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 of the Invention

FIELD OF INVENTION

This invention relates to soluble LAIT-protein (CD14) derived from mammals, and related proteins, that 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 CD 14.

Peptide Antibiotics and their Induction by Endotoxin

Antibiotic peptides are widely distributed in nature, and comprise a widespread mechanism of host defense (Lehrer, R. I. et. al. 1993. Ann. Rev. Immunol. 11:105; Boman, H. G. 1995. Ann. Rev. Immunol. 13:61; Lehrer, R. I., T. Ganz, and M. E. Selsted. 1991. Cell 64:229; Zasloff, M. 1992. Curr. Opin. Immunol. 4:3). An advantage of peptide antibiotics as factors of the innate immune system is their ability to function without specificity, and without memory. Their anti-bacterial, anti-viral, and anti-fungal activities permit the host to delay or possibly even avoid microbial growth shortly after infection, before the adaptive immune response can be mobilized (Lehrer, R. I. et al. 1993. Ann. Rev. Immunol. 111:105; Boman, H. G. 1995. Ann. Rev. Immunol. 13:61; Lehrer, 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 constitute greater than 5% of total cellular protein in human neutrophils (Boman, H. G. 1995. Ann. Rev. Immunol. 13:61; Lehrer, R. I., T. Ganz, and M. E. Selsted. 1991. Cell 64:229; Zasloff, M. 1992. Curr. Opin. Immunol. 4:3). In mammals defensins are also known to be produced by lung macrophages (Lehrer, R. I. et. al. 1993. Ann. Rev. Immunol. 11:105; Boman, H. G. 1995. Ann. Rev. Immunol. 13:61; Lehrer, R. I., T. Ganz, and M. E. Selsted. 1991. Cell 64:229; Zasloff, M. 1992. Curr. Opin. Immunol. 4:3), and have most recently been described in bovine epithelial cells of the trachea (Diamond, G. J. P. Russell, and C. L. Bevins. 1996. PNAS 93:5156) and tongue (Schonwetter, B. S., Stolzenberg, E. D. and M. A. Zasloff. 1995. Science 267:1645).

It has been demonstrated 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, termed tracheal antibiotic peptide (TAP), is of the .beta.-defensin class. The mechanism of TAP induction from epithelial cells of the respiratory mucosa was shown to be mediated through membrane CD14 (mCD14) expressed on the epithelial cells. The role of epithelial mCD14 was consistent with the observation that the activation process resulting in TAP expression was inhibited in the presence of monoclonal antibody (mAb) specific for CD14 (Diamond, G. J. P. Russell, and C. L. Bevins. 1996. PNAS93:5156).

While the expression of mCD14 was thought for some time to be an exclusive marker of monocytes macrophages (Zeigler-Heitbrock, H. W. L. and R. J. Ulevitch. 1993. Immunology Today 14:121), it is now known to be expressed by epithelial cells derived from many tissues (Fearns, C. et. al. 1995. J. Exp. Med. 181:857). It has also been found that epithelial linings of other tissues respond to endotoxin or inflammation through the local production of defensins. In particular, the squamous epithelial lining of the tongue has been shown to respond to infection or inflammation with the production of .beta.-defensin lingual antibiotic peptide (Schonwetter, B. S., Stolzenberg, E. D. and M. A. Zasloff. 1995. Science 267:1645). In this study, messenger RNA (mRNA) encoding lingual antimicrobial peptide was shown to be present in great abundance in epithelial cells of the tongue that surrounded naturally occurring lesions. There has apparently been no report of mCD14 involvement in this context.

The results described above provide the experimental basis for an immune response model in which innate immune response machinery is engaged at local sites of infection and/or inflammation so as to contribute to the initial defense of the host. The local production of antibiotic proteins and peptides in response to LPS derived from gram negative bacteria may thus come in to play in the prevention of bacterial colonization or subsequent infection prior to engagement of a clonal adaptive immune response. By way of background, a brief summary of the current understanding of mechanism(s) underlying endotoxin mediated responses in monocytes, macrophages, epithelial cells, and endothelial cells thus follows.

CD14 is a MEMBRANE Receptor on Monocytes for LPS:LBP Complexes.

Endotoxin in the form of LPS induces inflammatory cytokines by monocytes/macrophages both in vitro 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. Nature 330:662; Waage, A., Halstensen, A. and T. Espevik. 1987. Lancet 1:355). These monocyte-derived cytokines, including TNF.beta., IL-1, and IL-6, are associated with septic shock syndrome, ultimately leading to multi-organ failure. Recent work has characterized CD14 as a monocyte receptor for LPS (Wright, S. D. et. al. 1990. Science 249:1431), which in turn led to the initial characterization of a mechanism through which LPS activates monocytes/macrophages.

