermophilus is a gram-positive facultative anaerobe. It is a cytochrome, oxidase and catalase negative organism that is nonmotile, non-spore forming and homofermentative. Streptococcus thermophilus is an alpha-hemolytic species of the viridans group. It is also classified as a lactic acid bacteria (LAB). Steptococcus thermophilus is found in milk and milk products. Other Streptococci may be selected from Streptococcus cremoris, Streptococcus discetylactis, Streptococcus faecium, Streptococcus intermedius, Streptococcus lactis.

In one embodiment the probiotic according to the present invention include Streptococcus salivarus, such as subspecies thermophilus type 1 131 , Streptococcus cremoris, Streptococcus discetylactis, Streptococcus faecium, Streptococcus intermedius, Streptococcus lactis.

Enterococcus

Enterococci are gram-positive, facultative anaerobic cocci of the Streptococcaceae family. They are spherical to ovoid and occur in pairs or short chains. Enterococci are catalase-negative, non-spore forming and usually nonmotile. Enterococci are part of the intestinal microflora of humans and animals.

In one embodiment the probiotic according to the present invention include Enterococcus faecium SF68.

Combinations

In one embodiment the composition comprises one probiotic strain only, wherein said strain is selected according to a selection pressure in relation to one or more of the pathogenic bacteria involved in colonization of the airways.

In another embodiment the composition comprises two or more probiotic strains, such as three or more probiotics strains, such as four or more probiotic strains, such as five or more probiotics strains, such as ten or more probiotics strains, such as twenty or more probiotic strains. When using more than one probiotic strain it is important that those two or more strains do not compete thereby reducing the efficacy of either strain.

Thus, in one embodiment, the composition comprises one or more probiotics selected from the group consisting of: Bacillus subtilis, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifudum, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium thermophilum, Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus alactosus, Lactobacillus alimentarius, Lactobacillus amylophilus, Lactobacillus amylovorans, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus batatas, Lactobacillus bavaricus, Lactobacillus bifermentans, Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus buchnerii, Lactobacillus bulgaricus, Lactobacillus catenaforme, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus collinoides, Lactobacillus confusus, Lactobacillus coprophilus, Lactobacillus coryniformis, Lactobacillus corynoides, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus desidiosus, Lactobacillus divergens, Lactobacillus enterii, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus frigidus, Lactobacillus fructivorans, Lactobacillus fructosus, Lactobacillus gasseri, Lactobacillus halotolerans, Lactobacillus helveticus, Lactobacillus heterohiochii, Lactobacillus hilgardii, Lactobacillus hordniae, Lactobacillus inulinus, Lactobacillus jensenii, Lactobacillus jugurti, Lactobacillus kandleri, Lactobacillus kefir, Lactobacillus lactis, Lactobacillus leichmannii, Lactobacillus lindneri, Lactobacillus malefermentans, Lactobacillus mail, Lactobacillus maltaromicus, Lactobacillus minor, Lactobacillus minutus, Lactobacillus mobilis, Lactobacillus murinus, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pseudoplantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rogosae, Lactobacillus tolerans, Lactobacillus torquens, Lactobacillus ruminis, Lactobacillus sake, Lactobacillus salivarius, Lactobacillus sanfrancisco, Lactobacillus sharpeae, Lactobacillus trichodes, Lactobacillus vaccinostercus, Lactobacillus viridescens, Lactobacillus vitulinus, Lactobacillus xylosus, Lactobacillus yamanashiensis, Lactobacillus zeae, Pediococcus acidlactici, Pediococcus pentosaceus, Streptococcus cremoris, Streptococcus discetylactis, Streptococcus faecium, Streptococcus intermedius, Streptococcus lactis, Streptococcus thermophilus, and combinations thereof. In one embodiment, the lactic acid producing bacterium is Lactobacillus acidophilus. In another embodiment, the Lactobacillus acidophilus strains include the M35, LA45, LA51 and L41 1 strains. In another embodiment, the Lactobacillus acidophilus strain is LA51.

