In a preferred embodiment the invention provides a functional dendritic cell produced by the above described methods, wherein the functional dendritic cell is directed against the carbohydrate epitope an/or a carbohydrate structure, carbohydrate conjugate or a mammalian cell comprising said carbohydrate epitope. The nutraceutical, the pharmaceutical composition, the carbohydrate positive microorganism or the fraction thereof or formulations comprising those can also be used in the prophylaxis of carbohydrate positive positive tumor diseases, tumors or tumor cells. 10 shows the mean value of elisa signals against the PAA conjugate Gal beta1-3GalNAc alpha1-PAA relative to the elisa signal against GlcNAc1-2Gal-3GalNAcalpha-PAA, for sera from 4 mice per group at day 0 (pre-immune serum) and day 21 relative elisa signal is calculated after the equation. The formulation of the invention may also be in the form of oil-in-water emulsions. Said cellular immune response against the carbohydrate epitope is a T-cell response against the carbohydrate epitope which can be detected by at least one of the cellular immune response tests 1. Preferred are carbohydrate positive microorganisms which show a reduced binding of the carbohydrate binding molecule after treatment of the carbohydrate positive microorganism with enzymes or chemicals destroying the carbohydrate epitope as described elsewhere herein. Immune responses are followed by determination of the antibody and/or T cell response against Lewis-Y by using at least one of the humoral response tests 1 to 6 and/ or cellular immune response tests 1. 18: Cellular immune response test 4: immunofluorescence analysis of mNM-DC loaded with Core-1 negative (AG3) or Core-1 positive (AG6) bacteria or Core-1 negative (NM-wt) or Core-1 positive (NM-D4) human cell line. The invention further provides in a preferred embodiment a method for testing the potential of a Carbohydrate positive microorganism to induce a humoral and a cellular immune response which corresponds to a combination of the above described methods, comprising at least one of the humoral. In a further preferred embodiment the invention provides a Core-1 positive microorganism which can be used to reduce or even further preferred to prevent the occurrence of a Core-1 positive disease or tumor by orally administering it as a component of the nutraceutical of the.
Now few reports show that complex carbohydrate are not removed during processing of glycoproteins by antigen presenting cells and can be presented to major histocompatibility complex II restricted T cells together with the peptid. Cultures were plated aerobically on CBA to determine their ability to grow in the presence of oxygen and to check for the absence of aerobic contaminants. In a preferred embodiment the invention provides a method for generation of an activated T cell or T cells against the carbohydrate epitope comprising a) bringing into contact a suitable amount of at least one functional dendritic cell loaded with a suitable amount. Coli strain of serotyope O86 (32) was also tested in the humoral immune response tests after immunization of mice. Oral application of at least two different dosages is performed. Following incubation at 37C for 4 hours, the plate is centrifuged at 500 x g for 5 minutes, and 20 l of cell-free supernatant from every well are pipetted in 200l per well of enhancement solution (Perkin-Elmer Wallac) on the previously prepared flat-bottom plate (Nunc-Immunoplate. In a preferred embodiment of the invention the carbohydrate epitope directed immune cells are obtained by the administration of the formulation of the invention, the carbohydrate positive microorganism or the fraction or lysate thereof to a human or animal, more preferred by the administration. The invention relates also to a kit for the determination of the immune response against the carbohydrate epitope comprising at least one of the herein described immune response tests against Carbohydrate epitope, preferably at least two, and more preferably at least one humoral and one.
Samples were stored at -20 C until analysis. This suspension was split into two equal portions, one for fixation (7.1 the other for lyophilisation (7.2). For sequencing, we used either primer 27f, 338f (5' GCT GCC TCC CGT AGG AGT) (2 338r (5' ACT CCT ACG GGA GGC AGC 968f (5' AAC GCG AAG AAC CTT AC) (14 or 1492r. Elisa and elispot analysis of GM-CSF-release is known to those skilled in the art and described in detail in examples. Next, samples were washed with 40 ml PBS-c (8000g, 15 min, 4 C) and the pellets suspended in 15 ml PBS-c, followed by addition of an equal volume of 96 ice-cold ethanol. In a preferred embodiment of the invention the Core-1 microorganism is a non-pathogenic microorganism. In the experiments of Klaamas.
In a preferred embodiment the invention provides a method to treat a cancer patient comprising the administration of any of the activated T cell or T cells against Core-1, the cell composition comprising at least one T cell against Core-1, the T cell line against. Serum samples were stored in al" at -80C prior to analysis Faecal samples were collected, stored at -80. To determine spontaneous release, 100 l rpmi/5 FCS without effector cells are added. Niemand muss seinen Namen preisgeben, sich irgendwo eintragen oder etwas ausfüllen. The compositions disclosed herein may be formulated in a neutral or salt form. Said nutraceuticals or pharmaceutical compositions and preferred embodiments thereof are described in detail elsewhere herein. 3 x 105 cells per tube were pelleted and the pellet was resuspended in 50l murine serum (diluted 1:300 in PBS/1BSA control antibody or PBS/1BSA alone.
