Authentic hSAA related with AA amyloidosis: New method of purification, folding and amyloid polymorphism.

The secondary amyloidosis of humans is caused by the formation of hSAA fibrils in different organs and tissues. Until now hSAA was thought to have low amyloidogenicity in vitro and the majority of SAA aggregation experiments were done using murine protein or hSAA non-pathogenic isoforms. In this work a novel purification method for recombinant hSAA was introduced, enabling to obtain monomeric protein capable of amyloid aggregation under physiological conditions. The stability and amyloid aggregation of hSAA have been examined using a wide range of biophysical methods. It was shown that the unfolding of monomeric protein occurs through the formation of molten globule-like intermediate state. Polymorphism of hSAA amyloids was discovered to depend on the solution pH. At pH 8.5, rapid protein aggregation occurs, which leads to the formation of twisted short fibrils. Even a slight decrease of the pH to 7.8 results in delayed aggregation with the formation of long straight amyloids composed of laterally associated protofilaments. Limited proteolysis experiments have shown that full-length hSAA is involved in the formation of intermolecular interactions in both amyloid polymorphs. The results obtained, and the experimental approach used in this study can serve as a basis for further research on the mechanism of authentic hSAA amyloid formation.

Why working with porcine circulating serum amyloid A is a pig of a job.

Serum amyloid A (SAA) is a major acute phase protein in most species, and is widely employed as a health marker. Systemic SAA isoforms (SAA1, and SAA2) are apolipoproteins synthesized by the liver which associate with high density lipoproteins (HDL). Local SAA (SAA3) isoforms are synthesized in other tissues and are present in colostrums, mastitic milk and mammary dry secretions. Of systemic SAA the bulk is monomeric and bound to HDL, and a small proportion is found in serum in a multimeric form with a buried HDL binding site. In most species, systemic SAA could easily be studied by purifying it from serum of diseased individuals by hydrophobic interaction chromatography methods. For years, we were not able to isolate systemic pig SAA using the latter methods, and found that the bulk of pig SAA did not reside in the HDL-rich serum fractions but in the soluble protein fraction mainly as a multimeric protein. Based on these surprising results, we analysed in silico the theoretical properties and predicted the secondary structure of pig SAA by using the published pig primary SAA amino acid sequence. Results of the analysis confirmed that systemic pig SAA had the highest homology with local SAA3 which in other species is the isoform associated with non-hepatic production in tissues such as mammary gland and intestinal epithelium. Furthermore, the primary sequence of the pig SAA N-terminal HDL binding site did differ considerably from SAA1/2. Secondary structure analysis of the predicted alpha-helical structure of this HDL binding site showed a considerable reduction in hydrophobicity compared to SAA1/2. Based on these results, it is argued that systemic acute phase SAA in the pig has the structural properties of locally produced SAA (SAA3). It is proposed that in pig SAA multimers the charged N-terminal sequence is buried, which would explain their different properties. It is concluded that pig systemic SAA is unique compared to other species, which raises questions about the proposed importance of acute phase SAA in HDL metabolism during inflammation in this species.

Low density lipoprotein and very low density lipoprotein are selectively bound by aggregated C-reactive protein.

C-reactive protein (CRP), the classical acute-phase protein, can bind phospholipids by virtue of its specific, calcium-dependent reactivity with phosphorylcholine residues. However, analysis of acute-phase serum by gel filtration and by density gradient ultracentrifugation showed that the CRP was in a free, uncomplexed form, despite the coexistent presence of the various classes of serum lipoproteins, all of which contain phospholipids. In contrast, when isolated CRP was aggregated by immobilization at a sufficient density on a solid phase and then exposed to normal human serum, it selectively bound low density lipoprotein (LDL) and traces of very low density lipoprotein. The reaction was calcium dependent and reversible by free phosphorylcholine but not by heparin. LDL isolated from normal plasma was also bound by aggregated CRP. CRP reacts in vitro with a wide variety of different ligands both of extrinsic and of autogenous origin, e.g., microbial products and damaged cell membranes, respectively. If CRP aggregated in vivo by complexing with these ligands than acquires the capacity to selectively bind LDL, the phenomenon may have significant implications for the function of CRP and for the metabolism, clearance, and deposition of LDL.

Amyloid fibril in hereditary cerebral hemorrhage with amyloidosis (HCHWA) is related to the gastroentero-pancreatic neuroendocrine protein, gamma trace.