Composition

The composition according to the invention may be any suitable composition, such as a pharmaceutical composition or a health-promoting compound. Administration to the respiratory tract

The composition is administered to an individual to prevent or reduce asthma, eczema and/or allergy at a later stage in life. In one embodiment the composition is administered after diagnosis of a pathogenic colonization in the airways. In another embodiment the composition is administered prophylactic to infants without bacterial analysis. It is found that an early treatment is most effective in prevention of asthma, eczema and/or allergy. Preferably the composition is administered to an infant younger than 3 months, more preferably administered to the infant younger than 2 months, and more preferably to an infant younger than 1 month.

The composition prepared according to the invention is administered locally to the airways, ie. as local delivery via the nasal, buccal, sublingual and respiratory routes, for example in the form of solutions, suspensions, sprays, inhalants, and aerosols. Preferably the administration is by nasal or respiratory routes, more preferably by nasal routes. Without being bound by theory it is believed that nasal and/or buccal administration will lead to colonization of the remaining relevant parts of the airways.

The daily dosage typically depends on the age of the infant as well as the weight of the infant. A typical daily dosage is 0.1 to 100 billion viable bacteria, such as 0.02 - 20 billion live bacteria per kg body weight. Doses of probiotics are measured by the number of viable organisms, such as administration of at least 1x10⁸ CFU/day (CFU= colony-forming units). Thus, the daily dosage may be at least 1 x10⁸ CFU/day, such as at least 5x10⁸ CFU/day, such as at least 1x10⁹ CFU/day, such as at least 2x10⁹ CFU/day, such as at least 5x10⁹ CFU/day, such as at least 1x10¹⁰ CFU/day.

The daily dosage may be divided into two or more dosages administered two or more times per day.

In a preferred embodiment the composition is administered daily for a period of time being at least 3 days, more preferably at least 1 week, more preferably at least 2 weeks, more preferably at least 3 weeks, most preferably at least 4 weeks. In one embodiment it may be desired that the treatment is continued until no pathogenic bacteria are detectable in the airways. In another preferred embodiment the composition is administered pulsed, such as once a week or twice a week for a period of time as described above.

Administration to a pregnant woman

The composition is administered to a pregnant woman in the last part of the pregnancy, preferably shortly before the expected time of birth, such as in the last month of pregnancy in order to avoid the infant being colonized with pathogenic bacteria during birth, thereby preventing or reducing asthma, eczema and/or allergy at a later stage in life of the infant to be born. In one embodiment the composition is administered after diagnosis of a pathogenic colonization in the genital tract. In another embodiment the composition is administered prophylactically without bacterial analysis.

The composition prepared according to the invention may be administered by any suitable route to the mother, including orally or locally to the genital tract.

A typical daily dosage is 0.1 to 100 billion viable bacteria, such as 2-20 billion live bacteria per kg body weight. Doses of probiotics are measured by the number of viable organisms, such as administration of at least 1x10⁸ CFU/day (CFU= colony-forming units). Thus, the daily dosage may be at least 1x10⁸ CFU/day, such as at least 5x10⁸ CFU/day, such as at least 1x10⁹ CFU/day, such as at least 2x10⁹ CFU/day, such as at least 5x10⁹ CFU/day, such as at least 1 x10¹⁰ CFU/day.

In a preferred embodiment the composition is administered daily for a period of time being at least 3 days, more preferably at least 1 week, more preferably at least 2 weeks, more preferably at least 3 weeks, most preferably at least 4 weeks.