The current paradigm posits the involvement of the constitutively expressed plasma protein, lipopolysaccharide-binding protein (LBP), which forms high affinity complexes with LPS (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). LBP is a plasma glycoprotein produced by the liver, present constitutively in plasma of healthy adult humans at 5-10g/ml, which has been shown to increase in concentration up to 20-fold after an acute phase response (Schumann, R. R. et. al. 1990. Science 249:1429; Tobias, P. S. et. al. 1992. Cell. Mol. Biol. 7:239; Tobias, P. S., Mathison, J. C. and R. J. Ulevitch 1988. J. Biol. Chem. 263:13479; Tobias, P. S., Soldau, K. and R. J. Ulevitch 1986. J. Exp. Med. 164:777; Wright, S. D. et. al 1990. Science 249:1431; Wright, S. D. et. al. 1989. J. Exp. Med. 170:1231). Upon binding LBP, the ability of LPS to stimulate cytokine production in macrophages and monocytes is enhanced (Mathison, J. C., Tobias, P. S. and R. J. Ulevitch 1991. Pathobiology 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).

Membrane CD14 (mCD14), tethered through a glycosylphosphatidylinositol anchor (Zeigler-Heitbrock, H. W. L. and R. J. Ulevitch. 1993. Immunology Today 14:121), functions as a receptor for LPS-LBP complexes (Schumann, R. R. et. al 1990. Science 249:1429; Wright, S. D. et. al. 1990. Science 249:1431). CD 14 is expressed at high levels on monocytes and macrophages, and weakly on neutrophils (Ball, E. D. et. al. 1982. Proc. Natl. Acad. Sci. USA 79:5374; Buckle, A. M., Jayaram, Y. and N. Hogg 1990. Clin. Exp. Immunol. 81:339; Ferrero, E. et. al. 1990. J. Immunol. 145:331; Goyert, S. et. al. 1988. Science 239:497; Haziot, A. et. al. 1988. J. Immunol. 141:547). A murine pre-B cell line 70Z/3 (Paige, C. J. et. al. 1978. J. Immunol. 121:641), does not express detectable mCD14 by immunofluoresence, and is negative for message encoding CD14 as assessed by both northern blot analysis, and RT-PCR (Filipp, D. and M. Julius unpublished observation; Lee, J. D. et. al 1992. J. Exp. Med. 175:1697). This cell line has been used to provide compelling evidence for the role of mCD14 as a receptor for LPS-LBP complexes. Specifically, 70Z/3 responds to LPS with the expression of membrane immunoglobulin (mIg) (Paige, C. J. et. al. 1978. J. Immunol. 121:641). The concentration of LPS required to induce mIg expression in mCD14.sup.- 70Z/3 is orders of magnitude higher than that required to stimulate cytokine production by mCD14.sup.+ monocytes (Lee, J. D. et. al. 1992. J. Exp. Med. 175:1697). When 70Z/3 is transfected with cDNA encoding human CD14, it was demonstrated that the concentration of LPS required to induce mIg expression by mCD14.sup.+ clones was 10,000-fold lower than that required in the wild-type mCD14.sup.- parental line of 70Z/3 (Lee, J. D. et al. 1992. J. Exp. Med. 175:1697).

These results provide experimental evidence for the current model for in vivo LPS mediated activation of mCD14.sup.+ leukocytes. Upon exposure to LPS, LPS-LBP complexes form, and these complexes activate monocytes/macrophages through interaction with mCD14.

Soluble CD14 in Endotoxin Mediated Activation of Endothelial and Epithelial Cells.

In contrast to the suggested mechanisms involved in LPS mediated activation of mCD14.sup.+ leukocytes, less is understood about the mechanisms involved in LPS mediated activation of endothelial and epithelial cells. Until recently, endothelial and epithelial cells were thought to be mCD14.sup.+. Despite the undetectable expression of mCD14 in these cell types, LPS mediated activation has been shown to be serum dependent, and inhibited by monoclonal antibodies specific for CD14 (Patrick, D. et. al. 1992. J. Inf Dis. 165:865; Pugin, J. et. al. 1993. Proc. Natl. Acad. Sci. USA 90:2744; Arditi, M. et. al. 1993. Infect. Immun. 61:3149). This suggests that CD14 may play some role, albeit unknown, in endotoxin mediated activation of endothelial and epithelial cells.

It has been demonstrated that soluble CD14 (sCD14), lacking the glycosylphosphatidylinositol anchor, and present in serum of healthy adult humans (Bazil, V. et. al. 1986. Eur. J. Immunol. 16:1583), is involved in LPS mediated activation of both endothelial cells (Arditi, M. et. al. 1993. Infect. Immun. 61:3149; Pugin, J. et. al. 1993. Proc. Natl. Acad. Sci. USA 90:2744; Frey, E. A., et. al. 1992. J. Exp. Med. 176:1665; Read, M. A. et. al. 1993. Proc. Natl. Acad. Sci. USA 90:9887; Haziot, A. et. al. 1993. J. Immunol. 151:1500) and epithelial cells (Pugin, J. et. al. 1993. Proc. Natl. Acad. Sci. USA 90:2744). The serum dependence of the activation process was shown to be due to the presence of sCD14, and the requirement for serum could be replaced by sCD14. No role for LBP could be characterized in the case of LPS mediated endothelial cell activation in some studies, suggesting that sCD14 itself is an agonist for endothelial cell responses 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. Natl. Acad. Sci. USA 90:9887). In other studies, a dual role for the serum in the LPS response for both endothelial and epithelial cells was found. Specifically, both sCD14 and LBP appeared to be required for endotoxin mediated endothelial cell activation (Pugin, J. et. al. 1993. Proc. Natl. Acad. Sci. USA 90:2744; Haziot, A. et. al. 1993. J. Immunol. 151:1500), most pronouncedly when endotoxin was present at low concentrations (Haziot, A. et. al. 1993. J. Immunol. 151:1500).