Amyloid fibrils were isolated from the leptomeningeal blood vessels obtained at autopsy from three Icelandic patients dying of Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA) and verified by Congo red staining and electron microscopy. Gel filtration on Sephadex and Ultrogel columns yielded predominantly one component (molecular weight 11,500 daltons) and also another minor component (molecular weight 15,800 daltons). Automated amino terminal sequencing showed these proteins to be similar (36 residues) to a recently described human protein, gamma trace, beginning at its eleventh amino terminal residue. The amyloid deposits in all three patients stained with rabbit anti-gamma trace antiserum. Although the function of gamma trace is not known, it appears to have structural homology with several hormones and has been localized to the brain, pancreas and pituitary. The amyloid fibril subunits seem to have polymerized after cleavage of the amino terminal decapeptide from gamma trace-related proteins. Therefore, HCHWA appears to be the first genetically determined disease related to the gastroenteropancreatic neuroendocrine system.

Serum Amyloid A1 (SAA1) Revisited: Restricted Leukocyte-Activating Properties of Homogeneous SAA1.

Infection, sterile injury, and chronic inflammation trigger the acute phase response in order to re-establish homeostasis. This response includes production of positive acute phase proteins in the liver, such as members of the serum amyloid A (SAA) family. In humans the major acute phase SAAs comprise a group of closely related variants of SAA1 and SAA2. SAA1 was proven to be chemotactic for several leukocyte subtypes through activation of the G protein-coupled receptor FPRL1/FPR2. Several other biological activities of SAA1, such as cytokine induction, reported to be mediated via TLRs, have been debated recently. Especially commercial SAA1, recombinantly produced in Escherichia coli, was found to be contaminated with bacterial products confounding biological assays performed with this rSAA1. We purified rSAA1 by RP-HPLC to homogeneity, removing contaminants such as lipopolysaccharides, lipoproteins and formylated peptides, and re-assessed several biological activities attributed to SAA1 (chemotaxis, cytokine induction, MMP-9 release, ROS generation, and macrophage differentiation). The homogeneous rSAA1 (hrSAA1) lacked most cell-activating properties, but its leukocyte-recruiting capacity in vivo and it's in vitro synergy with other leukocyte attractants remained preserved. Furthermore, hrSAA1 maintained the ability to promote monocyte survival. This indicates that pure hrSAA1 retains its potential to activate FPR2, whereas TLR-mediated effects seem to be related to traces of bacterial TLR ligands in the E. coli-produced human rSAA1.

Autocrine induction of collagenase by serum amyloid A-like and beta 2-microglobulin-like proteins.

Two autocrine proteins of 14 and 12 kilodaltons that induce the synthesis of rabbit fibroblast collagenase were identified. The proteins were purified from serum-free culture medium taken from rabbit synovial fibroblasts stimulated with phorbol myristate acetate. The amino-terminal sequences of the 14- and 12-kilodalton species were approximately 60 to 80 percent homologous with serum amyloid A and beta 2 microglobulin, respectively. The polyacrylamide gel-eluted proteins retained the ability to induce collagenase synthesis in rabbit and human fibroblasts. These autocrine proteins may provide a means to modulate collagenase synthesis in normal remodeling as well as in inflammation and disease states.

Serum amyloid A does not affect high-density lipoprotein cholesterol measurement by a homogeneous assay.

BACKGROUND: Serum amyloid A (SAA), which is one of the acute phase proteins, alters the structure of HDL by associating with it during circulation. We focused on whether SAA influences the values of HDL-cholesterol (HDL-C) measurements when using a homogeneous assay. METHODS: HDLs were isolated by ultracentrifugation from serum samples of 248 patients that were stratified into three groups based on their serum SAA concentrations (low: SAA ≤ 8 µg/mL; middle: 8 < SAA ≤ 100 µg/mL; and high: SAA > 100 µg/mL). HDL-C concentrations of the serum samples measured by the homogeneous assay were compared with the total cholesterol concentrations of HDL fractions isolated by ultracentrifugation. RESULTS: HDLs obtained from patients with low SAA concentrations were separated into their general particle sizes and classified as HDL2 and HDL3 by native-gel electrophoresis. On the other hand, HDLs obtained from patients with high SAA concentrations occasionally showed distributions different from the typical sizes of HDL2 and HDL3, such as extremely small or large particles. Nevertheless, HDL-C concentrations measured using the homogeneous assay were strongly correlated with those measured using the ultracentrifugation method, regardless of the SAA concentrations. However, the ratios of HDL-C concentrations obtained by the homogeneous assay to those obtained by the ultracentrifugation method for patients with high SAA concentrations were significantly lower than those of patients with low SAA concentrations. CONCLUSIONS: A large amount of SAA attached to HDL altered the HDL particle size but did not essentially affect HDL-C measurement by homogeneous assay.