In another preferred embodiment the composition is administered pulsed, such as once a week or twice a week for a period of time as described above. Formulation for administration to the respiratory tract

The formulation of the composition should preserve as many viable probiotic organisms as possible. Generally the formulation may relate to dried probiotic organisms, such as freeze-dried organisms, or fresh probiotic organisms in solution. An example of producing the latter is described in WO 2005/017095. When administering dried probiotic organisms, they may be administered as such using an inhalant, or they may be resuspended in a suitable solution before administration. The formulations may include heat treated and/or irradiated probiotics.

Local delivery via the nasal, buccal and respiratory routes is contemplated for treatment according to the invention. Formulations of active ingredients in carriers suitable for nasal administration are therefore also included within the invention, for example, nasal solutions, sprays, aerosols and inhalants. Where the carrier is a solid, the formulations include a coarse powder having a particle size, for example, in the range of 1 to 500 microns, preferably in the range of 1 -10 microns, which is administered, e.g., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.

Thus, the formulations of the composition prepared according to the invention may be in the form of nasal solutions or sprays, aerosols, inhalants, topical formulations, liposomal forms and the like.

For intra-nasal administration, the therapeutic agent may be administered via nose drops, a liquid spray, such as via a plastic bottle atomizer or metered-dose inhaler.

Nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays and are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, the aqueous nasal solutions usually are isotonic and slightly buffered, preferably to maintain a pH of 5.5 to 6.5 taking into account the preferred pH for maintaining viable organisms in the composition.

Inhalations and inhalants are compositions designed for delivering a composition or compound into the respiratory tree of a patient. A vapour or mist is administered and reaches the affected area. This route can also be employed to deliver agents into the systemic circulation. Inhalations may be administered by the nasal or oral respiratory routes. The administration of inhalation solutions is only effective if the droplets are sufficiently fine and uniform in size so that the mist reaches the bronchioles.

Another group of products, also known as inhalations, and sometimes called insufflations, comprises finely powdered or liquid compositions that are carried into the respiratory passages by the use of special delivery systems, such as pharmaceutical aerosols, that hold a solution or suspension of the composition in a liquefied gas propellant. When released through a suitable valve and oral adapter, a metered does of the inhalation is propelled into the respiratory tract of the patient. Fine mists are produced by pressurized aerosols and hence their use in considered advantageous.

For administration to the upper (nasal) or lower respiratory tract by inhalation, the probiotics of the invention are conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray.

Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, the composition may take the form of a dry powder, for example, a powder mix of the probiotic and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatine or blister packs from which the powder may be administered with the aid of an inhalator, insufflator or a metered-dose inhaler.

The percent by weight of a probiotic of the invention present in a topical formulation will depend on various factors, but generally will be from 0.01% to 95% of the total weight of the formulation, and typically 0.1 % to 25% by weight.

According to a preferred embodiment a liquid formulation comprises between about 10⁶ composition and about 10¹⁰ CFU per ml composition, and the amount of viable organisms should preferably not diminish by more than a factor of 100, preferably no more than a factor of 10 during storage and period of use.

Formulation for administration to a pregnant woman

The formulation for administration to a pregnant woman may be in any form suitable for administration, such as orally for systemic use, and/or locally to the genital tract. In particular for local use the formulation suitable for vaginal administration may be presented as pessaries, tampons, suppositories, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Composition comprising a probiotic for the administration to the respiratory tract

In one aspect the present invention further relates to a composition comprising a probiotic suitable for the administration to the respiratory tract for the treatment of the airways, wherein the composition comprises at least one probiotic organism.

The composition may be formulated as described above, in that the probiotic formulation may be in powder form or it may be in a pharmaceutically acceptable liquid, suitable for administration to the airways. In another aspect the present invention relates to a composition comprising at least one probiotic suitable for the administration to the genital tract for the treatment of a pregnant woman.

EXAMPLES

EXAMPLE 1

Bacterial Airway Colonization In Asymptomatic Neonates Is Associated With Development Of Persistent Wheeze In Young Children.

