Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders

Definitions

• the invention relates to immunostimulatory oligo- and polysaccharides and to their use for modulating T helper (Th) cell mediated-immune responses and in the manufacture of a medicament, pharmaceutical, or nutritional preparation for prevention or treatment of type I immediate atopic allergies, infectious diseases, and cancer.
• Th T helper
• Type I immediate atopic allergies are one of the most common health problems in Western world, and constantly increasing in prevalence (ISAAC 1998). It has been speculated that this increase is associated with the reduced microbial load in the developed societies, which has weakened natural type 1 Th (Thi)-cell responses giving way to enhanced Th2 responses that characterize atopic diseases (Erb 1999, Hopkin 2002).
• Thi type 1 Th
• Th cells CD4+ T cells
• Th2 cells that secrete cytokines such as IL-4, IL-5 and IL-13 are important for development of allergic inflammation by inducing IgE-production by B cells, degranulation of mast cells and recruitment of eosinophils.
• Th1 cells in contrast, are essential for defence against microbes, in particular intracellular pathogens, and cancer by inducing activation of cytotoxic T (Tc) cells and natural killer (NK) cells.
• Tc cytotoxic T
• NK natural killer
• Th1-type cytokines including IFN- ⁇ , IL-12 and IL-18, are involved in suppression of allergen-induced Th2-type immune responses (Renz et al. 1992, Li et al.
• the regulatory cytokine IL-10 secreted by regulatory T (Tr) cells also has an important role in down- regulation of Th2-type responses towards allergens (Bellinghausen et al. 2001 ).
• allergen vaccination there is up-regulation of the cytokines IFN-y, IL- 10 and IL-18 indicating their crucial role in suppressing the allergic inflammation also in vivo (Varney et al. 1993, Savolainen et al. 2004).
• Th1-type immunity the crucial time for the development of either Th1- or Th2-type immunity is infancy. At birth, there is a weak Th2-type immunity, which in healthy individuals is conversed to a Th1-type response (Holt & Macaubas C 1997). It is thought that the priming of the immune system by microbial components in the environment and gut microflora (endotoxins, lactobacilli, mycobacteria) is essential in this conversion.
• MPL monophosphoryl lipid A
• CpG-ODN immunostimulatory bacterial DNA sequences
• Th1-type cytokine production by inducing strong Th 1 -type pattern of cytokine production by acting on various Toll-like receptors on antigen-presenting cells such as monocytes, macrophages and dendritic cells.
• Th1- enhancing effects of mycobacteria and bacterial lipopolysaccharide with subsequent suppression of Th2-type response, allergic inflammation, IgE- responses and bronchial hyperreactivity have been shown in several studies (Erb et al. 1998, Wang & Rook 1998, Herz et al. 1998).
• probiotic lactobacilli have been shown to reduce the prevalence of atopic eczema at the age of two years when administrated orally during pregnancy and immediately after birth (Kalliomaki et al. 2001 ).
• Immunotherapy with bacterial CpG-ODN oligonucleotides bound covalently to allergens has also been shown to decrease the nasal inflammatory response in allergic rhinitis patients (Tulic et al. 2004).
• MPL has been successfully tested as an adjuvant in allergen immunotherapy (Drachenberg et al. 2001 , Drachenberg et al. 2003).
• LPS is a toxin and as such unfit for therapeutic use.
• CpG-ODN is a gene and, in addition to being expensive for large-scale synthesis, implies potential problems with the public opinion (such as gene manipulated food products).
• MPL adjuvant is not a single chemical entity, but a mixture of analogues, with differences reflected in the number and length of fatty acid chains.
• Probiotic lactobacilli are live bacteria, comprising safety problems with respect to possible infections, and furthermore are difficult to standardize. Also mycobacterial lysates are crude mixtures of bacterial components out of range of biological standardization.
• An ideal therapeutic microbial component for allergy treatment would be a naturally occurring, non-toxic, safe purified molecule with as small molecular size as possible.
• carbohydrates are presented for prevention and treatment of a variety of over 80 diseases.
• carbohydrates are defined as any polymer comprising more than two sugar moieties and including such classes of compounds as polysaccharides (that include mucopolysaccharides and mannans) and oligosaccharides [that are comprised of branched oligosaccharides such as sialylated sugars including milk sugars; the key milk sugars (also called hexaoses) incorporated in the general class of complex carbohydrates being difucosyllacto-N-hexaose a and b, disialyl-monofucosyllacto- N-hexaose and monofucosyllacto-N-hexaose I, Il and II].