Amyloid fibril protein in familial amyloidotic polyneuropathy, Portuguese type. Definition of molecular abnormality in transthyretin (prealbumin).

Amyloid fibril protein in patients with familial amyloidotic polyneuropathy is known to be chemically related to transthyretin (TTR), the plasma protein that is usually referred to as prealbumin. A genetically abnormal TTR may be involved in this disease. Studies were conducted on amyloid fibril protein (AFp) isolated from tissues of two Portuguese patients who died with familial amyloidosis, and on TTR isolated from sera of patients with this disease. AFp, purified by affinity chromatography on retinol-binding protein linked to Sepharose, resembled plasma TTR in forming a stable tetrameric structure, and in its binding affinities for both thyroxine and retinol-binding protein. The structural studies included: (a) comparative peptide mappings by reverse-phase high performance liquid chromatography (HPLC) after trypsin digestion; (b) cyanogen bromide cleavage studies; and (c) amino acid microsequence analysis of selected tryptic and CNBr peptides. On the basis of the known amino acid sequence of TTR, comparative tryptic peptide maps showed the presence of a single aberrant tryptic peptide (peptide 4, residues 22-34) in AFp as compared with TTR. This aberrant peptide contained a methionine residue, not present in normal tryptic peptide 4. CNBr cleavage of AFp produced two extra peptide fragments, which were demonstrated, respectively, by HPLC analysis and by sodium dodecyl sulfate-gel electrophoresis. Sequence analyses indicated the presence of a methionine-for-valine substitution at position 30 in AFp as compared with TTR. Thus, the purified amyloid fibril protein comprised a TTR variant with a methionine-forvaline substitution at position 30. A single nucleotide change in a possible codon for valine 30 could explain the substitution. The variant TTR was also present in the TTR isolated from the pooled sera of amyloidoses patients, together with larger (four- to six-fold) amounts of the normal TTR. Thus, in these patients, the variant TTR was circulating in plasma, along with larger amounts of normal TTR. We suggest that the variant TTR represents the specific biochemical cause of the disease, and that this abnormal form of TTR selectively deposits in tissues as the amyloid characteristic of the disease.

Identification of lipopolysaccharide-binding proteins in porcine milk.

Septicemia and endotoxemia initiated by bacterial lipopolysaccharide (LPS) are relatively common in suckling and weaned piglets. Maternal milk is a source of both nutrition and immune protection for piglets. Passive transfer of colostral antibodies is necessary for protection of neonatal piglets against diseases, but the concentration of immunoglobulins in milk rapidly declines during the 1st wk of lactation in all mammals. We hypothesized, therefore, that nonimmunoglobulin substances in milk contribute to the innate protection of neonates against septicemia during the suckling period. Using LPS-affinity chromatography for isolation of LPS-binding proteins and liquid chromatography-mass spectrometry for their identification, we identified in porcine milk the following proteins with LPS-binding capacity: lactoferrin, soluble CD14, serum amyloid A, alpha-S1 casein, beta-casein, and kappa-casein. For lactoferrin, alpha-S1 casein, and kappa-casein, in vitro pepsin digestion did not inhibit LPS-binding activity, whereas combined digestion with pepsin and pancreatin abolished it. The biologic functions of these LPS-binding proteins and peptides were not determined.

Recombinant murine serum amyloid A from baculovirus-infected insect cells: purification and characterization.

