WO2001023882A1

WO2001023882A1 - DETECTION OF POLYPEPTIDE FRAGMENTS AND MODIFIED FORMS OF α1-ANTITRYPSIN FOR EARLY DIAGNOSIS OF INFLAMMATION

Info

Publication number: WO2001023882A1
Application number: PCT/US2000/024702
Authority: WO
Inventors: Sabina Janciauskiene
Original assignee: Biocache Pharmaceuticals, Llc
Priority date: 1999-09-28
Filing date: 2000-09-08
Publication date: 2001-04-05
Also published as: AU7827800A

Abstract

The present invention provides a method for the detection of a carboxyl-terminal proteolytic fragments of α-1-antitrypsin (AAT) or post-translationally modified forms of AAT (for example, oxidized AAT) as an indicator of chronic inflammation. This method is useful for the early diagnosis of chronic inflammation associated with such diseases as atherosclerosis.

Description

DETECTION OF POLYPEPTIDE FRAGMENTS AND MODIFIED FORMS

OF αj-ANTTTRYPSIN FOR EARLY DIAGNOSIS OF INFLAMMATION

DESCRIPTION

BACKGROUND OF THE INVENTION

Field of the Invention The invention generally relates to the early detection of inflammation, whose occult occurrence is linked to pathologies such as atherosclerosis and Alzheimer's disease. In particular, the invention provides a method for the early detection of inflammation by determining the amounts of modified forms of α,-antitrypsin (AAT) in samples of biological fluid.

Background of the Invention

Inflammation is a normal, inherent immunological response to tissue damage. Tissue damage may result from processes such as microbial invasion, auto-immune reactions, allograft rejection, or from external influences such as heat, cold, radiant energy, electrical or chemical stimuli, or mechanical trauma. Under normal circumstances, inflammation subsides upon healing of the damaged tissue. However, there are some pathological conditions in which the inflammatory response becomes chronic and self-perpetuating, and healing does not occur. For example, atherosclerosis is a chronic inflammatory disease of the blood vessel wall (1) and Alzheimer's disease is closely linked to chronic inflammation in the brain.

Inflarnmation is a complex, time-dependent response that follows infection, trauma, or idiopathic inflammatory states. The initial phases of the response are observed within hours, but the total duration may extend much longer. During acute or chronic inflammatory processes, circulating leukocytes and platelets are increased, metabolic changes may result in the increased synthesis of certain hormones, gluconeogenesis increases, and the concentration of certain plasma proteins derived from the liver increases.

The recruitment of blood monocytes into tissues is a central event in the inflammatory response and in atherogenesis. The mechanisms leading to monocyte adhesion to tissues are not completely understood (2). In the case of atherosclerosis, current studies suggest that, following endothelial injury, monocytes attach to the subendothelium and penetrate into the vessel wall (3). During the course of attachment and penetration, monocytes differentiate into macrophages (3). The release from macrophages of various cytotoxic products such as interleukins, complement factor fragments, tumor necrosis factors, oxidized cholesterol, proteases and free radicals, leads to still further endothelial injury (4) and can thus become self-peφetuating. It has also been shown that macrophages themselves accumulate lipids such as cholesterol and cholesterol esters and become foam cells, a hallmark of fatty streak atherosclerotic lesions. This contributes to the formation of the plaques characteristic of atherosclerotic disease. In addition to cardiac disease, atherosclerosis can also lead directly to stenosis of the renal artery, blockage of cerebrovascular circulation (resulting in stroke), and to compromised peripheral circulation.

Atherosclerosis typically develops over many years, but the growth of atherosclerotic plaques is more likely a discontinuous process with episodes of rapid evolution. Human atherosclerotic plaques are heterogenous tissues containing a number of cell types and proteins including immunoglobulins, albumin, transferrin, the protease inhibitors α-1-antitrypsin (AAT) and α-2-macroglobulin, and others (29,30). It has been suggested that the protease inhibitors AAT and α-2-macroglobulin, which are produced by monocytes and found in high concentration in plaques, may enhance fibrosis of the lesion by their inhibitory effects on collagenase and elastase, (27,30) proteases which would otherwise break down the material that plaques are made of.

The clinical expressions of atherosclerosis may be chronic, such as in the development of stable effort-induced angina pectoris. Alternatively, a dramatic acute clinical event, such as a myocardial infarction or cerebrovascular accident, may be the first manifestation of the disease. By that time, extensive damage may have already occurred. AAT is an acute phase protein, primarily synthesized by the liver, whose concentration in circulation rises from 3- to 5-fold that of normal levels (1.34 mg/mL) during acute phase processes (5). At inflammatory loci, it was shown in rats that the concentration of AAT rises by about 8-fold (6). Additional sources of AAT production in humans are peripheral blood monocytes and alveolar macrophages (7-9) which increase expression of AAT in response to inflammatory mediators such as IL-6 and lipopolysaccharide (10-12).

