MXPA97000209A - Medical use of bromela - Google Patents

Abstract

It has been found that bromelain, a mixture of enzymes derived from the stem of pineapple, is a modulator of intracellular signal transduction, in particular a path modulator, in which inositol phosphates play an important role and, therefore, therefore, it is useful in the treatment of various diseases and conditions, which are mediated by these intracellular signal trajectories

Description

MEDICAL USE OF BROMELAIN DESCRIPTION OF THE INVENTION The present invention relates to the use of bromelain for the treatment of a variety of diseases and conditions, which are mediated by intracellular signals. In particular, the invention relates to the use of bromelain for the treatment of diseases and conditions such as cancer and autoimmune diseases, and as an immunosuppressive agent. In addition, bromelain can be used as an adjunct to vaccines. Bromelain is the collective name for the proteolytic enzymes found in the tissues of the Bromeliaceae plant. Bromelain is a mixture of several portions derived from the stem of the pineapple (Ananas comosus). It contains at least two proteolytic enzymes, but also non-proteolytic enzymes, including an acid phosphatase and a peroxidase; it also contains amylase and cellulase activity. In addition, several other components may also be present. Bromelain has previously been used in the treatment of a variety of conditions, including inflammation and, in particular, has been used in the treatment of diarrhea. The use of bromelain in the treatment of infectious diarrhea is described in WO-A-9301800, where it is suggested that bromelain works by destroying intestinal receptors for pathogens by proteolysis, and in WO-A-8801506, which teaches that bromelain separates the pathogens of the intestinal receptors. It has now been discovered that, in addition to infectious diarrhea, bromelain is also useful in the treatment of non-infectious diarrhea and this, of course, can not be explained by the mechanism of action suggested in WO-A-9301800. Taussig et al., Medical Plant, 1985, 538-539, and Maurer et al., Medical Plant, 1988, 377-381, both suggest that bromelain may be used to inhibit tumor growth, and Taussig et al. Attribute this to the component or components of bromelain, which have peroxidase activity, but give no other explanation of the mechanism. However, Maurer and others teach that bromelain is capable of inducing the differentiation of leukemic cell lines and that this ability arises from proteolytic activity. Again, the mechanism of bromelain is unclear. The mechanisms of action, by which bromelain acts in the treatment of other conditions such as inflammation, have also not been satisfactorily explained. In WO-A-9400147, several experiments are described, which demonstrate that proteolytic enzymes and, in particular, bromelain, are capable of inhibiting secretion. The applications also describe that bromelain can reduce toxin binding activity and can inhibit the secretory effect of toxins such as heat labile toxin (LT) and cholera toxin (CT) and also toxins such as toxin. Heat stable (ST). This is despite the fact that ST has a very different mode of LT and CT action. These observations were explained by the fact that a component of the mixture of bromelain, bromelain protease of the stem, seems to be able to modulate cyclic nucleotide trajectories. In addition, bromelain has also been shown to inhibit the secretion caused by the calcium-dependent pathway (Ca2 *). LT and ST are both produced by enterotoxigenic strains of E. coli (ETEC). Some strains of ETEC also produce pilus adhesins called colonization factor antigens. These adhesins promote the union of the ETEC strains to the mucosa of the small intestine, facilitating colonization and the supply of enterotoxins. Diarrheal disease ultimately depends on the production and efficient supply of the enterotoxin. 15 Enterotoxins stimulate secretion through cells by activating signal paths. The internal signals inside the cells are carried by "secondary messengers". Every cell of the human body is constantly bombarded with various signals from its environment. The cells Normal people receive and process these signals, which can promote growth, differentiation or death, or control other functions of the cell, such as secretion of fluids in the cells of the intestinal epithelium. Therefore, the signals are the key to understanding the procedures in the cell, which finally determine their destiny. The signals are received through receptors with different biochemical activities on cell surfaces and transmit the messages to other response proteins. These proteins, in turn, process the signals and transduce them to other molecules within the cell. The series of biochemical events that take place after the interaction of a cell with a growth factor and before the cellular response occurs, is termed as the signal transduction. Most cellular signals are transmitted via GTP binding proteins, several protein kinases, protein phosphatases, lipid modifying enzymes, and secondary messengers such as Ca2 +, and cyclic adenosine monophosphate (cyclic AMP or cAMP). The instructions are finally interpreted in the nucleus by the transcription of factors that initiate the expression of genes and subsequently the conversion of cellular proteins. It is known that at least three signal paths are important for secretion. A path uses the secondary messenger, cyclic AMP. Another uses the secondary messenger, cyclic guanosine monophosphate (cyclic GMP or cGMP). These two messengers are referred to as cyclic nucleotics. The third signal path (trajectory dependent on Ca2 +) requires Ca2 + as the secondary messenger. WO-A-9400147 teaches that stem bromelain protease is capable of preventing diarrhea by interfering with the cyclic nucleotide and the Ca 2+ -dependent trajectories, and thus affect secretion.

The present invention has now investigated other intracellular signal trajectories and has surprisingly found that, in addition to its effect on the cyclic nucleotide trajectories, bromelain also appears to affect other intracellular signaling pathways, in particular the pathways that are modulated by phosphates of inositol, protein kinases and / or protein phosphatases. Therefore, in a first aspect of the present invention, the use of bromelain in the preparation of an agent for modulating intracellular signaling pathways, which depend on the action of inositol phosphates, protein kinases and / or phosphatases, is provided. of protein. In the context of the present invention, inositol phosphate refers to any phosphorylated inositol molecule, without considering the degree of phosphorylation or the positions of the phosphate groups. Examples of inositol phosphates include 4,5-phosphatidylphosphate (PI P2) and 1,4-inositol triphosphate (IP3). Protein kinases and protein phosphatases refer to any molecule capable of converting an inactive form of a protein to an active form, either by the addition or removal of phosphate molecules. It has been found that bromelain is particularly useful for controlling the signaling pathways that depend on inositol phosphate, protein kinase or protein phosphatase, which lead to the production of extracellular, synaptic signaling molecules, such as vasopressin and thrombin, and particularly signaling molecules, which affect the growth and proliferation of cells, for example interleukins and other growth factors. In order to grow or proliferate, normal cells require signals, which are provided by the growth factors produced by other cells, both near and in other parts of the body. This contrasts with the autonomous behavior of the cancer cell, which is governed by its own internally generated signals. The functions of several proteins involved in the control of cell growth, they can be intensified or modified by mutations, which can change the structure of the protein or produce normal proteins in abnormally large amounts. Therefore, in cancer cells, the cellular apparatus for receiving and processing signals becomes defective, and the cell is unable to process these signals and respond appropriately. Cells that have defective growth inhibitory signals, such as those that arise from defective tumor suppressor genes, are unable to balance growth-stimulating signals. Due to this defect, the growth suppressor signals in the signaling cascade are not transmitted, and the cells can not control their own proliferation. Cancer occurs when tumor suppressor genes are inactivated. Similarly, cells that receive hyperstimulation, which arise from defects in the stimulatory signaling cascade, exhibit excessive proliferation.

