MXPA00011511A - THE INDUCTION OF ANTIOBIOTIC PROTEINS AND PEPTIDES BY LAIT/sCD14-PROTEIN - Google Patents

Abstract

A method of ameliorating the symptoms of sepsis comprising directly exposing epithelial cells of a mammal in need thereof to soluble CD14, or active variants thereof. A method of obtaining CD14 from a stock solution containing protein of a mammary secretion is described. A method of directly activating B cells using a soluble polypeptide having the amino acid sequence selected from the group consisting of leu-leu-leu-leu-leu-leu-pro-ser, leu-leu-leu-leu-leu-leu-pro-leu;and leu-leu-leu-leu-leu-leu-val-his, and which is specifically recognized by the monoclonal antibody 3C10 and which activates B cells is described. Bovine CD14 genomic DNA is described.

Description

I DUCTION OF PROTEINS AND ANTIBIOTIC PEPTIDES BY PROTEIN DESCRIPTION OF THE INVENTION This invention relates to the soluble LAIT protein (CD14) derived from mammals and related proteins, which directly induce the expression of antibiotic polypeptides, particularly defensins in mammalian cells, particularly epithelial cells. This invention also relates to the identification of a portion of CD14 necessary for the direct activation of B cells by CD1. Antibiotic peptides and their induction by endotoxin Antibiotic peptides are widely distributed in nature and comprise a broad host defense mechanism (Lehrer, RI et al., 1993. Ann. Rev. Immunol., 11: 105; Boman, HG 1995. Ann. Rev. I Munol 13:61, Lehner, RI, T. Ganz, and ME Selsted, 1991. Cell 64: 229, Zasloff, M. 1992. Curr Opin Immunol 4: 3). One advantage of antibiotic peptides as factors of the innate immune system is their ability to function without specificity and without memory. Its anti-bacterial, anti-viral and anti-microbial activities allow the host to delay and possibly prevent microbial growth shortly after infection, before the adaptive immune response can be mobilized (Lehrer, RI et al., 1993. Ann. Rev.

? Immunol. 11: 105; Boman, H.G. 1995. Ann. Rev. Immunol. 13:61; Lehner, R.I. T. Ganz and M.E. Selsted. 1991. Cell. 64: 229; Zasloff, M. 1992. Curr. Opin. Immunol. 4: 3). Defensins are the largest family of antibiotic peptides and are composed of 29 to 35 amino acid residues and make up more than 5% of the total cellular protein in human neutrophils (Boman, HG 1995. Ann. Rev. Immunol., 13:61; Lehner, RIT Ganz, and ME Selsted, 1991. Cell 64: 229; Zasloff, M. 1992. Curr Opin. Immunol. 4: 3). It is also known that in mammals, defensins are produced by lung macrophages (Lehrer, RI et al., 1993. Ann. Rev. Immunol., 11: 105; Boman, HG, 1995. Ann, Rev. Immunol., 13:61; Lehner, RIT, Ganz, and ME Selsted, 1991. Cell 64: 229, Zasloff, M. 1992. Curr Opin. Immunol 4: 3) and have been described mostly in epithelial bovine cells of the trachea (Diamond, G.J.P. Russell and C.L. Bevins, 1996. PNAS 93: 5156) and the tongue (Schonwetter, B.S., Stolzenberg, E.D. and M.A. Zasloff, 199. Science, 267: 1645). It has been shown that endotoxin, in the form of Lipopolysaccharide (LPS), induces a ten-fold increase in the expression of messenger RNA (mRNA) encoding an antibiotic peptide in primary tracheal epithelial cells (Diamond, G.J.P. Russell and C.L. Bevins, 1996. PNAS 93: 5156). This peptide, called the antibiotic peptide trachea (TAP) is from the class of β-defensins. It was shown that the TAP induction mechanism of epithelial cells of the respiratory mucosa is mediated through the CD14 membrane (mCD14) expressed in the epithelial cells. The role of epithelial messenger CD14 was consistent with the observation that the activation process resulting in TAP expression was inhibited in the presence of the CD14-specific monoclonal antibody (mAb) (Diamond, GJP Russell and CL Bevins, 1996. PNAS 93: 5156). While it was thought that the expression of messenger CD14 was an exclusive marker of monocyte macrophages (Zeigler-Heitbrock, HWL and RJ Ulevitch, 1993. Immunology Today 14: 121), it is known to be expressed by epithelial cells derived from various tissues. (Fearns, C. et al., 1995. J. Exp. Med. 181: 857). It has also been found that epithelial coatings of other tissues respond to endotoxin or inflammation through the local production of defensins. In particular, it has been shown that the squamous epithelial lining of the tongue responds to infection or inflammation with the production of a β-defensin lingual antibiotic peptide (Schon ether, BS, Stolzenberg, ED and MA Zasloff, 1995. Science 267: 1645). In this study it has been shown that messenger RNA (mRNA) encodes the antimicrobial lingual peptide that is present in large quantities in the epithelial cells of the tongue that surrounded the naturally occurring lesions. Apparently there has not been any report about this context. The results described above provide the experimental basis for an immune response model in which the innate immune response machinery is involved in local sites of infection and / or inflammation to contribute to the host's initial defense. The production of antibiotic and peptide proteins in response to LPS derived from the gram-negative bacterium could then take a role for the prevention of bacterial colonization or subsequent infection prior to coupling an adaptive immune clonal response. As background, a brief summary of the current understanding of the mechanisms underlying the responses mediated by endotoxin in monocytes will be presented below., macrophages, epithelial and endothelial cells. CD14 is a membrane receptor in monocytes for LPS complexes: LBP Endotoxin in the LPS form induces inflammatory cytokines by monocytes / macrophages both in vi tro and in vivo (Beutler, B. et al 1986. Science 232: 977; Michie, H.R. et al. 1988. New Engl. J. Med. 318: 1481; Tracey, K. J. et al. 1987. Na ture 330: 662; Waage, A., Halstensen, A. and T. Espevik. 1987. Lancet 1: 355). These cytokines derived from monocytes including TNFa, IL-1 and IL-6, are associated with septic shock syndrome, ultimately leading to multiple organ failure. Recent work has characterized CD14 as a receptor monocyte for LPS (Wright, SD et al., 1990. Science 249: 1431), which led to the initial characterization of a mechanism through which LPS activates monocytes / macrophages. . The current paradigm places the envelopment of the constitutively expressed plasma protein, the lipopolysaccharide-binding protein (LBP), which forms complexes with high affinity with LPS (Schumann, RR et al., 1990. Science 249: 1429, Wright, SD et al. al., 1990. Science 249: 1431; Wright, SD et al., 1989. J. Exp. Med. 170: 1231). LBP is a plasma glycoprotein produced by the liver, constitutively present in the plasma of healthy human adults at 5 lOμg / ml, it has been shown that its concentration increases up to 20 times after an acute phase response (Schumann, RR et al. al., 1990. Science 249: 1429, Tobias, PS et al., 1992. Cell, Mol. Biol. 7: 239, Tobias, PS, Mathison, JC and RJ Ulevitch, 1988. J. Biol. Chem. 263-13479; Tobias, PS, Soldau, K. and RJ Ulevitch, 1986. J. Exp. Med. 164: 777; Wright, SD et al., 1990. Science 249: 1431; Wright, SD et al., 1989. J. Exp. Med. 170: 1231). When BINDING LBP increases the ability of LPS to stimulate cytokine production in macrophages and monocytes (Mathison, J.C., Tobias, P.S. and R.J. Ulevitch 1991. Pa thobiology 59: 185, Schumann, R.R. et al. 1990. Science 249: 1429; Wright, S.D. et al. 1990. Science 249: 1431; Wright, S.D. et al. 1989. J. Exp. Med.170: 1231). The CD14 membrane (mCD14), bound through a glycosylphosphatidylinositol anchor (Zeigler-Heilbrock, HWL and RJ Ulevitch, 1993. Immunology Today 14: 121), functions as a receptor for LPS-LBP complexes (Schumann, RR et al. 1990. Science 249: 1429; Wright, SD et al., 1990. Science 249: 1431). CD14 is expressed at high levels in monocytes and macrophages and at low levels in neutrophils (Ball, ED et al., 1982. Proc.Nat.Acid.Sci.USA 79: 5374; Buckle, AM, Jayaram, Y. And N. Hogg 19902. Clin Exp Immunol, 81: 339, Ferreri E. et al., 1990. J. Immunol., 145: 331, Goyet, S. et al., 1988. Science 239: 497, Haziot, A. et al., 1988. J. Immunol 141: 547). The murine pre-B 70Z / 3 cell line (Paige, CJ et al., 1978. J. Immunol 121: 641) does not express CD14 messenger that can be detected by immunofluorescence and is negative for CD14 message decoder as calculated by the analysis of aurorthern staining and RT-PCR (Filipp, D. and M. Julius unpublished observation, Lee, JD et al., 1992. J. Exp. Med. 175: 1697). This cell line has been used to provide evidence on the role of messenger CD14 as a receptor for LPS-LBP complexes. Specifically, 70Z / 3 responds to LPS with membrane immunoglobulin expression (mlg) (Paige, C. J. et al., 1978 J. Immunol., 121: 641). The concentration of LPS required to induce mlg expression in the CD14 messenger ~ 70Z / 3 are orders of magnitude higher than that required to stimulate cytokine production by monocytes of the messenger CD14 (Lee, JD et al., 1992. J Exp. Med. 175: 1697). When 70Z / 3 is transfected with human CD14 encoding cDNA, it has been shown that the concentration of LPS required to induce mlg expression by CD14 messenger clones was 10,000 times lower than that required in the WILD type CD14 parental line. messenger (Lee, JD et al., 1992. J. Exp. Med. 175: 1697). These results provide experimental evidence for the current model for the activation of CD14 leukocyte messenger-mediated LPS in vivo. Due to the expression of LPS, LPS-LBP complexes are formed and these complexes activate monocytes / macrophages through the interaction with messenger CD14. CDI4 soluble in endotoxin-mediated activation of endothelial and epithelial cells. In contrast to the suggested mechanisms involving the activation of LPS-mediated messenger CD14 leukocytes, less is understood about the mechanisms that involve the activation of endothelial and epithelial cells mediated by LPS. Until recently, it was thought that endothelial and epithelial cells were CD14 messenger. Despite the non-detectable expression of messenger CD14 in this cell type, it has been shown that LPS-mediated activation is serum dependent and is inhibited by monoclonal antibodies specific for CD14 (Patrick, D. et al., 1992. J Inf. Dis. 165: 865; Pugin, J. et al., 1993. Proc. Nati, Acad. Sci. USA 90: 2744; Arditi, M. et al., 1993. Infect. Immun., 61: 3149). This suggests that CD14 may play some role, although unknown, in the endotoxin-mediated endothelial and epithelial cell activation. It has been shown that soluble CD14 (sCD14) in the absence of a glycosyl phosphatidylinositol anchor and being present in the serum of healthy adult humans (Bazil, V. et al., 1986. Eur. J. Immunol. 16: 1583), is related in LPS-mediated activation of both endothelial cells (Arditi, M. et al 1993, Infect Immun 61: 3149, Pugin J. et al 1993. Proc Nati Acad Sci USA 90: 2744; Frey, EA et al., 1992. J. Exp. Med. 176: 1665; Read, MA et al., 1993. Proc. Nati, Acad. Sci. USA 90: 9887; Haziot, A. et al., 1993. J Immunol., 151: 1500) as epithelial cells (Pugin, J. et al., 1993. Proc. Nati, Acad. Sci. USA 90: 2744). It has been shown that the serum dependence of the activation process is due to the presence of soluble CD14 and that the serum requirement can be replaced by soluble CD14. In some studies, some role for LBP could not be characterized in the case of LPS-mediated endothelial cell activation, suggesting that the same soluble CD14 is an antagonist for the endothelial cell response to endotoxin (Arditi, M. et al. 1993, Infect. Immun 61: 3149; Frey, E. A. et al. 1992. J. Exp. Med. 176: 1665; Read, M. A. et al. 1993. Proc. Na ti. Acad. Sci. USA 90: 9887). In other studies, a double role for serum has been found in the LPS response for epithelial and endothelial cells. Specifically, both soluble CD14 and LBP appear to be required for endotoxin-mediated activation of endothelial cells (Pugin, J. et al., 1993. Proc.Nat.Acid.Sci.USA 90: 2744 Haziot, A. et. al., 1993. J. Immunol., 151: 1500), most pronounced when the endotoxin was present at low concentrations (Haziot, A. et al., 1993. J. Immunol., 151: 1500). The aforementioned experimental results lead us to the postulated role of CD14-LPS complexes soluble in activation of CD14 endothelial cells. It is thought that with high concentrations of LPS these complexes will be generated directly through the interaction of soluble CD14 and LPS. It is thought that with low concentrations of LPS the LBP interacts first with LPS, as in already characterized modes which is postulated to facilitate the generation of soluble CD14-LPS complexes. While the above results are potentially the same with respect to the suggested mechanisms that support endothelial and epithelial cell responses to LPS, they share a common element in that the mechanisms are different from those involving endotoxin-mediated activation of CD14 messenger cells. Both studies suggest a role for the functioning of soluble CD14 as an antagonist, allowing for the responses to the endotoxin of CD14 messenger cells rather than functioning as a receptor for LPS-LBP complexes in CD14 messenger cells. However, more recent studies have shown that this may not be the case, at least for epithelial cells. The CD14 membrane in an endotoxin-mediated defensin induction by epithelial cells. As discussed above, a recent study demonstrated that endotoxin induces the expression of defensins in bovine primary tracheal epithelial cells (Diamond, GJP Russell, and CL Bevins, 1996. PNAS 93: 5156) and that such expression involves CD14. messenger While the unstimulated epithelial cells were shown to be messenger CD14, they were induced to the messenger CD14 state subsequent to LPS-mediated activation (Diamond, G.J.P. Russell, and C.L. Bevins, 1996. PNAS 93: 5156). It was shown that the induction of messenger CD14 on the primary epithelial cells of the trachea correlates with the induction of specific messages for CD14 in the epithelial cells, suggesting that it probably had an endogenous origin. In addition, defensin induction mediated by LPS was inhibited by mAB specific for CD14 (Diamond, G.J.P. Russell, and C.L. Bevins, 1996. PNAS 93: 5156). The fundamental mechanisms for the activation of epithelial cells mediated by LPS have been shown to be parallel to those observed in monocytes and macrophages of the messenger CD14. The activation pathways of LPS in these two cell types appear to be different only at the base CD14 messenger levels expressed by the two cell types that are the target. No comparable studies have been reported on endothelial cells. Soluble CD14 directly activates monocytes in the absence of serum / LBP. The paradigm described so far is that endotoxin in the form of LPS mediates the activation of epithelial cells and monocytes / macrophages through its interaction with the messenger CD14 in the cell. It has been shown in all circumstances except in one serum dependence in this process, reflecting the involvement of LBP. As described above, CD14 soluble in the endotoxin-mediated activation / damage of endothelial cells has been implicated. Its function was postulated as potentialization of the interaction of Lps with the cell (Pugin, J. et al., 1993. Proc.Nat.Acid.Sci.USA 90: 2744). A subsequent study showed that soluble CD14 isolated from the urine of nephrotic human subjects was able to directly stimulate the production of inflammatory cytokines TNFI and IL-6, by human monocytes (Sundan, A. et al., 1994. Eur. J. Immunol., 24: 1779). Human monocytes are CD14 messengers and the LPS-mediated induction of these two cytokines is serum dependent (Espevik, T. et al., 1993. Eur. J. Immunol., 23: 255; Wright, S.D et al. 1992. J. Exp. Med. 176: 719). In contrast to the activity of the soluble CD14 isolated from the urine it showed to be independent serum and was not affected by LBP or by antibodies specific for LBP (Sundan, A. et al 1994, Eur. J. Immunol. 24: 1779) . In addition, it has been shown that the ability of soluble CD14 to stimulate the production of inflammatory cytokines by human monocytes is inhibited with mAb specific for CD14 (Sundan, A. et al 1994, Eur. J. Immunol. 24: 1779) . Thus, soluble CD14 appears to have the ability to directly interact with receptor structures not yet identified in monocytes in an independent serum mode and resulting in cytokine production. In this way endotoxin and soluble CD14 are capable of mediating similar biological responses in monocytes. In addition, the ability of the same mAb specific to CD14, 3C10 (Van Voorhis, WC et al., 1983, J. Exp. Med. 158: 126) to inhibit both modes of stipulation suggests at least that part of the identified pathways are shared. they involve endotoxin and soluble CD14, perhaps at the level of receptor structures. The CD14-specific mAb 3C10 recognizes the N-terminal portion of CD14, this recognition depends on the presence of 7 to 14 N-terminal amino acids of the CD14 molecule (Juan, TSC et al., 1995. J. Biol. Chem. 270 : 17237). The ability of 3C10 to inhibit monocyte activation mediated by LPS has been interpreted as reflecting the role of messenger CD14 residues 7 to 14 as an interaction site between messenger CD14 and LPS (Todd, SC et al., 1995). J. Biol. Chem. 270: 17237). In contrast, the basis for the ability of mAb 3C10 to inhibit the function of soluble CD14 on monocytes in the absence of LPS is not clear (Sundan, A. et al., 1994. Eur. J. Immunol., 24: 1779). . The simplest explanation suggests the presence of receptor structures not yet characterized for CD14 soluble in monocytes, the mAb interfering with the interaction between soluble CD14 and such structures. Soluble CD14 inhibits LPS-induced activation of monocytes and neutrophils ip vitro in a dose-dependent manner. The application for the international patent published under No. W093 / 19772 on October 14, 1993, describes the inhibition of LPS-induced activation of monocytes and neutrophils in vitro by recombining human CD14 in a dose-dependent manner, at least as shown by the data in Figure 1 of the published document. The experiments were carried out in the presence of the LPS-LBP complex and are consistent with the binding of the complex and the added CD14 present to reduce the degree of interaction between the complex CD14 and the membrane bond of monocytes and neutrophils. Such a result is curious since it is known that soluble CD14 is capable by itself of stimulating the production of inflammatory cytokines by monocytes. Lactationally Associated Protein Immunotropic (LAIT) and B cell activation. The description of the isolation, characterization of biological activities, molecular cloning and expression of recombinant LAIT protein (bovine CD14) is described in the Cooperative North American Patent Application Number of Series 08 / 746,883 dated November 18, 1996 and in the application for International Patent Serial Number. PCT / CA97 / 00880 dated November 18, 1997. Relevant portions of the aforementioned applications are reproduced herein. A protein was isolated from human milk and human and bovine colostrum, such protein has been called Protein-Immunotropic Associated with Lactation (LAIT).The biological activities of the LAIT protein distinguish it from other well-known cytokines that support the growth and differentiation of B cells in adult animals and therefore can play a unique role in regulating B cell activation. Most induction of humoral immune responses in the adult involve a sequence of cellular interactions between the "helpers" T lymphocytes, the cells that present antigen (APC) and B lymphocytes (Sprent, JJ 1978. J. Exp. Med. 147: 1159. Andersson J. et al., 1982. Proc. Na ti. Acad. Sci. USA 79: 1989) These increases are mandatory (Sprent, JJ 1978. J. Exp. Med. 147: 1159, Andersson J. et al., 1982 Proc. Na ti, Acad Sci. USA 77: 1612, Julius, MH et al., 1982 Proc. Na ti, Acad Sci USA 79: 1989, Julius, MH et al 1987. Immunol Rev. 95: 914). , and this reflects the role of molecules associated with the specific plasma membrane as transducers of activation signals n prerequisite. The essential molecular interaction reflected by the contact requirement T cell-B, is mediated by the CD40 expressed in the plasma membrane in the B cell and its affinal ligand, gp39 (or CD40L) expressed in the plasma membrane of the T cells (Noelle, RJ et al., 1992. Proc. Na ti, Acad. Sci. USA 89: 6550, Armitage, RJ et al., 1992. Na ture 357: 80). The interaction between CD40 and CD40L predicates the induction of B cell growth, differentiation of B cell into immunoglobulin secreting cells, and isotope switching of immunoglobulin (Foy, TM et al., 1993. J. Exp. Med. 178: 1567). However, the matrix of cytokines produced by these interaction cells are central to the regulation of growth and differentiation, activation of B lymphocyte (Andersson, J. et al., 1982. Proc. Na ti.Acid. Sci. USA 77: 1612; Noelle, RJ et al., 1983. Proc. Na ti., Acad Sci. USA 80: 6628; Hodgkin, PD 1990. J. Immunol., 145: 2025; Noelle, RJ, et al., 1991. J. Immunol., 146: 1118; Parker, DC 1980. Immunol., Rev. 52: 115; Howard, M., et al., 1982. J. Exp. Med. 155: 914). These soluble mediators of lymphocyte activation do not act in isolation. Rather, they are supplemented, each carrying the B lymphocyte to the next stage of activation, leaving them susceptible to progressive and subsequent activation signals (Julius, M.H. et al., 1987. Immunol.Rev.95: 914). In contrast, the LAIT protein is directly mitogenic for B cells at nM concentrations and functions as a co-stimulator of B cell growth in combination with stimulation through the B cell antigen receptor in the pM range. In these latter circumstances, these signals derived from the antigen transmute the B cell into a physiological state in which it can receive help from the T cell. The relevance of supplying the neonate with a factor that directly supports the growth and differentiation of the cell is significant. B in combination with an antigen when one considers the suppressed state of T cells in a developing neonate. It has been shown that while the thymus efficiently produces T cells at the beginning in ontogeny, unlike the adult thymus (Bill, J. et al 1989, J. Exp. Med 169: 1405, MacDonald, HR et al. 1988. Na ture 332: 4020), does not efficiently delete those potentially autoreactive antigen receptors that express T cells (Schneider, R. et al 1989, J. Exp. Med. 169: 2149, Smith, H. et al. 1989. Science 245: 749, Ceredig, R. 1990. In tl Immunol., 2: 859, Ceredig, R. and C. Waltzinger, 1990. In tl Immunol., 2: 869). At the same time, these neonates are healthy. Colostrum and early breast milk contain inhibitors of well-characterized T-cell functions, particularly TGFβ1 and TGFβ2, which are inhibitors of T cell activation (Sporn, MB et al., 1987. J. Cell. Biol. : 1039; Massagué, J. 1987. Cell 49: 437; Wrann, M. et al. 1987 EMBO J. 6: 1633; Stoeck, M. et al. 1989. J. Immunol. 143: 3258). It is therefore plausible that the functioning of T cells in neonates is actively suppressed by these cytokines to allow time for the maturation of thymus function. It is also of obvious importance for the neonate to initiate the production of their own protective antibodies, since the maternally derived and passively acquired Ig is transient and contains specificities that reflect the encounters with maternal antigens. It is expected that the functions of the LAIT protein as a substitute for T cells, supporting the growth and differentiation of B cells in the neonate newly exposed to environmental antigens. Therefore, the operation of the LAIT protein offers an alternative, a truncated route to activate the immune system, which is independent of the functioning of T cells. Sequential analysis of protein fragments Bovine LAIT revealed a high homology with human CD14. Subsequently CD14 was purified from human colostrum by affinity chromatography using available monoclonal antibodies and was shown to possess the same range of biological activities as bovine colostral LAIT protein. The gene encoding the bovine LAIT protein was cloned from a bovine cDNA library and showed to be highly homologous to human CD14 at protein and nucleotide levels. The recombinant human and mouse CD14, as well as the recombinant bovine LAIT / CD14 protein was prepared in expression systems in both mammary and insect cells and it was shown that each contains all the biological activities of the native bovine LAIT protein of colostral origin. , with specific activities with a factor of two of that observed in native material isolated from each of the three species. In the context of this invention "antibiotic proteins" or "antibiotic polypeptides" have antibiotic properties: (I) linear peptides mostly spiral without cysteine, with or without joint (cecropins); (II) linear peptides without cysteine and with a high proportion of certain residues such as proline and arginine; (III) antibacterial peptides with a disulfide bond; (IV) peptides with two or more SS bonds giving only or mostly ß-plate structures including but not limited to human defensins, HNP-1, rabbit defensin NP-1, rat defensin NP-1, bovine defensin ß, TAP , pork protegrin, PG-3, and taquiepiesin 1 of crab Hs; and (V) antibacterial peptides derived from major polypeptides with other known functions (Boman, H.G. 1995. Ann. Rev. Immunol., 13:61). "Defensins" are a subgroup of antibiotic polypeptides (Edwards, S.W. Biochemistry and Phisiology of the Neutrophil, 1994. Cambridge University Press pp. 67-70). The "asepsis" is a condition that manifests itself in a human patient when it is invaded by a microbial agent at a temperature higher than 38 ° C or lower than 36 ° C; a heart rate greater than 90 beats per minute; a respiratory rate greater than 20 breaths per minute or PaCO2 less than 32 mm Hg; a white blood cell count greater than 12,000 mm3, less than 4,000 mm3 or more than 10% of forms (gang) immature; organic dysfunction, hypoperfusion or hypotension. Hyperperfusion and perfusion abnormalities may include, but are not limited to lactic acidosis, or liguria or acute alteration of mental states (1992. Chest 101.1644). In the context of this application, CD14, unless otherwise indicated, means CD14 proteins from either bovine, human or murine, recombinant or isolated from a naturally occurring ("native") source, the sequences of which corresponds to SEC. FROM IDENT. DO NOT:; SEC. FROM IDENT. NO: 5 and SEC. FROM IDENT. NO: 6, respectively. The present invention provides a method to lessen the symptoms of asepsis. According to this first aspect of the invention, there is a step in which epithelial cells of a mammal are directly exposed to obtain an effective amount of a compound comprising (eg, that which is made of or includes) soluble CD14, or a a polypeptide fragment of CD14 that stimulates the expression of a defensin in epithelial cells, or a conservatively substituted variation of such CD14 or fragment that stimulates such expression.