Serum amyloid A (SAA) is an extremely sensitive acute-phase reactant and precursor to the subunit protein in reactive amyloid deposits. Although the mouse has long served as an informative experimental model, both the function of SAA and the pathogenic mechanism of amyloid formation remain unknown. The production of SAA by a heterologous system was pursued as means of generating readily-renewable amounts of SAA of defined sequence. Murine SAA2 has been expressed in and purified from baculovirus-infected insect cells. Using the transfer vector pBlueBac, SAA2 cDNA was cloned into baculovirus DNA such that expression was under the control of the polyhedrin promoter. Lysates prepared from infected cells contained three amyloid A-immunoreactive forms which accumulated intracellularly over a three day periods. The form having the lowest relative molecular mass, 12.5 kDa, co-migrated in SDS-polyacrylamide gels with the SAA2 present in murine acute-phase serum. Recombinant SAA2 was purified by Sepharose CL-6B chromatography followed by chromatofocusing between pH 8 and pH 5. Amino-terminal sequencing of the purified 12.5 kDa sample confirmed the first 20 residues of mature murine SAA2. After incubation with normal mouse serum, purified recombinant SAA2 fractionated exclusively with lipoprotein complexes, suggesting that it was bound to HDL. Based on this observation, we believe that recombinant SAA can serve as a suitable substitute for the native protein in physiologically relevant studies.

Identification of two novel amyloid A protein subsets coexisting in an individual patient of AA-amyloidosis.

Amyloid A protein (AA), the major fibril protein in AA-amyloidosis, is an N-terminal cleavage product of the precursor protein, serum amyloid A (SAA). Using mass spectrometry and amino-acid sequencing, we identified and characterized two novel AA protein subsets co-deposited as amyloid fibrils in an patient having AA-amyloidosis associated with rheumatoid arthritis. One of the AA proteins corresponded to positions 2-76 (or 75) of SAA2 alpha and the other corresponded to positions 2-76 (or 75) of known SAA1 subsets, except for position 52 or 57, where SAA1 alpha has valine and alanine and SAA1 beta has alanine and valine in position 52 and 57, respectively, whereas the AA protein had alanine at the both positions. Our findings (1), demonstrate that not only one but two SAA subsets could be deposited together as an AA-amyloid in a single individual and (2), support the existence of a novel SAA1 allotype, i.e., SAA152,57Ala.

Purification and separation of hydrophobic serum amyloid A precursor isoforms by a one-step preparative method.

The levels of two major serum amyloid A precursor isoforms, SAA1 and SAA2, which are associated with high-density lipoproteins (HDL) are increased during inflammation. The hydrophobic character and the small size difference--corresponding to just 0.8 kDa--between these two members of the SAA family hinder their separation and purification on a large scale by conventional methods. In the current work, both mouse SAA proteins were purified from HDL-SAA and acute-phase serum within 10 h in a one-step procedure using the high-resolution, continuous-elution preparative gel electrophoresis Prep-Cell system in combination with Tris/Glycine SDS-PAGE. Moreover, applying the Tris/Tricine system on the Prep-Cell resulted not only in purification of the SAA proteins, but also in their separation within 16 h. The SAA isoforms were freed from SDS using a Centricon concentrator and were identified using monoclonal antibodies. Optical density profile plots of gel protein or Western blot bands in combination with a colorimetric spectrophotometric protein assay showed that the recovery of the isoforms ranged from 38% to 60%. These results show that the preparative gel electrophoresis system Prep-Cell is a suitable device for separating SAA1 and SAA2 proteins in a simplified, convenient, and fast procedure, which can be applied on a small or large scale.

Purification of amyloid protein AA subspecies from amyloid-rich human tissues.

Protein AA, the major amyloid fibril protein in reactive (secondary) systemic amyloidosis is derived from the acute phase reactant liver-produced apolipoprotein serum AA (SAA) by proteolytic cleavage, usually in the C-terminal half of the 104 amino acid residues long precursor. The cleavage points in SAA vary between patients and the deposited protein AA is often quite heterogeneous. In this chapter, we describe methods to extract amyloid fibrils and to purify protein AA by sequential gel filtration. Further purification of subspecies of protein AA is best achieved by the use of differences in charge and chromatofocusing is described as the method of choice. Analytic methods include sodium dodecylsulfate polyacrylamide gel electrophoresis and analytic isoelectric focusing.

Finnish hereditary amyloidosis. Amino acid sequence homology between the amyloid fibril protein and human plasma gelsoline.