Oncogenes are genes that produce a protein with altered function and its activation provides the cell with a strong, inflexible growth momentum. An oncogene breaks down the carefully balanced molecular controls of cell proliferation to such an extent that malignant growth arises. Protein tyrosine kinases such as v-src and the related v-abl protein have proven to be among the proteins most frequently implicated in experimental and human cancer. C-src is a kinase, which is found in normal cells and is regulated by other kinases. This regulation is lost in v-src, found in cancer cells. The v-src kinase is persistently overactive as a result of some amino acid differences between the c-src and v-src proteins. The unbridled catalytic activity of the mutant protein tyrosine kinase can have a deleterious effect on the control of cell growth. Normal cells, which contain c-src, will only grow if stimulated to do so, by growth factors; cancer cells, which contain v-src, show an acquired independence of externally supplied growth factors, and, at the same time, may not respond to external growth inhibitory signals. Therefore, cancer results and tumors form. Protein tyrosine phosphorylation cascades (or kinase cascades) play a very important role in the regulation of cases through signal transduction. Many receptors for growth factors have tyrosine kinase activity and, when activated, activate the phosphorylation of multiple cellular proteins on tyrosine residues. The result of this phosphorylation procedure causes the target protein to gain or lose functions. P21 c-ras plays a critical role in the mediation of mitogenic and differentiation signals received from receptor tyrosine kinases (Wood et al., Cell, 68, 1041 -1050, 1992; Thomas et al., Cell, 68, 1031 -1040, 1992) for the activation of several kinases, including members of protein kinase C (PKC), Raf, mitogen-activated protein (MAP), and S6 kinase families, (Cantley et al., Cell, 64 , 281-302, 1991). These kinases can integrate signals from multiple membrane receptors. A key element in the signaling pathway involved in the transduction of receptor-initiated signals to the nucleus is now recognized as part of the family of protein kinases activated by mitogen (MAPk). MAPk are serine / threonine kinases that are activated by various growth factors and tumor promoters in cells. The best studied of these kinases are p42MAPK and p44MAPK, (also referred to as ERK2 and ERK1, respectively, pp42mapk / erk2, and pp44mapk / erk1 / mpk, also known as microtubule-associated protein kinase; myelin basic protein kinase (MBP) ), and RSK I and II). The substrates of the MA I i i nclude include pp90 and 70S rsk and several transcription factors, notably Jun (Pulverer et al., Nature, 353, 670-674, 1991), Myc and p62 TCF. The proteins that affect transcriptional activity are the most widely implicated in the cancer procedure. The activation mechanism of MAP kinases is very complex. MAPk exists as a dephosphorylated form in resting cells and is activated when the tyrosine and threonine residues are phosphorylated (Boulton et al., Cell, 65, 663-675, 1991). In vitro, this activation is almost completely reserved, if any residue is dephosphorylated (Anderson et al., Nature, 343, 651-653, 1990). It has recently been reported that PAC1 is a MAP kinase phosphatase, which inhibits the expression of MAP-regulated kinase report gene (Ward et al., Nature, 367: 651-653, 1994). Phosphorylation of both tyrosyl and threonyl regulatory sites, in the MAP kinase, is mediated by a MAP kinase-kinase of double specificity (M KK or MEK). MEK is, in turn, regulated through phosphorylation by MAP kinase-kinase kinases that include the proto-oncogene product, Raf, (Anderson et al., Biochem, J., 277, 573-576, 1991) and MEKK , which in turn are regulated by protein C kinase (PKC). The present invention has now found that bromelain is capable of interfering with signaling pathways, which are important for growth, in particular, the signaling pathways that lead to the production of growth factors such as I L-2, factor of platelet-derived growth (PDGF) and insulin-like growth factor (IGF). T-lymphocytes were used as a cell model to demonstrate the mode of action of the cell growth promoter mechanism. The growth of T-lymphocytes is regulated through the production of growth factor, receptor function, cytoplasmic signal processing, and gene responses in the nucleus. T-lymphocytes are a commonly used model to measure proliferation, due to their easy access to cells and the well-documented role of interleukin 2 (IL-2), the T cell growth factor, which is required for growth and proliferation. T cells and, in fact, other types of cells, require stimulation in order to initiate the series of events required for proliferation. The immune system contains billions of white blood cells or lymphocytes, which are divided into two classes, B lymphocytes and T lymphocytes. B cells function to protect the host from extracellular pathogens, and T cells protect the host from intracellular pathogens. B cells and T cells recognize the different forms of different antigens, using B cell receptors (BCR) and T cell receptors (TCR), respectively. Activation of T cells is a complex procedure that requires protein tyrosine kinase activity that results in cell growth and differentiation. The activation requires the recognition of the antigen by the TCR and interactions with other molecules on the surface of the T cell with antigen present in the cells. When a T cell is presented with an appropriate antigen and the secondary co-stimulatory signal, the T cell responds in two main ways. One is to enlarge and divide, thus increasing the number of cells that react to the antigen. The other is to secrete lymphokines or cytokines, proteins that directly inhibit the pathogen or that group other cells to bind in the immune response. Interleukin 2 (I L-2) cytokine is a T cell growth factor, which plays a pivotal role in the regulation of immune responses. Resting T cells can not normally respond to I L-2, since these cells do not express detectable high-affinity IL-2 receptors on the surface of their cells. Antigenic stimulation is required for the induction of high affinity I L-2 receptor expression, and thus confer sensitivity I L-2. Therefore, the initial activation signals provided by the stimulation of the T cell antigen receptor (TCR), and the co-stimulatory signal, initiate the activation of T cells through the induction of the production of I L-2. and the expression of the I L-2 receptor. The subsequent proliferation of T cells is driven by the interaction of I L-2 with its I L-2 receptor. If a T cell receives a signal only via the TCR, the T cell is anergized or can die (called apoptosis). If a T cell receives only the co-stimulatory signal, the T cell remains at rest (or does not respond).

All the above cases require tyrosine phosphorylation, as inhibitors of protein tyrosine kinases can divide most, if not all, of the latter cases associated with TCR stimulation (Mustelin et al., Science, 247, 1584 -1587, 1990; June and others, Proc. Nati, Acad. Sci. USA, 87, 7722-7726, 1990). Induction mediated by protein tyrosine kinase activity results in tyrosine phosphorylation of many cellular proteins including the TCR zeta chain (Baniyash et al., J. Biol. Chem., 263, 18225-18230, 1988) , phospholipase C g1 (PLC g1) (Weiss et al., Proc. Nati, Acad. Sci. USA, 88, 5484-5488, 1991), CD5 (Davies et al., Proc. Nati. Acad. Sci. USA, 89, 6368-6372, 1992), the vav proto-oncogene (Bustelo and Barbacid, Science, 256, 1196-1199, 1992), valosin-containing protein (VCP), ezrin (Ergoton et al., EMBO J., 11, 3533 -3540 and J. Immunol., 149, 1847-1852, 1992), ZAP-70 (Chan et al., Cell, 71, 649-662, 1992) and MAPk (Nel et al. J. Immunol., 144, 2683- 2689, 1990). The tyrosine phosphorylation of PLCgl results in its catalytic activation, resulting in the generation of secondary messengers from the path of phosphatidylinositol (Pl). The PLC unfolds phosphatidylinositol 4,5-bisphosphate (PIP2), resulting in the formation of inositol 1,4,5-triphosphate (IP3) and 1,2-diacyl gricerol (DAG). These molecules, in turn, function as secondary intracellular messengers to induce an increase in Ca2 + and the activation of PKC, respectively. Following the activation of PKC, the proto-oncogene, Ras, is activated, the kinase activity of Raf-1 is increased and MAPk is phosphorylated. Activation of MAPk causes the proto-oncogenes c-fos to form a dimer and with c-jun form the transcription complex, AP-1. The AP-1 complex binds to elements on DNA to initiate the transcription of I L-2. Figure 1 summarizes some of the cases associated with the TCR activation of the Pl path leading to the transcription of the I L-2 gene and the production of I L-2. It was found that bromelain inhibits the kinase cascade, which is associated with growth stimulation. A factor in this signaling pathway is ras protein, for which aberrant forms are found in 25 to 30% of human tumors. Bromelain is capable of blocking signals required for T cell proliferation, probably by blocking tyrosine phosphorylation of proteins, including MAP kinase. Due to its ability to block tyrosine phosphorylation of MAP kinase and other proteins, bromelain is able to act as an anticancer agent, as it will also block the overproduction of growth factors such as platelet-derived growth factor. (PGDF) and epidermal growth factor (EGF) in fibroblasts and epithelial cells. Furthermore, since it is able to block the proliferation of T cells, it is an immunosuppressive agent, which is useful to prevent the rejection, by a host, of a transplanted organ or in the treatment of autoimmune diseases, such as diabetes mellitus, sclerosis Multiple and rheumatoid arthritis. This discovery is a complete contrast to the teaching of WO-A-9301800, which is that compositions containing proteases, such as bromelain, have non-specific immunostimulatory activity. It has been found that bromelain, in fact, can be used either to stimulate or to inhibit cytokine production depending on whether it is used to treat activated cells (such as those that already received stimulation), or inactivated cells (i.e. rest or motionless). So it can be used as an immunosuppressive agent, v.gr. , to avoid rejection of tissue, or as an immunostimulant, v. gr. , as an assistant for vaccines. Bromelain can also be used to prevent or treat toxic shock through its ability to inhibit cytokine production and tyrosine phosphorylation. Bromelain can be used to treat allergies. As previously discussed, bromelain is a mixture of several components. Although it was taught in WO-A-9400147 that the stem bromelain protease is the bromelain component responsible for the cyclic nucleotide pathway mediation, it is not clear if the bromelain protease of such is also responsible for the action of bromelain on kinase trajectories or if some other component of the bromelain mixture may be responsible.