The invention also includes a method for increasing the expression of defensins in mammals in need thereof, by administering a compound containing soluble CD14 or a portion of CD14 polypeptide that increases such expression, or a conservatively substituted variant of such CD14 or the portion that increase such expression. The administration step preferably includes direct exposure of mammalian epithelial cells to such a compound. The invention includes a method for stimulating the expression of one or more defensins by epithelial cells by administering an effective amount of a compound containing soluble CD14 or a polypeptide fragment of CD14 that stimulates such expression.,? a conservatively substituted variant of such CD14 or the portion that stimulates such an expression. The invention includes stimulating the expression of defensins throughout the gastrointestinal tract, or along the respiratory tract of a mammal comprising exposure of the tract to an effective amount of a compound containing soluble CD14 or a polypeptide fragment of CD14 that stimulates such expression. , or a conservatively substituted variant of such CD14 or the portion that stimulates such an expression.

The expression of a defensin takes place in the tongue of a mammal according to a certain aspect. The expression of a defensin can be in the intestine, particularly, the small intestine of a mammal. According to a general aspect of the invention, the expression of defensins is induced by the epithelial cells of a mammal. The CD14 may have an amino acid sequence selected from the group consisting of SEC. FROM IDENT. NO: 4; SEC. FROM IDENT. N0: 5, SEC. FROM IDENT. NO: 6 or SEC. FROM IDENT. N0: 7, or a conservatively substituted variant thereof. In another aspect, the invention is a method for reducing the symptoms of asepsis comprising administering an effective amount of a soluble protein to a mammal in need thereof in order to directly expose the epithelial cells of the mammal to the protein, the protein having a sequence of amino acid that retains at least 63% in relation to the amino acid sequence identified as SEC. FROM IDENT. NO: 5 and having the ability to induce the expression of defensins in epithelial cells. Alternatively, the protein can have an amino acid sequence that is at least 68% or about 71% or about 73% or about 78% or about 83% or about 88% or about 93% or about 98% conserved in relation to the sequence of amino acid identified as SEC. FROM IDENT. NO: 5. The invention includes a method for prophylactically treating an inflammatory response induced by lipopolysaccharides in the host in a mammal, such method comprising administering a therapeutically effective amount of an effective amount of a protein to a mammal to directly expose the cells epithelial cells from the mammal to the protein, the protein having an amino acid sequence that retains at least approximately 63% relative to the amino acid sequence identified as SEC. FROM IDENT. NO: 4 or identified as SEC. FROM IDENT. NO: 5 or identified as SEC. FROM IDENT. NO: 6 and having the ability to increase the expression of one or more defensins in bovine epithelial cells. The invention includes a method for increasing the expression of defensins in a mammal in need thereof by administering to the mammal an effective amount of a soluble protein, the protein having an amino acid sequence that retains at least about 63% relative to the sequence of amino acid identified as SEC. FROM IDENT. NO: 4 or identified as SEC. FROM IDENT. NO: 5 or identified as SEC. FROM IDENT. NO: 6 and having the ability to increase the expression of defensins in mammalian epithelial cells.

The invention includes a method for stimulating the expression of one or more defensins by epithelial cells by exposing the cells to an effective amount of a soluble protein, the protein having an amino acid sequence that retains at least about 63% relative to the amino acid sequence identified as SEC. FROM IDENT. NO: 4 or identified as SEC. FROM IDENT. NO: 5 or identified as SEC. FROM IDENT. NO: 6 and having the ability to stimulate the expression of one or more defensins in the epithelial cells. The invention includes a method for stimulating the expression of a defensin along the tract to an effective amount of a soluble protein, the protein having an amino acid sequence that retains at least about 63% relative to the amino acid sequence identified as SEC. FROM IDENT. NO: 4 or identified as SEC. FROM IDENT. NO: 5 or identified as SEC. FROM IDENT. NO: 6 and having the ability to stimulate the expression of a defensin in bovine epithelial cells. The invention includes a method for stimulating the expression of a defensin at the lake of the respiratory tract and or the tongue or small intestine of a mammal comprising exposure of the tongue to an effective amount of a soluble protein., the protein having an amino acid sequence that retains at least about 63% relative to the amino acid sequence identified as SEC. FROM IDENT. NO: 4 or identified as SEC. FROM IDENT. NO: 5 or identified as SEC. FROM IDENT. NO: 6 and having the ability to stimulate the expression of a defensin in epithelial cells. The invention includes a method for inducing the expression of defensins by epithelial cells of a mammal in need, the method comprising administering an effective amount of a soluble protein, the protein having an amino acid sequence that retains at least about 63% in relation to the amino acid sequence identified as SEC. FROM IDENT. NO: 4 or identified as SEC. FROM IDENT. NO: 5 or identified as SEC. FROM IDENT. NO: 6 and having the ability to stimulate the expression of a defensin in epithelial cells. The CD14 or the polypeptide portion or the variant can be obtained by chemical or recombinant methods. In another aspect the invention is a method for preparing a concentrate of CD14. The method includes providing a solution of material that contains the protein of a mammary secretion, separating from the solution a concentrate that includes Endogenous of CD14; and determine the concentration of CD14 in the concentrate.

The mammary secretion can be milk, whole or a portion of whole milk that contains protein, or it can be colostrum or a portion of colostrum that contains protein. Preferably the secretion has previously been subjected to a treatment step and the treatment step is mild enough to allow the preservation of CD14 activity to induce or stimulate the defensin production and stimulate B cells. The mammary secretion can be human or bovine In situations where CD14 is obtained from a mammal in which it occurs endogenously (for example, in a mammal that has not been subjected to molecular genetic manipulations), the CD14 is preferably bovine. In cases where the solution is a liquid solution, the separation step may include putting a protein salt layer of the solution. Determining the concentration of CD14 can include exposing a sample obtained from the concentrate to a first antibody specific for CD14 to form a CD14 antibody complex and subsequently exposing the complex to a second antibody specific for CD14, where the second antibody includes a reporter molecule. ELISA assays are particularly convenient in this regard.

In another aspect, the invention is a method for obtaining CD14 which includes providing a solution of material containing the protein of a mammary secretion, precipitating from the solution of material, a fraction of protein containing CD14 and isolating the protein fraction. of the supernatant. Precipitation may include placing a layer of salt in a protein fraction containing CD14. Preferably, the concentration of the solution is increased to obtain an ionic strength at least as high as could be obtained by combining a saturated aqueous solution of ammonium sulfate with a volume of such mammary secretion (as naturally occurs), in which the volume of the ammonium sulfate solution is equal to 65 percent of the total volume of the combined solutions. The method often also includes a determinant amount of CD14 obtained in the isolation step. Again, the mammary secretion can be colostrum and / or milk and can be human or bovine, or of another type of mammals in which CD14 occurs in mammary secretions. The invention includes another method for obtaining CD14, this includes providing a solution material comprising protein from a mammary secretion: and isolating from the solution a fraction containing proteins that are soluble in mammary secretion by combining a saturated aqueous solution of sodium sulfate. ammonium with a volume of such mammary secretion, the volume of the ammonium sulfate solution being equal to 65 percent of the total volume of the combined solutions. Preferably, the method includes a step for determining the amount of CD14 obtained in the isolation step. The endogenous CD14 proteins obtained according to the methods of the invention can be used to directly activate the B cells when they are in a suitable soluble form. Likewise, they can be used to produce medicines for such uses. According to another aspect, the invention is a method for testing the presence of CD14 in a compound containing protein from a mammary secretion. The method includes exposing the compound to an antibody specific for CD14; and determining whether secretion of endogenous CD14 is present in the sample based on whether a CD14-antibody complex has been formed in the exposure step. The secretion may or may not have been subject to the treatment step, but if it has been subjected then the treatment step is mild enough to allow preservation of the activity of the CD14 to induce or stimulate defensin production and / or stimulate the B cells Preferably, the method includes determining the concentration of CD14 in the sample. In another aspect, the invention is a method for preventing, minimizing or treating the symptoms of asepsis in a mammal, comprising administering an effective amount to the mammal, of CD14 obtained from a mammalian mammary secretion. Preferably, CD14 is obtained from a mammary secretion according to one of the methods described herein. The CD14 may be contained in a liquid and the liquid may include a milk fraction enriched with CD14. The CD14 may be contained in a digestible product, such as a food bar (e.g., a chocolate or protein bar). In another aspect, the invention includes a method for determining the amount of endogenous CD14 contained in a compound that contains a protein from a mammary secretion, for example, in an animal that is not subject to molecular genetic manipulation as far as the production of CD14. The method includes providing the compound; exposing a sample of the compound to an antibody specific for CD14 and determining the amount of endogenous CD14 to the secretion present in the sample based on the amount of antibody-CD14 complexes formed in the exposure step.

