Anticachectin/tumor necrosis factor-alpha antibodies attenuate development of cachexia in tumor models.

C57BL/6 mice bearing either a transplantable methylcholanthrene-induced sarcoma or Lewis lung adenocarcinoma were passively immunized every other day with a rabbit immunoglobulin fraction raised against murine cachectin/tumor necrosis factor-alpha. Mice bearing methylcholanthrene-induced sarcoma developed tumor-associated hypophagia that was attenuated by anticachectin immunoglobulin treatment. In the same tumor-bearing animals, anticachectin treatment also significantly reduced the extent of carcass protein and fat loss, and reduced tumor weight. Mice bearing Lewis lung adenocarcinoma did not develop significant anorexia or carcass lean tissue depletion as tumor growth progressed, but they lost carcass lipid. Treatment of Lewis lung adenocarcinoma bearing mice with anticachectin antibodies diminished the degree of carcass lipid depletion and prevented plasma hypertriglyceridemia. However, in both tumor models, anticachectin treatment did not affect either the development of anemia, hypoalbuminemia or the increase in serum amyloid P concentrations seen with increasing tumor burden. We conclude that an endogenous cachectin response, inhibitable by exogenously administered antibody, contributes to anorexia and to changes in body fat and protein metabolism in these tumor-bearing animals. Neutralizing endogenous cachectin production with antibodies offers the potential to reduce tissue wasting that is frequently associated with neoplastic disease, but it does not appear to affect all of the hematologic and acute phase responses in these murine tumor models.

Appearance of hybridoma growth factor/interleukin-6 in the serum of mice bearing a methylcholanthrene-induced sarcoma.

Serum concentrations of hybridoma growth factor/interleukin-6 progressively increased in mice bearing a transplantable methylcholanthrene-induced sarcoma with tumor growth. Elevated HGF/interleukin-6 concentrations were also positively correlated with increased serum concentrations of the hepatic acute phase reactant protein, amyloid P. Daily Indomethacin treatment of sarcoma-bearing mice prolonged survival and reduced the magnitude of the serum amyloid P response, but failed to attenuate either tumor growth or serum HGF/interleukin-6 responses. Since previous studies have demonstrated that neither interleukin-1 nor tumor necrosis factor-alpha can be detected in the serum of these sarcoma-bearing mice, and that HGF/interleukin-6 is a principal mediator of the hepatic acute phase response, we conclude that circulating HGF/interleukin-6 may contribute significantly to the host responses which accompany experimentally-introduced cancer. Furthermore, prostanoid inhibition does not appear to regulate the synthesis and release of HGF/interleukin-6 during tumor growth.

Elevation of plasma fibronectin and serum amyloid P in autoimmune NZB, B/W, and MRL/1pr mice.

The acute-phase proteins, fibronectin (Fn) and serum amyloid P (SAP), are opsonins which by virtue of their adhesive properties may be involved in the glomerular nephritis associated with splenic lupus erythematosus (SLE). Because of their possible involvement in the pathophysiology of lupus, plasma Fn and SAP levels from three strains of autoimmune mice were measured over time to determine if Fn and SAP rose as the mice sickened and renal function degenerated. Baseline levels of Fn and SAP were measured when the mice were between 1.5 and 3 months of age. The characteristic rapid onset of autoimmune disease in MRL/1pr mice was accompanied by a two- to threefold increase in plasma Fn and SAP by Day 100. The B/W mice, which develop autoimmune disease more slowly, did not have a significant increase in plasma Fn and SAP until Day 240. The NZB mice, with the most delayed onset of disease, exhibited a modest but significant elevation of plasma Fn and SAP by Day 360. Histologic examination of the kidneys of B/W and NZB mice indicated that pathological abnormality of the glomeruli and tubules coincided with the elevation of plasma Fn and SAP levels. In contrast, blood samples taken over time from normal BALB/c mice did not possess abnormal levels of Fn or SAP. It appears that elevation of plasma Fn and SAP in the MRL/1 pr, B/W, and NZB mice is related to the onset and severity of autoimmune disease and the subsequent loss of renal function.

Isolation and analysis of murine serum amyloid P component cDNA clones.