Several lines of evidence suggest that AAT is important as a mediator of inflammation. For example, local regulation of AAT is crucial with respect to maintaining the "protease-antiprotease balance", and thus in preventing tissue damage induced by proteases in the micro environment of injury or inflammation where monocytes accumulate (12). Further, it has been suggested that intracellular regulation of the protease-antiprotease balance may play a central role in the phenomemon of cellular migration (10), which is known to be an important process in inflammation. Disruption of the proteinase-inhibitor balance as a result of mutational or oxidative inactivation of AAT has been shown to have major biological and clinical consequences in sustaining inflammation (13).

To date, the understanding of AAT function has been derived primarily from studies of native, functionally active AAT, while the possible biological roles of cleaved or oxidized, non-inhibitory forms of AAT have received little attention. Cleaved forms of AAT are generated when AAT forms an inhibitor complex with a target serine protease or, alternatively, when it is cleaved by non-target proteinases. The formation of a stable, covalent complex between AAT and a target serine protease occurs via cleavage of a single specific peptide bond in the serpin reactive center loop. This cleavage generates a 4 kD carboxyl-terminal fragment of 36 residues, corresponding to arnino acid sequence 358-396 (SEQ ID NO.l) and referred to as Peptide C-36. Peptide C-36 remains non-covalently bound to the cleaved AAT but it can be separated from the complex under denaturing conditions in vitro (19). In contrast, cleavage of AAT by non-target proteinases usually occurs at sites other than the specific target cleavage site without formation of proteinase-AAT complexes (14,15). Examples of non-target proteinases which inactivate AAT are human cathepsin L, collagenase and stromelysin (16,17); and bacterial proteinases from Staphylococcus aureus (18), Serratia marcescens metalloproteinase (19) and Pseudomonas aeruginosa elastase (20).

The few studies to elucidate possible roles of proteolytically cleaved forms of AAT have clearly shown that those forms are of biological significance. Joslin et al. have compared the properties of both cleaved AAT-elastase complexes and purified fragments of AAT (21). They have shown that proteolytically cleaved AAT binds to seφin-enzyme complex (SEC) receptors on HepG2 cells and monocytes and that binding is mediated by the same domain for both Peptide C-36 and the AAT-enzyme complex. Results from their studies confirmed that a domain within Peptide C-36 is necessary for binding to the SEC receptor(s), and that such a domain is available for receptor binding only after AAT forms a complex with a serine proteinase or after it has been proteolytically modified. Perlmutter and coworkers have demonstrated that upregulation of AAT synthesis by monocytes occurs following stimulation with cleaved AAT-elastase complexes (7).

Recently, hydrophobic C-terminal fragments of AAT have been isolated in free form from human plasma, bile and spleen, and have been shown to be associated with the phospholipid fraction of human bile (22). Studies utilizing immunohistochemical methods indicate that cleaved fragments of AAT, including a 44-residue C-terminal fragment, arising from non-target proteolytic cleavage within the reactive site loop of AAT, are present in a variety of human tissues, such as placenta, pancreas, stomach and small intestine (23-25). Also, the 44-residue C-terminal peptide of AAT was found to be associated with extracellular matrix proteins such as collagen and/or laminin-1, and it has been suggested that AAT peptide plays an important role in the protection of these proteins from inappropriate enzyme digestion (26). The demonstration that AAT proteolytically cleaved in its reactive site by the Pseudomonas metalloelastase promotes an increase in synthesis of AAT in human monocytes and mediates neutrophil chemotaxis (20,27) suggests that proteolytically inactivated AAT may play multiple roles at sites of inflammation.

Oxidized AAT (oAAT) is generated by free radicals and peroxides released by leukocytes and other cells at sites of inflammation. This is part of the initial, intrinsic immune response to inflammatory stimuli. Methionine residues in proteins are susceptible to oxidation by these reactive molecular species, whose local concentrations can reach high levels during inflammation. As mentioned above, methionine oxidation in AAT leads to inactivation of AAT as a protease inhibitor, which has been shown to have major biological and clinical consequences in sustaining inflammation (13).

Several inferences link AAT, its oxidized form and its proteolytic fragments to the process of inflammation and to atherogenesis. For example, AAT is an acute phase protein expressed by human monocytes at sites of inflammation, and is known to have the above-mentioned effects at those sites. Oxidized AAT and proteolytically modified forms of AAT are known to have multiple effects, including perturbation of lipid catabolism and induction of cell apoptosis. In addition, AAT is always present in atherosclerotic plaques and its circulating levels have been shown to correlate with the progression of the disease (28).

Further, elevated levels of o AAT or proteolytic fragments of AAT correlate and in some cases appear to be causative of conditions characteristic of inflammation. These include: (i) Elevated binding, uptake and accumulation of low density lipoprotein (LDL) in

HepG2, monocytes and others cells (31-33). (ii) Parallel increase in LDL receptor mRNA in monocytes and other cells; (iii) Formation of polymeric amyloid fibrils with properties of other amyloid forming proteins (34 - 37); (iv) Major changes in cellular cholesterol uptake and metabolism (32,33) in response to fibrils of C-36; (v) Decrease in DNA synthesis and increase in DNA fragmentation characteristic of apoptosis (32); (vi) Increased cytokine and glutathione reductase levels (32); (vii) Increased oxidation of LDL (32); (viii) Increased scavenger receptor expression (32); (ix) Moφhological changes in monocytes indicative of differentiation into lymphocytes (32). (x) Absence of atherogenesis in a small number of patients who are genetically deficient in AAT (38).