The above experimental results led to the postulated role of sCD14-LPS complexes in the activation of mCD14.sup.- endothelial cells. At high LPS concentrations these complexes are thought to be generated directly through the interaction of sCD14 and LPS. At low LPS concentrations LBP is thought to first interact with LPS, which in as yet uncharacterized ways is postulated to facilitate the generation of sCD14-LPS complexes.

While the above results are potentially at odds with each other in regard to the suggested mechanisms supporting endothelial/epithelial cell responses to LPS, they share a common element in that the mechanism(s) are distinct from those involving endotoxin mediated activation of mCD14.sup.+ cells. Both studies suggest a role for sCD14 functioning as an agonist, enabling responses to endotoxin by mCD14.sup.- cells rather than functioning as a receptor for LPS-LBP complexes on mCD14.sup.+ cells. However, more recent studies have demonstrated that this may not be the case, at least for epithelial cells.

Membrane CD14 in Endotoxin Mediated Induction of Defensins by Epithelial Cells.

As discussed above, a recent study demonstrated that endotoxin induces the expression of defensins in primary bovine tracheal epithelial cells (Diamond, G. J. P. Russell, and C. L. Bevins. 1996. PNAS 93:5156) and that the expression involved mCD14. While unstimulated epithelial cells were shown to be mCD14.sup.-, they were induced to a mCD14.sup.+ state subsequent to LPS mediated activation (Diamond, G. J. P. Russell, and C. L. Bevins. 1996. PNAS 93:5156). The induction of mCD14 on the primary tracheal epithelial cells was shown to correlate with the induction of message specific for CD14 in the epithelial cells, suggesting it was likely of endogenous origin. Further, LPS mediated induction of defensins was inhibited by mAb specific for CD14 (Diamond, G. J. P. Russell, and C. L. Bevins. 1996. PNAS93:5156).

The mechanism(s) underlying LPS mediated activation of epithelial cells was thus shown to parallel those observed in mCD14.sup.+ monocytes and macrophages. The LPS activation pathways in these two cell types appear to differ from each other only in the basal levels of mCD14 expressed by the two types of target cells. Comparable studies involving endothelial cells have not been reported.

Soluble CD14 Directly Activates Monocytes in the Absence of Serum/LBP.

The paradigm described thus far is that endotoxin, in the form of LPS, mediates the activation of monocytes/macrophages and epithelial cells through its interaction with mCD14 on the cell. The serum dependence of this process, reflecting the involvement of LBP, has been demonstrated in all but one circumstance.

As described above, sCD14 has been implicated in endotoxin mediated activation/injury of endothelial cells. Its function was postulated as potentiating the interaction of LPS with the cell (Pugin, J. et al. 1993. Proc. Natl. Acad. Sci USA 90:2744). A subsequent study demonstrated that sCD14 isolated from the urine of nephrotic human subjects was able to directly stimulate the production of inflammatory cytokines, TNF.beta. and IL-6, by human monocytes (Sundan, A. et. al. 1994. Eur. J. Immunol. 24:1779). Human monocytes are mCD14.sup.+, and 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, the activity of sCD14 isolated from urine in this regard was shown to be serum independent, and was not affected by LBP, or by antibodies specific for LBP (Sundan, A. et. al. 1994. Eur. J. Immunol. 24:1779). Further, the capacity of sCD14 to stimulate the production of inflammatory cytokines by human monocytes was shown to be inhibited with mAb specific for CD14 (Sundan, A. et. al. 1994. Eur. J. Immunol. 24:1779).

Thus, sCD14 appears to have the capacity to directly interact with as yet unidentified receptor structures on monocytes in a serum independent fashion, and result in cytokine production. Endotoxin and sCD14 are thus able to mediate the similar biological responses in monocytes. Further, the ability of the same CD14 specific mAb, 3C10 (Van Voorhis, W. C. et. al. 1983.J. Exp. Med. 158:126), to inhibit both of these modes of stimulation, suggests that at least in part, signaling pathways involving endotoxin and sCD14 are shared, perhaps at the level of receptor structures. CD14 specific mAb 3C10 recognizes the N-terminal portion of CD14, and this recognition is dependent on the presence of the N-terminal amino acids 7 to 14 of the CD14 molecule (Juan, T. S.-C. et. al. 1995. J. Biol. Chem. 270:17237). The capacity of 3C10 to inhibit LPS mediated monocyte activation has been interpreted as reflecting the role of mCD14 residues 7 to 14 as an interaction site between mCD14 and LPS (Todd, S. C. et. al. 1995. J. Biol. Chem. 270:17237). In contrast, the basis for the capacity of mAb 3C10 to inhibit the function of sCD14 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 as yet uncharacterized receptor structures for sCD14 on monocytes, the mAb interfering with the interaction between sCD14 and such structure(s).