In contrast to other animals, the biosynthesis of serum amyloid P component in mice is regulated as an acute-phase protein. As a first step in studying the regulation and biosynthesis of serum amyloid P component in the mouse, cDNA clones have been isolated from a liver cDNA library and sequenced. The largest of these clones was 960 bp in length, and contained an open reading frame encoding a protein of 224 amino acids. Comparison of the mouse cDNA sequence to that published for humans (Mantzouranis, E. C., S. B. Dowton, A. S. Whitehead, M. D. Edge, G. A. P. Bruns, and H. R. Colten, 1985. J. Biol. Chem. 260:7752.) revealed 74% identity for nucleotides in the translated region. Northern-blot analysis demonstrated that murine serum amyloid P component synthesis in the liver is directed by a 1.2-kb mRNA that is elevated in high responder (C57BL/6J) mice after thioglycollate-induced inflammation.

Effect of inhibitors of eicosanoid metabolism in murine collagen-induced arthritis.

The dual inhibitors of arachidonic acid metabolism, Smith Kline & French (SK&F) 86002, SK&F 104351, and phenidone; the corticosteroid, dexamethasone; and the selective cyclooxygenase inhibitors, ibuprofen, indomethacin, naproxen, and piroxicam were evaluated for their antiarthritic potency in the murine, collagen-induced arthritis model. The ability of these compounds to alter the severity of arthritic lesions and to reduce serum levels of the acute-phase reactant, serum amyloid P component (SAP) were monitored. Serum concentrations of SAP were found to correlate strongly (r = 0.985) with disease severity at day 35 postimmunization. Treatment with SK&F 86002, SK&F 104351, phenidone, or dexamethasone significantly reduced disease severity, as judged by clinical score (55%, 72%, 41%, and 45% inhibition, respectively) and SAP levels (62%, 94%, 52%, and 94% inhibition, respectively) in arthritic mice. This profile of activity was not shared by the selective cyclooxygenase inhibitors, which did not uniformly inhibit disease activity by both parameters. The results suggest that dual inhibitors of 5-lipoxygenase and cyclooxygenase may prove more effective than selective cyclooxygenase inhibitors as anti-arthritic agents.

Evidence that serum amyloid P component binds to mannose-terminated sequences of polysaccharides and glycoproteins.

Serum amyloid P component (SAP) is a normal human serum protein with pentraxin structure that has morphological and immunochemical identity to the amyloid P component found in normal tissue and amyloid deposits. In the presence of calcium, SAP binds to certain complex polysaccharides, including agarose and zymosan. While the binding of SAP to agarose involves interaction with a galactose pyruvate acetal, the ligand in zymosan has not been defined. In the present study we determined that SAP binds to ligand(s) in a soluble extract of zymosan prepared by alkaline hydrolysis, which contains the mannose oligosaccharide sequences alpha DMan1----3DMan and alpha DMan1----6DMan. SAP did not bind to the alkali-insoluble fraction of zymosan, which is predominantly a glucan polymer, and its binding to zymosan extract which had been absorbed with concanavalin A was markedly reduced, suggesting that mannose residues are involved in the binding of SAP to zymosan. We also demonstrated that SAP binds to the glycoproteins ovalbumin, thyroglobulin, beta-glucuronidase and C3bi, which contain mannose-terminated sequences, while it did not bind to native and desialized preparations of ovomucoid, alpha 1-acid glycoprotein and glycophorin, which lack terminal mannose residues. SAP did not bind to pneumococcal C polysaccharide or to N-acetylglucosamine oligosaccharides covalently linked to a protein carrier. The binding of SAP to ligand(s) in zymosan extract or ovalbumin was inhibited by the preincubation of SAP with either zymosan extract or ovalbumin glycopeptides, both of which share similar mannose oligosaccharide sequences. All of the SAP binding reactions required calcium, were maximal at approximately 1 mM calcium, and gave similar results whether purified SAP or SAP in serum was used. These findings indicate that mannose-terminated oligosaccharides of polysaccharides and glycoproteins represent a new class of ligands for SAP and suggest that SAP may function as a mannose-binding protein.

Diagnostic radionuclide imaging of amyloid: biological targeting by circulating human serum amyloid P component.