PATIENTS AND METHODS

Copenhagen Prospective Study on Asthma in Childhood (COPSAC) is a clinical prospective, longitudinal birth-cohort study.(8) Pregnant women from Greater Copenhagen with a history of physician-diagnosed asthma (N=798) were invited into COPSAC (N=452) and their newborns enrolled (41 1 ) between August 1998 and

December 2001 . Key exclusion criteria were severe congenital abnormality, gestational age <36 weeks, mechanical ventilation, and history of lower respiratory tract symptoms during the weeks prior to enrolment at 1 month of age.

The study followed the guiding principles of the Declaration of Helsinki, and was approved by the Ethics Committee for Copenhagen (KF 01 -289/96) and The Danish Data Protection Agency (2002-41 -2434). Data validity and quality control procedures followed "Good Clinical Practice" guidelines. Data were collected on-line into an SQL database by trained personnel. This database was double checked against source data by an external monitor and the database subsequently locked. An audit trail was run routinely.

Airway bacteria

Airway bacteria were investigated in the asymptomatic infant at 1 and 12 months of age. Hypopharyngeal aspirates were performed by the doctor in the COPSAC Clinical Research Unit (CRU) in the sedated 1 -month-old infant after lung function test(9) under aseptic conditions with a soft suction catheter passed through the nose into hypopharynx. Aspiration was done intermittently assuring not to apply suction while passing through the oro- and nasopharynx. Hypopharyngeal sampling was repeated in the 12 month old awake infant. Samples were transported to the microbiology laboratories within 2 hours from collection and included in the microbiology routine. Material representative for lower airways was cultured and standard laboratory techniques applied for identification of S. pneumoniae, H. influenzae, M. catarrhalis, S. pyogenes and S. aureus, chosen prior to the study.(10) The CRU personel were unaware of the outcome of the culture.

Clinical Endpoints

Respiratory symptoms were recorded in daily diaries for 4 years by the parents.

Wheeze was translated to the parents as wheeze or whistling sounds, breathlessness or persistent troublesome cough severely affecting the wellbeing of the infant, and was recorded as the composite dichotomized scores (yes/no) as previously described in details(1 1 ). The symptom description was supported by a dedicated book on early childhood wheeze with integrated dairy cards (see www.copsac.com). The CRU doctor reviewed symptom definition and the diary entries with the parents at the 6-monthly clinical sessions. The infants were given a full physical examination and parents were interviewed by the doctors at the CRU (authors) using structured questions and standardized response categories focusing on the child's lung symptoms, diagnoses, medication, healthcare utilization, lifestyle and home environment. Symptoms from airways or the skin were diagnosed and treated by the CRU doctor rather than by other health care providers. Wheezy episodes were defined as three consecutive days of wheeze, at which point the parents were requested to bring the child to the CRU for examination. Persistent wheeze was defined as five such episodes within 6 months or daily symptoms for four weeks. Differential diagnoses were excluded at that point by chest X-rays and sweat tests. Acute severe exacerbation of wheezy symptoms was diagnosed by the CRU doctors or from hospitalization for such symptoms.

Blood eosinophil concentration (10⁹/L) was measured by standard methods.

Total level of immunoglobulin E (Total IqE) was determined by ImmunoCAP™ (Pharmacia Diagnostics AB, Uppsala, Sweden) in serum(16); detection limit 2 kU/L.

Specific IqE was determined by a screening method (ImmunoCAP Phadiotop Infant™, Pharmacia Diagnostics AB, Uppsala, Sweden) for IgE against the most common food and inhalant allergens (hen's egg, cow's milk, peanut, shrimp, dust mite, cat, dog, birch, timothy, ragweed and wall pellitory). Values of Phadiotop Infant >= 0,35 kU/L were considered indicative of sensitisation(17) and was analyzed as the dichotomized measurement.