Soluble CD14 Inhibits the LPS-Induced Activation of Monocytes and Neutrophils in Vitro in a Dose Dependent Fashion

International patent application published under No. WO 93/19772 on Oct. 14, 1993 describes inhibition of LPS-induced activation of monocytes and neutrophils in vitro by recombinant human soluble CD14 in a dose-dependent fashion, at least as indicated by data shown in FIG. 1 (see Original Patent) of the published document. The experiments were carried out in the presence of LPS-LBP complex and are consistent with binding of the complex and the added CD14 present so as reduce the degree of interaction between the complex and membrane-bound CD14 of the monocytes and neutrophils. Such a result is curious in that it is known that sCD14 is itself capable of stimulating production of inflammatory cytokines by monocytes.

Lactation-Associated ImmunoTrophic (LAIT)-Protein 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 co-pending U.S. patent application Ser. No. 08/746,883 filed Nov. 18, 1996 and the International Patent Application Serial No. PCT/CA97/00880 filed Nov. 18, 1997. Relevant portions of these prior applications are reproduced herein.

A protein was isolated from bovine and human colostrum and breast milk, which protein has been termed Lactation-Associated Immuno-Trophic (LAIT)-protein.

The biological activities of LAIT-protein distinguish it from all other known cytokines that support B-cell growth and differentiation in adult animals, and thus may play a unique role in the regulation of B cell activation. The induction of most humoral immune responses in the adult involves a sequence of cellular interactions among "helper" T lymphocytes, antigen presenting cells (APC), and B lymphocytes (Sprent, J. J. 1978. J. Exp. Med. 147:1159; Andersson, J. et. al. 1982. Proc. Natl. Acad. Sci. USA 77:1612; Julius, M. H. et. al. 1982. Proc. Natl. Acad. Sci. USA 79:1989). These interactions are mandatory (Sprent, J. J. 1978. J. Exp. Med. 147:1159; Andersson, J. et. al. 1982. Proc. Natl. Acad. Sci. USA 77:1612; Julius, M. H. et. al. 1982. Proc. Natl. Acad. Sci. USA 79:1989; Julius, M. H. et. al. 1987. Immunol. Rev. 95:914), and thus reflect the role of specific plasma membrane associated molecules as transducers of prerequisite activation signals. The essential molecular interaction, reflected by the requirement for T cell-B cell contact, is mediated by CD40 expressed on the plasma membrane of the B cell, and its cognate ligand, gp39 (or CD40L), expressed on the plasma membrane of the T cell (Noelle, R. J. et. al. 1992. Proc. Natl. Acad. Sci. USA 89:6550; Armitage, R. J. et. al. 1992. Nature 357:80). The interaction between CD40 and CD40L predicates the induction of B cell growth, B cell differentiation into immunoglobulin secreting cells, and immunoglobulin isotype switching (Foy, T. M. et. al. 1993. J. Exp. Med. 178:1567). Moreover, the array of cytokines produced by these interacting cells are central to the regulation of B lymphocyte activation, growth, and differentiation (Andersson, J. et. al. 1982. Proc. Natl. Acad. Sci. USA77:1612; Noelle, R. J. et. al. 1983. Proc. Natl. Acad. Sci. USA80:6628; Hodgkin, P. D. 1990. J. Immunol. 145:2025; Noelle, R. J. et. al. 1991. J. Immunol. 146:1118; Parker, D. C. 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 supplement one another, each driving the B lymphocyte to the next stage of activation, rendering them susceptible to subsequent and progressive activation signals (Julius, M. H. et. al. 1987. Immunol. Rev. 95:914).

LAIT-protein, in contrast, is directly mitogenic for B cells at nM concentrations, and functions as a co-stimualtor of B cell growth in combination with stimulation through the B cell antigen receptor in the pM range. In these latter circumstances, those signals derived from antigen convert the B cell into a physiological state in which it can receive T cell help. The pertinence of supplying the neonate with a factor that directly supports B cell growth and differentiation in combination with antigen is significant when one considers the suppressed state of T cells in a developing neonate.