At the present time, substances which can be monitored as general indicators of systemic inflammation include plasma C-reactive protein and serum amyloid A-protein. With respect to atherosclerosis in particular, several factors are currently associated with the diagnosis of the disease. They include high triglyceride and total serum cholesterol levels, the ratio of high to low density lipoproteins, high systolic blood pressure, body mass index, lipoprotein A levels, diabetes with sustained hyperglycemia, nonenzymatic glycation of apolipoproteins, and others. However, these methods have the drawback that they are non-specific for atherosclerosis and many of them depend on detecting conditions which manifest relatively late in the course of the disease.

It would be highly desirable to have a specific means of detecting inflammation (for example the inflammation which accompanies plaque formation) at an earlier stage. An early indication of a predisposition toward disease conditions characterized by inflammation would allow prompt and thus more effective treatment of those conditions in their presymptomatic stages.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method for detecting inflammation and infection in mammals. In particular, it has been discovered that modified forms of AAT, including oxidized forms of AAT and proteolytic fragments of AAT, can be used as early symptomatic markers for the occurrence of a state of inflammation and/or infectious disease which may not yet have manifested any other readily detectable symptoms. In a preferred embodiment of the present invention, the modified form of AAT is a carboxyl-terminal proteolytic fragment of AAT (Peptide C- 36), corresponding to residues 358-396 of AAT. In another preferred embodiment of the present invention, the modified form of AAT that is detected is oxidized AAT (oAAT), that is AAT in which one or more amino acids, most commonly methionine, are oxidized. The oAAT may be either full-length or shorter fragments of oAAT generated by proteolysis. In a preferred embodiment of the present invention, the disease state that is detected is atherosclerosis, though the same methods can be applied to the detection of inflammation as a symptom of other pathologies.

In a preferred embodiment of the present invention, the concentration of a modified form of AAT in biological fluid is determined. The level of the modified form of AAT detected is then compared to the level of the modified form of AAT found in normal control samples to determine whether or not the level in the sample is higher than normal. In a preferred embodiment of the invention, the means of detection includes the use of monoclonal antibodies specific for the modified form of AAT.

Diseases characterized by chronic inflammation may be detected or monitored by the method of the present invention. Such diseases include but are not limited to atherogenesis, Alzheimer's disease, microbial infections, autoimmune diseases, rheumatoid arthritis, gallstones, inflammatory bowel disease, and glaucoma. The biological fluid to be sampled may be any appropriate biological fluid, such as blood, plasma, synovial fluid, ascitic fluid, seminal fluid, cerebrospinal fluid, eye fluid and the like.

The present invention also provides a kit for the detection of modified forms of AAT which, in a preferred embodiment, includes a specific binding antibody and an indicator.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Ascitic fluid samples from 10 normal, non-inflamed controls and from 10 patients with chronic inflammation were analysed on sodium-dodecyl sulfate polyacrylamide gels (SDS-PAGE). Lanes 1-10 (right panel) are those of the test patients; samples on the left were from controls (excluding lanes 6 and 7, which are molecular weight markers and AAT). The gel was blotted to paper (Western blot analysis) and the blot developed with polyclonal antibodies to peptide C-36. Bands corresponding to intact AAT and peptides are indicated. Higher bands are multimers of AAT.

DETAILED DESCRD?TTON OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides a method for assessing asymptomatic, occult inflammation in mammals which may predispose to multiple pathologies. The method is based on the ability to detect modified forms of AAT in biological fluids. Modified forms of AAT include posttranslationally modified forms of AAT (e.g. oxidized AAT) and proteolytic fragments of AAT. As will be seen in the Example below, we have discovered that proteolytic fragments of AAT are readily detectable in the ascitic fluid of patients with chronic inflammation of various organs at levels significantly higher than those of normal controls.

The method will aid in the early diagnosis of diseases which are characterized by chronic inflammation, such as atherosclerosis. Early diagnosis followed by prompt treatment of such conditions will greatly improve the prognosis for the health of a patient suffering from such a disease. Those skilled in the art will recognize that the method can be used as an aid for the early diagnosis of any disease characterized by chronic inflammation and accompanied by elevated levels of modified forms of AAT. Those diseases include but are not limited to atherosclerosis, Alzheimer • s disease, microbial infections, autoimmune diseases, rheumatoid arthritis, gallstones, glaucoma and inflammatory bowel disease. The method of the present invention may be used to screen individuals thought to be susceptible to such diseases, such as patients known to be at risk for atherosclerosis due to genetic factors, age, lifestyle, etc. prior to the onset of other detectable symptoms. Alternatively, the method of the present invention may be used in conjunction with other diagnostic methods to confirm the state of the progression of a disease state characterized by chronic inflammation in patients already displaying other symptoms. Further, the method can also be used to monitor the state of inflammation after diagnosis, for example, during treatment of the disease to aid in determining the efficacy of treatment.