However, this does not affect the work of the invention, since the raw bromelain mixture, at least, is capable of affecting the phosphorylation (or activation) of the MAP kinase. Bromelain can be administered by a variety of routes including enteric, for example oral, nasal, buccal, or anal administration, or parenteral administration, for example, by intravenous, intramuscular, or intraperitoneal injection. However, the preferred route is oral. To aid the survival of bromelain through the stomach, when administered orally, it may be desirable to formulate the enzyme in a preparation protected with enteric coating. Other orally administrable formulations include syrups, elixirs, and hard and soft gelatin capsules, which can also be enteric coated. The activity of bromelain is stable over a wide range of pH (pH 2-9). Therefore, it is not necessary to protect bromelain with enteric coating (or enteric coating) from the acid conditions in the stomach. However, it may be necessary to protect the enzyme from digestion by acid protease in the intestines. In this way, bromelain can be administered with a pH regulating agent, for example, bicarbonate. The dose of bromelain is conventionally measured in Rorer units, FI P units, BTU (bromelain tyrosine units), CDU (casein digestion units), G DU (gelatin digestion units), or MCU (cluster units) of milk). A Rorer unit of protease activity is defined as that amount of enzyme, which hydrolyses a standardization casein substrate at a pH of 7 and at 25 ° C, in order to cause an increase in absorbance of 0.00001 per minute to 280 nm. An FI P unit of the bromelain activity is contained in that amount of a normal preparation, which hydrolyzes a suitable casein preparation (FI P controlled) under normal conditions at an initial rate, so that a minute preparation is released per minute. amount of peptide, not precipitated by a specific protein precipitation reagent, which gives the same absorbance of 1 μmol of tyrosine at 275 nm. BTUs, CDUs, GDUs, and MCUs are defined in the literature, as follows: BTU A tyrosine unit of bromelain is that amount of enzyme, which will release a micromole of tyrosine per minute under the conditions of analysis (eg, after digestion of a denatured hemoglobin substrate, acid, at a pH of 5 and 30 ° C).

CPU That amount of enzyme that will release in microgram of tyrosine after one minute of digestion, at 37 ° C, from a normal casein substrate at a pH of 7.0.

GPU The enzyme activity that releases a milligram (10"3g) of aminonitrogen from a normal gelatin solution after 20 minutes of digestion at 45 ° C and at a pH of 4.5. 1100 BTU / g = 750 CDU / mg = 1200 GDU / g.

Since the precise dose will be under the control of the doctor or practitioner, it can be found that daily doses of 50 to 4000 GDU / day, for example, 100 to 1000 GDU / day, are appropriate. The daily dose can be given in one or more aliquots per day, for example, twice, three times or four times a day. A particularly preferred dose could be 10 mg / kg, (giving a dose of 700 mg for an average adult human being, equivalent to 2800 BTU). The invention will now be explained by the following examples. The examples refer to the accompanying drawings, in which: Figure 1 is a diagram illustrating the cases associated with the activation of the T cell receptor of the path of phosphatidylinositol (Pl), which leads to transcription of the IL gene -2 and the production of IL-2. Figure 2 shows an immunostaining for the detection of MAP kinase in proteins obtained from the T-cell GA15 hybridoma.

GA 15 were stimulated with either calcium ionophore, PMA or ionophore in combination with PMA or imitation treated with PBS (control not stimulated). The cells were treated either with bromelain or with PBS. Figure 3 shows an immunostaining for MAP kinase detection. The proteins were obtained from GA15 activated with calcium ionophore in combination with PMA or, treated PBS (unstimulated control), which was treated either with bromelain or with PBS. Samples were tested for the presence of MPA kinase at various intervals. Figure 4 is a graph showing the appearance with time of MPA kinase in stimulated T cells, treated either with bromelain or with PBS. Figure 5 shows an immunostaining, which shows that a polyclonal antibody, raised against a highly conserved peptide of MAPk Erk 1), recognizes two proteins of Mr 42000 and 44000. Figure 6 shows an immunostaining with MPAk antibody, which indicates that changes in electrophoretic mobility, normally observed in protein phosphorylation, are partially blocked in cells treated with bromelain. Figure 7 shows the effect of bromelain a treatment on the accumulation of I L-2, I L-4, and I FN-? MRNA in GA 15 cells, in vitro. GA 15 cells treated with bromelain accumulate less IL-2, IL-4 and IFN-α MRNA, when stimulated with PMA (20 ng / ml) and calcium ionophore A23187 (500 ng / ml). Figure 8 shows that splenic T cells treated with bromelain produce less I L-2, when stimulated with 2C 1 1 (aCD3e) and CD28 (aCD28). Figure 9 shows the effect of treatment with bromelain on the accumulation of I L-2, IL-4 and? -INF mRNA in splenic T cells, in vitro. The splenic T cells treated with bromelain accumulate less I L-2, I L-4 and I FN-? MRNA, when stimulated with immobilized anti-CD3e (4μg / ml) and soluble anti-CD28 (10μg / ml) of mAbs. Figure 10 shows that treatment with bromelain increases the proliferation of splenic T cells, when stimulated with immobilized anti-CD3e (4 μg / ml) and with soluble anti-CD28 (10 μg / ml) of mAbs. Figures 1 1 a and b show that bromelam treatment increases the binding of both anti-CD3e (a) and anti-CD28 (b) mAbs to the splenic T cell surface, as indicated by a change in the profiles of FACS to the right. Figure 12 shows that bromelain increases the binding of anti-CD3e of mAb to the surface of GA15 cells. Figures 13a and b show immunostaining of tyrosine phosphorylated proteins in splenic T cells. Figure 13a shows that treatment with bromelain induces protein tyrosine phosphorylation of proteins 56 and 58 kDa. Figure 13b shows that treatment with bromelain inhibits protein tyrosine phosphorylation of a 16 kDa protein. Figure 14 shows the effect of bromelain treatment on the accumulation of I L-2, I L-4 and IFN-α MRNA in GA15 cells, in vitro. The cells treated with bromelain accumulate more IL-2, IL-4 and IFN-? MRNA, when stimulated with immobilized anti-CD3e (4 μg / ml) and soluble anti-CD28 (10 μg / ml) of mAbs. Figure 15 shows the effect of bromelain on sheep red blood cell antibody (SRBC) responses, in vivo. Mice that were treated with bromelain have more B cells, which produce antibodies directed against SRBC.

MATERIALS AND METHODS Cell Line GA15 of ThO cell hybridoma was used for experiments investigating tyrosine phosphorylation. GA15 was generated from the fusion of thymoma BW5147 with clone F4 of Th2, specific for KLH in association with l-Ab (Fox, Int. Immunol., 5, 323-330, 1993). Cells were maintained in tissue culture media (TCM) consisting of RPM I 1640 medium (Biofluids, Rockville, MD), supplemented with 10% fetal bovine serum (Inovar Biologicals, Gaithersburg, MD) 50 mM 2 -mercaptoethanol, 4 mM glutamine, and 50 μg / ml gentam icin.

Animals The effect of pretreatment with bromelain on the production of IL-2 by splenic murine T cells was investigated, isolated Male C57BL / 6NCrlBR mice were purchased from Charles River Laboratories (Wilmington, MA, USA). Young mice (3 to 4 months old) and older mice (20 to 26 months old) were used in pairs experiments. Mice that had tumors, visible lesions on the skin, or significant pathology were not used. For experiments and studies, in vivo, that investigate the accumulation of I L-2, I L-4 and I FN-? MRNA, mice were purchased from A. Tuck and Son Ltd (U K). Mice with an age of 6 and 10 weeks were used.

Agonists The phorbol esters are structurally related to 1,2-diacylglycerol (DAG) and therefore occasional activation of PKG, which induces hyperphosphorylation of Raf-1 (Morrison et al., Proc. Nati, Acad. Sci. USA, 85, 8855-8859, 1988), as well as the activation of MPA kinases (Chung et al., Mol.Cell. Biol., 11, 1868-1874, 1991). The ionophore increases free cytoplasmic calcium, which in turn binds calmodulin and PKC. For tyrosine phosphorylation experiments, phorbol myristate 13-acetate (PMA) and calcium ionophore A23187 were used to stimulate cells. The phorbol esters and the ionophore treatment of T-lymphocytes act synergistically to simulate the effect of the secondary messengers, diacylglycerol (DAG) and 1,4-inositol triphosphate (IP3), and therefore, reproduce many aspects of TCR stimulation (Truneh et al., Nature, 313, 318-329, 1985) such as secretion of I L-2, expression of the IL-2 receptor and proliferation of T cells.

Isolation of Splenic T Cells from Mice The animals were sacrificed by cervical dislocation and the spleens were removed aseptically. Single cell suspensions were prepared in the tissue culture medium (TCM), consisting of RPMI 1640 (GI BCO Laboratories, Grand Island, NY) supplemented with fetal bovine serum, 10% inactivated heart (FCS) (GI BCO Laboratories) and 2 mM L-glutamine, 5 x 10"5 M 2 -ME, and antibiotics (50 mg / ml gentamicin and 10 U / ml penicillin) .The erythrocytes were lysed at 5 x 10 7 lymphocytes / ml , in a pH regulating solution to lyse (140 mM NH4CI, 17 mM Tris, pH 7.2) for 2-5 minutes The lysis was terminated by adding TCM, and the T cells were purified by incubation on nylon wood ( Polysciences, Warrington, PA) for one hour at 37 ° C (J ulius et al., Eur J J Immunol 3: 645 (1973)). T cells were collected in the effluent and contained> 90% cells of Thy-1 + and < 5% MHC class I I +, as analyzed by flow cytometry.