It has been demonstrated that while the thymus efficiently produces T cells early in ontogeny, unlike the adult thymus (Bill, J. et. al. 1989. J. Exp. Med. 169:1405; MacDonald, H. R. et. al. 1988. Nature 332:4020), it does not efficiently delete those T cells expressing potentially autoreactive antigen receptors (Schneider, R. et. al. 1989. J. Exp. Med. 169:2149; Smith, H. et. al. 1989. Science 245: 749; Ceredig, R. 1990. Intl. Immunol. 2:859; Ceredig, R. and C. Waltzinger. 1990. Intl. Immunol. 2:869). At the same time, these neonates are healthy. Colostrum and early breast milk contain well characterized inhibitors of T cell function, particularly, TGF.beta.1 and TGF.beta.2, which are inhibitors of T cell activation (Spom, M. B. et. al. 1987. J. Cell. Biol. 105:1039; Massague, 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 T cell function in neonates is actively suppressed by these cytokines to allow time for the maturation of thymic function. It is also of obvious importance for the neonate to initiate the production of its own protective antibodies, given that maternally-derived and passively acquired Ig is both transient and contains specificities that reflect maternal antigen encounter. It is expected that LAIT-protein functions as a T cell surrogate, supporting the growth and differentiation of B cells in the neonate freshly exposed to environmental antigens. The operation of LAIT-protein thus offers an alternative, truncated route for activating the immune system, which is independent of T cell function.

Sequencing analysis of bovine LAIT-protein fragments revealed high homology with human CD14. CD14 was subsequently purified from human colostrum by affinity chromatography using available monoclonal antibodies, and was shown to possess the same range of biological activities as colostral bovine LAIT-protein. The gene encoding bovine LAIT-protein was cloned from a bovine cDNA library, and shown to be highly homologous to human CD14, at both nucleotide and protein levels.

Recombinant human and mouse CD14, as well as recombinant bovine LAIT-protein/CD14 was prepared in both insect cell and mammalian cell expression systems, and each were shown to contain all of the biological activities of native bovine LAIT-protein of colostral origin, with specific activities within a factor of two of that observed with native material isolated from each of the three species.

In the context of this invention, "antibiotic proteins" or "antibiotic polypeptides" are with antibiotic properties: (i) linear, mostly helical peptides without cysteine, with or without a hinge (cecropins); (ii) linear peptides without cysteine and with a high proportion of certain residues such as proline and arginine; (iii) antibacterial peptides with one disulfide bond; (iv) peptides with two-or more S--S bonds giving mainly or only .beta.-sheet structures including but not limited to human defensin, HNP-1, rabbit defensin NP-1, rat defensin NP-1, bovine .beta.-defensin, TAP, pig protegrin, PG-3, and H-s crab tachypiesin 1; and (v) antibacterial peptides derived from larger 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 Physiology of the Neutrophil, 1994. Cambridge University Press pp 67-70).

"Sepsis" is condition which manifests itself in a human patient, when invaded by a microbial agent, a temperature of greater than 38.degree. C. or less than 36.degree. C.; a heart rate of greater than 90 beats per minute; a respiratory rate of greater than 20 breaths per minute or PaCO.sub.2 less than 32 mm Hg; a white blood cell count of greater than 12,000 mm.sup.-3, less than 4,000 mm.sup.-3 or greater than 10% immature (band) forms; organ dysfunction, hypoperfusion, or hypotension. Hypoperfusion and perfusion abnormalities may include, but are not limited to lactic acidosis, oliguria, or an acute alteration of mental states (1992. Chest 101:1644).

In the context of this application, CD14, unless otherwise indicated means any of bovine, human and murine CD14 proteins, recombinant or isolated from a naturally occurring source ("native"), the sequences of which correspond to SEQ ID NO:4; SEQ ID NO:5 and SEQ ID NO:6, respectively.

The present invention provides a method of ameliorating the symptoms of sepsis. According to this first aspect of the invention, there is a step of directly exposing epithelial cells of a mammal in need thereof to an effective amount of a compound comprising (i.e., which is made up of or includes) soluble CD14, or a polypeptide fragment of the CD14 that stimulates expression of a defensin in epithelial cells, or a conservatively substituted variant of said CD14 or the fragment that stimulates said expression.

The invention also includes a method of enhancing expression of defensins in a mammal in need thereof, by administering a compound comprising soluble CD14 or a polypeptide portion of CD14 that enhances said expression, or a conservatively substituted variant of said CD14 or the portion that enhances said expression.

The administering step includes preferably includes directly exposing epithelial cells of the mammal to said compound.

The invention includes a method of stimulating expression of one or more defensins by epithelial cells by administering thereto an effective amount of a compound comprising soluble CD14 or a polypeptide fragment of CD 14 that stimulates said expression, or a conservatively substituted variant of said CD14 or the fragment that enhances said expression.

The invention includes stimulating expression of a defensin along the gastrointestinal tract, or along the respiratory tract, of a mammal comprising exposing the tract to an effective amount of a compound comprising soluble CD14 or a polypeptide fragment of CD14 that stimulates said expression, or a conservatively substituted variant of said CD14 or the fragment that stimulates said expression.

Expression of a defensin takes place on the tongue of a mammal according to a certain aspect. Expression of a defensin can be in the intestine, particularly, the small intestine of a mammal.

According to a general aspect of the invention, expression of defensins by epithelial cells of a mammal is induced.

The CD14 can have an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, or a conservatively substituted variant thereof.