In one embodiment of the present invention, the modified form of AAT which is detected is a proteolytic fragment of AAT. In a preferred embodiment of the present invention, the proteolytic fragment of AAT that is detected is peptide C-36 (SEQ ID #1). However, it will be understood by those of skill in the art that the detection of other proteolytic fragments known to be present in amounts higher than normal during chronic inflammation could also be practiced within the scope of the present invention. Any appropriate proteolytic fragment of AAT may be detected according to the method of the present invention. By appropriate proteolytic fragment of AAT, we mean any proteolytic fragment of AAT, the concentration of which rises during chronic inflammation. Proteolytic fragments of AAT are most commonly carboxyl terminal peptides ranging from 5 to 50 amino acids in length. They are formed by non- specific cleavage of AAT by microbial or leukocyte proteases at sites of inflammation, thus their concentration reflects the existence of inflammatory processes. Peptides of length at the higher end of this range (for example, peptide C-36) are thought to be stable to further degradation because of their protective β-sheet secondary structure. They therefore persist in serum and are likely candidates for detection. In a preferred embodiment of the present invention, such C-terminal peptides(s) are detected in a sample of biological fluid, and an increase in their concentration, as compared to their concentration in a normal control sample of equivalent biological fluid, is indicative of a state of inflammation.

In another embodiment of the present invention, the modified form of AAT which is detected is a posttranslationally modified form of AAT. In a preferred embodiment of the present invention, the posttranslationally modified form of AAT that is detected is oxidized AAT (AAT or a fragment thereof wherein at least one of the amino acids is oxidized). Any appropriate posttranslationally modified form of AAT known to be present in amounts higher than normal during chronic inflammation may be detected by the methods of the present invention. The methods of detection may be directed toward the full length of the posttranslationally modified protein (to assure inclusion of all modified amino acids which uniquely distinguish modified from wild type AAT) or to shorter proteolytic fragments of posttranslationally modified AAT, for example, proteolytic fragments of oxidized AAT (e.g. oxidized carboxyl terminal peptides from 5 - 50 amino acids in length).

The modified forms of AAT which are detected by the methods of the present invention may be naturally occurring (such as in a biological fluid), synthetic (such as reference peptides in a kit), bound to another biological molecule (e.g. a protease), or in the free state.

By "biological fluid' we mean those biological fluids in mammals typically accessible for sampling. For example, whole blood, serum, ascitic fluid, seminal fluid, amniotic fluid, spinal fluid, eye fluids, etc. Samples will be withdrawn from the patient by means well known to those of skill in the art and analyzed according to the method of the present invention.

In a preferred embodiment of the present invention, the method of detecting the modified form of AAT is immunological in nature. By 'immunological', we mean that a first monoclonal antibody specific for the modified form to be detected will be used. The monoclonal antibodies which can be used according to the method of the invention can be prepared using hybridoma fusion techniques (39,40) or can be derived from known secreting myeloma cell lines. Those of skill in the art will recognize that many techniques are available for the production of monoclonal antibodies, for example, those described in references 39-44.

Many antibody detection systems are known to those of skill in the art and can be employed within the scope of the present invention. For example, the monoclonal antibody can be diagnostically labeled. The term "diagnostically labeled" means that the antibody has attached to it a diagnostically detectable label. Many such labels and methods of conjugating labels to antibodies are well-known to those of skill in the art. Examples of types of labels which can be used in the practice of the present invention include but are not limited to fluorescent labels, enzyme labels, and radionuclide labels, specific binding pair components, colloidal dye substances, fluorochromes, reducing substances, latexes, digoxigenin, metals, particulates, dansyl lysine, antibodies, protein A, protein G, electron dense materials, chromophores and the like. Any suitable label, whether directly or indirectly detectable, may be employed. One skilled in the art will recognize that these labels set forth above are merely illustrative of the different labels that could be utilized in this invention.

Many methods employing antibodies which specifically bind target substances are known in the art. Preferred methods include immunochemical methods, such as enzyme-linked immunosorbent assay (ELISA) methods, immunonophelometry methods, agglutination methods, precipitation methods, immunodiffusion methods, immunoelectrophoresis methods, immunofluorescent methods, and radioimmunoassay methods. Assays for detecting the presence of proteins and/or peptides with antibodies have been previously described and follow known formats, such as a standard blot and ELISA formats. These formats are normally based on incubating an antibody with a sample suspected of containing the protein or peptide and detecting the presence of a complex between the antibody and the protein or peptide. The antibody is labeled either before, during or after the incubation step. Immobilization is usually required and may be accomplished by immobilizing the protein or peptide to a solid surface, such as a microtiter well, or by binding the protein to immobilized antibodies. In a preferred embodiment, the modified form of AAT is bound to an immobilized first antibody. A second labeled antibody, also specific for the modified form of AAT, or specific for the first antibody, is then bound, unbound material is washed away, and the complex is detectable due to the immobilized label of the second antibody. Such assays are well-known to those of skill in the art and include such assays as simultaneous sandwich, forward sandwich and reverse sandwich immunoassays, terms which are well-known to those of skill in the art.