To measure the production of I L-2 by murine T cells, monoclonal antibodies (mAb) were used as agonists to simulate the molecular interactions that occur between the surface of the T cell and the cell presenting the antigen. The monoclonal antibody 2C 1 1 (anti-CD3e chain) was used to cross-link the TCR receptor and simulate the agonistic effects of the antigen / major histocompatibility peptide (MHC) stimulation. A co-stimulatory signal was provided through the CD28 molecule using anti-CD28 mAb (37.51). The binding of the antibody of the CD28 molecule and the TCR entanglement have shown that they initiate the cases of specific signal transduction, which lead to the production of I L-2 and to the proliferation of T cells.

Other Reagents Reagents were purchased from the indicated sources: sheep red blood cells (SRBC) from TCS Biologicals (Buckingham, U K); anti-CD3e-mAb chain (PV-1) and a polyclonal hamster IgG (control hamster IgG) was a generous donation from Dr. C. June (NMRI, Bethesda, MD); goat anti-hamster IgG Ab, 12-myristate and phorbol 13-acetate (PMA), calcium ionophore A23187, all from Sigma (Dorset, U K); mouse antifostothiosin mAb (4G 10) from Upstate Biotechnology (Lake Placid, NY); Goat anti-mouse IgG Ab conjugated to horseradish peroxidase from Southern Biotechnology Associates (Birmingham, AL); Goat anti-hamster IgG Ab conjugated to fluorescein isothiocyanate (FITC) from Cappel (Durham, NC); RNAzol B from Cinna / Biotecx Laboratories (Houston, TX); RNAguard, dNTPs both from Pharmacia Biotech (St Albans, Herts); MMLV reverse transcriptase, random primers both from GIBCO BRL (Gaithersberg, MD); Biotaq polymerase from Bioline (London, U K); raw bromelain extract (E .C 3.4.22.4), (Lot No. TAD8TK1 125) from Miles Scientific (Elkhart, I N).

Treatment and Stimulation of the GA15 Cell For the phosphoprotein experiments, GA15 (1 x 106 cells) were pretreated with bromelain (15 μg / ml or 50 μg / ml, diluted in phosphate buffered saline, pH 7.4) for 30 days. minutes before the stimulation of the secondary messenger. The cells were then washed twice with repeated centrifugation (1500 rpm, Sorvall RT 6000B refrigerated centrifuge, DuPont) and resuspended in RPMI. The control cells were treated only with a PBS vehicle. The cells were stimulated for several times with either calcium ionophore (1 μM), PMA (10 ng / ml), ionophore and PMA combined. After stimulation, the cells were used and analyzed for tyrosine phosphorylated proteins, as described below.

Measurement of IL-2 Production in Splenic T Cells To measure the production of I L-2, splenic murine T cells were cultured in flat-bottomed microculture plates with 96 wells (Corning, Corning, NY, USA) at 106 cells per well. Cells were stimulated with immobilized anti-mouse mAb (145-2C 1 1) (plate-binding) (Leo et al, Proc. Nati, Acad Sci. USA., 84, 1374, 1987) at 100 μg / ml , and CD28 mAb (37.51) of soluble anti-mouse (Gross et al., J. Immunol., 144, 3201, 1990) at 10 μg / ml. For the presentation of immobilized anti-mouse CD3e mAb, 145-2C1 1 ascites were diluted in PBS, added to microculture plates in 50 μl, incubated for 16 hours at 4 ° C, and then the wells were washed three times in PBS. Cultures were incubated in triplicate at 37 ° C in humidified 5% CO2 for 24 hours before the culture supernatants were harvested to analyze the activity of IL-2. The activity of I L-2 was measured using IL-2-dependent cell line CTL-L, as previously described (Gillis et al., J .. I mmunol., 120, 2027, 1978). In summary, the supernatants were serially diluted with 4 x 10 3 CTL-L cells / well in 96-well, flat-bottom microculture plates. After 24 hours, the cells were pulsed with 0.5 μCi / well of [3H ] -thymidine for an additional 16 hours. Cells were harvested and proliferation was determined by counting by scintillation. Units were calculated using I L-2 of recombinant murine (Pharmigen, San Diego, CA, USA) as normal. In the experiments to measure the accumulation of I L-2, IL-4 and IFN-? MRNA and in the FACS analysis, cells were incubated at 37 ° C for 30 minutes in RPMI 1640, containing 50 μg / ml bromelain at 107 cells / ml. False treated cells were incubated with an equal volume of saline regulated at their pH (0.1 M, pH 7.4, PBS), (diluent for bromelain). After incubation, the cells were washed three times in RPM I 1640, and then resuspended in TCM.

Cell Culture for the Determination of mRNA In the experiments investigating the accumulation of cytokine mRNA, GA15 hybridomas were cultured in 24-well flat bottom plates (Nunc, Rosskilde, Denmark) at 2.5 x 106 cells per well in one volume 2 ml culture for 4 hours. Splenic T cells were cultured in 6-well round bottom plates (Flow Laboratories, McLean, VA) at 5 x 106 cells per well in a culture volume of 4 ml for 24 hours. In the experiments investigating proliferation, splenic T cells were cultured in triplicate, in 96-well round bottom plates (Nunc) to cells per well in a culture volume of 200 μl for 36 hours. These cultures were pulsed with 0.5 μCi of [3 H] TdR, 16 hours before harvesting on glass fiber filters and the incorporated count of [3 H] TdR. All reagents were diluted in TCM, except for anti-CD3e immobilized mAb, which was prepared by diluting mAb in PBS, adding to the culture plates to cover the bottom of the wells, incubating for 16 hours at 4 ° C and then washing the wells three times with PBS. All cells were incubated at 37 ° C in humidified 5% CO2.

Flow Cytometry Before staining, the cells (106) were incubated in 1 ml of an activated, fluorescent, cell distribution pH regulator (FACS), (1% FCS heat inactivated in PBS, 0.02% NaN3 ) containing 50% horse serum filtered for 30 minutes on ice and then washed once with the FACS regulator. The cells were stained in 100 μl of FACS buffer for 30 minutes on ice, either with anti-CD3e mAb, anti-CD28 mAb or control Ab of hamster IgG (all at 10 μg / ml) and washed once with FACS regulator. Ab was specifically detected bound to the cell surfaces, incubating the cells in 100 μl of FACS regulator for 30 minutes on ice with FITC-conjugated anti-hamster IgG (10 μg / ml) and then washed once in buffer. FACS. The cells were resuspended in 1% paraformaldehyde (diluted in the FACS regulator) and stored at 4 ° C. The cells were analyzed in a Becton Dickinson FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Apertures were placed on lymphocytes using a front and side scrutinizer and the data were plotted on a logarithmic scale.

PCR analysis of Cytokine mRNA The accumulation I L-2, I L-4 and I FN-? RNA m, using an adaptation of a semi-quantitative RT-PCR analysis, previously described (Svetic et al., J. Immunol 147: 2391-237, 1991). Briefly, RNA is isolated from cells and splenic tissue (Chomczynski and Sacchi, Anal. Biochem. 162: 156-159, 1987) using RNAzol B according to the manufacturers' instructions (Cinna / Biotecx Laboratories), and mRNA was transcribed in reverse form by normal procedures (Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd from Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), using 3 μg of total recovered RNA of the samples in a final reaction volume of 25 μl. PCRs were performed in duplicate for each sample in a final volume of 25 μl, using 2.5 μl of the mRNA sample transcribed in reverse form as a cDNA template. The oligonucleotides specific for IL-2, I L-4, IFN-α and the handling gene, hypoxanthine-guaninosporibosyltransferase (HPRT) and other PCR components were as previously described (Svetic et al., 1991, supra). All PCRs consisted of a denaturation step at 95 ° C for one minute, an annealing step at 55 ° C for one minute, and an extension step at 72 ° C for 2 minutes. The number of PCR cycles, used to ensure such amplification, was determined when the amplified product could be detected, but was well below the saturation concentrations, determined for each analyzed sample of cytokine and RNA. The PCR products were detected by separating amplified DNA by means of agarose-gel electrophoresis and transferring the DNA to the membrane of Hybond N + nylon, according to the instructions of the manufacturers (Amersham, Buckinghamshire, U K). The amplified cytokine mRNA was revealed using cytokine specific oligonucleotides (Svetic et al., 1991, supra) labeled with horseradish peroxidase (H RP) and reacted with the chemiluminescence detection system SL, as described by the manufacturer (Amersham ). Specific signals were recorded on a self-radiographic film (Kodak, Rochester, NY) and analyzed on a Sharp JX-3F6 computer densitometer (Sharp, Japan), using software (programs) Phoretix 1 -D (Phoretix International, Newcastle, UK). The intensity of the signals generated by the HPRT products was used to ensure an even load of the target cDNA towards the PCRs. Generally, the signals generated by the H PRT products were within 10% of the intensity of the corresponding signals of the isolated samples from cells stimulated in the same way from the different treatment groups. The intensity of the signals generated by the cytokine products was calculated in relation to the signals generated by the H PRT products for each sample.