In another aspect, the invention is a method of ameliorating the symptoms of sepsis comprising administering to a mammal in need thereof an effective amount of a soluble protein so as to directly expose epithelial cells of the mammal to the protein, the protein having an amino acid sequence which is at least about 63% conserved in relation to the amino acid sequence identified as SEQ ID NO:5 and having the ability to induce expression of defensins in epithelial cells.

Alternatively, the protein can have an amino acid sequence which 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 SEQ ID NO:5.

The invention includes a method for prophylactically treating a lipopolysaccharide-induced host inflammatory response in a mammal, which method comprises administering a therapeutically effective amount of an effective amount of a protein to the mammal so as to directly expose epithelial cells of the mammal to the protein, the protein having an amino acid sequence which is at least about 63% conserved in relation to the amino acid sequence identified as SEQ ID NO:4 or identified as SEQ ID NO:5 or identified as SEQ ID NO:6 and having the ability to enhance expression of one or more defensins in bovine epithelial cells.

The invention includes a method of enhancing expression of defensins in a mammal in need thereof, by administering an effective amount of a soluble protein to the mammal, the protein having an amino acid sequence which is at least about 63% conserved in relation to the amino acid sequence identified as SEQ ID NO:4 or identified as SEQ ID NO:5 or identified as SEQ ID NO:6 and having the ability enhance expression of defensins in mammalian epithelial cells.

The invention includes a method of stimulating 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 which is at least about 63% conserved in relation to the amino acid sequence identified as SEQ ID NO:4 or identified as SEQ ID NO:5 or identified as SEQ ID NO:6 and having the ability to stimulate expression of one or more defensins in epithelial cells.

The invention includes a method of stimulating expression of a defensin along the gastrointestinal tract of a mammal comprising exposing the tract to an effective amount of a soluble protein, the protein having an amino acid sequence which is at least about 63% conserved in relation to the amino acid sequence identified as SEQ ID NO:4 or identified as SEQ ID NO:5 or identified as SEQ ID NO:6 and having the ability to stimulate expression of a defensin in bovine epithelial cells.

The invention includes a method of stimulating expression of a defensin along respiratory tract and/or on the tongue, or in the small intestine, of a mammal comprising exposing the tongue to an effective amount of a soluble protein, the protein having an amino acid sequence which is at least about 63% conserved in relation to the amino acid sequence identified as SEQ ID NO:4 or identified as SEQ ID NO:5 or identified as SEQ ID NO:6 and having the ability to stimulate expression of a defensin in epithelial cells.

The invention includes a method of inducing expression of defensins by epithelial cells of a mammal in need thereof, the method comprising administering and effective amount of a protein, the protein having an amino acid sequence which is at least about 63% conserved in relation to the amino acid sequence identified as SEQ ID NO:4 or identified as SEQ ID NO:5 or identified as SEQ ID NO:6 and having the ability to induce expression of defensins in epithelial cells.

The CD14 or the polypeptide portion or the variant can be obtained recombinant or chemical methods.

In another aspect, the invention is a method of preparing a CD14 concentrate. The method includes providing a stock solution containing protein of a mammary secretion, separating from the solution a concentrate comprising endogenous CD14; and determining the concentration of CD14 in the concentrate.

The mammary secretion can be milk, whole milk or a protein-containing portion of whole milk, or it can be colostrum or a protein-containing portion of colostrum.

Preferably, the secretion has been previously subjected to a treatment step, and the treatment step is sufficiently mild to permit preservation of the CD14 activity for inducing or stimulating defensin production and/or for stimulating B cells.

The mammary secretion can be human or it can be bovine. In situations in which the CD14 is obtained from a mammal in which it occurs endogenously (i.e., in a mammal which has not been the subject of molecular genetic manipulations), the CD14 is preferably bovine.

In cases where the solution is a liquid solution the separating step can include salting out of proteins from 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 an antibody-CD14 complex and subsequently exposing the complex to a second antibody specific for CD14, wherein the second antibody includes a reporter molecule. ELISA assays are particularly convenient in this regard.

Determining the concentration of CD14 can include exposing a sample obtained from the concentrate to a first antibody specific for CD14 to form an antibody-CD14 complex and subsequently exposing the complex to a second antibody specific for the first antibody, wherein the second antibody includes a reporter molecule.

In another aspect, the invention is a method of obtaining CD14 which includes providing a stock solution containing protein of a mammary secretion, precipitating from the stock solution a protein fraction containing CD14 and isolating the protein fraction from the supernatant.

Precipitation can include salting out a protein fraction containing CD14. Preferably, the salt concentration of the solution is increased to obtain an ionic strength at least as high as would be obtained by combining a saturated aqueous solution of ammonium sulphate with a volume of a said mammary secretion (as it occurs naturally), in which the volume of the ammonium sulphate solution being equal to 65 percent of the total volume of the combined solutions.

The method often includes also determining the amount of CD14 obtained in the isolating step.

Again, the mammary secretion can be colostrum and/or milk and can be bovine or human, or from another type of mammal in which CD14 occurs in mammary secretions.