• US 2002/0054886 and US 2002/016009 disclose a vaccine, comprising ⁇ -1 , 2- linked straight chain oligo-mannosyl residues for the treatment of candidiasis.
• the oligo-mannosyl residues are used as an epitope to elicit a protective antibody response against candidiasis or to prevent Candida albicans adhesion to mammalian cells.
• One object of the present invention is to provide an immunostimulant for the preparation of a medicament for treating a mammal, including human, suffering from or susceptible to a condition which can be improved or prevented by inducing a Th 1 -type, and/or inhibiting a Th2-type immune response.
• Another object of the present invention is to provide an immunostimulatory composition.
• a further object of the present invention is to provide a immunostimulatory food.
• Yet another object of the present invention is to provide for use of an immunostimulatory compound as a food additive.
• a yet further object of the present invention is to provide a method for inducing of Th1-type immune response in a subject and a method for inhibiting of a Th2-type immune response in a subject.
• the present invention provides use of an immunostimulant selected from the group consisting of mannan polysaccharides comprising ⁇ -1 ,2-Iinked chains, ⁇ -1 ,2-(D)-mannooligosaccharides, and any combination thereof for the preparation of a medicament for treating a mammal, including human, suffering from or susceptible to a condition which can be improved or prevented by inducing a Th1- type, and/or inhibiting a Th2-type immune response.
• an immunostimulant selected from the group consisting of mannan polysaccharides comprising ⁇ -1 ,2-Iinked chains, ⁇ -1 ,2-(D)-mannooligosaccharides, and any combination thereof for the preparation of a medicament for treating a mammal, including human, suffering from or susceptible to a condition which can be improved or prevented by inducing a Th1- type, and/or inhibiting a Th2-type immune response.
• the present invention also provides an immunostimulatory composition comprising at least one component selected from the group consisting of mannan polysaccharides comprising ⁇ -1 ,2-Iinked chains and ⁇ -1 ,2-(D)-manno- oligosaccharides; and a carrier.
• the present invention further provides a food comprising at least one component selected from the group consisting of mannan polysaccharides comprising ⁇ -1 ,2- linked chains and ⁇ -1 ,2-(D)-mannooligosaccharides.
• the present invention also provides use of mannan polysaccharides comprising ⁇ -1 ,2-linked chains, ⁇ -1 ,2-(D)-mannooligosaccharides and/or any combination thereof as a food additive.
• the present invention also provides a method for inducing of Th1-type immune response comprising administering to a subject the composition of the invention or the food of the invention in an amount effective to induce a Th1-type immune response, and a method for inhibition of Th2-type immune response comprising administering to a subject the composition of the invention or the food of the invention in an amount effective to partially or completely inhibit development of Th2-type immune response.
• Figure 1 illustrates the chemical structure of ⁇ -1 ,2-(D)-mannotriose.
• Figure 2 illustrates the chemical structure of ⁇ -1 ,2-(D)-mannobiose.
• Figures 3a and 3b depict the proliferation of (3a) and IFN- ⁇ production in (3b) human white blood cell cultures following stimulation with 200 ⁇ g/ml of Candida albicans mannan hydrolyzed at mild acidic conditions for 15, 30, or 60 minutes.
• Figures 4a, 4b and 4c depict the expression of Th2-attenuating cytokines IFN- ⁇ (4a), IL-10 (4b) and IL-18 (4c) in human white blood cells following stimulation with 0, 0.2, 2 and 20 ⁇ g/ml of ⁇ -1 ,2-(D)-manno-oligosaccharides, from ⁇ -1 ,2-(D)-mannohexaose to ⁇ -1 ,2-(D)-mannobiose.
• the y-axis indicates fold increase in mRNA expression in comparison to culture without oligomannoside.
• Figure 5 illustrates the effect of 0, 0.2, 2 and 20 ⁇ g/ml of ⁇ -1 ,2-(D)-mannotriose on production of interleukin-4 (Th2 cytokine) in allergen (birch) stimulated white blood cell cultures of eleven patients with allergic rhinoconjunctivitis.
• the y-axis indicates IL-4 mRNA production as the percentage of allergen-induced response.
• ⁇ -1 ,2-(D)-manno- oligosaccharide -mediated modulation of Th-immune response as presented in Scheme 1.