Immunostaining of Cells Phosphotyrosine blots were performed on cells, as described by Thomas et al. (Cell, 68, 1031-1040, 1992). Briefly, cell lysates were prepared from 1 x 106 GA15 cells, using the cells in ice-cold lysis pH buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 4 mM EDTA, 1% of Triton X-100, 4 mM of sodium orthovanadate, 1 mM of PMSF, 50 mM of NaF, 10 μg / ml of leupeptin) for 30 minutes with continuous rotation. The lysates were clarified (14,000 xg, for 2 minutes), and suspended in a SDS-PAGE sample buffer, and inhibited for 5 minutes. Samples containing equal amounts of protein were resolved on a 12.5% SDS-polyacrylamide gel. The separated proteins were electrophoretically transferred to nitrocellulose (BioRad), and unoccupied binding sites were blocked in the blocking solution containing 5% bovine serum albumin (Sigma, Fraction V; BSA), 0.05% N P- 40, 170 mM NaCl and 50 mM Tris (pH 7.5). Immunostains were incubated with anti-phosphotyrosine monoclonal antibody (4G 10; IgG2bk) at 1 μg / ml (Upstate Biochemical Industries, NY) to detect tyrosine phosphorylated proteins. The monoclonal binding antibody was detected by goat anti-mouse IgG conjugated with horseradish peroxidase. Immuno-reactivity was determined using the chemiluminescence reaction of ECL (Amersham Corp., Arlington Heights, IL) and the specific signals were recorded on a self-radiographic film. Immunostaining of separated proteins was conducted for the detection of MAP kinase, using polyclonal IgG (erk1-CT) MAP of rat anti-rat kinase R2, followed by goat anti-rabbit IgG conjugated with horseradish peroxidase. The anti-MAP kinase antibody used recognizes the 42 kda, 43 kda and 44 kda MPA kinases encoded by the erki gene, the mapk gene, and the mpk gene, respectively.

Tyrosine Phosphorylation Analysis of Protein followed by Stimulation of aCD3e and aCP28 mAb Cells were suspended at 5-10 x 10 7 cells / ml in RPMI 1640 at 37 ° C, 5 minutes before stimulation. PMA and calcium ionophore A23187 were used at 20 and 500 ng / ml, respectively. The entanglement of CD3e and CD28 mAbs was carried out by incubating T cells on ice for 30 minutes in the presence of 10 μg / ml of each mAb. After the excess antibodies were washed, the cells were suspended at 5-10 x 10 7 cells / ml in RPMI 1640 (all at 4 ° C). The entanglement was performed at 37 ° C with 10 μg / ml of goat anti-hamster IgG. The cells were stimulated at the times indicated in the legends of the figure and the text. The stimulation was terminated by the addition of ice-cooled buffer, producing a final concentration of 0.5% Triton X-100, 25 mM Tris, pH 7.2, 75 mM NaCl, 400 mM EDTA, 10 mM of sodium fluoride, 400 mM of sodium orthovanadate, 10 mM of sodium pyrophosphate, 74 mg / ml of leupeptin, 740 mM of PMSF and 74 μg / ml of aprotinin. After lysis at 4 ° C, the samples were centrifuged at 12,000 rpm (13,200 g), 4 ° C for 15 minutes. Post-nuclear supernatants were collected and an equal volume of 2 x pH regulator was added from the SDS-PAGE sample (50 mM Tris, pH 7, 700 mM 2-ME, 50% (V / V) glycerol , 2% (P / V) of SDS, 0.01% (P / V) of bromophenol blue). The proteins were solubilized at 100 ° C for 5 minutes and resolved by SDS-PAGE. Red Blood Cell Analysis of Borrego Mice received 3 x 200 μl intravenous (iv) injections of 200 μg bromelain or an equal volume of 0.9% NaCl (diluent for bromelain) on days 1, 4 and 6 of the experiment, both treatment groups received either an intraperitoneal (ip) injection of 100 μl of 107 SRBC or an equal volume of 0.9% NaCl (diluent for SRBC) on day 4 of the experiment. The mice were sacrificed on day 7, their spleens were removed and the splenocytes were isolated, as described in the cell preparation section. The number of B-cell secretion antibodies, specific for the SRBC antigen, was determined by an analysis based on the original method of Jeme and Nordin, described by Weir (1986). In summary, the analyzes were performed in 160 μl, consisting of 5 x 10s of splenocytes, 6 x 106 of SRBC and 1: 27 of a complement of guinea pigs in RPMI 1640. The reaction mixture was placed in a chamber created by joining two slides of glass, together, with a double-sided tape, and then sealed with wax. The samples were incubated at 37 ° C for one hour, before counting the plaque-forming cells (PFC), (i.e., a B-secreting Ab, specific for SRBC).

RESULTS EXAMPLE 1 Effects of Bromelain on Phosphorylation of Tyrosine Induced by Lonophore and Ester of Forbol in GA15 The effects of pretreatment with T-cell bromelain on the stimulation of secondary messengers of cellular substrates of tyrosine kinases were analyzed. Immunostains, using .anti-phosphotyrosine-specific antibodies, revealed the tyrosine phosphorylation of several protein bands after 5 minutes of stimulation with combined PMA, ionophore, or PMA and ionophore (PMA + Ca). A larger protein band of approximately 42 kda was the most visible. A synergistic effect of PMA + Ca increased the intensity of the phosphorylated band (Figure 2). The effect of bromelain on tyrosine phosphorylation was analyzed by its ability to inhibit the presence of the 42 kda band, as detected by anti-phosphotyrosine immunostaining. The pretreatment with bromelain of GA15 cells blocked the tyrosine phosphorylation of the 42 kda protein, when stimulated by all secondary messenger agonists. This band was not detected by phosphotyrosine immunostaining. Due to the synergistic effect of combined PMA + Ca, all experiments were subsequently conducted with this combination. The kinetics of phosphorylation were then examined and it was determined whether the ability of bromelain to inhibit tyrosine phosphorylation in 5 minutes was a function of time, or whether bromelain completely inhibited tyrosine phosphorylation. It is said that bromelain did not completely inhibit phosphorylation, but delayed tyrosine phosphorylation (Figure 3). Then, the syn- thetics of the phosphorylation was quantified by means of densitometric measurements of autoradiograms (see Figure 4). Despite the delay in tyrosine phosphorylation, the phosphorylation of this band did not reach the same intensity as that observed in the untreated controls, and was completely dephosphorylated in 180 minutes (data not shown). We also observed the inhibition of some other proteins not yet identified, in addition to the 42 kda protein, when the cells were stimulated with secondary messenger agonists. However, bromelain did not affect the total phosphorylation of abundant cellular phosphoproteins in unstimulated cells, as estimated by anti-phosphotyrosine immunostaining.

Identification of the Greater Phosphorylated Band, 42 kda As mentioned above, several members of the MAPk-r- family are phosphorylated on tyrosine residues, following the treatment with growth factors, and also with phorbol esters and calcium ionophores. Since the molecular weights of MAPk described in the literature correspond to the band tyrosine of major protein, phosphorylated in the experiments, we hypothesized that the 42 kda protein, observed in GA15 cells, was MAPk. Therefore, anti-MAPk antibodies were used to detect MAPk in GA15 cells. Figure 5 shows that a polyclonal antibody, emerged against a highly conserved MAPk peptide (Erk 1), recognizes two proteins of Mr 42,000 and 44,000 (presumably, erk 1 and erk 2, respectively). The 42 kda protein exhibits an electrophoretic mobility similar to the 42 kda phosphotyrosine-containing protein detected in stimulated T cells. The band 44 kda also correlates with a protein, which is phosphorylated tyrosine independent of stimulation with PMA + ionophore. A 48 kDa protein and other low molecular weight proteins were also detected, however, since these bands were also detected in control immunostains without the addition of anti-MAPk, the reactivity was thought to be nonspecific. Immunostaining with anti-MAPk antibodies revealed similar intensities in all treated samples, indicating that the reduction in the intensity of tyrosine phosphorylation, observed in cells treated with bromelain, was not due to differences at the level of the protein present.

Electrophoretic Mobility It was also investigated whether, in the experiments, the 42 kda phosphorylated band was MAPk, detecting phosphorylated MAP kinase through its characteristic delay in electrophoretic mobility. The cell lysates were examined by immunostaining analysis with anti-MAPk antibody. Immunostaining with anti-body MAPK indicated a change in electrophoretic mobility consistent with that normally observed under phosphorylation of MAPk. This change in mobility was also partially blocked in cells treated with bromelain (Figure 6).