The invention includes another method of obtaining CD14, that involves providing a stock solution comprising protein of a mammary secretion; and isolating from the solution a fraction containing proteins that are insoluble in the mammary secretion upon combining a saturated aqueous solution of ammonium sulphate with a volume of a said mammary secretion, the volume of the ammonium sulphate solution being equal to 65 percent of the total volume of the combined solutions. Preferably, the method includes a step of determining the amount of CD14 obtained in the isolating step.

Endogenous CD14 proteins obtained according to methods of the invention can be used for directly activating B cells when in a suitably soluble form. Likewise, they can be used in producing medicaments for such use.

According to another aspect, the invention is a method for testing for the presence of CD14 in a composition containing protein of a mammary secretion. The method includes exposing the composition to an antibody which is specific for CD14; and determining whether CD14 endogenous to the secretion is present in the sample based on whether CD14-antibody complex has formed in the exposing step.

The secretion may or may not have been previously subjected to a treatment step, but if it has the treatment step is sufficiently mild to permit preservation of the CD14 activity for inducing or stimulating defensin production and/or for stimulating B cells.

Again, preferably, the method includes determining the concentration of CD14 in the sample.

In another aspect, the invention is a method of preventing, ameliorating or treating the symptoms of sepsis in a mammal, comprising administering to the mammal an effective amount of CD14 obtained from a mammalian mammary secretion.

Preferably, the CD14 is obtained from a mammary secretion according to one of the methods therefor described herein.

The CD14 can be contained in a liquid and the liquid can include a fraction of the milk enriched in CD14. The CD14 can be contained in an edible product, such as a food bar (e.g., chocolate or protein bar).

In another aspect, the invention includes a method for determining the amount of endogenous CD14 contained in a composition containing protein of a mammary secretion, i.e., on an animal not subject to molecular genetic manipulation as far as CD14 production is concerned. The method includes providing the composition; exposing a sample of the composition to an antibody which is specific for CD14 and determining the amount CD14 endogenous to the secretion present in the sample based on the amount of CD14-antibody complex formed in the exposing step.

The invention includes a method for determining the suitability of a product derived from a mammary secretion for use in inducing or stimulating defensin production in mammals, the method comprising the steps of: providing a sample of the product; and determining the amount of CD14 present in the sample.

This aspect of the invention could thus be used as a preliminary step in determining the amount of such product is to be incorporated into a medicament or food product etc., to be used according to a method of this invention, or other method for which soluble CD14 is known to be useful.

Accordingly, it is preferable that, if the secretion has been previously subjected to a treatment step, the treatment step is sufficiently mild to permit preservation of the CD14 activity for inducing or stimulating said defensin production.

Likewise, the invention includes a method for determining the suitability of a product derived from a mammary secretion for use in stimulating B cells in mammals, the method comprising the steps of: providing a sample of the product; and determining the amount of endogenous CD14 present in the sample.

In preferred aspects, an antibody is used in the determining step and more preferably, the antibody is mAb 3C10 and/or a mAb that recognizes the same amino acid sequence as mAb 3C10.

The invention includes the use of compounds described herein for the preparation of a medicament for use in ameliorating the symptoms of sepsis, for use in enhancing expression of defensins in a mammal, etc.

Another aspect of the invention includes a method of enhancing expression of defensins in a mammal in need thereof, comprising administering to a mammal in need thereof an effective amount of a recombinant polypeptide CD14 encoded by a non-naturally occurring recombinant DNA molecule comprising a first DNA sequence selected from the group consisting of: (a) a cDNA sequence encoding CD14 according to SEQ ID NO:2; (b) a DNA sequence which specifically hybridizes to the noncoding strand of (a) and which codes on expression for a polypeptide specifically recognized by an antibody which also specifically recognizes human CD14; and (c) a DNA sequence which encodes the same polypeptide as is encoded by a DNA sequence of (a) or (b) above; wherein the polypeptide encoded by (b) or (c) enhances said expression.

Yet another aspect of the invention is a method of stimulating expression of one or more defensins by epithelial cells comprising administering to a mammal in need thereof an effective amount of a recombinant polypeptide CD14 encoded by a non-naturally occurring recombinant DNA molecule comprising a first DNA sequence selected from the group consisting of: (a) a cDNA sequence encoding CD14 according to SEQ ID NO:2; (b) a DNA sequence which specifically hybridizes to the noncoding strand of (a) and which codes on expression for a polypeptide specifically recognized by an antibody which also specifically recognizes human CD14; and (c) a DNA sequence which encodes the same polypeptide as is encoded by a DNA sequence of (a) or (b) above; wherein the polypeptide encoded by (b) or (c) stimulates said expression.

Preferably, the specific hybridization is under stringent hybridization conditions.