Treatment

Positive cultures for airway bacteria were not treated because the infants were asymptomatic at the time of sampling. All subjects participated in the randomized controlled clinical trial of intermittent treatment with inhaled budesonide versus placebo for 2-weeks during wheezy episodes in the first 3 years of life, showing no short-term or long-term treatment eff ect( 1 1 ). Wheezy symptoms were treated by the CRU doctors in accordance with the following algorithm: for symptomatic relief, parents were provided with terbutaline (Bricanyl® AstraZeneca, Lund, Sweden) pMDI with a spacer(18) to be administered as needed. Persistent wheeze defined the threshold for daily treatment with 400 microgram of inhaled budesonide pMDI with a spacer for 3 months increasing to 6 and 12 months at subsequent relapses. Montelukast 4 mg daily was added to children with recurrent wheeze despite budesonide maintenance treatment. Acute severe exacerbation of wheeze was treated with budesonide 1600 μg daily for 2 weeks or oral prednisolone 1 -2 mg/kg daily for 3 days. No other treatment was allowed for wheezy symptoms.

Study objectives

The primary outcomes were the indicators of recurrent wheeze: wheezy episodes; persistent wheeze; acute severe exacerbations and acute hospitalization for wheeze. The secondary outcomes were lung function; blood eosinophils; total-lgE and specific IgE.

Statistical analyses

Age accumulated risks of first wheezy episode; diagnosis of persistent wheeze; acute severe wheezy exacerbation and hospitalization stratified on colonization of bacteria were estimated by the Kaplan-Meier estimator. Change in risk due to bacterial colonization was quantified as hazard ratios obtained by Cox regression. Confounder adjusted hazard ratios were calculated for the sub-cohort of children with full records of the confounders in question. The effect of bacterial colonization on the logarithmic lung function and logarithmic total IgE at four years of age was assessed by ANOVA methods. The event specific allergy at age 4 years was modelled by logistic regression. Logarithmic measurements of blood eosinophils at ages 6 months, 18 months, and 4 years were modelled by a mixed linear model including a random effect for each child.

RESULTS

Bacterial cultures from lower airways were collected from 321 of 324 eligible infants at the 1 -month-visit (3 lost for technical reasons) as 87 infants had been enrolled into the COPSAC cohort of 41 1 infants before this protocol was enacted (Figure 1 ). Of those 321 children with valid bacterial cultures 306 (95%) completed 1 year, 291 (91%) 2 years, 280 (87%) 3 years and 266 (83%) 4 years of this clinical study. Two hundred and thirty-one of the 321 infants had a second bacterial culture collected at the 12- month-visit.

Colonization

At one month of age 223 were colonized with S. pneumoniae, H. influenzae, M. catarrhalis, S. aureus or S. pyogenes in 30 (9%), 28 (9%), 27 (8%), 196 (61%) and 1 (0%) cases respectively. Prevalence of S.p or M. c or H.i. was 66/321 (21%). Multiple strains were found in 49 infants (2 strains), 15 (3 strains) and 2 (4 strains). S. aureus colonization comprised 73 different Fag-patterns within the 170 colonies, the most common pattern repeating in 14%.

Colonization vs. wheeze (Table 1 and 2, Figure 2-5)

The risk of wheeze increased in neonates colonized with the bacteria S. pneumoniae, M. catarrhalis and H. influenzae (S.p., M.c. or H.i.) but not Staphylococcus aureus

(Table 1 ). The hazard ratio being 1.92 (1.43;2.57) for a first wheeze; 2.46 (1 .44;4.18) for development of persistent wheeze; 2.73 (1.49; 5.00) for acute severe exacerbation and 3.87 (1.87;8.02) for acute hospitalization.