Effect of Bromelain on the production of IL-2, IL-4 and IFN-α? of mRNA, in GA15, in vitro. It was previously shown that treatment of the T-cell hybridoma of GA15, with bromelain, resulted in alterations in tyrosine phosphorylation, when the cells were stimulated with PMA and calcium ionomer A23187. In particular, bromelain markedly reduced the tyrosine phosphorylation of a 42 kda protein, named MAP kinase. To determine whether alterations, in the tyrosine phosphorylation patterns, induced by bromelain, resulted in any functional change, the accumulation of cytokine mRNA in GA15 was measured. The effect of bromelain on the production of I L-2, I L-4 and IFN-α was tested by measuring the accumulation of mRNA coding for these cytokines, after 4 hours of culture using an RT-PCR analysis. GA15 cells were stimulated with PMA and A23187. This combination of stimuli (PMA and A23187) directly activates cell signaling trajectories and therefore derives all cell surface molecules. The relatively short culture period (4 hours) used was to ensure that the accumulated mRNA cytokine was a result of the specific stimuli used, rather than an autocrine effect of the newly synthesized cytokine. The mRNA cytokine was not detected in cells cultured only in TCM. The accumulation of all the mRNA cytokine species, after stimulation with PMA and A23187, was consistently lower in GA15 cells treated with bromelain, compared to control cells (PBS), (Figure 7). The difference in mRNA cytokine accumulation was more evident for IL-4, which was significantly lower (n = 3, p <0.05) in cells treated with bromelain. Since tyrosine phosphorylation of the MAP kinase is associated with the transcription of the cytokine gene (Pelech, Curr. Biol. 3: 513, 1993), we hypothesized that the reduction in cytokine accumulation of mRNA resulted of reduced tyrosine phosphorylation of MPA kinase.

Effect of Bromelain on the Production of IL-2 in Splenic T Cells We next investigated whether bromelain could affect cytokine production in normal T cells isolated from mouse (murine) spleens, when stimulated via cell surface molecules. The first stimulus combination (PMA + A23187) directly activates the cell signaling pathways, and therefore diverts all cell surface molecules. A combination of immobilized aCD3e or soluble anti-CD28 mAbs could provide a primary signal through the T-cell receptor (via the CD3e chain) and a co-stimulatory signal through CD28 to activate cell signaling cascades. Both stimulus combinations (PMA + A23187; aCD3e + aCD28) have previously shown to provide optimal activation of T cells (Truneh et al., Nature, 313: 318-320, 1985; Linsley and Ledbetter, Ann. Rev. Immunol., 1 1: 191, 1993), however, by a different mechanism, that is, a direct and an indirect action, respectively. It was measured in the secretion of IL-2 in culture supernatants by simple, murine splenic T cells. T cells were stimulated for 24 hours with anti-CD3e immobilized mAb, in the presence of anti-CD28 mAb, as described above. T cells were cultured in the absence of antibody, or in the presence of anti-CD28 or anti-CD3e, only, to serve as controls. Figure 8 shows the results of a single experiment. T cells cultured in the absence of antibody or only with anti-CD28, did not produce detectable levels of I L-2 (data not shown). Similarly, immobilized anti-CD3e alone produced barely detectable levels of I L-2. Since both a primary signal, via TCR, and a co-stimulatory signal are required for optimal activation of T cells, T cells were cultured in the presence of both antibodies. The combination of anti-CD28 and anti-CD3e, in the cultures, resulted in a substantial increase in the production of IL-2 by T cells. A difference of I L-2, after stimulation, was observed among the mice young and old. This phenomenon has been widely reported in the literature (reviewed by Thoman and Weigle, 1989). Again, as previously observed in the GA15 experiments, T cells that were pretreated with bromelain produced markedly less IL-2 than T cells that were pretreated only with PBS. A similar reduction was observed in both young and older mice. This effect was observed when the T cells were optimally stimulated with a primary signal through TCR and a costimulatory signal via the access molecule CD28. The effect of bromelain did not appear to be related to the older mice, since the T cells of both the young and older mice exhibited reductions in IL-2 production.

Effect of Bromelain on the Production of IL-2, IL-4, and IFN-α MRNA in splenic T cells, In Vitro It was then investigated whether bromelain-induced alterations, in patterns of tyrosine phosphorylation, can affect other cytokines produced by splenic T cells. The effect of bromelain on the production of I L-2, I L-4 and I FN- ?, was investigated by measuring the accumulation of AR N m that codes for these cytokines. The T cells were cultured for 24 hours in the presence of single media or anti-CD3e and soluble Anti-CD28 mAbs, combined. The mRNA of cytokine was not detected in the cells cultured only in TCM (data not shown). The accumulation of all species of cytokine mRNA, after stimulation, was consistently lower in T cells treated with bromelain, compared to the controls (Figure 9). The differences in the accumulation of mRNA from cytokine was not apparent for I L-4, which was significantly lower (n = 3, p <0.05) in cells treated with bromelain stimulated with anti-CD3e and anti-/ "". CD28 mAbs.

Effect of Bromelain on the Proliferation of Splenic T Cells, In vitro Given the reduced accumulation of cytokine mRNA, observed in splenic T cells pretreated with bromelain, and the importance of these cytokines, particularly I L-2 and I L-4, for the proliferation of T cells, one would expect that bromelain would cause a reduction in T cells. Therefore, the effect of bromelain on the proliferation of T cells, midiend or 3H-TdR, incorporated into cells stimulated with anti-CD3e and anti-C28 mAbs. Surprisingly, the pretreatment with bromelain of the T cells caused a significant increase (n = 6; p <0.05) in the proliferation of T cells, instead of a reduced proliferation, as expected (Figure 10). One possible explanation for these contradictory effects is that bromelain may cause reduced cytokine production, but it increases the sensitivity of growth factors, such as IL-2 and I L-4 (possibly by modifying the cell surface receptors for said growth factor, or increasing expression via the effects on the signaling cascades), in T cells.

Effect of Bromelain on Surface Molecules of Splenic T Cells Since it has previously been shown that bromelain removes specific molecules from the surface of T cells (Hale and Haynes, J. Immunol., 149: 3809-3816, 1992), investigated whether bromelain removed CD3e and CD28 molecules in splenic T cells, treated with bromelain, by flow cytometry. The mAbs used in these studies were the same used in the stimulation analyzes described above. It was observed that the binding to CD3e on the surface of splenic T cells was consistently higher when the cells were treated with bromelain (Figure 1 1 a). An increase in the binding of anti-C28 mAb to CD28 (expressed at detectable levels in splenic T cells) was also found (Figure 11b). These results indicate that the removal of cell surface molecules, by treatment with bromelain, was not responsible for the reduced accumulation of cytokine mRNA, observed, although the increased binding of mAbs to molecules of CD3e and CD28 could have contributed to the increased proliferation of cells treated with bromelain.

Effect of Bromelain on GA15 Cell Surface Molecules Since an increase in the binding of aCD3e to splenic T cells has been observed, after treatment with bromelain, it was also investigated whether bromelain affected the CD3e and CD28 molecules in cells of GA15. The mAbs used in these studies were the same used in the stimulation analyzes described above. Again, the binding of mAb to CD3e, on the surface of GA15, appeared to increase slightly (indicated by the increase in mean fluorescence intensity (profile changing to the right)), followed by treatment with bromelain (Figure 12). This result is consistent with that observed previously with splenic T cells. The observed increase was a specific case, since a control mAb showed no increase in binding above the levels of background in GA15 treated with both bromelain and PBS. It is remote that the increase in the binding of anti-CD3e mAb to the surface of GA15, which results from the increased expression of CD3e, since the cells were only treated with bromelain for 30 minutes at 37 ° C, and all subsequent procedures of staining were conducted at 4 ° C, where a very small cell activity could be expected. It is possible that the increased binding of mAb to CD3e may have resulted from bromelain, which modifies the molecule, to expose more antigenic determinants.Effect of Bromelain on Tyrosine Phosphorylation in Murine Splenic T Cells, In Vitro The effect of bromelain on tyrosine phosphorylation on GA1 5 cells was initially investigated. The effect of treatment with bromelain on phosphorylation of tyrosine in normal murine T cells. T cells were isolated from the spleens of healthy Balb / c mice, and stimulated with either PMA and A231 87 or immobilized anti-CD3e, and with soluble anti-CD28 mAbs, as described above for GA1 5 cells. Splenic T cells were isolated and then cultured for 48 hours in TCM containing 5 ng / ml of PMA, as described by Vandenberghe et al., J. Exp. Med. 1 75: 951-960, 1992), prior to stimulation for tyrosine phosphorylation analysis. The reason for this pre-culture in PMA is that it is very difficult to detect the increased tyrosine phosphorylation in resting T cells (Vandenberghe et al., 1992, supra). A different pattern of tyrosine phosphorylation was observed in splenic T cells, than that previously observed in GA15 (Figure 13a and 13b). Bromelain inhibited tyrosine phosphorylation of a low molecular weight protein (approximately 16 kDa), regardless of whether the cells were stimulated or not (Figure 13b). The band of MAP kinase phosphorylated with 42 kda tyrosine, previously observed in GA15, was not detected. A more sensitive assay to measure MAP kinase activity may be required for these cells. Another striking difference between tyrosine phosphorylation patterns in splenic T cells and GA15 was that treatment with bromelain resulted in protein tyrosine phosphorylation (Figure 13a), whereas in GA15 cells it had an inhibitory effect, followed for the stimulation. When T cells were stimulated with either PMA and A23187 or anti-CD3e, and anti-CD28 mAbs, a prominent 56 kda tyrosine phosphorylated protein was observed. In addition, a slightly higher 58 kda tyrosine phosphorylated protein was also observed in these cells. The tyrosine phosphorylation of these proteins was not detected unless the T cells were treated with bromelain. These proteins were phosphorylated with tyrosine for 2 and 5 minutes after stimulation with anti-CD3e and anti-CD28 mAbs, and remained phosphorylated for at least 30 minutes after stimulation (Figures 13a and 13b). Since the phosphorylation of some proteins is associated with the activation of cellular responses, the ability of bromelain to induce tyrosine phosphorylation in T cells suggests that bromelain can activate these cells.