The invention includes a method for determining whether a product derived from a mammary secretion is suitable for use in the induction or stimulation of defensin production in mammals, the method comprising the steps of: providing a sample of the product and; determine the amount of CD14 present in the sample. This aspect of the invention can be used as a preliminary step to determine the amount of such a product that will be incorporated into the medicament, food product, etc., to be used according to a method of this invention or another method for which it is known that CD14 is useful. Accordingly, it is preferable that, if the secretion has been previously subjected to the treatment step, the treatment is mild enough to allow the preservation of CD14 activity to induce or stimulate the production of such defensins. Also, the invention includes a method for determining whether a product derived from a mammary secretion is suitable for use in the stimulation of B cells in mammals, the method comprising the steps of: providing a sample of the product and; determine the amount of CD14 present in the sample.

In preferred aspects, the antibody is used in the determinant step and preferably, the antibody is a 3C10 mAb and / or a mAb that recognizes the same amino acid sequence as the 3C10 mAb. The invention includes the use of the compounds described herein for the preparation of a medicament for use in ameliorating the symptoms of asepsis, for its use to increase the expression of defensins in a mammal, etc. Another aspect of the invention includes a method for increasing the expression of defensins in a mammal in need, comprising administering, to a mammal in need thereof, an effective amount of a recombinant CD14 polypeptide encoded by a recombinant DNA molecule that does not occur. naturally, which comprises a primary DNA sequence selected from the group consisting of: (a) a cDNA sequence encoding CD14 according to SEQ. FROM IDENT. NO: 2; (b) a cDNA sequence that specifically hybridizes to the unencoded strain of (a) and which encodes an expression of a polypeptide specifically recognized by an antibody which also specifically recognizes human CD14; and (c) a cDNA sequence encoding the same polypeptide that is encoded by the aforementioned DNA sequence of (a) or (b); wherein the polypeptide encoded by (a) or (b) increases such expression. Another aspect of the invention is a method for stimulating the expression of one or more defensins by epithelial cells comprising administering, to a mammal in need thereof, an effective amount of a recombinant CD14 polypeptide encoded by a recombinant DNA molecule that does not occur naturally comprising a first DNA sequence selected from a group consisting of: (a) a cDNA sequence encoding CD14 according to SEQ. FROM IDENT. NO: 2; (b) a cDNA sequence that specifically hybridizes to the unencoded strain of (a) and which encodes expression of a polypeptide specifically recognized by an antibody which also specifically recognizes human CD14; and (c) a cDNA sequence encoding the same polypeptide that is encoded by the above-mentioned DNA sequence of (a) or (b); wherein the polypeptide encoded by (a) or (b) stimulates such expression. Preferably, the specific hybridization is under stringent hybridization conditions.

The "conditions of strict hybridization" has its common meaning in a person skilled in the art in the aforementioned. Appropriate conditions of stringency are known to those skilled in the art and promote nucleic acid hybridization, for example, 6x sodium chloride / sodium citrate (SSC) at about 45 ° C. The following examples are found in the current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6: for 50 ml of a first appropriate hybridization solution, mix together 24 ml of formamide, 12 ml 20 x SSC, 0.5 ml 2 M Tris-HCl pH 7.6, or 5 ml lOOx Denhardt solution, 2.5 H20 deionized, lOml 50% dextran sulfate and 0.5 ml 10% SDS. A second appropriate hybridization could be 1% crystalline BSA (portion V), lmM EDTA, 0.5 M Na2HP04pH 7.2, 7% SDS. The concentration of salts in the wash step can be selected from a low stringency of approximately 2x SSC at 50 ° C to a high stringency of approximately 0.2x SSC at 50 ° C. Both solutions washed may contain 0.1% SDS. In addition, the temperature of the washing step can be increased from conditions of low stringency of room temperature, about 22 ° C, to high stringency conditions, about 65 ° C. The cited reference offers more details, but the appropriate rigor to wash depends on the degree of homology and the length of the probe. If the homology is 100%, a high temperature (65 ° C to 75 ° C) can be used. If the homology is low, lower wash temperatures should be used. However, if the probe is very short (< 100bp), low temperatures should be used even with 100% homology. In general, it begins to wash at low temperature (37 ° C to 40 ° C) and the temperature is increased at intervals of 3-5 ° C until the supernatant is low enough not to be a major factor in autoradiography. Preferably, such polypeptide is specifically recognized by an antibody which also specifically recognizes human CD14, such as mAb 3C10. The methods of intervention may include a direct topical exposure of the epithelium of the trachea or other epidermis of the mammal, particularly wounds, to the protein or polypeptide, as the case may be. Therefore, the invention also includes a method for preparing an ointment for its direct topical application to a wound in the skin of a human to reduce the effects of infection, particularly a bacterial infection, for this, it comprises the incorporation of the ointment of an effective amount of a concentrate or other compound of the invention having CD14 activity to induce the defensins. Also part of the invention is a formula for infants, milk or other liquid to which a portion of a dairy product has been added, the portion including a higher concentration of CD14 than that naturally occurring in unfractionated milk products, where the product it is one that has not been treated with a process that denatures CD14 contained to the extent that CD14 loses its desired activity. The compounds and methods of the invention can be used by a mammal in need of protection against a microbial pathogen selected from the group consisting of virus, bacteria, fungus and yeast, particularly when the mammal is a human suffering from immune deficiency. Induced defensins include RtNPl, RtNP2, RtNP3, RtNP4, HNP1, HNP2 and HNP3 and any combination thereof or HNP1, HNP2 and HNP3 and any combination thereof. Preferably, the protein or polypeptide of the invention, as the case may be, is administered in an amount between about 250 μg per kg of mammalian weight per day or in an amount between 300 μg per approximately lmg per kilo of weight per day. In another aspect, the invention is a method for directly activating B cells using a soluble polypeptide containing the amino acid sequence selected from the group consisting of leu-leu-leu-leu-leu-leu-pro-ser; leu-leu-leu-leu-leu-leu-pro-leu; and leu-leu-leu-leu-leu-leu-val-his and which is specifically recognized by monoclonal antibody 3C10 and which activates B cells. Preferably in such a method, the amino acid comprises a sequence selected from the group consisting of . FROM IDENT. NO: 4, SEC. FROM IDENT. NO: 5, or SEC. FROM IDENT. NO: 6 or a conservatively substituted variant thereof that activates B cells, or a fragment thereof that activates B cells or a conservatively substituted variant thereof that activates B cells. The invention includes a transgenic mammal having introduced in its genome a nucleic acid sequence encoding a pilpeptide containing the amino acid sequence identified as SEQ. FROM IDENT. NO: 4, SEC. FROM IDENT. NO: 5, or SEC. FROM IDENT. NO: 6 or a fragment of such a polypeptide that directly activates B cells; a conservatively substituted variant of a polypeptide; or linked fragment or variant thereof that directly activates B cells, where the nucleic acid sequence is under the control of an endogenous CD14 promoter to the mammal and the nucleic acid sequence is in addition to the nucleic acid sequences that occur naturally in the mammalian DNA. The nucleic acid sequence optionally encodes a polypeptide having the amino acid sequence identified as SEQ. FROM IDENT. NO: 4, SEC. FROM IDENT.

NO: 5, or SEC. FROM IDENT. NO: 6, or a fragment of such a polypeptide that directly activates B cells, or a conservatively substituted variant of the polypeptide and even more preferably, a polypeptide having the amino acid sequence identified as SEQ. FROM IDENT. NO: 4, SEC. FROM IDENT. N0: 5, or SEC. FROM IDENT. NO: 6. Preferably, the nucleic acid sequence has the sequence identified as SEQ. FROM IDENT. N0: 1 or SEC. FROM IDENT. NO: 2. Preferably, the transgenic mammal has introduced into its genome a nucleic acid sequence that encodes a protein capable of directly activating B cells using a soluble polypeptide having the amino acid sequence selected from the group consisting of leu-leu leu-leu-leu-leu-pro-ser; leu-leu-leu-leu-leu-leu-pro-leu; and leu-leu-leu-leu-leu-leu-val-his, and which is specifically recognized by monoclonal antibody 3C10 and which activates B cells A transgenic mammal may have introduced into its genome alternatively a nucleic acid encoding other proteins of this invention. The nucleic acid sequence can be a heterologous sequence. In another aspect, the invention is a transgenic mammal having introduced into its genome a nucleic acid sequence identified as SEQ. FROM IDENT. NO: 8, wherein the nucleic acid sequence is an addition to the nucleic acid sequences that occur naturally in the DNA of a mammal. The SEC. FROM IDENT. NO: 8 includes coding sequences (see SEQ ID NO: 1) as non-coding portions of the sequence, such non-coding function is excised during the production of messenger RNA containing only coding bases. Preferably, the nucleic acid sequence has been introduced into the mammalian or progenitor of the mammal by recombinant technology. Preferably, the mammal is a bovine.

BRIEF DESCRIPTION OF THE DRAWINGS Figure Ia shows the differential inhibition of B cell activation mediated by native human LAIT (Hu-LAIT, nhCD14), mAbs 3C10 and by MEM-18 specific for CD14 (Todd S.-C. Juan, et al., 1995. J. Biol. Chem. 270: 5219). The indicated concentration of mAbs 3C10 (), MEM-18 () or its nonspecific mAb isotypes, 12CA5 (IgG2b) (- -) (J. Field, et al., 1988. Mol. Cell, Biol. 8: 2159), and W3 / 25 (Igd) () (AF Williams, 1977. Cell 12: 663), respectively, was added to 0.2. ml in a serum-free culture medium in a 96-well culture plate with the flattened (Costar) plate, which contains either: lμg / ml of (n) Native Hu-LAIT (B) or 5 μg / ml LPS ( •). The following 5 hours of incubation at 37 ° C, 1.5xl05 were added to each of the cultures mouse splenic B cells buoyant high density (Ratcliffe, MJ et al 1983. J. Immunol. 131:. 581) isolated as It was described earlier. At 40 hours, the cultures were pulsed with 1 μCi of 3H-TdR, 6 hours later harvested inside filter pellets and a thymidine intake determined by the titration spectroscope. The results are expressed as percentages of the response induced by each of the two stimuli in the absence of mAbs. The antecedent response in the absence of stimuli ranged from 0.7 to 1.7xl03cpm; responses to 5 μg / ml LPS and lμg / ml of (n) Hu-LAIT in the absence of mAbs were 77.8x103 cpm and 82.3x103 cpm, respectively. The error bars indicate a standard deviation over the mean of triplicate cultures. Figure IB shows the inhibition of activation of B cells mediated by (r) Hu-recombinant and rBo-LAIT by mAbs, 3C10 specific for CD14. The indicated concentration of mAb 3C10, or an IgG2b isotype non-specific, mAb OX40 (Paterson, DJ et al 1987 Mol Immunol. 24:.. 1281), was added to 0.2 ml of a culture medium free of serum on a plate culture 96 with the flattened floor (Costar), which contained either: 15 μg / ml human recombinant soluble CD14 (rHu-LAIT) (•); 2 μg / ml bovine recombinant soluble CD14 (rBo-LAIT) (), or 5μg / ml LPS (O). The following 5 hours of incubation at 37 ° C, 1.5xl05 were added to each of the cultures splenic B cells of buoyant high density mouse, (Ratcliffe, MJ et al., 1983. J. Immunol., 131: 581) isolated as described previously. At 40 hours, the cultures were pulsed with 1 μCi of 3H-TdR, 6 hours later harvested within filter meshes and the predetermined thymidine capture by the titration spectroscope. The results are expressed as percentages of the response induced by each of the three stimuli in the absence of any mAb. The antecedent response in the absence of stimuli ranged from 0.7 to 1.7xl03cpm; responses to 5μg / ml LPS, 15μg / ml rHu-LAIT, and 2μg / ml rBo-LAIT, in the absence of mAb 3C10, were 77.8x103cpm, 10.9x103cpm and 82.3x103cpm, respectively, with one standard deviation over triplicate cultures of < 10% The inhibition mediated by OX40 was less than 15% of all concentrations tested. Figures 2A and 2B illustrate a comparative analysis of the induction of membrane expression Ig? (mlg?) by induction of LPS and rBo-LAIT in the murine pre-B cell line, 70Z / 3. Cells 8xl0470Z / 3 were cultured in 0. lml culture dishes 96 (Costar) with flat, serum-free flat for 20 hours in the presence of no stimulus, or the indicated concentration of rBo-LAIT (), LPS derived from S. typhimurium (Sigma) (o) or a deep basal LPS derived from a mutant E. coli D31m4 (•) (Kirkland, TN, Qureshi, N. and K. Takayama 1991. Inf. and Imm. 59: 131). the cells were harvested and stained with mAb 187.1 (f187.1) conjugated with fluorination (Yelton, D, E. et al., 1981. Hybridoma 7: 5), specific for Ig? murine, and the proportion of Ig? * cells determined fluxitometrically using a B. -D. FACScan. The three histograms in the upper part of Figure 2A illustrate the proportions of mIg? * 70Z / 3 cells after the culture period of 20 hours at 37 ° C: in the absence of stimuli (left), with the presence of 3μg / ml S. typhimurium LPS (in the middle) and the presence of 0. lμg / ml rBo-LAIT (right). Figure 2B shows the percentage of mIg? * 70Z / 3 cells induced by the indicated concentrations of the three stimuli. Figure 3A shows the inhibition of mIg? * 70Z / 3 cells mediated by rBo-LAIT by mAb 3C10. The indicated concentration of mAb 3C10 was added to 0.1 ml of serum free medium without containing an additional stimulus [), 3 μg / ml of S. typhimurium LPS (0), or 0. lμg / ml rBo-LAIT (•) and the mixtures that are in dish 96 (Costar) and that are incubated at 37 ° C for 2 hours. Subsequent to this pre-incubation period, 8xl0470Z / 3 cells are added to each culture plate, followed by a culture period of 20 hours at 37 ° C, after which the cells are harvested and stained with f187.1 and the proportion of mlg? * cells determined flux ocitometrically using a B. -D. FACScan. The% of Control responses are illustrated, for example, the proportion of mIg? * 70Z / 3 cells observed in the presence of the indicated concentration of mAb 3C10 divided by the proportion of mIgk * 70/3 observed in the absence of the indicated concentration of mAb 3C10 for each induction of rBo-LAIT and LPS. Nonspecific OX40 mAb isotypes did not mediate more than 15% inhibition when cultured at any of the concentrations at which mAb 3C10 was used for either stimulus. Figure 3B shows the differential inhibition of mIg? * 70Z / 3 cells mediated by rHu-LAIT by mAb specific for CD14. The indicated concentrations of mAb 3C10 (•), MEM-18 (B) or its nonspecific mAb isotypes, 12CA5 (0) and W3 / 25 () were added to 0.1 ml of serum-free medium containing 0.75 μg / ml rHu- LAIT. After incubation for two hours at 37 ° C, 8xl0470Z / 3 cells are added to each culture plate, followed by a culture period of 20 hours at 37 ° C, after which the cells are harvested and stained with f187 .1 and the proportion of mlg? * Cells was determined fluxitometrically using a B. -D. FACScan. The% of Control responses are illustrated, for example, the proportion of mIg? * 70Z / 3 cells observed in the presence of the indicated concentration of mAbs divided by the proportion of mIg? * 70Z / 3 cells observed in the presence of 0.75 μg. / ml nHu-LAIT in the absence of any mAb.