• ⁇ -1 ,2-(D)-mannooligo- saccharide refers to a carbohydrate consisting of two to nine (D)-mannose units comprising at least one ⁇ -1 ,2-glycosidic link, preferably two or more ⁇ -1 ,2- glycosidic linkages and most preferably only ⁇ -1 ,2-glycosidic linkages.
• polysaccharide refers to a corresponding carbohydrate consisting of at least ten monosaccharides.
• Allergic inflammation is characterized by IgE antibody production, mast cell degranulation and eosinofilic inflammation. These responses are mediated by allergen-specific Th2-type immune cells that secrete cytokines such as IL-4 and IL-5.
• Th2-type immune cells that secrete cytokines such as IL-4 and IL-5.
• the Th1 cells that secrete cytokines, including IFN- ⁇ are involved in suppression of allergen-induced Th2-type immune responses.
• the regulatory cytokine IL-10 is also important in down-regulation of Th2-type immune responses.
• the present invention describes immunostimulatory ⁇ -1 ,2-(D)-manno- oligosaccharides, and the use of such molecules or their prodrugs for modulation of Th-mediated immune responses and in the manufacture of a medicament, pharmaceutical and nutritional preparation for prevention or treatment of type I atopic allergies, infectious diseases, and cancer in a subject.
• immunostimulant refers to a biologically active substance whose activities affects or plays a role in the functioning of the host immune system by stimulating T helper type 1 and regulatory type T cell responses.
• subject refers to a mammal, including human.
• a composition according to the invention typically includes at least one component selected from the group consisting of ⁇ -1 ,2-(D)-mannooligosaccharides, preferably ⁇ -1 ,2-(D)-mannotriose and/or ⁇ -1 ,2-(D)-mannobiose, which have the chemical formulas depicted in Fig. 1 and Fig. 2, respectively, ⁇ -1 ,2-(D)- mannotriose thus refers to a structure: ⁇ Man(beta.1-2)Man(beta.1-2)Man ⁇ (Fig. 1) and ⁇ -1 ,2-(D)-mannobiose to a structure: ⁇ Man(beta.1-2)Man ⁇ (Fig. 2). It should be noted that the although Fig.
• compositions can also consist of or comprise prodrugs of ⁇ -1 ,2-(D)- mannooligosaccharides, preferably ⁇ -1 ,2-(D)-mannotriose and ⁇ -1 ,2-(D)- mannobiose, e.g.
• a mannan polysaccharide comprising ⁇ -1 ,2-linked chains, ⁇ -1 ,2- (D)-mannooligosaccharides, preferably ⁇ -1 ,2-(D)-mannotriose and ⁇ -1 ,2-(D)- mannobiose, can be prepared from mannan of Candida albicans yeast or other fungi, synthesized chemically, or produced enzymatically by any method known in the art.
• Mannan of Candida albicans yeast typically comprises about 10 % ⁇ -1 ,2- linked chains and its hydrolysis results in an amount of ⁇ -1 ,2-(D)-mannotriose and ⁇ -1 ,2-(D)-mannobiose that corresponds to about 1 % of the amount of the mannan.
• the invention also encompasses a method for inducing a Th1-type immune response. The method involves administrating to a subject compositions according to the invention in an effective amount to induce the synthesis of TM -type cytokines. In preferred embodiments, the method involves, but is not limited to, the induction of IFN- ⁇ synthesis in T cells.
• the method involves the induction of IL-12 and IL-18 production in antigen-presenting cells.
• the antigen-presenting cell is a macrophage, monocyte or dendritic cell.
• the method involves activation of cytolytic activity of Tc cells and NK cells.
• the invention further involves a method for inhibiting a Th2-type immune response.
• the method involves administrating to a subject, the composition according to the invention in an effective amount to partially, or completely inhibit the development of Th2-type immune response to an allergen.
• the mechanisms of inhibition include, but are not limited to, induction of 1L-10 production in T cells.
• the method can also involve suppression of Th2-type immune response by inhibition or suppression of the function of Th2- type T cells, mast cells and eosinophil and basophil granulocytes.
• the present invention also provides a method for modulating a Th-mediated immune response.
• the immune response stimulated according to the invention is biased toward the Th1-type response and away from the Th2-type response.
• the method involves administrating to a subject, said composition in an effective amount to stimulate the production of Th1-type cytokines.
• the method involves, but is not limited to, the induction of lFN- ⁇ synthesis in T cells.
• the method involves, but is not restricted to, the induction IL-12 and IL-18 production in antigen-presenting cells.
• antigen-presenting cell used herein denotes to a macrophage, monocyte or dendritic cell.