Previous results obtained, which investigated the effect of bromelain on the proliferation of splenic T cells, could suggest that these cells were activated by bromelain.

EXAMPLE 2 In the previous example it is observed that bromelain caused reduced phosphorylation of proteins, particularly MAP kinase, in GA 15 cells (a hybridoma of the T cell). It was also observed that the bromelain treatment of GA15 cells resulted in IL-2, I L-4 and IFN-α. reduced, when stimulated with phorbol ester and ionophore. Furthermore, it was observed that bromelain treatment of splenic, murine, normal T cells caused a similar reduction in IL-2, IL-4 and I FN- ?, when it was provided with a more physiological stimulus, that is, aCD3e and aCD28, which provides a signal via the T cell receptor and a co-stimulatory signal via CD28, respectively. Taken together, these data suggest that reduced tyrosine phosphorylation correlates with a reduction in cytokine production. However, the data obtained in Figure 13a, which shows increased protein tyrosine phosphorylation, and the observed increase in proliferation of splenic T cells (Figure 10) could suggest that bromelain also stimulates or activates T cells. Additional experiments were conducted and the effect of bromelain on the cytokine mRNA was investigated, produced after stimulation of GA15 cells with aCD3e and aCD28 (first experiments in example 1 investigated the effect of bromelain and PMA + A23187 on cytokine production). Interestingly, an increase in the production of cytokine mRNA was observed, when the cells were treated with bromelain and stimulated with anti-CD3e and Anti-CD28 mAbs (Figure 14). This pattern of accumulation of cytokine mRNA is opposite to that observed in Example 1, where bromelain reduced the accumulation of mRNA in GA15 cells, when stimulated with phorbol ester plus ionophore. Again, the difference in the accumulation of cytokine mRNA was very evident for IL-4, which was significantly higher (n = 3, p <0.05) in cells treated with bromelain, stimulated with anti-CD3e and anti-CD28 in mAbs (in the beginning, bromelain induced a significantly lower accumulation (n = 3, p <0.05) of mRNA, IL-4, in GA15 cells stimulated with PMA + A23187). One possible explanation for the different results described above is that the bromelain treatment of GA 15 cells inhibits signaling pathways directly stimulated by PMA + A23187, but may increase the alternating signaling cascades associated with cell surface molecules. , such as CD3e and CD28.

The Effect of Bromelain on Mice, In Vivo The data presented in this way suggest that bromelain can have multiple effects on T cells (both stimulants and inhibitors, depending on the type of cell studied). Different effects have been shown in tyrosine phosphorylation patterns, between normal splenic T cells (mainly natural or inactivated T cells), and GA15 of the T-cell hybridoma (representative of an activated T cell). In addition, when ligands are used for cell surface molecules, to stimulate cells (ie, anti-CD3e and anti-CD28 mAbs), treatment with bromelain increased the accumulation of cytokine mRNA in GA 15, but reduced the cytokine mRNA in normal splenic T cells, despite causing an increase in proliferation in these cells. Given the differences observed between cells cultured in vitro, the effect of bromelain on the function of T cells in intact animals was investigated. The effects of bromelain on T cell activation were investigated, in a well-described in vivo model of T cell-dependent antibody responses to sheep red blood cells (SRBC), (Weir, Handbook of Experimental Immunology, 1-4, 4a., Blackwell Scientific Publications, Oxford, UK, 1986). This analysis directly measures the number of B cells, which produce SRBC-specific Ab (ie, platelet-forming cells (PFCs)), after immunization with this antigen. This response depends on the proliferation of SRBC-specific T cells and the production of cytokines, particularly I L-4.

Mice immunized with saline (control) produced very few PFCs, regardless of whether they were pretreated with bromelain or with saline (control) (Figure 15). This indicated that treatment with bromelain did not cause the spontaneous production of PFC. In mice immunized with SRBC, administration of bromelain caused a significant increase (n = 11, p <0.05) in PFC, compared to the control animals (Figure 15). One possible explanation for this result is that treatment with bromelain caused an increased proliferation of SRBC-specific T cells (similar to the increased proliferation seen in the experiments conducted with splenic T cells, in vitro, Figure 10), resulting in a improved help of T cells for the production of SRBC-Ab specific by B cells. However, the possibility that bromelain directly stimulated B cells or some other cells involved in the antibody response can not be excluded. Regardless of the precise mechanism involved, this result suggests a novel application for bromelain as an adjuvant.

DISCUSSION It has been reported that bromelain inhibits the secretory effects of several secondary messengers, such as cyclic AMP, Cyclic GMP and Ca2 +, in intestinal cells. The mechanism of action of bromelain is unknown, but it is believed to act very close to the accumulation of cyclic nucleotides in cells. In view of the effects of bromelain on intestinal cells, and the role played, in these cases, by secondary messengers and protein phosphorylation, it was tested whether bromelain could inhibit signal transduction systems in other cells. Specifically, the effect of bromelain on the inositol trajectory and the tyrosine phosphorylation required for the production of cytokines (IL-2, IL-4 and IFN-α) and proliferation in T cells was examined. The hypothesis was presented that if bromelain affects signal transduction in cells, then this inhibition could be accompanied by a failure of T cells to produce cytokines, increased autocrine growth factors and proliferation. The results indicate that bromelain can either inhibit or stimulate tyrosine phosphorylation of proteins, when stimulated by phorbol esters and calcium ionophores, and antibodies directed against surface molecules. One of the affected proteins that were identified is presumed to be MAPk (activated mitogen protein kinase). Several observations support this identification: 1) MAPk is phosphorylated under the stimulation of T cells with phorbol esters and calcium ionophores, and is also phosphorylated under the binding of the T cell receptor and a co-stretching molecule. 2) A 42 kda protein, which corresponds to the literature value of * 42 kda, is phosphorylated in GA 15 cells under stimulation with phorbol ester and ionophore. This protein was not phosphorylated or the phosphorylation was markedly reduced when the cells were treated with bromelain. 3) Immunostaining with antibodies specific for anti-MAPk resulted in the recognition of a 42 kDa protein band. 4) Immunostaining revealed that bromelain caused a delay in electrophoretic mobility of the 42 kda protein, which is characteristic of MAPk phosphorylation.