Many solid phase immunoabsorbents for immobilization are known and can be used in the practice of the present invention. Well-known immunoabsorbents include beads formed from glass, polystyrene, polypropylene, dextran, nylon and other material; and tubes formed from or coated with such materials, and the like. The immobilized antibodies may be covalently or physically linked to the solid phase immunosorbent by techniques such as covalent bonding via an amide or ester linkage or by absoφtion.

In each of the above assays, the exact details of the assay protocol, such as time and temperature of incubation, may vary according to the concentration of antibodies used, the source of the biological fluid, the affinity of the antibodies for their target molecules, etc. In a preferred embodiment of the present invention, an ELISA assay may be carried out as follows: 96-well microtiter plates are coated with a monoclonal first antibody specific for a modified form of AAT. (The first antibody is immobilized in the wells.) Standards and samples are pipetted into wells in, for example, duplicate or triplicate, and any modified form of AAT present in the standards and samples will be bound by the immobilized antibody. The standards are composed of known concentrations of the modified form of AAT from the region of interest, which is known to be crossreactive with the first antibody. After incubation at room temperature for 2 hours, the wells are washed with an appropriate buffer to remove any unbound substances. Then a second enzyme-linked polyclonal (or monoclonal) antibody specific for a defining epitope of the modified form of AAT is added to the wells. The antibody will bind any modified forms of AAT which have been sequestered by the first antibody. After a 1 hour incubation at room temperature, the wells are again washed with an appropriate buffer to remove unbound antibody- enzyme reagent, and a solution which contains a substrate for the enzyme is added to the wells. The substrate is such that when it is acted on by the enzyme, a characteristic color is produced. Color will develop in proportion to the amount of enzyme present in the wells, which is directly proportional to the amount of bound modified form of AAT. After an appropriate period of time, the color development is stopped and the intensity of the color will be measured spectrophotometrically. The amount of modified form of AAT in the samples will be determined by comparing the color intensity of the sample wells to that of the control wells which contain a known amount of the modified form of AAT.

The monoclonal antibodies to be employed in the practice of the present invention are specific for a modified form of AAT and may be specific for any epitope of a modified form of AAT whose concentration in biological fluid increases as a result of chronic inflammation. For example, the monoclonal antibodies will be raised against purified samples of a synthetic peptide having a sequence identical to that of a proteolytic fragment of AAT, or to full-length oAAT, or to proteolytic fragments thereof. In a preferred embodiment of the invention, antibodies will be raised against a synthetic peptide having a sequence identical to that of carboxyl terminal Peptide-36 of AAT (SEQ ID 1) or to oAAT in which Met359 has been oxidized. Purified preparations of appropriate synthetic peptides are readily available commercially, and oAAT can be prepared by a simple oxidative incubation. Those of skill in the art will readily recognize that there are numerous established protocols available for generating monoclonal antibodies to specific peptides and proteins.

While in a preferred embodiment of the present invention the method of detection of modified forms of AAT is by utilizing monoclonal antibodies, those of skill in the art will recognize that other methods of detection can also be used in the practice of the invention. For example, polyclonal antibodies raised against a modified form of AAT such as oAAT (or proteolytic fragments thereof) or the C-terminal portion of AAT, might also be used in various immuno assays. Various types of immuno assays which might be utilized in the practice of the present invention include but are not limited to immunoelectrophoresis, nephelometry, gel electrophoresis followed by Western blot, dot blots, affinity chromatography, immuno-fluorescence, and the like. In addition, other methods of detection of peptides known to those of skill in the art may be used in the practice of the current invention, such as gas chromatography/mass spectrometry, HPLC, and gel electrophoresis followed by sequencing.

The present invention also provides kits for diagnostic use. In these kits, antibodies may be provided with means for binding to detectable marker moieties or substrate surfaces. Alternatively, the kits may include antibodies already bound to marker moieties or substrates. The kits may further include positive and/or negative control reagents as well as other reagents for carrying out diagnostic techniques. For example, kits containing antibody bound to multiwell microtiter plates can be provided.

The kit will include a standard or multiple standard solutions containing a known concentration of the modified form of AAT for calibration of the assays.

A large number of control samples will be assayed to establish the threshold, mode and width of the distribution of modified forms of AAT (peptide fragments and oAAT, both full-length and peptide fragments thereof) in normal fluid against which test samples will be compared. These data will be provided to users of the kit. Slightly elevated values for any of the markers in patients test samples may lie within the distribution range of normal patients, and their significance will be evaluated by standard statistical methods. Existing data indicate that the distributions for peptide marker from normal and inflamed patient samples are well separated. The level of C-36 peptide marker in normal controls is below detectability in Western blots (<about 3 pg mL^"1) while that for inflamed test samples is about 0.05 ng mL^"1

EXAMPLE

Analysis of ascitic fluid from patients with chronic inflammation

Ascitic fluid from 10 patients with chronic inflammation of various origins was analyzed for the presence of Peptide C-36. The analysis was carried out by SDS- PAGE electrophoresis with Western blot analysis, and by crossed immunoelectrophoresis. Polyclonal antibodies to AAT and to Peptide C-36 were used. The results are given in Figure 1. Quantitation of the data of Figure 1 revealed that the levels of Peptide C-36 detected in samples from patients with chronic inflammation were between 40-60% greater than the level of Peptide C-36 detected in normal controls. While the invention has been described in terms of its preferred embodiments, the invention can be practiced with modification and variation within the spirit and scope of the appended claims.