Figures 4A to 4C show the effect on the induction of mlg? * In 70Z / 3 cells by nBo-LAIT or by LPS of a lipid diphosphoryl A derived from Rhodopseudomonas sphaeroides (RSDPLA). The 8xl0470Z / 3 cells were cultured for two hours in 0. lml of a serum-free medium containing lOμg / ml RSDPLA in 96 culture dishes (Costar) at 37 ° C. Subsequent to this pre-incubation period, the indicated concentration of ReLPS (, Figure 4A), or native Bo-LAIT (0, Figure 4B) was added, followed by a 20-hour culture at 37 ° C. The cells were harvested and stained with f187.1 and the proportion of mlg? * Cells determined fluxitometrically using a B. -D. FACScan. Two controls were used to compare the results obtained with each stimulus. RSDLPA (A) was added to the indicated concentration of cells which had been similarly pre-treated with RSDLPA. ReLPS was added to cells that had been pre-incubated similarly but without RSDLPA [M, Figure 4A). N-Bo-LAIT was also added to cells that had been pre-incubated similarly but without RSDLPA (•, Figure 4B). In Figure 4C, 96 replicates of flat bottom plates (Costar) with 8xl0470Z / 3 cells in 0.1 of a serum-free medium containing the indicated concentration of RSDLPA at 37 ° C were seeded for 2 hours. Subsequent to this pre-incubation period, the cultures were provided with no stimulus (A), 5μg / ml LPS (•) or 0.3μLg / ml nBo-LAIT [M), as indicated in Figure 4C . A plate was harvested 20 hours later, and the cells were stained with phycoerythrin (PE) an Ig specific antibody? conjugate of goat anti-mouse (Southern Biotechnology). The proportion of mlg? * Cells was determined using a B. -D. FACScan. The% of Control responses are illustrated, for example, the proportion of cells observed in the presence of the indicated concentration of RSDLPA divided by the proportion of cells observed in the presence of the indicated stimulus in the absence of RSDLPA (vertical axis left-hand) . The second plate was pulsed with lμCi of 3H-TdR 14 hours after adding the stimulus, it was harvested within filter meshes 6 hours later and the thymidine intake determined by the titration spectroscope (vertical axis right hand). Figure 5A is a diagrammatic representation of nHu-LAIT / CD14. The two regions that characterize the epitopes recognized by mAb 3C10 are represented (amino acids 7 to 14) and MEM18 (amino acids 57 to 65). Figure 5B is a schematic of how, the RSDPLA can function to inhibit the 70Z / 3 differentiation mediated by Hu-LAIT. The RSDPLA may be interacting with the binding site of the Hu-LAIT protein, or the RSDPLA may be interacting with the putative receptor for LAIT in the 70Z / 3 cell. Some elements of the cellular activation mediated by the LAIT protein or by LPS can be shared. Also shown is mAb MEM-18, which could block the interaction of nHu-LAIT and RSDPLA according to one of the possible modes of interaction. Figure 5C shows the effect of various concentrations of mAb MEM-18 on induction of mlgk * in 70Z / 3 cells by nHu-LAIT in the presence of RSDPLA. 8xl04 70Z / 3 cells were cultured in 0.ml of serum-free medium containing 30μg / ml of RSDPLA for 12 hours at 37 ° C. After another 20 hours of incubation at 37 ° C, the cells were harvested and stained with phycoerythrin (PE) an Ig-specific antibody. conjugate of goat anti-mouse (Southern Biotechnology). The proportion of mlg? * Cells was determined using a B. -D. FACScan. The% of Control responses are illustrated, for example, the proportion of mIg? * 70Z / 3 cells observed in the presence of the indicated concentration of RSDLPA divided by the proportion of cells observed (90%) in the presence of 0.75 μg / ml of nHu-LAIT in the absence of RSDLPA. Figure 6A shows the quantification of soluble nHu-LAIT / CD14 in samples in pairs of milk and human serum obtained from 9-subjects at the indicated postpartum time. The soluble CD14 was quantified using a commercially available ELISA kit. (IBL, Hamburg). The results are presented as the proportion of the total soluble CD14 protein in milk (open symbols) and serum (closed symbols), each form of symbols represents another different subject. The total protein was determined by using a colorimetric detection system. (BioRad). Figure 6B shows an analysis of the B-cell growth promoting activity of nBo-LAIT denatured by heat. Splenic high density B-cells were prepared from conventional C57B1 / 6 mice as described above (Ratcliffe, MJH and Julius, MH 1983. J. Immunol 131: 581) and cultured in 0.2 ml of serum-free medium in the presence of the indicated concentrations of stimuli. The indicated concentrations of nBo-LAIT were achieved by diluting a 10X solution that has been subjected to 99.9 ° C for 10 minutes in a 9600 Perkin Elmer GeneAmp PCR system, or by leaving them untreated. Subsequent to this treatment, the samples were cooled on ice for 5 minutes and added to the cultures containing B cells. The cultures were pulsed with lμCi of 3H-TdR at 40 hours, harvested 6 hours later and the determined thymidine intake by the titration spectroscope. Figures 7A to 7C show the partial purification of the bioactive nBoLA.IT / CD14 using a combination of putting a salt layer and size exclusion chromatography. Bovine milk was prepared and placed in a layer of salt as described in the text. In Figure 7A the specific immunostaining of CD14 of clarified milk whey (CM) is shown, the affinity of nBo-LAIT (nBo) and each of the fractions (NH4) 2S04 was tested. Immunostaining was carried out as described in the above for Figure 7D. The resolution of the proteins in each of the fractions described in Figure 7A is shown in Figure 7B using 10% SDS-PAGE followed by silver staining. The 62% (NH4) 2S0 fraction containing the highest proportion of nBo-LAIT / CD14 was subjected to molecular screening in an FPLC Superdex-75 size exclusion column (Pharmacia) balanced in TN buffer (lOmM Tris pH 8.0, 150mM NaCl). The TN buffer was used to extract the proteins at a flow rate of 0.4ml / min, and fractions of 0.2ml were collected over a period of 40 minutes using an OD2a or nm detector, to verify the protein elution profile. Each of the fractions obtained was determined by nBo-LAIT / CD14 by immunostaining as described in Figure 7D. Fractions 47 to 49 of this separation process contained the highest concentration of nBo-LAIT / CD14 by immunostaining analysis. Figure 7C shows a comparative analysis of the induction of the expression of mlg? in the pre-B cell murine line, 70Z / 3, by cognate nBo-LAIT / CD14 (nBo-LAIT). The 62% (NH4) 2S04 portion (o) The 62% (NH4) 2S04 (O) fraction, used as the starting material for sieve; and fractions 47 (B), 48 (A), and 49 (#) isolated from the Superdex-75 that contained the most nBo-LAIT / sCD14 determined by an immunostaining analysis. The 8xl04 70Z / 3 cells were cultured in 0. lml of serum-free medium in 96-well flat bottom culture dishes (Costar) for 20 hours in the presence of the indicated concentrations of each stimulus. The cells were harvested and stained with a specific antibody Ig? goat anti mouse conjugated with PE (Southern Biotechnology) and the proportion of mlg? * cells was determined flow-cytometrically using a B.-D. FACScan. Figure 7D shows the comparative activity of B cell stimulation of nBo-LAIT purified affinity of bovine milk and colostrum. The clarified colostrum and whey were subjected to sequential salping using increased concentrations of NH4S02 as described in Figure 7A. The 62% (NH4) 2S04 fraction was solubilized and desalted and the sCD14 was purified affinity in 3C10 mAb conjugated to Sephadex 4B. Material with purified affinity of colostrum (o) and milk (•) was added at the indicated concentrations to 0.2 ml cultures of serum-free medium containing 1.5xl05 of splenic B-cells floating high density isolated as described above. At 40 hours the cultures were shaken with 1 μCi of 3H-TdR, harvested in filter nets 6 hours later and the increase of Thymidine determined by the titration spectroscope.

The annex in Figure 7D represents immunostaining of sCD14 derived from milk (M) and colostrum (C) from bovine. 250ng of protein was resolved by 10% SDS-PAGE and then the protein was transferred to the PVDF membrane. After blockage of 5% skim milk for one hour, the protein was revealed using rabbit polyclonal CD14 antiboin in combination with strong root peroxidase conjugated with goat anti-rabbit IgG (BioRad). The signals were detected with ECL (Amersham) Figure 8A shows the sequences of the oligonucleotide probes used to detect the mRNA specific for bovine tracheal antimicrobial peptides (TAP) Figure 8B shows the sequences of the oligonucleotide probes used to detect the mRNA Specific for bovine tubulin used as load control Figure 9A shows the induction of a tracheal antimicrobial peptide (TAP) mRNA in primary tracheal epithelial cells by LPS, the native LAIT protein derived from bovine (nBo-LAIT) and human (nHu) LAIT) and the recombinant bovine LATÍ protein derived from a mammalian expression system (rBo-C127) or a baculovirus expression system (rBo-Sf9) Primary cultures of bovine tracheal epithelial cells were prepared according to previously published methods (Diamond, Gy col. 1996. Proc.Nat.Acad.Sci.USA 93: 5156.) Tracheal epithelial cells 5xl05 were cultured in lml. of serum-free medium containing the indicated concentration of various stimuli. After 16 hours of culture at 37 °, the total RNA of each culture was isolated using the Trizol (Gibco) method and 20μg and loaded to 1.2% formaldehyde / agarose gel. The RNA was then transferred to a nylon membrane (GeneScreen, DuPont) using a vacuum blotter (pharmacy) in lOxSSC and UV- and crosslinked according to the manufacturer's recommendations. TAP labeled oligo-probes were mixed 5'-end (see Figures 8A and 8B 1: 1 and hybridized to RNA immobilized in 50% (vol / vol) formamide / 6X standard saline citrate (SSC) / 5X Denharts / 0.5% (wt / vol) SDS / 10% (wt / vol) Dextran sulfate / 100 μg / ml Salmon sperm DNA at 42 ° C for 16 to 20 hours and then washed in 2X SSC, 0.1% SDS at 65 ° C for 30 min. Normalized the load by determining the levels of bovine tubulin in each row.Hybridization was performed with OLIGO-specific probe for bovine tubulin using very stringent washing conditions consisting of 0.1X SSC, 1% SDS at 65 ° C for 2 hours. The intensity of signals for TAP was normalized to the relative amount of RNA measured by determining the signal intensity of the charge controller probe using a PHOSPHORIMAGER (molecular dynamics). Figure 9B shows the kinetics of tracheal antimicrobial peptide mRNA (TAP) induction by LPS and nBo-LAIT / sCD14 in primary tracheal epithelial cells. The primary tracheal epithelial cells were prepared and cultured as described in Figure 9A. All replicate cultures contained either lμg / ml LPS or lμg / ml nBo-LAIT / sCD14. At the indicated time the total RNA was isolated, resolved on agarose gel, and D-probe first with OLIGO-specific probes for TAP followed by a OLIGO-specific probe for tubulin, as described in Figure 9A. The signal intensity for TAP was normalized to the relative amount of RNA measured by determining the signal intensity of the charge controller probe using a phosphorimager (molecular dynamics). The experiments described below demonstrate that the induction of splenic floating-derived high density B cells are induced to enter and progress through a cell cycle in response to recombinant forms of bovine or human-in vitro LAIT / sCD14 protein, in a defined serum-free medium. It has been shown in this assay that a specific 3cl0 mAb for amino terminal residues 7-14 of human CD14, but not mAb MEM-18, specific for residues 57-65, specifically inhibits the growth promoting activity of human LAIT / sCD14 proteins. and recombinant native bovine cells for mouse B cells.

The in-vitro induction of differentiation of mCD14, murine preB murine mlg 70Z / 3 cell line to a mlg * state, was demonstrated by recombinant human or bovine LAIT / sCD14 protein in a serum-free defined medium. It is shown that this process is inhibited by mAb 3C10 but not by mAb MEM-18. It is shown that the induction of murine preB cell line mlg 70Z / 3 to a mlg * state mediated by LPS and recombinant bovine LAIT / sCD14 protein, is inhibited by a lipid difoforil A derived from Rhodopseudomonas sphaeroides (RSDPLA). It is shown that human colostrum contains 100-400 times higher concentrations of native human LAIT / sCD14 protein compared to serum samples taken from the same subjects at the same time postpartum. It is shown that the increase in the concentration of human LAIT / sCD14 protein in colostrum and milk relative to that observed in serum persists up to 400 days postpartum. It is shown that the biological activity of native bovine LAIT / sCD14 protein in milk obtained up to 200 days postpartum is comparable to that observed in bovine colostrum. The LAIT / sCD14 protein derived from bovine milk shows an induction of splenic B-derived high density floating B cells to enter and progress through the cell cycle, in vitro, in a defined serum-free medium, with specific activity comparable to that observed in the LAIT / sCD14 native bovine protein derived from colostrum. It is shown that the growth promoter activity of nBo-LAIT B cells of purified affinity is severely reduced after boiling at 99.9 C per lOminm. It is shown that the sequential salping of bovine whey proteins using (NH4) 2S04 as described below in detail, results in enrichment of the native bovine LAIT / sCD14 protein in the 62% (NH4) 2S04 fraction. . The 62% (NH4) 2S04 fraction of bovine serum stimulates the induction of mCD14 differentiation, the preB murine mlg 70Z / 3 cell line to a mlg * in-vitro state. It is shown that protein molecular sieve in the 62% (NH) 2S04 fraction of bovine serum shows fractions enriched with the native bioactive LAIT / sCD14 bovine protein. These enriched fractions show to have specific activity hardly more than the 62% (NH4) 2S04 fraction of bovine milk in the induction of the differentiation of mCD14, the line of preB murine cells mlg 70Z / 3 to a mlg * in-vitro state.

It is shown that LPS, the human and bovine LAIT / sCD14 proteins in their native form and the recombinant forms of bovine LAIT / sCD14 protein produced in the baculovirus and mammalian expression systems, each induce the expression of tracheal antimicrobial peptide ( TAP) in primary tracheal epithelial cells of cattle. EXPERIMENTS Activation of B cells mediated by protein LAIT / sCDl4 in inhibited by MAb 3C10. The mAbs, 3C10 and MEM-18 specific for CD14 inhibit the activation of monocytes and their subsequent production of inflammatory cytokines mediated by LPS-LBP complexes. In addition, mAb 3C10 has been shown to inhibit monocyte activation mediated by sCD14 and that the sCD14 region that interacts with these putative receptors is the same or very close to the mCD14 region in monocytes that interact with the LPS-LBP complexes. Since monocytes are mediated by mCD14, 3C10, the inhibition of the sCD14 function could be due to their interaction with sCD14, mCD14 expressed in the subject monocytes. The results illustrated in Figure IA show the differential ability of mAbs specific for CD14 to inhibit murine B cell activation mediated by the native human LAIT / sCD14 protein. The 3C10 MAb, but not the mAb MEM-18 or its respective control mAb isotypes, inhibits the induction of B cell growth by the LAIT protein, while none of the mAbs inhibits the growth of B cells induced by LPS. The results illustrated in Figure IB show that the CD14-specific mAb 3C10 inhibits the growth of murine B cells mediated by rBo-LAIT / sCD14 and rHu-LAIT / sCD14, while they do not affect the growth of B cells induced by LPS. Since the expression of mCD14 by B cells is unstable, these results do not distinguish between possible mechanisms for carrying out the inhibition of B cell activation mediated by sCD14 due to mAb 3C10. In addition, while the purity of the high density floating murine B cells used as objects in this experiment varies from 88% to 95%, the contaminating cells are mCD14. Specifically, it has been shown that these populations of B cells contain CD14 encoding mRNA (PCT application number No. PCT / CA97 / 00880). In an attempt to clarify the mechanisms that carry out the activation of B cells mediated by LAIT / sCD14 2 experiments were carried out. B cells were isolated based on their expression of mlgP by a fluorescently activated cell computer at more than 99% purity. This population of B cells has been shown to be negative because it contains Cd14 specifically mRNA determined by northern bolt analysis. It was subsequently demonstrated that this population of "CD14" B cells responds robustly to native Bo-LAIT / sCD14 (nBo-LAIT) derived from colostrum as B cells were not purified by this technique. As per this criterion, it appears that nBo-LAIT interacts with a putative receptor on B cells, in an independent way mCD14 (PCT Application No. PCT / CA97 / 00880). A second approach is used to determine the involvement of mCD14 in the LAIT protein by activating B cells involved in demonstrating their ability to induce differentiation of the CD14 cells pre-B line 70Z / 3. LAIT / sCDl4 Includes the differentiation of mCD14 pre-B cells line The negative immunoglobulin membrane (mlg) line 70Z / 3 murine pre-B cells (Paige, CJ et.al. 1'978, J. Immunol .121: 641), does not express mCD14 detectable by imunofluorescence, it is negative for mRNA encoded CD14 as demonstrated by the analysis by both norteen bolt, and RT_PCR (Filipp, D. and M. Julius observation not published: Lee, JD et.al. 1992. J. Exp. .Med, 175: 1697). 70Z / 3 was introduced to the state of simulations mlgp * with LPS or IFN-K) Paig, C.J. et.al. 1978. J. Immunol .121: 641) the results illustrated in Figure 2 show that rBo-LAIT / sCD14 induces differentiation from 70Z / 3 to a mlgk * state as efficiently as LPS. This result indicates that there is a receptor for rBo-LAIT in 70Z / 3 cells, and further, that the receiver can not be mCD14. This point of view provides a means to demonstrate the dependence of mCD14 on the inhibitory mediation of mAb 3C10 of biological activity LAIT / sCD14. The LAIT / sCD14 induction of 70Z / 3 differentiation is inhibited by mAb 310 The results illustrated in Figure 3A show that mAb 3C10 inhibits the induction of LAIT / sCD14 of the expression mlg? in 70Z / 3. The induction by LPS of mlg? it is not inhibited by mAb 3C10, illustrating that the inhibition is specific for LAIT / sCD14 by induction, and further, that the mechanism supporting mAb 2C10 by inhibitors does not involve its direct interaction with 70Z / 3. The results illustrated in Figure 3B show that not all specific CD14 mAbs inhibit LAIT / sCD14 by 70Z / 3 differentiation to a mlg? State. Specifically, the mAb 3C10, but not the mAb MEM-18, also specific for Cdl4 or any of its respective control isotype mAbs, inhibits the induction of 70z / 3 differentiation by recombinant human LAIT / sCDl4 proteins. The results indicate that the mAb 3C10 interacts with the LAIT / sCD14 and conceals the determinants that interact with the putative receptors for LAIT / sCD14 expressed by 70Z / 3. Endotoxin and the LAIT / sCD14 protein share signal pathways.