The baseline characteristics gender, gestational age, mothers smoking and antibiotic use during 3^rd trimester, infant solely breastfeed for a minimum of 4 weeks, older siblings at home, and the neonatal baseline lung function (FEVO.5) and bronchial responsiveness (PD(TcO2) were included in a confounder adjusted analysis of the primary outcome. Univariate relationships between these baseline characteristics and colonization with S.p., M.c. or H.i. are given in Table 2. Hazard Ratios adjusted for all of the above characteristics did not cause any material change (table 1 ). Extended Cox regression analyses for primary outcomes showed no additional effect from presence of multiple species or specific species of the bacteria strains S.p., M.c. or Hl

The Kaplan-Meier curves (Figure 2-5) suggest that the risk change of wheeze associated with S.p., M.c. or Hl colonization increases during the first two years of life stabilizing thereafter. The 0-2 year hazard ratio for persistent wheeze was 3.24 (1.73;6.07) and the 2-4 year hazard ratio was 1.23 (0.41 ;3.69), but was not significantly different (WaId test for equality: p=0.13).

To assess the interplay between colonization at 4 weeks and colonization at 12 months a Cox regression analysis was performed including an additional 1 -4 year effect of colonization at 12 months and only including follow-up in the 0-1 year age period for children with a missing record of colonization at 12 months. For persistent wheeze the hazard ratio of colonization at 4 weeks adjusted for colonization at 12 months was estimated to 2.53 (1 .38;4.65) and a WaId test showed no significant effect of colonization at 12 months (p=0.32).

There was no confounding effect of the intermittent treatment with inhaled budesonide of wheezy episodes in the nested randomized controlled trial previously reported (1 1 ). Sub-analyses of specimens with and without representative columnar epithelium did not affect the conclusions of the outcome analyses.

Colonization vs. lung function, blood-eosinophils and allergy

Lung function (sRaw) baseline by age 4 was 1.25 vs 1.23 kPa^*L^*s (p=0.81 ; N=25+97) and the response to cold-dry air hyperventilation was +8% vs +7% (p=0.90; N=25+97) in children with and without S.p., M.c. or /-/./ colonization.

Blood-eosinophil level was significantly increasing with time in children colonized neonatally with S. p., M.c. or H.i. (p=0.0071 ), (figure 6).

Total IgE was significantly increased by 51% (3%;121%) by age 4 in children colonized neonatally with S.p., M.c. or H.i., (p=0.036; N=245).

Specific IgE at 4 years was not significantly affected by colonization status as neonate; OR 1.48 (0.80;2.75) (p=0.21 ; N=250).

DISCUSSION

Persistent wheeze during the first 4 years of life present a high risk of representing early asthma in this cohort borne of asthmatic mothers(3), but because there is no international consensus on the specific diagnosis of asthma in young children, we have instead analyzed intermediate asthma phenotypes which are contributing to the clinical asthma diagnoses(21 ). Neonatal airway colonization with S.p.; M.c. or H./was associated with several independent intermediate asthma phenotypes including symptoms of persistent wheeze and acute severe exacerbations of wheeze as well as raised blood eosinophils and raised total-lgE. Blood eosinophilia is a marker of asthma in children distinct from that of sensitization, (22) whereas total IgE is a marker of sensitization and wheeze(23). Therefore the association between neonatal colonization and these independent intermediate phenotypes implicates bacterial colonization in the causal pathway of early childhood asthma.

This study suggests a sub-group of infants and young children with persistent wheeze driven by an inflammatory process associated with bacteria. This role of early bacterial colonization is compatible with the observation of a predominantly neutrophilic inflammation in young children with persistent wheeze. (4;5;7;25)

The adherence rate in this longitudinal clinical study was 83% for all 4 study years. The single-centre study-design assures consistency in procedures, definitions of conditions and data capture methods. Children visited the CRU every 6 months as well as acutely at episodes of wheeze. Symptoms were monitored in diaries and data capture was assessed in personal interviews at each visit. Physicians at the CRU, but not the family physician, control the diagnosis and day-to-day management. This reduces the risk of misclassification of symptoms, which is of particular importance in the clinical evaluation of wheezy symptoms in young children where between-observer variation is significant.(41 ;42)

In conclusion, the association of early bacterial colonization of the lower airways of neonates and the four independent intermediate asthma phenotypes (wheeze from diary cards, hospitalization, blood eosinophils and total-lgE) suggests a causal role of bacterial colonization in the pathogenesis of early asthma, eczema and allergy.