"Stringent hybridization conditions" takes on its common meaning to a person skilled in the art here. Appropriate stringency conditions which promote nucleic acid hybridization, for example, 6.times. sodium chloride/sodium citrate (SSC) at about 45.degree. C. are known to those skilled in the art. The following examples are found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6: For 50 ml of a first suitable hybridization solution, mix together 24 ml formamide, 12 ml 20.times.SSC, 0.5 ml 2 M Tris-HCl pH 7.6, 0.5 ml 100.times. Denhardt's solution, 2.5 ml deionized H.sub.2O, 10 ml 50% dextran sulfate, and 0.5 ml 10% SDS. A second suitable hybridization solution can be 1% crystalline BSA (fraction V), 1 mM EDTA, 0.5 M Na.sub.2HPO.sub.4 pH 7.2, 7% SDS. The salt concentration in the wash step can be selected from a low stringency of about 2.times.SSC at 50.degree. C. to a high stringency of about 0.2.times.SSC at 50.degree. C. Both of these wash solutions may contain 0.1% SDS. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22.degree. C., to high stringency conditions, at about 65.degree. C. The cited reference gives more detail, but appropriate wash stringency depends on degree of homology and length of probe. If homology is 100%, a high temperature (65.degree. C. to 75.degree. C.) may be used. If homology is low, lower wash temperatures must be used. However, if the probe is very short (<100 bp), lower temperatures must be used even with 100% homology. In general, one starts washing at low temperatures (37.degree. C. to 40.degree. C.), and raises the temperature by 3-5.degree. C. intervals until background is low enough not to be a major factor in autoradiography.

Preferably, such a polypeptide is specifically recognized by an antibody which also specifically recognizes human CD14, such as mAb 3C10.

Methods of the invention can include direct topical exposure of the epithelium of the trachea, or of the outer epidermis of a mammal, particularly of wounds, to the polypeptide or protein, as the case may be.

The invention thus also includes a method of preparing an ointment for direct topical application to a wound of human skin for ameliorating the effects of infection, particularly bacterial infection, thereof, comprising incorporating into the ointment an effective amount of a concentrate or other compound of the invention having CD14 defensin inducing activity.

Likewise, an infant formula, milk or other liquid having added thereto a fraction of a milk product, the fraction including a higher concentration of CD14 than occurs naturally in the unfractionated milk product, wherein the milk product is one which has not been treated by a process which denatures the CD14 contained therein to the extent that CD14 loses the desired activity, is part of the invention.

Compositions and methods of the invention can be used for a mammal that is in need of protection against a microbial pathogen selected from the group consisting of virus, bacteria, fungus and yeast, particularly where the mammal a human suffering from immune deficiency.

Induced defensins include RtNP1, RtNP2, RtNP3, RtNP4, HNP1, HNP2, and HNP3 and any combination thereof, or of 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 of between about 250 .mu.g to about 2500 .mu.g per kg of bodyweight of the mammal per day or in an amount of between about 300 .mu.g to about 1 mg per kg of bodyweight per day.

In another aspect, the invention is 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 (SEQ ID NO:9); leu-leu-leu-leu-leu-leu-pro-leu (SEQ ID NO:10); and leu-leu-leu-leu-leu-leu-val-his (SEQ ID NO:11), and which is specifically recognized by the 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 SEQ ID NO:4, SEQ ID NO:5 or SEQ ID 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.

The invention includes a transgenic mammal having introduced into its genome a nucleic acid sequence encoding a polypeptide having the amino acid sequence identified as SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or fragment of said polypeptide which directly activates B cells; or a variant of said polypeptide which directly activates B cells; a conservatively substituted variant of the polypeptide; or conjugates of the fragment or variant thereof which directly activates B cells, wherein the nucleic acid sequence is under control of a CD14 promoter endogenous to the mammal and the nucleic acid sequence is in addition to nucleic acid sequences which naturally occur in the DNA of the mammal.

The nucleic acid sequence optionally encodes a polypeptide having the amino acid sequence identified as SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or fragment of said polypeptide which directly activates B cells; or a conservatively substituted variant of the polypeptide, more preferably, a polypeptide having the amino acid sequence identified as SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or a conservatively substituted variant of the polypeptide and even more preferably, a polypeptide having the amino acid sequence identified as SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. Most preferably, the nucleic acid sequence has the sequence identified as SEQ ID NO:1 or SEQ ID NO:2.

Preferably, the transgenic mammal has introduced into its genome a nucleic acid sequence encoding 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 the monoclonal antibody 3C10 and which activates B cells.

A transgenic mammal can alternatively have introduced into its genome a nucleic acid sequence encoding other proteins of the 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 ID NO:8, wherein the nucleic acid sequence is in addition to nucleic acid sequences which naturally occur in the DNA of the mammal. SEQ ID NO:8 includes both coding sequences (see SEQ ID NO:1) and a non-coding sequence portion, the non-coding of which is excised during the production of mRNA which contains only coding bases. Preferably, the nucleic acid sequence has been introduced into the mammal or a progenitor of the mammal by recombinant technology. Preferably, the mammal is bovine.

Claim 1 of 22 Claims

1. A method of stimulating expression of at least one defensin in a mammal in need thereof, by orally administering to the mammal a masticable product comprising isolated soluble CD14 obtained from a mammalian mammary secretion, so as to directly expose the CD14 to epithelial cells of the mammal wherein the CD14 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5 , and SEQ ID NO:6.

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