Due to the inhibitory effect of bromelain on the phosphorylation of tyrosine and MAPk, it was postulated that this inhibition could be associated with damaged signal transduction. MAPk is important for the production of IL-2, since it is capable of phosphorylating other proteins such as c-Jun, which is required for the initiation of transcription of IL-2. Therefore, the ability of bromelain to inhibit the production of IL-2 in splenic T cells of murine was tested. Interestingly, T cells, which were pretreated with bromelain, produced markedly less I L-2 than T cells that were pretreated only with PBS, after stimulation with anti-CD3e mAb and anti-CD28 mAb. The precise mechanism of action of bromelain is unknown. It is possible that it inhibits tyrosine phosphorylation by inhibiting kinase-tyrosine activity, or by stimulating a phosphatase that results in the dephosphorylation of proteins (for example by activating CD45). Regardless of the mechanism of action, modulators of protein kinase-tyrosine activity have been proposed to have immunosuppressive properties (June et al, Proc Nati Acad Sci USA, 87, 7722-7726, 1990) . It has been shown that Herbimycin A inhibits the early biochemical cases of T cell activation stimulated by the antigen receptor, and correlates with the inhibition of protein kinase-tyrosine activity. However, herbimycin can not inhibit the stimulation effects of phorbol and ionophore esters. The effects of bromelain are different from those previously reported for herbimycin A, in that bromelain can inhibit the effects of T-cell receptor stimulation (demonstrated in antibody ligation experiments), and the effects of secondary messengers (antigens). phorbol and ionophore). Bromelain has different effects, compared to rapamycin, which is also reported to have immunosuppressive properties. Unlike rapamycin, bromelain inhibits tyrosine phosphorylation of MAP kinase (Chung et al., Cell, 69, 1227-1236, 1992). Cyclosporin A, another immunosuppressant, inhibits calcinuerin, again in a different form to the action of bromelain. Due to the ability of herbimycin and rapamycin to inhibit protein tyrosine activity and signal transduction, they are proposed to be used as immunosuppressive agents. In this study, it was shown that bromelain inhibits tyrosine phosphorylation in T cells, therefore, it can be said that bromelain can also be potential as an immunosuppressive agent. Herbomycin A has also been shown to induce differentiation in a number of cell lines, and in one case, this has been correlated with the inhibition of protein tyrosine kinase activity (Kondo et al., J. Cell. Biol. 190.285-293, 1989). The results discussed above indicate a number of potential applications for bromelain. the fact that bromelain performs tyrosine phosphorylation patterns in T cells indicates that cellular cases, which result from this signaling mechanism, can be manipulated through the use of bromelain. In addition, given the various effects of bromelain on different types of mammalian cells, and the importance of tyrosine phosphorylation in all these cells, bromelain can modulate cellular cases, resulting from signaling mechanisms, on a wide range of cells . In addition to the effect of bromelain on tyrosine phosphorylation, it was found that bromelain treatment of T cells can modify cell surface receptors to improve ligand binding. Previously, bromelain was thought to affect only T cells by cleaving surface receptors (Hale and Haynes, 1992, supra). Therefore, it is believed that two more mechanisms have been found (in addition to cell surface molecule cleavage), whereby bromelain affects T cells (ie, modifies tyrosine phosphorylation and modifies receptors) specific cell surfaces to increase binding to their physiological ligands). Based on these studies, it is believed that bromelain can be used to modify the following cellular procedures: Modification of Cytokine Production There is evidence that bromelain can stimulate (Figure 14) or inhibit (Figures 7, 8 and 9) the production of cytokine in T cells. Potential applications to use bromelain to stimulate cytokine production , include as an immunomodulator in immunocompromised individuals or those infected with a parasite / pathogen, and as an adjuvant for vaccines (direct evidence of this in Figure 15) or chemotherapies.

Potential applications in the use of bromelain to inhibit cytokine production, as an immunosuppressant, to prevent tissue rejection after transplantation and to avoid autoimmune responses. Also, bromelain may have application to avoid toxic shock (production of inflammatory cytokine by an individual is a major contributor to toxic shock). Protein phosphorylation, including MAP kinase, is thought to be important in toxic shock. It has been shown that tyrosine kinase inhibitors inhibit the spatial shock, in vivo, (Novogrodsky et al., Science, 264: 1319-1322, 1994). Similarly, bromelain can be used to prevent allergic reactions. Inflammatory cytokines and other cellular products, such as histamine, are released from cells after exposure to allergens. Signaling cascades, which lead to the secretion of inflammatory products from cells, involve tyrosine phosphorylation.

Stimulation of Proliferation or Differentiation of T Cells There is evidence that bromelain can stimulate the proliferation of splenic, normal T cells (Figure 10).

By presenting the fact that a natural T cell proliferates, it can be differentiated to a specific type of cytokine production cell, it is believed that bromelain may be able to effect this differentiation procedure. The potential application to use bromelain to stimulate T cell proliferation is the same as to increase cytokine production. However, in addition to the increased proliferation of T cells, which could lead to more cells being able to produce cytokines, there will also be more cells to provide cell-cell interactions, which, in addition to the production of cytokines, they are also a vital component of an immune response. Another advantage to promote cell differentiation is in the leukemias, or T cell cancers, by which the disease results due to an increased population of undifferentiated T cells (NB is not anticipated that bromelain could stimulate the proliferation of abnormal T cells , as data generated in GA 15 cells, a T-cell hybridoma, demonstrate that bromelain inhibits the proliferation of these cells.

Prevention of T Cell Death A possible explanation for bromelain that causes an increase in normal proliferation of T cells (Figure 10), may prevent programmed cell death (apoptosis). A reduction in cell death could lead to more cells becoming available to incorporate 3H-TdR (used to measure proliferation). Apoptosis is a specific case, whereby cells are stimulated to destroy their own DNA and die. It is an essential case, in most immune responses (to prevent the accumulation of too many cells), but it can also have immunosuppressive consequences in some cases, such as in HIV infection and aging (that is, too many cells die and they do not remain sufficient to fight the infection), (Perandones et al., J. I mmunol., 151: 3521 -3529, 1993).

Since the initiation of apoptosis depends on specific cell signaling cases, the evidence that r-bromelaine effects tyrosine phosphorylation (Figure 13a), which may be involved in the stimulation or prevention of apoptosis, also supports a possible explanation that bromelain prevents apoptosis. 5 Prevention of Invasion of Parasites / Pathogens and Survival in Cells The invasion of parasites and pathogens, and the subsequent survival in cells, depends on these organisms that use host cell signaling pathways (Bliska et al., Cell 73: 903-920, 1993). In particular, it has been shown that Salmonella can phosphorylate the MAP kinase, which allows the bacteria to be endocytosed by macrophages (Galán et al., Nature 357: 588: 589, 1992). Afterwards, the bacteria proliferate and destroys the cell. Since bromelain has been shown to modify the host signaling trajectories (Figure 13a), and in particular inhibits tyrosine phosphorylation of MAP kinase (Figure 2), it is believed that another potential application for bromelain may be that it inhibits either invasion of parasites / pathogens or their survival in cells.

Claims (27)

1. CLAIMS 1 .- The use of bromelain in the preparation of an agent to modulate the intracellular signal trajectories, which depends on the inositol phosphates, protein kinases and / or protein phosphatases.
2. 2 - The use according to claim 1, wherein the signal path depends on the inositol phosphates.
3. 3. The use according to claim 1 or claim 2, wherein the agent modulates the trajectories to control the growth and proliferation of cells.
4. 4. The use according to claim 3, in the preparation of an agent to reduce or prevent the production of growth factors by cells.
5. 5. The use according to claim 4, in the preparation of an agent to prevent the formation of MAP kinase.
6. 6. The use of bromelain in the preparation of an agent for the treatment or control of a disease mediated by intracellular signal transduction mediated by inositol phosphate, protein kinase and / or protein phosphatase.
7. 7 - The use of bromelain in the preparation of an agent to modulate the production of cytokine.
8. 8. The use according to claim 7, wherein the agent stimulates the production of cytokine.
9. 9. The use according to claim 8, wherein the agent is used as an immuno-enhancer and / or as an adjunct to a vaccine.
10. 10. The use according to claim 7, wherein the agent inhibits the production of cytokine.
11. 11. The use according to claim 10, wherein the agent is an immunosuppressant.
12. 12. The use according to claim 1, wherein the agent is to be used to prevent or treat rejection of tissues, or to avoid autoimmune responses.
13. 13. The use according to claim 10, wherein the agent is to be used to prevent or treat toxic shock.
14. 14. The use of bromelain in the preparation of an agent for the treatment or prevention of an autoimmune disease or the rejection of transplants by a host.
15. 15. The use according to claim 14, wherein the autoimmune disease is multiple sclerosis or rheumatoid arthritis.
16. 16.- The use of bromelain in the preparation of an agent to be used to prevent or treat toxic shock.
17. 17. The use of bromelain in the preparation of an agent to be used as an adjuvant for a vaccine.
18. 18.- The use of bromelain in the preparation of an agent for the treatment of cancer.
19. 19. The use of bromelain in the preparation of an agent to be used in the prevention or treatment of allergies.
20. 20. - A method for the treatment or prevention of an autoimmune disease, the method comprising, administering to a patient, an effective amount of bromelain.
21. 21 .- A method for the treatment or prevention of rejection of a transplant, the method comprising, administering to a patient, an effective amount of bromelain.
22. 22. A method for the treatment or prevention of toxic shock, the method comprising, administering to a patient, an effective amount of bromelain.
23. 23. A method for inducing immunostimulation, the method comprising, administering to a patient, an effective amount of bromelain together with a vaccine.
24. 24.- The use of bromelain in the preparation of an agent to prevent apoptosis.
25. 25. A method to prevent apoptosis, the method comprising, administering to a patient, an effective amount of bromelain.
26. 26.- The use of bromelain in the preparation of an agent to be used to inhibit, prevent or treat infection by parasites / pathogens.
27. 27. A method for inhibiting, preventing or treating infections by parasites / pathogens, the method comprising, administering to a patient, an effective amount of bromelain. 28 - A method for the treatment of cancer, the method comprising, administering to a patient, an effective amount of bromelain. 29.- A method to prevent or treat allergies, the method comprising, administering to a patient, an effective amount of bromelain.