Amyloid fibrils were isolated from the kidney of a patient with Finnish hereditary amyloidosis. After solubilization of the fibrils in guanidine-HCl, fractionation by gel filtration, and purification by reverse-phase high-performance liquid chromatography, a homogeneous amyloid protein with an apparent Mr of 9000 was obtained. The protein was subjected to enzymatic digestion by trypsin and endoproteinase Lys-C. The amino acid sequences were determined for 6 of the released peptides and they were all found to be identical to the reported, deduced primary structure of human plasma gelsoline in the region of amino acids 235-269. The results show that the amyloid fibril protein in Finnish hereditary amyloidosis represents a new type of amyloid protein that shows amino acid sequence homology with gelsoline, an actin-modulating protein.

Quantification and mapping of antigenic determinants of serum amyloid A (SAA) protein utilizing sequence-specific immunoglobulins and Eu3+ as a specific probe for time-resolved fluorometric immunoassay.

Serum amyloid A (SAA) protein, the most prominent amongst acute-phase proteins, is the specific precursor protein of secondary reactive amyloidosis. The fact that SAA once released into the circulation as a 'free' protein rapidly associates with lipoproteins of the high-density range indicates a specific role in lipoprotein metabolism. In this study a new sensitive assay for quantification of human SAA protein in biological specimens using affinity-purified polyclonal antibodies and Eu3+ as a specific probe for time-resolved fluorometric immunoassay is presented. Both purified SAA and SAA-rich high-density lipoprotein particles served as reliable standards in the indirect and the direct sandwich dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA). The detection limit of the DELFIA technique presented was 4-10 ng after sample dilution of 1/2500. The intra-assay coefficient of variation averaged 4.3% whereas the inter-assay coefficient of variation averaged 6.2%. Comparison with the nephelometric assay, a widely and commonly used assay for SAA quantification in plasma, revealed correlation coefficients of 0.9428. In addition to polyclonal anti-human SAA antibodies sequence-specific antibodies raised against synthetic peptides corresponding to region; 1-17, 14-30, 27-44, 40-63, 59-72, 68-84, 79-94, and 89-104 of the human SAA amino acid sequence were studied. Sequence-specific antibodies raised against epitopes 27-44, 59-72, 68-84, and 89-104 recognize human SAA protein in the DELFIA assay whereas antibodies raised against epitopes 1-17, 14-30, 40-63 and 79-94 failed to recognize the corresponding epitopes. Results obtained from these studies indicate that the N-terminal domain (1-30) as well as epitopes 40-63 and 79-94 of human SAA are apparently masked by the environment of the lipoprotein particle. From our studies it is proposed that the epitopes 31-39, 64-78, and 95-104 may be responsible for the interaction of SAA-rich high density lipoprotein particles with peripheral cells.

A high resolution ultrastructural comparison of isolated and in situ murine AA amyloid fibrils.

There is an inconsistency between the ultrastructural organization of AA amyloid fibrils that have been isolated, which are composed of a slowly twisting set of two or more protofibrils, and those seen in situ, which are tubular entities with a tight helical substructure. In this study, the ultrastructure of fibrils isolated from experimental murine AA amyloid were observed at high resolution and compared with those seen in situ in the hope of clarifying the reason for this inconsistency. The fibrils in situ were composed of a microfibril-like 8-9 nm wide core covered by a layer of heparan sulfate proteoglycan (HSPG) to which 1 nm wide filaments, immunohistochemically identified as AA protein, were externally associated. Following isolation with the standard distilled water washing procedure, the HSPG layer and AA protein filaments detached from their core and dispersed into the water. The remaining denuded, variously loosened cores lost their typical appearance. In distilled water the detached 1 nm wide AA protein filaments became quite conspicuous and coiled themselves into 3 nm wide tight helices which in turn assembled into the characteristic slowly twisting sets of two parallel protofibrils similar to that previously reported as "isolated amyloid fibrils". The results emphasize that great caution must be taken in extrapolating amyloid fibril structure from isolated preparations to in situ tissue conditions.

N-terminal sequence analysis of SAA-derivatives purified from murine inflammatory macrophages.