• the method also involves activation of Tc and NK cells.
• the method involves administrating to a subject said composition in an effective amount to partially, or completely inhibit the development of Th2-type immune response to allergen.
• the mechanisms of inhibition include, but are not limited to, induction of IL-10 production in T cells.
• the method can also involve suppression of Th2-type immune response by inhibition or suppression of the function of Th2- type T cells, mast cells and eosinophil and basophil granulocytes.
• the invention further relates to the use of a composition according to the invention for the manufacture of a medicament, pharmaceutical, or nutritional preparation for prevention or treatment of type I immediate atopic allergies.
• the type I immediate atopic allergy is selected from the group consisting of atopic eczema/dermatitis syndrome (AEDS), allergic asthma, allergic urticaria, food allergy, venom allergy, and allergic rhinoconjunctivitis.
• AEDS atopic eczema/dermatitis syndrome
• the composition of the invention can be used for prevention and treatment of infectious diseases and cancer.
• prevent used herein refers to inhibiting completely or partially the development of the disorder in a subject that has, or is at high risk of developing, said disorder.
• treating is defined as administering a subject an effective amount of the said composition to prevent the onset of, alleviate the symptoms of, or stop the progression of the disorder.
• effective amount used herein refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
• the therapeutically effective amount typically varies from about 1 ⁇ g to several grams depending on the composition and especially the mode of administration. E.g.
• the typical amount for parenteral administration of ⁇ -1 ,2 ⁇ (D)-mannooligosaccharide, preferably ⁇ -1 ,2-(D)-mannotriose and/or ⁇ -1 ,2-(D)-mannobiose is from 1 ⁇ g to 100 ⁇ g, preferably 2 ⁇ g to 30 ⁇ g, most preferably 3 ⁇ g to 10 ⁇ g, and for oral administration the typically amount can be much higher from a few mg up to several grams. If a prodrug is administered the effective amount depends on how much ⁇ -1 ,2-(D)-mannotriose and/or ⁇ -1 ,2-(D)-mannobiose it can result in and how much of is actually released.
• the medicament or pharmaceutical preparation of the invention may be administered to a subject by any known route in art, including enteral, mucosal, parenteral and topical routes.
• enteral routes include oral and any route involving absorption from the gastrointestinal tract.
• the mucosal routes include, but are not restricted to, oral, nasal, sublingual, buccal, pulmonary, transdermal and ocular routes.
• the parenteral routes include, but are not restricted to, intravenous, intradermal, intramuscular, and subcutaneous routes.
• the composition of the invention is used in conjugation with a pharmaceutically acceptable carrier.
• pharmaceutically acceptable carrier refers to a carrier substance with which the active ingredient is combined to facilitate the application to a subject and that is physiologically acceptable to the recipient.
• the pharmaceutically acceptable carrier can be selected from the group consisting of, but not restricted to, transdermal carriers, transmucosal carriers, oral carriers and parenteral carriers based upon the intended route of administration.
• a mannan polysaccharide comprising ⁇ -1 ,2-linked chains, can optionally serve as the natural carrier of immunostimulatory ⁇ -1 ,2-(D)- mannotriose and ⁇ -1 ,2-(D)-mannobiose.
• the pharmaceutical composition of the invention may include another therapeutic compound.
• therapeutic compound used herein is preferentially an allergy medicament, an asthma medicament, an anticancer medicament, or antimicrobial agent.
• the pharmaceutical composition of the invention comprises an antigen.
• antigen broadly includes any type of molecule (e.g. protein, peptide, polysaccharide, glycoprotein, nucleic acid, or combination thereof) that is recognized by a host immune system and is capable of eliciting a specific immune response.
• the antigen is an allergen preparation for specific allergen immunotherapy (allergen vaccination or sublingual immunotherapy).
• allergen used herein refers to a substance that can induce an allergic or asthmatic response in a susceptible subject, and includes but is not limited to pollens, insect venoms, animal dander, fungal spores and house dust mite.
• specific allergen immunotherapy refers to treatment of a subject with atopic disorders by administrating gradually increasing amounts of allergen by any of the known routes to induce tolerization to the allergen to prevent further allergic reactions.
• the pharmaceutical composition of the invention comprises an antigen preparation for cancer vaccination or immunization against microbial infections.
• Cancer antigens used herein are compounds associated with tumour or cancer cell surface, and include but are not limited to antigens that are prepared by crude extracts of cancer cells, by recombinant technology, or by de novo synthesis of known antigens.