References

I. Watanabe, T., Haraoka, S., Shimokama, T. 1996. Inflammatory and immunological nature of atherosclerosis. Int. J. Cardiol. 54, suppl: S5 1-60.

2. McEvoy, L.M., Jutila, M.A., Tsao, P.S., Cooke, J.P., Butcher E C. 1997. Anti- CD43 inhibits monocyte-endothelial adhesion in inflammation and atherogenesis. Blood. 90:3587-3594.

3. Schmitz, G., Herr AS., Rothe, G. 1998. T-lymphocytes and monocytes in atherogenesis. Herz. 23: 168-177.

4. Murakami, T., Yamada, N. 1996. Modification of macrophage function and effects on atherosclerosis. Curr. Opin. Lipidol. 7:320-323.

5. Travis J, Salvesen GS. 1983. Human plasma proteinase inhibitors. Annu Rev Biochem. 52:655-709.

6. Zay, K., Loo, S., Xie, C, Devine, D.V., Wright, J. and Churg, A. 1999. Role of neutrophils and α,-antitrypsin in coal- and silica-induced connective tissue breakdown. Amer. J. Physiol. 276 (2 Pt 1), L269-L279

7. Perlmutter, D.H., Cole, F.S., Kilbridge, T.H., Rossing, T.H., Colten, H.R. 1985, Expression of alpha- 1-proteinase inhibitor gene in human monocytes and macrophages. Proc. Natl. Acad. Sci. 82: 795-799.

8. Issacson, P.D., Jones, D.B., Milward-Sadler, G.H., Judd, M.A., Payne, S. 1981. Alpha- 1-antitrypsin in human macrophages. J.Clin. Pathol. 34: 982-990.

9. Paakko, P., Kirby, M., du Bois, R. M., A, Gillisen, A., Ferrans, J., Crystal, R.G. 1996. Activated neutrophils secrete stored alphal-antitrypsin. Am. J. Respir. Crit. Med. 154: 1829-1833.

10. Knoell, D.L., Ralston, D.R., Coulter, K. R., Wewers, M.D. 1998. Alpha- lantitrypsin and protease complexation is induced by lipopolysacchride, interleukin lβ in monocytes. Am. J. Respir. Crit. Care Med. 157: 246-255.

I I. Perlmutter, D.H., Punsal, P.I. 1988. Distinct and additive effects of elastase and endotoxin on expression of alpha 1 proteinase inhibitor in mononuclear phagocytes. J. Biol. Chem. 263:16499-503. 12. Perlmutter, D.H., May L.t., Sehgal, P.B. 1989. Interferon beta2/interleukin 6 modulates synthesis of alpha- 1-antitrypsin in human mononuclear phagocytes and in human hepatoma cells. J. Clin. Invest. 84: 138-144.

13. Carrell RW, Lomas DA. 1997. Conformational disease. The Lancet. 350: 134-138.

14. Bruch M, Weiss V, Engel I 1988. Plasma serine proteinase inhibitors (seφins) exhibit major conformational changes and large increase in conformational stability upon cleavage at their reactive sites J. Biol. Chem. 263: 166626-166630.

15. Engh RA, Schulze AJ, Huber R, Bode W. 1993. Seφin structures. Behring. Inst. Mit. 93 :41-62.

16. Michaelis J, Vissers MC, Winterbourn CC. 1990. Human neutrophil collagenase cleaves alpha- 1-antitrypsin 2?/ocAem. J. 270:809-814.

17. Winyard PG, Zhang Z, Chidwick K, Blake DR, Carrell RW, Muφhy G. 1991. Proteolytic inactivation of human alpha- 1-antitrypsin by human stromelysin FEBS Lett. 279:91-94.

18. Potempa J, Watorek W, Travis J. 1979. The inactivation of human plasma alpha-1 proteinase inhibitor by proteinases from Staphylococcus aureus. J. Biol. Chem. 254:5317-5320.

19. Virca GD, Lyerly D, Kreger A, Travis J.1982. Inactivation of human plasma alpha-1 proteinase inhibitor by a metalloproteinase from Serratia marcescens Biochim. Biophys. Ada. 704:267-271.

20. Morihara K, Tsuzuki H, Harada M, Iwata T. 1984. Purification of human plasma alpha-1 proteinase inhibitor and its inactivation by Pseudomonas aeruginosa elastase. J. Biochem. Tokyo 95:795-804.