** "*« - - «- As discussed in the previous section, a number of models have been proposed to explain the mechanisms by which LPS induces the activation of monocytes and their subsequent production of proinflammatory cytokines. of the postulated mechanisms, mCD14 plays a central role in its putative endotoxin receptor function, while the expression of mCD14 reduces the required concentration of LPS to induce cellular activation by order of magnitude, it does not predict LPS-mediated activation. , the concentration of LPS required to induce the expression of mlg in mCD14 * 70/3 is greater than that required to stimulate cytokine production by mCD14 * monocytes (Lee, JD et al., 1992. J. Exp. : 1697) Furthermore, when 70Z / 3 is transfected with cDNA encoding human Cdl4, it has been shown that the required concentration of LPS to induce the expression of mlg by mCD14 * clones was 10,000 times less than the one required in the mCD14 * wild line of 70Z / 3. In addition, the efficacy of the stimulation by LPS in these circumstances was serum dependent, reflecting the participation of LBP (Lee, J.D. and col.1992.J. Exp. Med. 175: 1697). Thus, while emphasizing the central role of mCD14 in mediating cell interaction with LPS-LBP complexes, the results also show that there is an activation path independent of mCD14 used by LPS, which is serum, and therefore, Independent LBP. This questioning leads us to the question of whether the independent paths of mCdl4 related to the activation of 70Z / 3 by means of LPS and LAIT / sCD14 share signal elements. The results shown in Figure 4 indicate that this may be the case. It has previously been shown that lipid diphosphoryl A derived from LPS inhibits the activation of 70Z / 3 by LPS (Kirkland, T.N., Quershi,., And Takayama, K.1991, Infection and I munity 59: 131). In particular, pre-incubation of 70Z / 3 with diphosphoryl lipid A (RSDPLA) inhibits the subsequent expressions of mlg induced by LPS. As shown in Figure 4A, the pre-incubation of 70Z / 3 in a medium containing RSDPLA at 10 μg / ml resulted in a 3 to 4-fold inhibition of the expression of Ig? mediated by LPS. Also shown in Figure 4A that the RSDPLA by itself does not induce the expression Ig? in 70Z / 3 over the tested concentration range, for example 0.1 to 30μg / ml. Figure 4B illustrates that pre-incubation of 70Z / 3 in a medium containing RSDPLA at 10 μg / ml not only inhibits the induction of Ig expression? by nBo-LAIT, but it does so much more effectively than when LPS is used as a stimulus, resulting in at least 10-fold inhibition of nBo-LAIT-mediated induction over all the nBo-LAIT concentration ranges tested. It is illustrated in Figure 4C that while the RSDPLA inhibits the induction of the expression mlg? caused by LPS and by nBo-LAIT / sCD14 in 70Z / 3. The ability of RSDPLA to inhibit activation mediated by LAIT / sCD14 could indicate that LAIT / sCD14 and LPS share common receptor elements and / or could be due to RSDPLA interacting with the LPS interaction sequence previously characterized in CD14, residues 57/64 ( Juan, TS-Cy col 1995, J. Biol Chem 270: 5219), who in turn could inhibit LAIT / sCD14 activity in these trials, despite the LPS independence of the LAIT / sCD14 function. The possibility that RSDPLA inhibits the function of the LAIT / sCD14 protein by directly binding LAIT / sCD14 protein was determined as shown in Figure 5. Figure 5A shows a LAIT / sCD14 protein scheme and highlights the two regions of the molecule that characterize the binding sites of mAbs 3C10 and MEM-18. It has been shown that MEM-18 blocks the binding of CD14 with LPS and thus residues 57-65 are indicated as the LPS binding site on CD14. Figure 5B illustrates the two potential mechanisms that could lead to the inhibition of the function of the LAIT / sCD14 protein mediated by RSDPLA. RSDPLA interacts directly with putative receptor elements shared by LPS and LAIT / sCD14 protein, or BIND protein LAIT / sCD14 can interact directly. If the latter were the case, then blocking the interaction of RSDPLA with the LAIT / sCD14 protein using mAb MEM-18 (Figure 5B), should interfere with the RSDLPA inhibition of the function of the LAIT / sCD14 protein. As shown in Figure 5C, pre-incubation of the native human LAIT / sCD14 protein with mAb MEM-18 does not alter the ability of RSDPLA to inhibit the induction of mlg expression. of 70Z / 3 caused by LAIT protein. Here it has been shown that mAb 3C10 but not mAb MEM-18 inhibits the function of LAIT / sCD14 in mature murine B cells (FIG. IA) and in line 70Z / 3 of murine pre-B cells (FIGS. 3A and 3B) . As described above, mAb 3C10 recognizes a sequence in CD14, residues 7 to 14, which is essential for signaling mediated by LPS but not LPS binding (Juan, TS-Cy col. 1995. J. Biol. Chem.270 : 17237). Since mAb 3C10 inhibits the independent LAIT / sCD14 LPS signaling, it follows that residues 7 to 14 in LAIT / sCD14 are related to the interaction of this ligand with putative membrane receptor structures. Furthermore, it has been demonstrated that the MEM-18 mAb that is specific for the sequence in LPS binding related to CD14, and is capable of blocking the LPS-Co14 interaction, has no effect on the inhibition of LAIT / sCD14-mediated protein activation. RSDPLA. Thus, even if it is not shown definitively, the interpretation that the RSDPLA blocks the LAIT / sCD14 signaling as a consequence of competition for common receiving elements is favored. LAIT / sCDl4 is enriched in human milk and colostrum Given the possible importance of the role of soluble Hu-LAIT in infants neonates, the expression pattern of Hu-LAIT in human females after parturition was examined. Colostrum and milk samples were obtained from 9 human subjects at various post-partum times. As it is known, that the serum of healthy human subjects contains between 1-5 μg / ml sCD14, serum samples were taken from the aforementioned subjects to determine if they contained sCD14 concentrations parallel to those observed in mammary secretions. Cd14 was quantified using commercially available ELISA equipment and the total protein was determined using a commercially available colorimetric detection system. The results shown in Figure 6A are presented as μg of CD14 / mg total protein of each of the paired samples obtained from milk and serum as illustrated, human milk contains 100-400 times more sCD14 than the serum of the same individual. It is also shown that the enrichment of sCD14 in milk vs serum persists more than 400 days postpartum. Heat instability of affinity-purified nBo-LAIT Care should be taken to avoid conditions that decrease the desired activity of variant CD14 in the polypeptide of interest. Figure 6B shows the effects of boiling nBo-LAIT of purified affinity at 99 ° C for 10 minutes. The wide reduction in the thymidine consumption by B cells, shows a dramatic reduction if not total of the stimulating activity of B cells. Partial Purification of Protein LAIT / sCD14 Bioactive Milk Bovine; Colostrum- and Milk Derivatives LAIT-Protein / sCD14 Bovine have Comparable Biological Activity. It was previously known that soluble CD14 is present in cow's milk. In the context of this invention, the term "mammary secretion" includes colostrum and milk. Colostrum is a breast secretion that begins at the time a shoot is born and continues for a relatively normal period of time, not longer than 24 hours. "Milk" is the mammary secretion that follows the colostrum. An expert person can distinguish between colostrum and milk. Colostrum is usually obtained during the first hours postpartum and before the start of lactation. In the experiments with PCT / CA 97/00880 described here, in which bovine colostrum was used as a source of bo-LAIT, colostrum was obtained during the next postpartum hour and before breast-feeding. The method previously used to isolate LAIT from bovine colostrum (PCT / CA 97/00880) here was used to obtain bo-LAIT from Milk. Whole unpasteurized cow's milk was obtained from a research ranch. It was determined that the milk was sterile to determine the growth in liquid broth and in platelets from blood perforations. Only aseptic material was used in the experiments described here. The clarified milk was obtained by centrifuging colostrum, first at 4420g for 30 minutes to remove cells and cell debris. The supernatant of this spin was when centrifuged at 250,000g for two hours. The floating lipids and the casein in pellets were discarded, and the clarified colostral was subjected to later fragmentations. Salting the proteins contained in the milk the preparations were achieved using sequential precipitation in (NH4) 2S04 adding a saturated solution of ammonium sulfate (AS). The ascending sequence of salt concentrations used were 42%: 50%: 62%: 65% (v / v) ammonium sulfate (AS) Although the concentration of AS in the supernatant of the material precipitated at 42% was increased to 50%; the material precipitated at 50% was rescued, and the concentration of AS in the remaining supernatant increased to 62%, and so on. Each PELLET precipitation of AS was soluble in lOmM Tris-HCL pH 8.0, containing 0.15M MaCl and 1 mM AEBSF (TNAEBSF). Salting the proteins contained in the bovine milk using (NH4) 2S0 resulted in enrichment of the native bovine LAIT-proteins / sCDl4 in the 62% action (NH) 2S04 (compare Figures 7A and 7B). Protein concentrations in 62% (NH4) 2S04 fractions derived from bovine and colostral milk ARE 8-15mg / ml and 47-65mg / ml, respectively. The concentrations fr LAIT-Proteins / sCD14 in 62% (NH4) 2S04 fractions derived from milk and colostrum are 1-5μg / ml and 5-12μg / ml respectively. Although LAIT-protein / sCD14 yields from these two sources is comparable at 0.15-0.26μg / mg protein. The activity of the 62% fraction was subsequently enriched. Fifty milligrams of the 62% enriched fraction of AS was applied to an exchangeable anion column, and the material separated by the use of the gradient salt of 50mM up to 400mM NaCl in lOmM Bis-tris propane, simultaneously, gradient pH from 7.5 to 9.5 . Shown in Figure 7C, it is a comparative analysis of partially purified LAIT-protein / sCD14 bovine milk fractions, with that of the affinity of the purified material derived from the same source to induce the expression of mlgk in 70Z / 3. As illustrated, the 62% (NH4) 2S04 fraction had specific activity almost 10,000-fold lower than the affinities of the purified milk derivative LAIT-protein / sCD14. Also shown in Figure 7C is the biological activity of the fractions containing the majority of LAIT-protein / sCD14 (determined by immunostaining analysis) obtaining subsequent fractions of the 62% (NH4) 2S04 fraction in a Sephadex 75 molecular sieve resin . The specific activity of fractions 47-49 were 100-fold higher than those of 62% (NH4) 2S04 fraction and correlated with comparable increases in the concentration of LAIT-protein / sCD1. Figure 7D shows a comparative analysis of milk- and cow milk colostrum derivatives LAIT-protein / sCD14 by introducing B-cell growth. As illustrated, the specific activity of the material derived from bovine milk was as high as the material Colostrum derivative. The annex in Figure 7D illustrates an immunostaining with an affinity of LAIT-protein / sCD14 milk derivatives and colostrum. It has been shown that the LAIT-protein / sCD14 occurs naturally in bovine milk and it is possible to concentrate and isolate the milk protein with retention of the activation activity of the B cell. LAIT-protein / sCDl4 induces specific TAP mRNA in Primary tracheal epithelial cells in Bovine. The possibility that LPS and LAIT / sCD14 share common receptive elements, as described above, leads to the possibility that they may have common biological activities. The possibility that LAIT / sCD14 has a role in the introduction of defenses was explored. Specific defense molecules were recently described in bovines (Diamond, G.J.P. Russell, and C.L. Bevins, 1996. PNAS 93: 5156, Schonwetter, B.S., Stolzenberg, E.D. and M.A. Zasloff, 1995. Science 267: 1645). OLIGONUCLEOTIDE tests for bovine TAP were prepared to determine the relative capacities of LPS and LAIT / sCD14 to induce the message for this antibiotic peptide in vitro. Figure 8A shows the sequence of oligonucleotide probes used to detect tracheal antimicrobial peptide (TAP) in bovine tracheal epithelial cells. TAP is a 38 amino acid polypeptide and its expression appears to be restricted to columnar epithelial cells of the bovine respiratory tract (Diamond, G. et al 1996. Proc.Nat.Acid.Sci.USA 93: 5156). Primary tracheal epithelial cattle were prepared according to the methods described above (Diamond, G. et. al 1996. Proc. Na ti. Acad. Sci. USA 93: 5156). Wells containing approximately 5x005 epithelial cells in 1 ml of serum-free medium per well, 24 well-culture well plates were prepared and stimulated for 16 hours at the indicated concentrations (Figure 9A) of LPS, natural forms in human LAIT-protein / sCD14 and bovine, or recombined forms Bo-LAIT / sCD14 prepared using either, mammalian expression system (C127) or a baculovirus expression system (Sf9). In the experiments, bovine LAIT obtained from milk was used. As illustrated in Figure 9A, 1 μg / ml of any LAIT / sCD14, either natural or combined, includes comparable levels of TAP specifically mRNA as well as 1 μg / ml of LPS, and as a result increases the observed signal by 15-20 times. of non-simulated cells. Figure 9A also shows the signals obtained for the mRNA specific for bovine tubulin, which indicated that comparable amounts of RNA were loaded in each route, which were used to normalize TAP mRNA signals. Figure 9B shows a comparative analysis of kinetics in which 1 μg / ml of any LPS or LAIT-protein / sCD14 from natural bovine, induces TAP mRNA in cultures of primary tracheal epithelial cells of bovine. The epithelial cells were cultures as described in Figure 9A and the expression of TAP specifically mRNA was determined at the indicated time points. As illustrated, both induce a peak expression of TAP mRNA, normalizing to the level of tubulin mRNA in 16 hours (Figure 9B). The present invention provides a method for preparing CD14 concentrate from mammary secretion. The secretion may be from Colostrum or milk, or both. The secretion can be human or cow or other species. With respect to the industrial process, the production of CD14 endogenous bovine concentrate, as occurs naturally in cow's milk, seems promising. Under certain circumstances it would be preferable to manipulate an organism genetically to produce CD14. This is described soon. As well as it is possible to diminish or destroy beneficial qualities (induction of defensin activity, and / or stimulation of activity of B cells) of CD14 through the heat treatment that is given to milk. It would be necessary or desirable to obtain CD14 without this previous treatment. One way in which CD14 can be "concentrated" is by salping the procedure described above. However, this is not the only method available to a specialist. For example, ion chromatographic exchange, chromatographic sieving of molecules can be used. The methods described herein can be improved and optimized to commercialize the production of concentrates or CD14 isolates. Additionally, other components of colostrum or milk, which would be undesirable, can be removed in accordance with procedures known to the specialists. Here some examples are described, in which the milk is subjected to a centrifugation and floating lipids are removed, and the liquid containing protein decanted from the pelletcasein formed during the centrifugation. CD14"isolated" is a CD14 protein that has been identified and separated from at least one contaminant with which it is normally associated in nature, as it is formed by animals or the human source of CD14. In preferred embodiments in which CD14 is intended to be used as a medicine, CD14 will be isolated to the pharmaceutically acceptable standard levels of purity with respect to proteins in the original species. In preferred embodiments, the CD14 protein will be purified (1) at a maximum of 95% by weight of protein determined in the Lowry Method, and preferably more than 99% by weight. These are the preferable degrees of isolation. A "concentrate" CD14 is a CD14 protein obtained from a natural source, has been treated to present in the concentrate in higher concentration than in the one obtained from the natural source. By CD14"soluble", it is understood, a molecule of CD14 that is not bound to the membrane, such as by a glycosylphosphatidyl-inositol anchor. As far as the treatment is concerned, it is important that the desired activity of CD14 is retained in a CD14 concentrate. By producing CD14 in order to activate B cells, a solution containing CD14 that is not heated above 99 C for 10 minutes, for example, these conditions are known to severely reduce this type of CD14 activity. See Figure 6B in which it is shown that the purity affinity activity n-bo-LAIT is destroyed under those conditions.