• microbial antigens used herein include intact microorganisms, as well as natural isolates and fragments or derivates thereof, and also synthetic compounds, which are identical to or similar to natural microbial antigens.
• infectious disease refers to a disease arising from the presence of foreign microorganisms or infectious pathogens in the body.
• infectious pathogens i.e. microbes, refers to viruses, bacteria and parasites.
• infectious pathogens also includes normal flora, which is not desirable.
• the combined administration of the pharmaceutical composition of the invention and cancer or microbial antigen is useful for stimulating enhanced immune response to malignant cells and pathogens.
• the pharmaceutical composition of the invention comprises antibodies or antibody fragments which specifically bind or recognize cancer or microbial antigens.
• the present invention also relates to use of immunostimulatory composition
• immunostimulatory composition comprising of ⁇ -1 ,2-(D)-mannooligosaccharide, preferably ⁇ -1 ,2-(D)-mannotriose, ⁇ -1 ,2-(D)-mannobiose and/or prodrugs, e.g. mannan oligo- or polysaccharides comprising ⁇ -1 ,2-linked chains, thereof in the manufacture of a medicament, pharmaceutical, or nutritional preparation for prevention or treatment of type I immediate atopic allergy.
• the type I immediate atopic allergies include, but are not limited to, atopic eczema/dermatitis syndrome (AEDS), allergic asthma, allergic urticaria, food allergy, venom allergy and allergic rhinoconjunctivitis.
• AEDS atopic eczema/dermatitis syndrome
• allergic asthma allergic asthma
• allergic urticaria allergic urticaria
• food allergy venom allergy
• allergic rhinoconjunctivitis allergic rhinoconjunctivitis
• the composition of the invention is used as a food additive.
• the composition of the invention can also be a nutritional preparation.
• the nutritional composition is preferentially enterally administrable, e.g. powder, tablet, capsule, a liquid concentrate, solid product, or ready-to drink beverage.
• the composition of the embodiment is used as an additive of usual food products.
• the nutritional composition of the invention is used for enrichment of infant formulas and other functional food products.
• a further embodiment comprising the composition of the invention could be a chewing gum.
• CTAB-mannan was prepared according to Nakajima and Ballou (1974). Briefly, fungi were autoclaved for 2 hours at 14O 0 C in neutral citrate buffer (0.02 M CeH 5 Na 3 O 7 x 2 H 2 O). The mannan was isolated from the primary extract by methanol precipitation (4 days in 70 % methanol) and with a final precipitation with CTAB(cetyltrimethylammonium bromide, Sigma, MO, USA) (2.7 % CTAB and 2.7 % lyophilised methanol precipitate in distilled water overnight).
• CTAB cetyltrimethylammonium bromide
• the long ⁇ -1 ,2-linked polysaccharide side chains of Candida albicans mannan were degraded by subjecting CTAB-mannan to mild acid hydrolysis (20 mg/ml of lyophilized mannan in 0.1 N HCI) for 60 minutes at 100 0 C.
• the human PBMC were isolated from heparinized blood samples with Ficoll (Ficoll-Paque, Amersham Pharmacia Biotech, Sweden) density gradient centrifugation.
• the PBMC were washed twice with Hanks' balanced salt solution (HBSS) and suspended in RPMI medium supplemented 5 % autologous serum, 2 mM L-glutamine (Fluka Biochemica, Germany) and 100 ⁇ g/ml gentamycin (Biological Industries, Israel).
• HBSS Hanks' balanced salt solution
• RPMI medium supplemented 5 % autologous serum, 2 mM L-glutamine (Fluka Biochemica, Germany) and 100 ⁇ g/ml gentamycin (Biological Industries, Israel).
• the cell suspension was diluted in the RPMI-based culture medium to 10 cells/ml and applied on 48-well culture plates (Costar, Cambridge, MA, USA), 400 ⁇ l/well in duplicate for cytokine assays and on 96-well culture plates (Costar), 100 ⁇ l suspension/well in triplicate for proliferation assays.
• the cultures were incubated at 37 0 C, in humidified atmosphere with 5 % CO 2 for 5-7 days.
• tritium-labelled thymidine (Amersham Pharmacia Biotech) was used in the culture medium during the last 18 hours and the incorporated radioactivity was measured from the cells with liquid scintillation counter.
• PBMC peripheral blood mononuclear cells
• RNA was suspended to 20 ⁇ l of diethylpyrocarbonate (DEPC)-treated water (Promega, Wl, USA).