The pathogenesis of secondary amyloidosis in vivo is not well-understood. Experimental studies suggest that incomplete degradation of acute phase serum amyloid A (SAA), presumably endocytosed by activated monocytoid cells, may lead to intralysosomal formation of amyloid A (AA). To establish a possible link between these two events, we have carried out partial N-terminal sequence analysis of affinity purified SAA derivatives from peritoneal macrophages isolated at 4 weeks post-infection from alveolar hydatid cyst infected C57BL/6 mice. The macrophage lysates yielded five N-terminally intact SAA derivatives of approximately 5 to approximately 12 kDa which reacted with anti-mouse AA IgG, and contained a mixture of SAA1 and SAA2 isoforms. The SAA2:SAA1 ratio, evaluated from their proportion present in each M(r) SAA derivative, showed a decrease with the decreasing apparent mass of the N-terminally infected SAA material. These results not only confirm that both SAA1 and SAA2 are processed by activated monocytoid cells but, more importantly, establish a plausible link between N-terminally intact SAA derivatives and formation of AA within activated monocytoid cells.

Family studies of the genetic abnormality in transthyretin (prealbumin) in Portuguese patients with familial amyloidotic polyneuropathy.

Amyloid deposits in several heredofamilial forms of amyloidosis are chemically related to transthyretin (TTR, the protein usually referred to as prealbumin). A genetically abnormal TTR may be involved. Studies were conducted on TTR isolated from sera of patients with familial amyloidotic polyneuropathy (FAP), and on amyloid fibril protein (AFp) isolated from tissues of two Portuguese patients who died with FAP. AFp, purified by affinity chromatography on retinol-binding protein (RBP), resembled plasma TTR in forming a stable tetrameric structure, and in its binding affinities for both thyroxine and RBP. Purified AFp was found to comprise a TTR variant with a methionine for valine substitution at position 30. This conclusion was based upon studies that included: (i) comparative peptide mapping by reverse-phase high-performance liquid chromatography after trypsin digestion; (ii) cyanogen bromide (CNBr) cleavage studies; and (iii) amino acid microsequence analysis of selected tryptic and CNBr peptides. The variant TTR was also found to be present in serum samples from FAP patients, along with larger amounts of normal TTR. An effective, small-scale procedure was developed to determine whether or not the variant TTR was present in the plasma of an individual subject. This procedure involved isolation of TTR by affinity chromatography on RBP, followed by CNBr cleavage, and analysis for the presence of specific aberrant CNBr peptides. Studies with six kindreds, including 21 asymptomatic children of 6 patients with FAP, showed that the "abnormal" TTR can be detected and used as a preclinical marker of the disease in affected children of patients with FAP. It is likely that the variant TTR represents a point mutation within the TTR structural gene, and that the normal and mutant genes act as co-dominant alleles at a single locus in FAP. The distribution of the mutant TTR within the six families was consistent with the autosomal dominant mode of inheritance of FAP. The mutant TTR apparently selectively deposits in tissues as the amyloid characteristic of the disease.

Enrichment of serum amyloid proteins by hydrophobic interaction chromatography combined with two-dimensional electrophoresis with immobilised pH gradients.

Serum amyloid A protein was subjected to one-step octyl-Sepharose extraction in three different dimensions. Elution was performed partly without UV recording, and with urea or guanidine-based buffers. The eluent was applied directly to denaturing two-dimensional electrophoresis with immobilised pH gradient, or octyl-Sepharose extracted fractions were pooled and lyophilised before application. Proteins were characterised by N-terminal analysis or mass spectrometry. In most of the species that were studied, previously undescribed serum amyloid proteins were detected. Compared to conventional strategies, the presented techniques are more rational and yield more comprehensive information. The presented data also provide a basis for novel perspectives regarding certain inflammatory conditions.

Serum amyloid protein (SAP) as a marker of autoimmune disease in mice.

Acute phase proteins are good markers of inflammatory processes. To clarify whether Serum Amyloid Protein (SAP) can be a marker for the onset of SLE disease in mice, we measured constitutive and inducible SAP levels in normal mice of different strains, in C57Bl/6 lpr/lpr (B6lpr) and [NZB x NZW]F1 (NZB/W) SLE-prone mice, in mice that develop Lupus-like syndrome during chronic Graft versus Host (GvH) reaction and in mice suffering acute GvH reaction. In comparison to B6lpr, NZB/W mice showed higher blood levels of SAP but those levels did not correlate with autoimmune parameters. In B6lpr, the SAP levels steadily increased with age and correlated with some of the parameters used for monitoring the SLE disease. High levels of SAP were also found in mice suffering acute GvH reaction whereas the lupus-like chronic GvH disease was associated with limited increase of SAP levels.