Using the techniques described here, a specialist can determine conditions that are harmful to a desirable activity or that are relatively benign. For example, the effects of variation in time and temperature can be applied (eg 40 ° C, 50 ° C, 60 ° C, 70 ° C, etc.), it can be applied to solutions containing CD14 for several periods of time ( for example 1 minute, 2 minutes, 3 minutes ... 10 minutes) and the effect on the simulated activity of B cells determined in accordance with the methods set forth herein. Thus, the effect of these conditions on the defensin epithelial cell induction can be quickly determined using a similar scheme. The effect of other conditions, say the effects of salt, or other chemicals that can be used in the treatment of milk can be determined promptly. Care should be taken to prevent exposure of CD14 to conditions that are adverse to the desired activity. Since CD14 is a concentrate, the amount of concentration of CD14 can be determined according to routine methods. A popular assay is the ELISA assay based on chemiluminescence. For the detection of human CD14, commercially available packages (IBL, Hamburg, Germany) can be used. For the detection of bovine CD14, or any other species. As such an ELISA can be quickly developed by a specialist, an example of this follows. A recombination of CD14 is prepared, as described by PCT application No. PCT / CA 97/00880. The recombined material is generally used to generate a standard curve. Ployclonal antibodies are generated in a rabbit, against the recombined molecule and isolated fractions of IgG. The IgG is then immobilized on an ELISA plate and washed to remove excess (non-adhered) immunoglobulin. This is preceded by a sum of BSA (bovine serum albumin) to "block" inactivated sites on the ELISA plate. The recombinant protein is titrated on a table of concentration ranges, e.j. from 100 μg to 1 ng per ml in 10-fold increments. The plate was washed. An antibody is labeled by conjugating it with an enzyme, for example, strong root peroxide, and its exposure to the titration plates, incubated and washed with antibodies. The antibody binding is revealed through the substrate of an additional enzyme and developed and read in an ELISA plate reader. A linear portion in the curve (absorption against protein concentrates) is taken as standard since it has generally been found to be an appropriate range. The amount of bound antibodies (as indicated by optical density) corresponds to a known protein concentration. To determine the concentration of a sample of an unknown protein, serially dissolved samples of the unknown protein are added to a new plate where an acceptable absorption is found (one that falls within the range of the standard curve) and the amount of the sample present unknown protein calculated. This type of assay can be routinely developed for CD14 of any species in which a polyclonal is obtained. When it is said that an antibody is "specific for" a polypeptide having a particular amino acid sequence (or other antigen), it means, as would be understood by a skilled person, that the antibody and the polypeptide are each mixed in a selective manner and the polypeptide will not mix with a multitude of other antibodies that can be evoked by other antigens. In such a case, it can be said equitably that the polypeptide is "specifically recognized by" the antibody. Once the concentration of a concentrated CD14 die is determined, provided it is free of other unwanted components, it is then useful in incorporation into medicaments, foodstuffs, etc., in desirable quantities. For example, 0.1 (5) g of a concentrate containing 100 Tg / g of CD14 would be incorporated into the food bar which contains 10 (500) Tg of CD14.

It would be desirable to convert a concentrated liquid to a solid form, for example, by subjecting the liquid to evaporation, lyophilization, or some other technique. There will be times when it would be desirable to include one or more condoms to maintain CD14 activity, to be included in a liquid, such as infant formula, for example. The aspects of this invention are surprisingly simple, but powerful. For example, one aspect in the invention is the method for detecting the presence of CD14 in a composition containing protein from a mammary secretion. Such a method, being a direct application, it was not possible to know a priori that CD14 occurs naturally in mammary secretions, particularly in bovine milk. In accordance with the method, the composition is exposed to an antibody specific for CD14 and it is determined whether CD14 endogenous to secretion is present in the sample based on the fact that the antibody-CD14 complex was formed in the step of exposure, using for example, an ELISA assay. The invention also provides a method for determining the amount of endogenous CD14 contained in a composition containing mammary secretion proteins, particularly bovine milk. The composition is provided. The sample is exposed to an antibody that is specific for CD14 and the amount of endogenous CD14 to secretion is present in the sample based on the amount of complex forms of CD14-antibody formed in the exposure stage. Again, the ELISA assay can be used. According to these methods, colostrum or human or bovine milk can be used as a source of obtaining CD14. In another aspect, the invention is a method for determining the appropriateness of a product derived from mammary secretion to be used properly. E.j. its use for the induction or stimulation of defensin production (or activation of B cells) in mammals. The method includes a sample of the product and determines the amount of CD14 in the current sample. Knowing the amount of CD14 of a sample, a qualified person, could incorporate an appropriate amount of the product to the composition of its use, in accordance with the desired activity. For example, a use of soluble human CD14 is described in "U.S. Patent No. 5,804,189 and determines the amount of bovine CD14 the results will be comparable within the capabilities of the skilled person." In accordance with certain aspects of this invention , the protein that has activity similar to CD14, (eg B cells stimulating activity, or defensive induction activity), can occur naturally, as in mammary secretions, or can be manufactured according to chemical or recombined techniques. "Recombinant" or polypeptide is that produced using molecular genetic techniques to express an isolated nucleic acid sequence, as would be understood by a specialist.In this specification, homology is calculated by standard methods that involve aligning two sequences to be compared and occur. maximum compatibility, calculating the percentage of coincidences In a preferable aspect, the invention ion is a method to lessen the symptoms of asepsis comprises the administration to a mammal in need of this, an effective amount of a soluble protein to thereby expose mammalian epithelial cells directly to the protein, where the protein has an amino acid sequence that is preserved at least 63% in relation to the amino acid sequence identified as SEC. IDENT. NO: 5 and has the ability to induce defensin expressions in epithelial cells. In other words, the homologous of the sequence in question has at least 63% homology with SEC. IDENT. NO: 5. Substances, substantially equivalent to these, include those in which one or more residues of the natural sequence is deleted, substituted or inserted by an amino acid or a different acid.

It is possible to vary the polypeptide sequences described herein while retaining at least a portion of the desired activity. Preferred variants include conservative substitutions for example ones in which one residue is replaced by another of the same general type. As it is understood, naturally occurring amino acids can be subclassified as acidic, neutral and polar, or neutral or non-polar. It will also be understood, without intending to be limited, that the variety of amino acid substitutes is possible as long as the structure responsible for the bone simulating effect of the polypeptides described herein is "preserved". It is expected, for example, would be possible the exchange between non-polar neutral aliphatic amino acids, glycine, alanine, proline, valine and isoleucine. As well as it would be possible to make the substitutions between neutral aliphatic polar amino acids, serine, trenina, methionine, cysteine, asparagine and glutamine. Substitutions between charged basic acidic amino acids, aspartic acid, and glutamic acid, could be made, as well as substitutions between charged basic amino acids, lisien, and arginine. Substitutions between aromatic amino acids could be made, including phenialanine, histidine, tryptophan and tyrosine. This type of substitutions and exchanges are well known to specialists. Other substitutions might well be possible. This type of substitutions and exchanges are well known to the specialists, as exemplified by U.S. Patent No. 5,487,983. Instruction to determine which amino acid residue could be substituted, inserted, or deleted without abolishing any biological or immunological activity, can be found using a computer program known in the medium as DNASTAR software. "Sequence of Identity or Homology" refers to the sequence similarity between two polypeptide molecules. When a position in any of the compared polypeptides is, for example, occupied by the same amino acid (for example, if the position in each of the two polypeptide molecules is an alanine residue, then the molecules are homologous or their sequences are identical in The percentage of homology between two molecules or sequential identity between two sequences is a function in the number of such position points shared by the two sequences divided by the number of positions compared xlOO For example, if 6 of 10, of the positions in two sequences are the same then both sequences are 60% homologous or have 60% sequential identity.For example, the METLIA sequence of polypeptide and MPTWIF are homogeneous or sequentially identical by 50%. - It is done when two sequences are aligned to give maximum homogeneity.

The comparison of sequences and the determination of the percentage of homogeneity between two sequences can be achieved using mathematical logarithms. In this specification, the alignment can be done according to the Clustal method. The Clustal logarithm (as applied here using the software available from DNASTAR Inc., 1228 South Park Street, Madison, WI, USA, 1994) is recommended to align sequences in which their similarities are not necessarily evolutionary. The logarithm is described by Higgins, D.G. et ai. 1989. CABIOS 5: 151. The same software program provides to align the sequences according to the Jotun Hein method, which is recommended for the alignment of sequences of highly evolved families where they clearly have an evolutionary relationship. The logarithm is described by Hein, J. 1990 Methods in Enzymology 183: 626. Circumstances where the program fails (standard parameters) are used. In the case of heavy residues amino acids based on evolved patterns of substitution with similarity, load, structural and chemical, mutation of acceptable percentage (MAP) circumstances of 250 is selected. For protein alignments, the parameters of alignment in pairs are K-tuple = l, Gap penalty = 3, Window = 5, and Diagonals Saved = 5 are used.

In a preferred aspect, the present invention is a method of ameliorating aseptic symptoms comprising administering to a mammal in need of this an effective amount of a soluble protein to be directly exposed to the epithelial cells of the mammal, the protein having a sequence of amino acids which is at least 63% conserved in relation to the sequence of amino acids identified as SEC. IDENT. NO: 5 and having the ability to induce expression of defensins in epithelial cells. In other words, the homogeneity of the sequences in question has at least 63% homogeneity with SEC. IDENT. NO: 5. The present invention also provides compositions containing an effective amount of compounds of the present invention, including additional non-toxic salts, t-amides, which alone can serve to provide the benefits of the treatment described above. Such compositions can also be provided together with physiologically tolerable liquid, gel or solids diluents, adjuvants and excipients. In situations involving CD14 proteins, approximately 250 to 300 μg of polypeptide per kg body weight of a mammal can be fed, as an example, a mammal has an average consumption per day between 18 and 36 ml of fluid per day of fluid. It will be appreciated that the consumption of young mammals increases over time. The dose administered will be based on measurements of CD14 concentration in adult breast milk from 10 to 20 μg per ml, considering that a human increases their milk consumption from around 0.11 to 11 per day for the first six months after the birth and assuming a weight ratio of around 28 between humans and rats. In practice, particularly as it concerns human subjects, the daily dose may be formed around 250 μg and about 2500 μg or more per kg of body weight per day. Preferably, the dosage would be in the vicinity of about 300 μg to about 1 mg per kg of body weight per day. It could be that the preferable frequency of administration is greater or less than once per day, depending on the route of administration, convenience, and the variation in the effectiveness of the treatment with frequency of and amount used per administration. The dose administered also depends on the subject to which such administration will give it. The dose of one or more of the compounds will depend on many factors including the specific compound or the combination of compounds being used, the route of administration, and the treated mammal. Dosages of a particular compound or combination of compounds can be determined using conventional considerations, for example, by comparing the differential activities of the subject compounds and the known agent, which is, by an appropriate pharmaceutical protocol. The pharmaceutical preparations include any of the compounds prepared just before use. An injectable dose can be a liquid solution or a suspension; Suitable solid forms for solution in, or suspension in, the liquid can be prepared before injection. The preparation can be emulsifying day. The active protein is often mixed with diluents and excipients that are physiologically tolerable and compatible with the polypeptide. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. Also, if desired, the compositions may contain lesser amount of auxiliary substances as well as emulsifying or wetting agents, stabilizers or pH buffering agents, and the like. The pharmaceutical compositions include the use of compounds mixed with conventional excipients, these being carriers of pharmaceutically acceptable organic or inorganic substances which do not deleteriously react with the compounds, which possibly increases storage and provides stability of the compounds. Preparative procedures may include sterilization of pharmaceutical preparations. The compounds can be mixed with auxiliary agents such as lubricants, preservatives, salts to influence the osmotic pressure, etc., which do not react deleteriously with the compounds. The compositions are perenteral and conventionally administered, by injection, for example, subcutaneous or intravenous. Additional formulas that are appropriate for other routes of administration include suppositories, intranasal sprays, and in some cases, oral formulas. For suppositories, traditional mixers and excipients may be included, for example, polyalkylene glycols or triglycerides; as some suppositories can be made of mixtures containing active ingredients in the range of 0.5% to 10%, preferably l% -2%. Oral formulations include such excipients normally used as, for example, pharmaceutical grades of mannitol, lactose, cornstarch, magnesium stearates, sodium saccharin, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, lozenges, capsules, sustained sustained formulas, or powders, and active ingredients containing 10% -95%, preferably 25% -70%. Oral formulations may include formulas designed to protect the protein while reaching the appropriate site for the intended action.