• DEPC diethylpyrocarbonate
• the reverse transcription (RT) reaction was performed with First Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech) using poly(dT)12-18 primers, and cDNA was stored at -70 0 C.
• the amplification of ⁇ -actin, IFN- ⁇ , IL-10, IL-18 and IL-4 cDNAs were performed in MicroAmp ® optical 96-well reaction plate (Applied Biosystems, CA, USA).
• the 25 ⁇ l reaction in each well contained 1 ⁇ l of total cDNA, 300 nM of sequence specific primers and 200 nM of dual-labelled fluorogenic probe in 1x Taqman ® Universal PCR master mix (Applied Biosystems).
• the primer and probe sequences were designed using Primer Express software (Applied Biosystems) and were labelled with FAM (6-carboxyfluorescein) at the 5'-end and with TAMRA (6-carboxytetramethylrhodamine) at the 3'-end.
• FAM 6-carboxyfluorescein
• TAMRA 6-carboxytetramethylrhodamine
• a negative PCR control without template and a positive PCR control with a template of known amplification were included in each assay.
• the reaction was performed in ABI PRISM 7700 Sequence Detection System (Applied Biosystems) according to manufacturer's instructions.
• the Ct values of the PCRs (the cycle number at which the detected fluorescence exceeds the threshold) for each amplification product were determined using a threshold value of 0.03.
• IFN- ⁇ , IL-10, IL-18 and IL-4 specific signals were normalized by constitutively expressed ⁇ -actin signals using formula (I).
• CTAB-mannan was hydrolyzed with 0.1 M HCI at 100 0 C for 15, 30, 45, or 60 min, and the lyophilized fractions were used for stimulation of human PBMC as described above.
• Fig. 3a prolonged incubation of CTAB-mannan at mild acidic conditions resulted in a progressive degradation of mannan polysaccharide according to decrease in optic density on TLC (thin layer chromatogram).
• IFN- ⁇ production mRNA and protein
• Th2-attenuating cytokines by human PBMC in response to ⁇ -1 ,2-(D)-mannotriose and ⁇ -1 ,2-(D)-mannobiose
• ⁇ -1 ,2-linked side chains of mannan polysaccharide were fractionated with Bio-Gel P-2 column (Bio-Rad Laboratories, CA, USA) chromatography.
• the potency of the isolated ⁇ -1 ,2-(D)-manno-oligosaccharides to induce production of the Th2-attenuating cytokines IFN- ⁇ , IL-10 and IL-18 were thereafter tested in vitro in PBMC cultures of allergic subjects.
• IFN- ⁇ , IL-10 and IL-18 mRNA were measured by real-time RT-PCR as described above. As illustrated in Figs. 4a ⁇ 4c, strong inductions of IFN- ⁇ , IL-10 and IL-18 production were elicited by ⁇ -1 ,2-(D)-manno-oligosaccharides.
• ⁇ -1 ,2-(D)-mannotriose and ⁇ -1 ,2-(D)-mannobiose induced strong Th1-type cytokine responses in human PBMC are naturally occurring, non-toxic, safe, purified molecules with small molecular size, and thus ideal therapeutic microbial components for allergy treatment.
• ⁇ -1 ,2-(D)-mannotriose on the production of IL-4, the main Th2-type cytokine promoting allergic inflammation, was studied in an allergen stimulated PBMC culture of eleven patients with allergic rhinitis.
• the PBMC were isolated and cultured as described above.
• Birch whole allergen extract was used at a concentration of 50 ⁇ g/ml and ⁇ -1 ,2-(D)-mannotriose in concentrations 0.2, 2 and 20 ⁇ g/ml. The cultures were incubated for 3 days. Following stimulation, the expression of IL-4 and IFN- ⁇ mRNA were measured by real-time RT-PCR. In the cultures of both atopic patients ⁇ -1 ,2-(D)-mannotriose effectively suppressed the birch allergen induced IL-4 response in a dose-dependent manner (Fig. 5). The p- values in Fig. 5 refer to a non-parametric paired test (Wilcoxon rank sum test).
• Drachenberg KJ, Wheeler AW, Stuebner P, Horak F (2001 ) A well-tolerated grass pollen-specific allergy vaccine containing a novel adjuvant, monophosphoryl lipid A, reduces allergic symptoms after only four preseasonal injections. Allergy 56:498-505.
• Hopkin JM (2002) The rise of atopy and links to infection. Allergy 57(Suppl 72):5-9.

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