21. Joslin G, Fallon RJ, Bullock J, Adams SP, Perlmutter DH. 1991. The SEC receptor recognizes a pentapeptide neodomain of alpha 1-antitrypsin-protease complexes. J. Biol. Chem. 266: 11281-11288.

22. Johansson J, Grondal S, Sjovall J, Jornvall H, Curstedt T. 1992. Identification of hydrophobic fragments of α-antitrypsin and Cl protease inhibitor in human bile, plasma and spleen. FEBS Lett. 299: 146-148.

23. Niemann M Narkates AJ, Miller AJ. 1992. Isolation and serine proteinase activity of the 44-residue C-terminal fragment of alpha- 1-antitrypsin from human placenta. Matrix. 12:233-241.

24. Ray MB, Geboes K, Callea F, Desmer VJ. 1997. Alpha- 1-antitrypsin immunoreactivity in islet cells of adult human pancreas Cell. Tissues Res. 185:63-68. 25. Geboes K, Ray MB, Rutgeerts P, Callea F, Desmet VJ, Vantrappen G. 1982. Moφhological identification of alpha- 1-antitrypsin in the human small intestine Histopathology 6: 55-60.

26. Niemann MA, Baggott JE, Miller EJ. 1997. Binding of SPAAT, the 44-residue C-terminal peptide of alpha- 1-antitrypsin, to proteins of the extracellular matrix. J. Cell. Biochem. 66:346-357.

27. Barbey-Morel C, Perlmutter DH. 1991. Effect of Pseudomonas elastase on human mononucleaφhagocyte alpha- 1-antitrypsin expression. Pediatr. Res. 29: 133-140.

28. Mori, T., Sasaki, J., Kawaguchi, H, Handa, K., Takada,Y., Matsunaga, A, Kono, S., Arakawa, K. (1995) Serum Glycoproteins and severity of coronary atherosclerosis. Amer. Heart J. 129, 234-238.

29. Smith, E.B. 1977. Molecular interactions in human atherosclerotic plaques. Am. J. Pathol. 86:665-674.

30. Hollander, W., Colombo, MA., Kirkpatrick, B., Paddock, J. 1979. Soluble proteins in the human atherosclerotic plaque. With special reference to immunoglobulins,

C3 -complement component, alpha-1-antitrypsin and alpha2-macroglobulin. Atherosclerosis. 34: 391-405.

31. Janciauskiene S, Al Rayyes O, Floren C-H, Eriksson S. 1997. Low density lipoprotein catabolism is enhanced by the cleaved form of alpha- 1-antitrypsin Scand. J. Clin. Lab. Invest. 57:325-336.

32. Janciauskiene S, Lindgren S, Wright HT. 1998. The C-terminal peptide of α-1- antitrypsin increases low density lipoprotein binding in HepG2 cells Eur. J. Biochem. 254:460-467.

33. Janciauskiene, S., Lindgren, S. 1998. Effects of fibrillar C-terminal fragment of cleaved alpha- 1-antitrypsin on cholesterol homeostasis in HepG2 cells. Hepatology. In press

34. Janciauskiene S, Carlemalm E, Eriksson S. 1995. In vitro fibril formation from alpha- lantitrypsin-derived C-terminal peptides. Biol. Chem. Hoppe-Seyler 376:415- 423.

35. Mirzabekov TA, Lin M, Kagan BL. 1996. Pore formation by the cytotoxic islet amyloid peptide amylin. J. Biol. Chem. 271:1988-1992.

36. Behl C, Davis JB, Lesley R, Schubert D. 1994. Hydrogen peroxide mediates amyloid β protein toxicity. Cell. 77:817-827.

37. Lorenzo A, Yankner BA. 1996. Amyloid fibril toxicity in Alzheimer s disease and diabetes Ann. NYAcad. Sci. 777:89-95. 38. Elzouki, AN., Ahlgren, A.R., Lanne, T., Sonesson, B. and Eriksson, S. (1998) Is there a relationship between abdominal aortic aneurysims and α,-antitrypsin deficiency (PiZ) Ewr. J. Vase. Endovasc. Surg. 17, 149-154.

39. Kohler et al, 1976. European Journal of Immunology, 6, 292.

40. Abbink j, Kamp AM, Swaak AJ, Hack CΕ. 1991. Production of monoclonal antibodies against inactivated alpha- 1-antitrypsin. Cross-reactivity with complexed alpha- lantitrypsin and aplication in an assay to determine inactivated and complexed alpha- 1-antitrypsin in biological fluids. J. Immunol. Methods 143:197-208.

41. Niedbala, W.G. and Stott, D.I. 1998. Hybridoma, 17 (3), 299-304.

42. Kreutz, F.T., Xu, D.Z. and Siresh, M.R. 1998 Hybridoma, 17 (3), 267-273.

43. Kenney, J.S., Gray, F., Ancel, M.H. and Dunne, J.F. 1995. Biotechnology, 13 (8), 787-790.

44. Sado, Y. and Okigaki, T. 1996. Cell Biol. Int. 20 (1), 7-14.

Claims

Having thus described our invention, what we wish to claim by Letters Patent is:

A method for assessing an inflammatory disease or condition in a patient, comprising the steps of: detecting a concentration of proteolytic fragments of α- 1-antitrypsin in a biological sample; comparing a detected concentration of proteolytic fragments of α- 1-antitrypsin in said biological sample to a standard concentration of proteolytic fragments of α-1 - antitrypsin for an equivalent biological sample; and determining from a comparison made in said comparing step a patient • s predisposition towards an inflammatory disease or condition.