Selected Core-1 positive strains as well as strains that were not Core-1 positive were characterized by their sensitivity against different antibiotics (see table 1FIG. Both rough CPS extracts were prepared from different cultures. The carbohydrate epitope positive cells are labeled with europium or chromium-51 which allows the measurement of cells which are lysed. 3 2 The accumulation of fucose, GalNH2 / GalNAc and galactose might be an indication, that these monosaccharides are components of the repeating units of the core-1-positive polysaccharide. Bringing into contact a suitable amount of dendritic cells comprising at least one dendritic cell, dendritic cells, or a mixture of cells comprising at least one dendritic cell, loaded with a suitable amount of the carbohydrate positive microorganism, a lysate or a fraction thereof, formulations. The invention provides a carbohydrate positive microorganism which induces an effective carbohydrate-specific cellular immune response against said carbohydrate epitope, carbohydrate epitope positive tumor cells or carbohydrate epitope positive diseases, preferably in at least one humanor animaland/or in vitro. The Core-1 positive microorganism or fraction thereof represents the active ingredient which induces the specificity of the immune response against Core-1, the Core-1 antigen and/or a Core-1 positive tumor cell and/or disease due to the fact that it carries an antigen resembling Core-1. Gallen, bifidobacteria BSM Medium Switzerland K-F Streptococcus Oxoid streptococci KF Agar Nutrient Agar Oxoid non-selective N Schaedler Anaerobe Oxoid non-selective S Agar Wilkins Chalgren Oxoid non-selective WC Anaerobe Agar Brain Heart Biomérieux, Marcy non-selective BHI Infusion Agar l'Etoile, France Columbia Agar with Biomérieux non-selective CBA.
Most preferably is a cellular immune response against Core-1 which is a cytotoxic T cell response and Th1 type helper T cell response against Core-1 which can be detected by cellular immune response tests 1, 2, 3, 4 and. Said Core-1 specific microorganism and/or Core-1 positive lysate or fraction thereof effectuates a specific immunisation against core 1 upon administration of said Core-1 specific microorganism. Incubations with different dilutions of mouse sera were performed for 1,5h. Detection of core-1-containing polysaccharige on the western blot was performed using the core-1-specific antibody nemod-TF2 (culture supernatant) showing a core-1-positive band at 37kDa (fig. Hence, the terms Core-1 and Thomsen-Friedenreich do not necessarily refer to identical structures. There are no reports documenting a cellular immune response and especially a cytotoxic cellular immune response against a carbohydrate tumor antigen and especially a small non-charged carbohydrate such as Core-1...
In a further preferred embodiment the invention provides a nutraceutical or a pharmaceutical formulation comprising at least one carbohydrate positive microorganism or fraction or lysate thereof which induces or enhances a carbohydrate specific immune response in at least one human or animal functioning. Galacturonic acid and glucuronic acid, which are characteristics components of capsular polysaccharides, could not be identified in the purified core-1-positive polysaccharide extract. In a preferred embodiment the invention provides a method to treat a cancer patient wherein the patient has or had a cancer cell positive for Core-1. In a preferred embodiment of the invention the administration according to the previous methods under (i) is performed once. Following addition of 20l of Corel-specific antibodies at varying concentrations (0.05 to 50 g/ml final concentration in 200 l incubation volume) or the corresponding controls (medium, isotype control IgG pbmc (human peripheral blood mononucleare cells, 80 l) are added as effector cells, using different effector. Measuring the amount of secreted IFNgamma and/or secreted TNFalpha by elisa or elispot, whereby a positive cellular immune response against the carbohydrate epitope shows (i) a significantly higher IFNgamma and/or TNFalpha secretion of said immune cells restimulated with said dendritic cells loaded with a carbohydrate.
In another embodiment the invention provides a cellular immune response test (cellular immune response test 5) against Core-1 comprising a) incubating a suitable amount of target cells from the cell lines ZR-75-1, NM-D4, NM-F9, NM-H9 and/or NM-wt labelled with a suitable amount of europium. Gooseberries-, redcurrants-, blackcurrants-, citrus fruits-, apples-, raspberries-, strawberries- and ripe blackberries-jam or royal jelly pasta.g. 6: Sequence of repeating units of core-1-positive capsular polysaccharide. The purity of cultures was checked by comparing cell morphology, as well as Gram staining behaviour. Preferred are cells which are able to stably produce the antibodies meaning that the cells can be passaged over a suitable amount of cycles for production of the antibodies such as but not limited to hybridoma cells and otherwise immortalised cells or by recombinantly stably. In a further preferred embodiment the invention provides a method for generation of a T cell clone against Core-1 wherein said functional dendritic cell and the T cell or T cells are human cells.
In order to enhance the specificity, one may use two or more of these antibodies in order to determine/test that a microorganism is Core-1 positive and thus a Core-1 positive microorganism according to the present invention. In 800 l of europium buffer (50 mM hepes,.4, 93 mM NaCI, 5 mM KCI, 2 mM MgCl2, 10 mM diethylentriaminepentaacetic acid, 2 mM europium (III) acetate electroporated (710V, 1 pulse, 30p) in a Multiporator (Eppendorf and subsequently incubated on ice for another. Primers 27f (5 AGA GTT TGA TCC TGG CTC AG) and 1492r (5 TAC CTT GTT ACG ACT T) (10) were used to amplify the bacterial 16S ribosomal RNA gene. Even more important, there is no agent available to patients which can prevent the development of Core-1 positive tumors. Dynabeads prepared in this manner were either used immediately or within two weeks of preparation after a repeated three-fold wash step with 2 ml PBS-a.
Without intending to be limiting, examples of carbohydrate structures and corresponding carbohydrate binding molecules are listed in table. These include a reliance on mechanised tools which have been developed, from the threshing machine, seed drill, through to the tractor and combine, etc. GM2, GD2, GD3, fucosylated GM1, Globo H, LeY and the mucin core structures Tn, Sialyl-Tn and the Thomson Friedenreich antigen. Com and further preferred immature dendritic cells were generated from mutz-3 cells or NemodDC under suitable conditions comprising using IL-4 and GM-CSF typically for about one week, the resulting immature dendritic cells or inmdc are brought into contact with said suitable amount of at least. Preferable, the capsular polysaccharidee.
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