Table 1 : Hazard Ratio (presence/absence of bacterium strain) for primary outcomes wheezy episode, persistent wheeze, exacerbation and hospitalization with and without adjustment for the possible confounders listed in table 2.

Table 2. Perinatal and neonatal characteristics of neonates with and without colonization with S. pneumoniae, H. influenzae and/or M. catarrhalis in the lower airways at one month of age. Frequencies are calculated for the 288 neonates with non-missing characteristics. P-values correspond to Fishers exact test for independence. Baseline lung function and bronchial responsiveness was measured by the Raised Volume Rapid Thoracic Compression technique at 1 month. (9)

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Claims

1 . Use of a probiotic for the preparation of a composition for the administration to the respiratory tract of an individual for the treatment of asthma, eczema and/or allergy in an infant, or for the preparation of a composition for the administration to a pregnant woman for the treatment of asthma, eczema and/or allergy in her infant to be born.

2. The use according to claim 1 , wherein the individual is an infant.

3. The use according to claim 1 , wherein the treatment is prevention or inhibition of asthma, eczema and/or allergy.

4. The use according to claim 1 , wherein the treatment is reduction of asthma, eczema and/or allergy symptoms.

5. The use according to any of the preceding claims, wherein the infant is treated before an age of 3 months.

6. The use according to any of the preceding claims, wherein the infant is treated before an age of 2 months.

7. The use according to any of the preceding claims, wherein the composition is for administration to the respiratory tract.

8. The use according to any of the preceding claims, wherein the composition is for nasal administration.

9. The use according to any of the preceding claims, wherein the composition is in the form of nasal spray or nasal drops.

10. The use according to any of the preceding claims 1 -7, wherein the composition is in the form of an aerosol, a nebuliser, or an inhalant.

1 1. The use according to any of the claims 1 -3, wherein the mother is treated during the last month of pregnancy.

12. The use according to any of the claims 1 -3 or 10, wherein the mother is treated locally in the genital tract.

13. The use according to claim 1 , wherein the composition comprises two or more non-pathogenic organism strains.

14. The use according to any of the preceding claims, wherein the non-pathogenic organism is selected from the group of Bifidobacterium, Lactobacillus, Lactococcus, Saccharomyces and Streptococcus.

15. The use according to any of the preceding claims, wherein the composition is administered at least one time daily.

16. The use according to any of the preceding claims wherein the composition is administered daily for a period, wherein said period is at least 4 weeks.

17. A non-pathogenic organism formulation for administration to the respiratory tract, said non-pathogenic organism formulation comprising at least one nonpathogenic organism organism.

18. The non-pathogenic organism formulation according to claim 16, wherein the non-pathogenic organism formulation is in powder form.

19. The non-pathogenic organism formulation according to claim 16, wherein the non-pathogenic organism formulation is in a pharmaceutically acceptable liquid.

20. The non-pathogenic organism formulation according to any of the claims 16-18, wherein formulation is as the composition as defined in any of claims 7-1 1 .

21. The non-pathogenic organism formulation according to any of the claims 16-19, comprising 0.1 to 100 billion viable bacteria per dosage unit.

22. A non-pathogenic organism formulation for administration to the genital tract, said non-pathogenic organism formulation comprising at least one nonpathogenic organism.

23. The non-pathogenic organism formulation according to claim 21 , wherein the non-pathogenic organism formulation is selected from the group consisting of pessaries, tampons, suppositories, creams, gels, pastes, foams and sprays.

24. The non-pathogenic organism formulation according to any of the claims 16-19, comprising comprises 0.1 to 100 billion viable bacteria per dosage unit.