2. The method of claim 1 wherein said inflammatory disease or condition is atherosclerosis.

3. The method of claim 1 wherein said detecting step includes the step of selectively binding said fragments of α-1 -antitrypsin.

4. The method of claim 1 wherein said proteolytic fragment of α-1 -antitrypsin is Peptide C-36.

5. A diagnostic kit, comprising: a sample container for receiving a biological sample; means for detecting a concentration of proteolytic fragments of α-1 -antitrypsin in a biological sample; and means for comparing a detected concentration of proteolytic fragments of α-1 - antitrypsin in a biological sample to a standard concentration of proteolytic fragments of α-1 -antitrypsin for an equivalent biological sample to said biological sample.

6. The diagnostic kit of claim 4 wherein said means for detecting includes a means for selectively binding said proteolytic fragments of α-1 -antitrypsin.

7. The diagnostic kit of claim 4 wherein said means for selectively binding includes at least one monoclonal antibody for said proteolytic fragments.

8. The diagnostic kit of claim 4 wherein said proteolytic fragment is the C-36 peptide of α-1 -antitrypsin.

9. The diagnostic kit of claim 4 wherein said sample container is a cuvette.

10. The diagnostic kit of claim 4 wherein said sample container is a test tube.

11. The diagnostic kit of claim 4 wherein said means for detecting includes a colorimetric reagent.

12. The diagnostic kit of claim 4 wherein said means for detecting includes a fluorescent reagent.

13. The diagnostic kit of claim 4 wherein said means for detecting includes a chemiluminescent reagent.

14. The diagnostic kit of claim 4 wherein said means for detecting includes an enzyme- cleavable label.

15. The diagnostic kit of claim 4 wherein said means for comparing includes a graph.

16. The diagnostic kit of claim 4 wherein said means for comparing includes a table.

17. The diagnostic kit of claim 4 wherein said means for comparing includes a computerized look-up table.

18. A method for assessing an inflammatory disease or condition in a patient, comprising the steps of: detecting a concentration of a post-translationally modified form of α- 1 - antitrypsin in a biological sample; comparing a detected concentration of said post-translationally modified form of α-1 -antitrypsin in said biological sample to a standard concentration of a post- translationally modified form of α-1 -antitrypsin for an equivalent biological sample; and determining from a comparison made in said comparing step a patient • s predisposition towards an inflammatory disease or condition.

19. The method of claim 18 wherein said inflammatory disease or condition is atherosclerosis.

20. The method of claim 18 wherein said detecting step includes the step of selectively binding said modified form of of α-1 -antitrypsin.

21. The method of claim 18 wherein said post-translationally modified form of α-1 - antitrypsin is oxidized α-1 -antitrypsin.

22. The method of claim 18 wherein said post-translationally modified form of α-1 - antitrypsin is a proteolytic fragment of oxidized α-1 -antitrypsin.

23. A diagnostic kit, comprising: a sample container for receiving a biological sample; means for detecting a concentration of a post-translationally modified form of α-1 -antitrypsin in a biological sample; and means for comparing a detected concentration of a post-translationally modified form of α-1 -antitrypsin in a biological sample to a standard concentration of a post-translationally modified form of α-1 -antitrypsin for an equivalent biological sample to said biological sample.

24. The diagnostic kit of claim 22 wherein said means for detecting includes a means for selectively binding said post-translationally modified form of α-1 -antitrypsin.

25. The diagnostic kit of claim 22 wherein said means for selectively binding includes at least one monoclonal antibody for said post-translationally modified form of α-1 - antitrypsin.

26. The diagnostic kit of claim 22 wherein said modified form of α-1 -antitrypsin is oxidized α-1 -antitrypsin.

27. The diagnostic kit of claim 22 wherein said modified form of α-1 -antitrypsin is a proteolytic fragment of oxidized α- 1 -antitrypsin.

28. The diagnostic kit of claim 22 wherein said sample container is a cuvette.

29. The diagnostic kit of claim 22 wherein said sample container is a test tube.

30. The diagnostic kit of claim 22 wherein said means for detecting includes a colorimetric reagent.

31. The diagnostic kit of claim 22 wherein said means for detecting includes a fluorescent reagent.

32. The diagnostic kit of claim 22 wherein said means for detecting includes a chemiluminescent reagent.

33. The diagnostic kit of claim 22 wherein said means for detecting includes an enzyme-cleavable label.

34. The diagnostic kit of claim 22 wherein said means for comparing includes a graph.

35. The diagnostic kit of claim 22 wherein said means for comparing includes a table.

36. The diagnostic kit of claim 22 wherein said means for comparing includes a computerized look-up table.