Protein compounds can be formulated in compositions such as neutral or salt forms. Pharmaceutically acceptable non-toxic salts (formed with free amino groups) and which are formed by inorganic acids such as, for example, hydrochloric acid or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, and the like. The salts formed by the free carboxyl groups are derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, ethanolamine-2, histidine, procaine, or the like. The compounds of the invention can be homopolymerized to themselves. The compounds can also be conjugated to biocompatible polymer compounds, such as BIOPOL ™ (WR Grace &Co. Conn.). It is possible to find methods known in the specialty of those who make formulas in, for example, "Remington's Pharmaceutical Sciences". In the case of the general health promotion regime, an oral route of administration is preferable. CD14 being present in a liquid such as Gl the trachea is exposed to CD14 at the time of swallowing the liquid. It could be contained in chewy and malleable substances which protect the CD14 from degradation but which releases the CD14 when it is chewed, for subsequent expositions of it to the Gl tract. The compositions of the present invention could be applied topically to the wound site or to any suitable pharmaceutically acceptable carrier, for example, a liquid carrier such as ethanol, propylene glycol, chloroform, ethanol, propylene glycol, or the like. A possible concentration of an active compound in these compositions is at least 0.01% by weight, probably between 0.1% and 0.5% by weight and still more likely between 0.05% and 0.2% by weight, but any therapeutically effective concentration can be used. The composition presented in this invention can also be formulated in many other ways, depending on whether it is an aqueous solution, cream or ointment if so desired, and whether it would be used / or its site of use is on the surface of the skin or in the eye. Compositions in cream formula may contain a stabilizing cream such as xanthene gum, and preferably non-ionic emulsifier, at least one liquid and a solid hydrophobic material selected from liquids and fatty acids, fatty alcohols, fatty acid esters, pharmaceutical tiers of wax and hydrocarbons, the later range goes from liquids to semi-liquids such as petrolatum, to solids or similar, preservatives, antioxidants and water. Methods that promote the healing of a wound by reducing skin infection, include applying or contacting the composition of the present invention which promotes the production of defensins in epithelial cells directly in the wound. The composition can remain in contact with the wound for a period of time sufficient to help reduce infection, for example, such methods include incorporating compositions of the present invention into a cream formula or soaking a gauze dressing with a solution of the composition and then applying the cream or wetting a gauze on the wound is a burn, donor site, ulcer, or any type of skin wound. Additionally, staples or sutures may be covered or submerged in the composition of the invention and used to close an open wound. The type of wounds in which the composition of this invention can be used includes those that result from an accidental or medical wound that causes epithelial damage such as ophthalmic wounds, such as those resulting from a cornea ulcer, skin wounds, like burns, DONOR SITE wounds, like those that result from a transplant and ulcers. Additionally, dermatological conditions in which the skin has been abused can be treated with the composition of this invention. Foot and foot ulcers can be treated with the composition of this invention. The aerosol delivery route of the compositions of the present invention would be particularly suitable for exposing the trachea and lungs to CD14. In any case, the advantage of exposing a bacterium (or virus, fungus, etc.) to one or more defensins during ingestion or inhalation, etc., would be generally understood. This would be especially true in the case of subjects not necessarily capable of responding immunologically totally or normally. In the case of gram-negative bacteria form LPS may be generated, the presence of one or more defensins in the Gl tract or trachea would be especially advantageous for those who may be at high risk of septic shock. In ortho appearance, the present invention includes a transgeneric mammal, which has been genetically altered to provide increased production of CD14 in milk. Although the technology goes on to produce transgeneric mice has been used in the last two decades, the ease of introducing genes foreign to the cattle genome, including goats, sheep, cows and pigs has only recently been demonstrated. Although technically it is more difficult, the introduction of molecules encoding cloned genes of pharmaceutical importance to cattle provides the advantage of being possible to increase the scale of production of recombinant proteins. This could dramatically lower the cost of production. In this way transgenic cattle can be considered as a "bioreactor" producing pharmacologically useful proteins in milk, blood or urine. Over recent years the presence of proteins in blood and tissue has significantly expanded the potential of transgenic animal applications in medicine. For example, the creation of "stealth animal organs" for the use of transplants in humans has recently received a lot of attention. Significant and significant progress has been made in the ability to express transgenic proteins in milk. Studies have focused on the direct expression of proteins to the mammary gland, and thus improving its productive level. The production technique of transgeneric animals follows established principles (Marki, U and Harri, A. 1996. Int. J. Exp. Pa 77: 247). The gene of interest is combined with a promoter sequence that directs and regulates the expression of that gene in a tissue or model cell specific in the transgeneric animal. The expression of construction (DNA) is injected into the pronucleus of a fertilized ova, and the injected ova are implanted into the hormonally synchronized adoptive mother. The animal resulting from this artificial insemination are controlled in the integration of the DNA construct. The offspring of the founders are tested for the expression of the gene, and its product and the inheritance pattern of the gene to ensure the line of transmission of germs, that is, that the transgene has been incorporated into the line of germs of the animal. A preferred aspect, the invention is a transgenic bovine mammal in which CD14 (or analogue thereof which directly activates B cells or which directly induces defensin production in epithelial cells) is introduced by technologically recombining the DNA to introduce it to the mammal. be under the control of the endogenous CD14 promoter. Another preferred aspect, the transgenic mammal is a bovine having a genome to which additional copies of the genomic sequence (exon and intron, for example SEQ ID NO: 8) have been introduced from which endogenous bovine CD14 is derived. encodes the mRNA. Soluble CD14 is a natural component of milk and colostrum of three species that have been tested: human, cow, and mouse. Although the molecular basis leading to the appearance of CD14 soluble in these secretions is not well understood, it is possible that the endogenous CD14 promoter has an important role in this respect. Therefore, the endogenous CD14 promoter sequence is used to achieve expression of the transgenic CD14, as well as the tissue-specific promoter, which selectively activates the expression of transgenic CD14 in mammary tissues. The use of genomic sequences instead of cDNA in the preparation of transgenic animals has been shown to produce a more efficient expression of the transgene (Janne, J. Et al 1994. Int. J Biochem. 26: 859). In this manner genomic DNA encoding CD14, bovine and / or human can be cloned in combination with endogenous promoter sequences of CD14, or alternatively, the ovine? -lactoglobulin promoter (Wright, G. et al., 1991. Biotechnology 9: 830 ) that directs and regulates the expression of the gene predominantly to the mammary gland. A vector can be added that transports the gene construct with efficient transcription terminating factors as well as regions adhered to the matrix and specialized chromatin sequences that serve to increase the expression of the transgene and isolate it from the negative regulatory effect of the chromatin environment. This construct is injected into the pronucleus of an ovule of the desired species and implanted in a container of the same hormonally synchronized species. Each of the citations referred to in this application is incorporated herein by reference in its entirety. Also incorporated by reference are the complete specifications and claims of U.S. Provisional Patent Application No. 60 / 086,884, received on May 27, 1999, of which this application claims priority and which is currently pending (as of date of this application) U.S. Patent Application Serial No. 08 / 746,883 received November 18, 1996. The polypeptide and protein sequences discussed herein are identified as follows: SEC. FROM IDENT. NO: 1: nucleic acid coding sequence for bovine CD14. SEC. IDENT. NO: 2: Nucleic acid coding sequence for human CD14 SEC. IDENT. NO: 3: Sequence encoding nucleic acids for murine CD14 SEC. IDENT. NO: 4: Sequence of amino acids for bovine CD14 SEC. IDENT. NO: 5: Sequence of amino acids for human CD14 SEC. IDENT. NO: 6: Sequence of amino acids for murine CD14 SEC. IDENT. NO: 7: Amino acid sequence for Rabbit CD14 SEC. IDENT. NO: 8: Genomic nucleic acid sequence for bovine CD14. In SEC. FROM IDENT. NO: 8 Bases 1 to 83 are the 5 'untranslated region, 84 to 86 is the initial ATG codon. 87 to 163 in an intron, bases 164 to 1282 is a coding sequence and bases 1283 to 1405 are the 3 'untranslated region. The amino acid sequence for rabbit CD14 is previously known (Ikeda, A. et al 1997 J. Vet. Med. Sci. 59: 715) The homology found between human and cattle was 70.8%, between human and mouse 63.4% , between human and rabbit of 71.8% and between cattle and mice was 58.7%.

Claims (137)

1. CLAIMS 1. A method for ameliorating the symptoms of asepsis comprising directly exposing the epithelial cells of a mammal in need thereof in an effective amount of a compound comprising soluble CD14, or a fragment of the CD14 polypeptide that stimulates the expression of a defensin in epithelial cells, or a conservatively substituted variant of CD14 that stimulates expression.
2. 2. A method that increases the expression of defensins in a mammal that requires it by administering a compound characterized in that it comprises soluble CD14 or a polypeptide portion of CD14 that stimulates such expression, or a conservatively substituted variant of CD14 or the that increases such expression.
3. 3. The method according to claim 2, characterized in that the administration step includes direct exposure of the epithelial cells of the mammal to the compound.
4. 4. A method that stimulates the expression of one or more defensins by epithelial cells by administering thereto an effective amount of a compound characterized in that it comprises soluble CD14 or a fragment of CD14 polypeptide that stimulates such expression, or a variant conservatively substituted of such CD14 or the portion that stimulates such expression.
5. 5. A method for stimulating the expression of a defensin in the digestive tract of a mammal characterized in that it comprises exposing the tract to an effective amount of a compound comprising soluble CD14 or a fragment of CD14 polypeptide that stimulates such expression, or a conservatively substituted variant of CD14 or the portion that stimulates such expression.
6. 6. A method for stimulating the expression of a defensin in the respiratory tract of a mammal comprising tract exposure to an effective amount of a compound comprising soluble CD14 or a portion of CD14 polypeptide that stimulates such expression, or a variant conservatively substituted of CD14 or the portion that stimulates such expression.
7. 7. A method for stimulating the expression of a defensin in the tongue of a mammal comprising exposing the tongue to an effective amount of a compound comprising soluble CD14 or a fragment of CD14 polypeptide that stimulates such expression, or a variant conservatively substituted of CD14 or the portion that stimulates such expression.
8. 8. A method for stimulating the expression of a defensin α in the small intestine of a mammal comprising exposing the intestine to an effective amount of a compound comprising soluble CD14 or a fragment of CD14 polypeptide that stimulates such expression, or a conservatively substituted variant of CD14 or the portion that stimulates such expression.
9. 9. A method for inducing expression by epithelial cells of a mammal that requires it, the method comprising administering an effective amount of a compound comprising soluble CD14 or a fragment of CD14 polypeptide that induces such expression, or a conservatively variant substituted for CD14 or the fragment that induces such an expression.
10. 10. The method according to any of the preceding claims characterized in that the CD14 has a sequence of amino acids selected from a group consisting of SEC. IDENT. NO: 4, SEC. IDENT. NO: 5, SEC. IDENT. NO: 6 or SEC. IDENT. NO: 7. The method according to claim 10, characterized in that the compound comprises an amino acid sequence selected from the group consisting of SEC. IDENT. NO: 4, SEC. IDENT. NO: 5, SEC. IDENT. NO: 6 or SEC. ID NO: 7, or a conservatively substituted variant thereof. The method according to claim 11, characterized in that the compound comprises an amino acid sequence selected from the group consisting of SEC. IDENT. N0: 4, SEC. IDENT. NO: 5, SEC. IDENT. NO: 6 or SEc. IDENT. NO: 7. 13. A method that improves the symptoms of asepsis and characterized in that it comprises administration to a mammal that requires it of an effective amount of a soluble protein so that it is directly exposed to the epithelial cells of the mammal to the protein, the protein containing an amino acid sequence that is conserved at least 63% in relation to the sequence of amino acids identified as SEC. IDENT. N0: 5, and having the ability to induce the expression of defensins in epithelial cells. The method according to claim 13, characterized in that the protein has an amino acid sequence that is at least about 68% or about 71% or about 73% or about 78% or about 83% or about 88% or about 93% or approximately 98% conserved in relation to the amino acid sequence identified as SEC. IDENT. NO: 15. A method for prophylactically treating a host inflammatory response induced by lipopolysaccharide in a mammal, which method is characterized in that it comprises administering a therapeutically effective amount of the protein to the mammal in order to directly expose the epithelial cells of the mammal. to the protein, the protein containing an amino acid sequence that is at least 63% conserved in relation to the amino acid sequence identified as SEC. IDENT. NO: 4 or identified as SEC. ID NO: 5 or identified as SEC. IDENT. NO: 6 and having the ability to increase the expression of one or more defensins in bovine epithelial cells. 16. The method of compliance with the claim 15, characterized in that the protein has an amino acid sequence that is at least about 68% or about 71% or about 73% or about 78% or about 83% or about 88% or about 93% or about 98% conserved in relation to the amino acid sequence identified as SEC. IDENT. NO: 5 17. A method for increasing the expression of defensins in a mammal that requires it, by administering an effective amount of soluble protein to the mammal, the protein is characterized by having an amino acid sequence that is at least approximately 63% conserved in relation to the amino acid sequence identified as SEC. IDENT. NO: 4 or identified as SEC. IDENT. NO: 5 or identified as SEC. IDENT. NO: 6 and with the ability to increase the expression of defensins in mammalian epithelial cells. The method according to claim 17, characterized in that the protein has an amino acid sequence that is at least about 68% or about 71% or about 73% or about 78% or about 83% or about 88% or about 93% or approximately 98% conserved in relation to the amino acid sequence identified as SEC. IDENT. NO: 5 19. A method for stimulating the expression of one or more defensins by epithelial cells, by exposing the cells to an effective amount of a soluble protein, the protein is characterized in that it has an amino acid sequence that is at least 63% conserved in relation to the amino acid sequence identified as SEC. IDENT. NO: 4 or identified as SEC. IDENT. NO: 5 or identify as SEC. IDENT. NO: 6 and having the ability to increase the expression of one or more defensins in epithelial cells 20. The method according to claim 19, characterized in that the protein has an amino acid sequence that is at least about 68% or about 71% or approximately 73% or approximately 78% or approximately 83% or approximately 88% or approximately 93% or approximately 98% conserved in relation to the amino acid sequence identified as SEC. IDENT. NO: 5 21. A method for stimulating the expression of a defensin along the gastrointestinal tract of a mammal by exposing the tract to an effective amount of a soluble protein, the protein is characterized in that it has an amino acid sequence that is at least 63% conserved in relation to the amino acid sequence identified as SEC. IDENT. NO: 4 or identified as SEC. ID NO: 5 or identified as SEC. IDENT. NO: 6 and having the ability to increase the expression of one or more defensins in bovine epithelial cells. The method according to claim 21, characterized in that the protein has an amino acid sequence that is at least about 68% or about 71% or about 73% or about 78% or about 83% or about 88% or about 93% or approximately 98% conserved in relation to the amino acid sequence identified as SEC. IDENT. NO: 5. 23. A method for stimulating the expression of one or more defensins in the respiratory tract of a mammal by exposing the tract to an effective amount of a soluble protein, the protein is characterized in that it has an amino acid sequence that is at least 63% conserved in relation to the amino acid sequence identified as SEC. IDENT. NO: 4 or identified as SEC. IDENT. NO: 5 or identified as SEC. IDENT. NO: 6 and having the ability to increase the expression of one or more defensins in epithelial cells. The method according to claim 23, characterized in that the protein has an amino acid sequence that is at least about 68% or about 71% or about 73% or about 78% or about 83% or about 88% or about 93% or approximately 98% conserved in relation to the amino acid sequence identified as SEC. IDENT. NO: 5 25. A method to stimulate the expression of one or more defensins in the tongue of a mammal by exposing the tongue to an effective amount of a soluble protein, the protein is characterized in that it has an amino acid sequence that is at least 63% conserved in relation to the amino acid sequence identified as SEC. IDENT. NO: 4 or identified as SEC. FROM IDENT. NO :: 5 or identified as SEC. IDENT. NO: 6 and having the ability to increase the expression of one or more defensins in the epithelial cells. 26. The method according to claim 25, characterized in that the protein has an amino acid sequence that is at least about 68% or about 71% or about 73% or about 78% or about 83% or about 88% or about 93% or approximately 98% conserved in relation to the amino acid sequence identified as SEC. IDENT. NO: 5. 27. A method for stimulating the expression of one or more defensins in the small intestine of a mammal by exposing the intestine to an effective amount of a soluble protein, the protein is characterized in that it has an amino acid sequence that is at least 63% conserved in relation to the amino acid sequence identified as SEC. IDENT. NO: 4 or identified as SEC. FROM IDENT. NO :: 5 or identified as SEC. IDENT. NO: 6 and having the ability to increase the expression of one or more defensins in the epithelial cells. The method according to claim 27, characterized in that the protein has an amino acid sequence that is at least about 68% or about 71% or about 73% or about 78% or about 83% or about 88% or about 93% or approximately 98% conserved in relation to the amino acid sequence identified as SEC. IDENT. NO: 5. 29. A method for inducing the expression of defensins in the epithelial cells of a mammal that requires it, the method is characterized in that it includes the administration of an effective amount of a protein, the protein containing an amino acid sequence that it is at least 63% conserved in relation to the amino acid sequence identified as SEC. IDENT. NO: 4 or identified as SEC. FROM IDENT. NO: 5 or identified as SEC. IDENT. NO: 6 and having the ability to increase the expression of one or more defensins in the epithelial cells. The method according to claim 29, characterized in that the protein has an amino acid sequence that is at least about 68% or about 71% or about 73% or about 78% or about 83% or about 88% or about 93% or approximately 98% conserved in relation to the amino acid sequence identified as SEC. IDENT. NO: 5. 31. The method according to any of claims 1 to 14, characterized in that the CD14 or the polypeptide portion, or the variant is recombinant. 32. The method according to any of claims 1 to 9, characterized in that the compound includes CD14 obtained from mammary secretion of a mammal. 33. The method according to claim 32, characterized in that the CD14 is obtained from bovine milk. 34. The method according to claim 32 or 33, characterized in that if the secretion has previously been subjected to treatment, the treatment is sufficiently mild to allow the preservation of the activity of the CD14 to induce or stimulate the production of defensins by the epithelial cells . 35. The method according to claim 32, characterized in that the CD14 is contained in a liquid. 36. The method according to claim 35, characterized in that the liquid includes a milk fraction enriched in CD14. 37. The method according to claim 32, characterized in that the CD14 is contained in an edible product. 38. The method according to any of claims 32 to 37, wherein the administration of CD14 includes exposure of the gastrointestinal tract to CD14. 39. The method according to any of claims 32 to 38, characterized in that it includes the administration of CD14 to the mammal orally. 40. A method to determine the suitability of a product derived from a mammary secretion for its use in the induction or stimulation of the production of defensins in mammals, the method is characterized in that it includes the steps of: providing a sample of the product and determining the amount of CD14 present in the sample 41. The method according to claim 40, characterized in that the secretion has previously been subjected to treatment, the treatment it is sufficiently mild to allow the preservation of CD14 activity to induce or stimulate such production of defensins. 42. The method according to claim 40 or claim 41, characterized in that determining the amount of CD14 present in the sample includes exposing the sample to a specific antibody to CD14, and checking whether complexes of CD14 antibody are formed in this step. 43. The method according to any of claims 1 to 12, characterized in that the compound is specifically recognized by an antibody that also specifically recognizes human CD14. 44. The method according to claim 43, characterized in that the antibody is mAb 3C10 and / or a mAb that recognizes the same amino acid sequence as mAb 3C10 45. The method according to any of claims 1 to 12 or 43 or CD14 is Human CD14. 46. The method according to any of claims 1 to 12 or 43 to 45, is characterized in that it includes the administration of the compound orally. 47. The method according to claim 46, characterized in that the compound is administered to an infant as a component of an infant formula. 48. The method according to any of claims 1 to 12 or 43 or 44 is characterized in that it includes the administration of the compound in the form of an aerosol. 49. The use of a concentrate in the method of preparing a medicament, a dietary substance or a chewable product for use in directly stimulating the production of defensin in a mammal, method is characterized in that it comprises the steps of: providing a stock solution containing mammary secretion protein separates from the solution a concentrate comprising endogenous CD14; determine the concentration of CD14 in the compound. 50. The method according to claim 49, characterized in that the mammary secretion includes milk, whole milk, a portion of whole milk containing protein or colostrum. 51. The method of compliance with the claim 49 or 50, characterized in that if the secretion has been previously subjected to treatment, the treatment has been sufficiently mild to allow the preservation of CD14 activity in the induction or stimulation of the defensins production. 52. The method according to any of claims 49 to 51, characterized in that the mammary secretion is human. 53. The method according to any of claims 49 to 52, characterized in that the mammary secretion is bovine 54. The method according to any of claims 49 to 53, characterized in that the solution is a liquid solution and the separation step. includes the extraction of proteins from the liquid by means of salt. 55. The method according to any of claims 49 to 54, characterized in that determining the concentration of CD14 includes exposing a sample obtained from the concentrate to a first antibody specific for CD14 to form a CD14 antibody complex and subsequently exposing the complex to a second antibody specific for CD14, where the second antibody includes an indicator molecule. 56. The method according to any of claims 49 to 54, characterized in that determining the concentration of CD14 includes exposing a sample obtained from the concentrate to a first antibody specific for CD14 to form a CD14 antibody complex and subsequently exposing the complex to a second antibody specific for the first antibody, wherein the second antibody includes an indicator molecule. 57. The use of a concentrate in the method of preparing a medicament, a dietary substance or a chewable product for use in directly stimulating the production of defensin in a mammal, the method is characterized in that it comprises the steps of: providing a mammary solution containing mammary secretion protein precipitate from the material solution a fraction of protein containing CD14 isolate the protein fraction from the supernatant. 58. The method according to claim 57, characterized in that the step of precipitating includes extracting the fraction of the protein containing CD14 by means of salt. 59. The method according to claim 58, characterized in that the step of precipitating includes increasing the salt concentration of the solution to obtain an ionic strength at least as high as that which would be obtained by the combination of a saturated aqueous solution of sodium sulfate. ammonium with a volume of such mammary secretion, the volume of the sulphate solution being equal to 65 percent of the total volume of the combined solutions. 60. The method according to any of claims 57 to 59, characterized in that it further comprises the step of determining the amount of CD14 obtained in the isolation step. 61. The method according to any of claims 57 to 60, characterized in that the mammary secretion is colostrum. 62. The method according to any of claims 57 to 61, characterized in that the mammary secretion is milk. 63. The method according to any of claims 57 to 62, characterized in that the mammary secretion is bovine. 64. The method according to any of claims 57 to 62, characterized in that the mammary secretion is human. 65. The use of a concentrate in the method of preparing a medicament, a dietary substance or a chewable product for use in directly stimulating the production of defensin in a mammal, the method is characterized in that it comprises the steps: providing a stock solution containing protein from breast secretion; isolate from the solution a fraction containing proteins that are insoluble in mammary secretion by combining an aqueous solution of ammonium sulfate with a volume of such mammary secretion, the volume of the sulfate solution being equal to 65 percent of the total volume of the combined solutions. 66. The method according to claim 65, further characterized in that it comprises the step of determining the amount of CD14 obtained in the isolation step. 67. The method according to claim 65 or claim 66, characterized in that the mammary secretion is colostrum. 68. The method according to claim 65 or 66, characterized by the mammary secretion is milk. 69. The method according to any of claims 65 to 68, characterized in that the mammary secretion is bovine. 70. The method according to any of claims 65 to 68, characterized in that the mammary secretion is human. 71. The use of a mammary secretion in the method of preparing a medicament, a dietary substance or a chewable product for use in directly stimulating the production of defensin in a mammal, the method is characterized in that it comprises the steps: to provide a compound that contain mammary secretion protein exposing the compound to an antibody specific for CD14 to determine if the endogenous CD14 secretion is present in the sample based on whether a CD14 antibody complex has been formed in the exposure step. 72. The method according to claim 71, characterized in that if the secretion has been previously exposed to a treatment, it is sufficiently mild to allow the preservation of the activity of CD14 to induce or stimulate the production of defensin. 73. The method according to claim 71 or claim 72, further characterized in that it comprises the step of determining the amount of CD14 in the sample. 74. The method according to any of claims 71 to 73, characterized in that the mammary secretion is colostrum. 75. The method according to any of claims 71 to 73, characterized in that the mammary secretion is milk. 76. The method according to any of claims 71 to 75, characterized in that the mammary secretion is bovine. 77. The method according to any of claims 71 to 75, characterized in that the mammary secretion is human 78. The use of a CD14 or a compound according to claim 1, in the preparation of a medicament for use in improving the aseptic symptoms. 79. The use of a CD14 or a compound according to claim 2, in the preparation of a medicament for use in increasing the expression of defensins in a mammal. 80. The use of a CD14 or a compound according to claim 4, in the preparation of a medicament for use stimulating the expression of one or more defensins by epithelial cells. 81. The use of a CD14 or a compound according to claim 5, in the preparation of a medicament for use in stimulating the expression of a defensin in the gastrointestinal tract of a mammal. 82. The use of a CD14 or a compound according to claim 6, in the preparation of a medicament for use in stimulating the expression of a defensin in the respiratory tract of a mammal. 83. The use of a CD14 or a compound according to claim 7, in the preparation of a medicament for use in stimulating the expression of a defensin in the tongue of a mammal. 84. The use of a CD14 or a compound according to claim 8, in the preparation of a medicament for use in stimulating the expression of a defensin in the small intestine of a mammal. 85. The use of a CD14 or a compound according to claim 9, in the preparation of a medicament for use in inducing the expression of defensins by epithelial cells of a mammal. 86. The use of a protein according to claim 13 or claim 14, in the preparation of a medicament for use in ameliorating the symptoms of asepsis. 87. The use of a protein according to claim 15 or 16, in the preparation of a medicament for use in treating prophylactically an inflammatory reaction induced in the host by lipopolysaccharides, in a mammal. 88. The use of a protein according to claim 17 or 18, in the preparation of a medicament for use in increasing the expression of defensins in a mammal. 89. The use of a protein according to claim 19 or 20, in the preparation of a medicament for use in stimulating the expression of one or more defensins by epithelial cells. 90. The use of a protein according to claim 21 or 22 in the preparation of a medicament for use in stimulating the expression of a defensin along the gastrointestinal tract of a mammal. 91. The use of a protein according to claim 23 or 24, in the preparation of a medicament for use in stimulating the expression of a defensin along the respiratory tract of a mammal. 92. The use of a protein according to claim 25 or 26, in the preparation of a medicament for use in stimulating the expression of a defensin in the tongue of a mammal 93. The use of a protein according to claim 27 or 28 in the preparation of a medicament for use in stimulating the expression of a defensin in the thin intetin of a mammal. 94. The use of a protein according to claim 29 or 30 in the preparation of a medicament for use in inducing the expression of defensins by epithelial cells of a mammal. 95. A method of increasing the expression of defensins in a mammal that requires it, characterized in that it includes the administration to the mammal of an effective amount of recombinant CD14 polypeptide encoded by a recombinant DNA molecule of non-natural occurrence, comprising a first sequence of DNA selected by the group consisting of: (a) a cDNA sequence coding for CD14 according to SEC. FROM IDENT. NO: 2 (b) a sequence that specifically hybridizes to the non-coding strand of (a) and which encodes expression for a polypeptide specifically recognized by an antibody which in turn specifically recognizes human CD14 (c) a DNA sequence encoding the same polypeptide that is encoded by the DNA sequence of (a) or (b), wherein the polypeptide encoded by (b) or (c) increases such expression. 96. A method that stimulates the expression of one or more defensins by epithelial cells characterized in that it includes the administration to the mammal of an effective amount of recombinant CD14 polypeptide encoded by an unnaturally occurring recombinant DNA molecule, comprising a first DNA sequence selected by the group consisting of: (a) a cDNA sequence 'which codes for CD14 according to SEC. FROM IDENT. NO: 2 (b) a sequence that specifically hybridizes to the non-coding strand of (a) and which encodes expression for a polypeptide specifically recognized by an antibody which in turn specifically recognizes human CD14 (c) a DNA sequence encoding the same polypeptide that is encoded by the DNA sequence of (a) or (b), wherein the polypeptide encoded by (b) or (c) stimulates such expression. 97. The method according to claim 95 or 96, characterized in that the polypeptide is specifically recognized by an antibody that also specifically recognizes human CD14. 98. The method according to claim 97, characterized in that the antibody is mAb 3C10 and / or a mAb that recognizes the same amino acid sequence as mAb 3C10. 99. The method of any of claims 95 to 98, characterized in that it includes oral administration of the polypeptide. 100. The method according to any of claim 98, characterized in that the polypeptide is administered to an infant as a component of a infant formula. 101. The method according to any of claims 95 to 98, characterized in that it includes the administration in aerosol form of the polypeptide. 102. The method according to any of claim 95 to 98, characterized in that the polypeptide is contained in the concentrated milk. 103. The use of claim 95 of a polypeptide, in the preparation of a medicament for increasing the expression of defensins in a mammal. 104. The use of claim 96 of a polypeptide in the preparation of a medicament that stimulates the expression of one or more defensins by epithelial cells. 105. The method according to any of claim 1 to 31 or 95 to 100, characterized in that the compound, polypeptide, protein, concentrate, or CD14 as the case requires, is added as a supplement to a nutritional source. 106. The method according to any of claims 1 to 4, 6, 7, 9 to 20, 23 to 26 or 29 or 30, characterized in that it includes direct topical exposure of the epithelium of the trachea to the polypeptide or protein, such as case it requires it. 107. The method according to any of claims 1 to 4, 13, 14, 17 to 20 or 29 or 30, characterized in that it includes the topical exposure of the external epidermis of a mammal in particular, the wounds thereof. 108. A method for preparing an ointment for direct topical application to a wound in human skin to reduce the effects of infection, in particular bacterial infection thereof, characterized in that it comprises incorporating into the ointment an effective amount of concentrate comprising CD14 obtained from mammary secretions. 109. A nutritional source such as infant formula, milk or any other liquid that has added a fraction of milk product, the fraction including a high concentration of CD14 that occurs naturally in the milk product, characterized by the milk product it has not been treated by processes that denaturate the CD14 contained therein to the extent that CD14 loses the desired activity. 110. The nutritional source according to claim 109, characterized in that the CD14 is obtained from bovine milk. 111. Administering the nutritional source according to claim 109 or 110, to a human to prevent, reduce or treat the symptoms of asepsis in humans. 112. Administer the nutritional source according to claim 109 or 110 to a human to increase the expression of defensins in the human. 113. The method according to any of claims 1 to 31 and 95 to 102, characterized in that the mammal is human. 114. The method according to any of claims 1 to 31 and 95 to 102, characterized in that the mammal has the need for protection against a select group of pathogenic microbes consisting of viruses, bacteria, fungi and yeast. 115. The method according to any of claims 1 to 31 and 95 to 102, characterized in that the mammal is a human suffering from immunological deficiency. 116. The method according to any of claims 1 to 31, 34, 78 to 94 or 95 or 104, characterized in that the defensins are selected from the group consisting of RtNP1, RtNP2, RtNP3, RtNP4, HNP1, HNP2, and HNP3 and any combination thereof or the group consisting of HNP1, HNP2, and HNP3. 117. The method according to any of claims 1 to 31, 34, 78 to 94 or 95 or 104, characterized in that the protein or polypeptide, as the case requires, is administered in amounts between about 250μg to about 2500μg per kg of body weight of the mammal per day or in an amount between about 300μg to about lmg per kg of body weight per day. 118. The method according to claim 95 or 96, characterized in that the nucleic acid molecule has the DNA sequence of hybridizers (B) under stringent conditions for the strand of (a) non-coding. 119. A method for directly activating B cells using a soluble polypeptide having the amino acid sequence selected from the group consisting of leu-leu-leu-leu-leu-leu-pro-ser; leu-leu-leu-leu-leu-leu-pro-leu and leu-leu-leu-leu-leu-leu-val-his; , and which is specifically recognized by the monoclonal antibody 3C10 which activates B cells, when administered to a mammal in need thereof, an effective amount of the polypeptide. 120. The method according to claim 119, characterized in that the amino acid comprises a sequence selected from a group consisting of SEC. FROM IDENT. NO: 4, SEC. FROM IDENT. NO: 5 or SEC. FROM IDENT. NO: 6, or a conservatively substituted variant thereof, which activates B cells, or a fragment thereof which activates B cells or a conservatively substituted variant thereof, which activates B cells. 121. A transgenic mammal to which a nucleic acid sequence encoding a polypeptide characterized by having the amino acid sequence identified as SEQ. FROM IDENT. NO: 4, SEC. FROM IDENT. NO: 5 or SEC. FROM IDENT. NO: 6, or a fragment of such polypeptide which directly activates B cells; or a variant of such polypeptide that directly activates B cells; or a variant of a conservatively substituted form of the polypeptide or conjugates of the fragment or variant thereof that directly activates the B cells, where the nucleic acid sequence is under the control of a CD14 promoter endogenous to the mammal and the nucleic acid sequence is added to the nucleic acid sequences that occur naturally in the mammalian DNA. 122. The transgenic animal according to claim 121, characterized in that the nucleic acid sequence encoding a polypeptide having the amino acid sequence identified as SEQ. FROM IDENT. NO: 4, SEC. FROM IDENT. NO: 5 or SEC. FROM IDENT. NO: 6, or a fragment of such polypeptide which directly activates B cells; or a variant of such a polypeptide. 123. The transgenic animal according to claim 122, characterized in that the nucleic acid sequence encoding a polypeptide having the amino acid sequence identified as SEQ. FROM IDENT. NO: 4, SEC. FROM IDENT. NO: 5 or SEC. FROM IDENT. NO: 6 a conservatively substituted variant of the polypeptide. 124. The transgenic animal according to claim 123, characterized in that the nucleic acid sequence encoding a polypeptide having the amino acid sequence identified as SEQ. FROM IDENT. NO: 4, SEC. FROM IDENT. NO: 5 or SEC. FROM IDENT. NO: 6. 125. The transgenic animal according to claim 124, characterized in that the nucleic acid sequence encoding a polypeptide having the amino acid sequence identified as SEQ. FROM IDENT. NO: 4 or SEC. FROM IDENT. NO: 5. 126. The transgenic animal according to claim 125, characterized in that the nucleic acid sequence encoding a polypeptide having the amino acid sequence identified as SEQ. FROM IDENT. I do not know: c. FROM IDENT. NO: 2 127. The transgenic mammal to which a nucleic acid sequence encoding a protein according to claim 139 or 140 is introduced into its genome, characterized in that the nucleic acid sequence is under the control of a CD14 promoter endogenous to the mammal and the Nucleic acid sequence is added to the nucleic acid sequences that occur naturally in the mammalian DNA. 128. The transgenic mammal that has been introduced into its genome a nucleic acid sequence encoding a protein according to claims 1 to 31, 34, 78 to 94 or 95 to 104, characterized in that the nucleic acid sequence is under control of a CD14 promoter endogenous to the mammal and the nucleic acid sequence is added to nucleic acid sequences that occur naturally in the mammalian DNA. 129. The transgenic mammal according to any of claim 121 to 148, characterized in that the nucleic acid sequence is a heterologous sequence. 130. The transgenic mammal according to any of claims 121 to 129, characterized in that the nucleic acid sequence has been introduced into the mammalian or progenitor of the mammal by recombinant technology. 131. The transgenic mammal according to claim 130, characterized in that the nucleic acid sequence has been introduced into the mammal or progenitor of the mammal by recombinant technology. 132. The transgenic mammal according to any of claim 121 to 131, characterized in that the CD14 promoter is a bovine promoter. 133. The transgenic mammal according to any of claims 121 to 131, characterized in that the mammal is bovine. 134. A transgenic mammal that has been introduced into its genome a nucleic acid sequence identified as SEC. IDENT. NO: 8 where the nucleic acid sequence is added to the nucleic acid sequences that occur naturally in the mammalian DNA. 135. The mammal according to claim 134, characterized in that the nucleic acid sequence has been introduced into the mammal or the progenitor of the mammal by recombinant technology. 136. The mammal according to claim 134, characterized in that the nucleic acid sequence has been introduced to the mammal by recombinant technology. 137. The mammal according to any claim 134 to 136, characterized in that the mammal is bovine. A method for improving aseptic symptoms comprising directly exposing the epithelial cells of a mammal in need thereof for soluble CD14, or active variants thereof. A method for obtaining Cdl4 from a solution of matter containing proteins of a mammalian secretion is described. A method for directly activating B cells using a soluble peptide having the amino acid sequence selected from the group consisting of leu-leu-leu-leu-leu-leu-pro-ser, leu-leu-leu, is described. -leu-leu-leu-pro-leu; and leu-leu-leu-leu-leu-leu-val-his, and which is specifically recognized by monoclonal antibody 3C10 and which activates B cells. Bovine CD14 genomic DNA is described.