The specific molecular affinity of the normal plasma protein, serum amyloid P component (SAP), for all known types of amyloid fibrils was used to develop a new general diagnostic method for in-vivo radionuclide imaging of amyloid deposits. After intravenous injection of 123I-labelled purified human SAP there was specific uptake into amyloid deposits in all affected patients, 7 with systemic AL amyloid, 5 with AA amyloid, and 2 with beta 2M amyloid, in contrast to the complete absence of any tissue localisation in 5 control subjects. Distinctive high-resolution scintigraphic images, even of minor deposits in the carpal regions, bone marrow, or adrenals, were obtained. This procedure should yield much information on the natural history and the management of amyloidosis, the presence of which has hitherto been confirmed only by biopsy. Clearance and metabolic studies indicated that, in the presence of extensive amyloidosis, the rate of synthesis of SAP was greatly increased despite maintenance of normal plasma levels. Furthermore, once localised to amyloid deposits the 123I-SAP persisted for long periods and was apparently protected from its normal rapid degradation. These findings shed new light on the pathophysiology of amyloid and may have implications for therapeutic strategies based upon specific molecular targeting with SAP.

The carbohydrate composition of human serum amyloid P component.

The carbohydrate moiety of human serum amyloid P component was analyzed and found to consist of equal amounts of galactose and mannose (total 4.0%), of glucosamine and galactosamine in a ratio of 7:1 (total 2.7%) and sialic acid (3.9%). It should be noted that this is the first report on the separate quantification of the neutral hexoses and the demonstration of the presence of galactosamine. The contents of glucosamine and galactosamine suggest that this protein possesses both an N- and an O-glycan.

Observations on the delay in onset of the acute phase protein response.

The early time course of the acute phase protein response (APPR) and mediators involved in its control were investigated in the rat and mouse. After turpentine-induced inflammation in the rat C-reactive protein and fibrinogen increased in concentration peaking at 48 h and 18-24 h, respectively. A 9 h delay prior to elevation of these protein was observed. After injection of endotoxin into mice, a 4-6 h delay was observed prior to any increase in the concentration of the acute phase protein serum amyloid P-component. This delay was shortened to 2 h after injection of leucocytic endogenous mediators (LEM) produced from rabbit peritoneal exudate cells. It is concluded that the delay between the initiating stimulus and the increases in the acute phase proteins is due to some obligatory intermediate steps which lead to the production of the final mediators of the APPR, and that these mediators are present in LEM.

The serum amyloid P response in the mouse air pouch.

Levels of the acute phase reactant serum amyloid P (SAP) have been measured in the mouse pouch model of rheumatoid arthritis. Implantation of cartilage resulted in a significant and rapid elevation in the SAP concentration, which remained high for the duration of the experiment (14 days). Initial studies with several clinically employed antirheumatic drugs indicated that dexamethasone and cyclosporin A had a marked inhibitory effect.

Dietary fish oil modulation of macrophage amyloid P component responses in mice.

Arthritis-susceptible B10.RIII mice, maintained on either fish oil (FO) or corn oil (CO) diets (5% by weight), and amyloid-susceptible CBA/J mice fed chow diets were given 20 micrograms purified LPS by i.p. injection. Both strains of mice responded to LPS with a 20- to 30-fold increase in plasma amyloid P component (AP) levels. There were no differences in the response between males and females or between FO and CO treatment groups. The data demonstrated that cultured peritoneal macrophages (M phi) respond to LPS stimulation with increased secretion of AP. In contrast to plasma AP levels, the MO response to LPS stimulation, as measured by production of AP, was influenced by both gender and diet. Although M phi from both male and female mice on the CO diet and male mice on the FO diet responded similarly, those from female mice on the FO diet secreted only 25 to 35% as much AP as did the other three groups. There were no dietary effects on the LPS-induced serum amyloid A protein response nor was there detectable serum amyloid A protein produced by the M phi. These results demonstrate that unstimulated, resident peritoneal M phi secrete AP as a normal constituent and in increasing amounts in response to LPS stimulation.

Serum amyloid P component induction by immunomodulators.

The capacity of immunoadjuvants to enhance serum amyloid P component (SAP) levels and to modulate the humoral immune response to sheep red blood cells in a number of different mouse strains was investigated. Although the synthetic adjuvants dimethyldioctadecylammonium bromide, dextran sulphate and bacterial-derived lipopolysaccharide did not enhance SAP levels in some of the mouse strains tested, these strains responded normally to the immunomodulating effects of the adjuvants. We conclude that increased SAP levels and modulation of immune responses are induced via at least partially different pathways. For these reasons, it is impossible to screen drugs for potential adjuvant activity by only measuring SAP levels in mice.

T-2 toxin effects on the serum amyloid P-component (SAP) response of Listeria monocytogenes- and Salmonella typhimurium-infected mice.

T-2 mycotoxin, given to mice 4 days prior to an intraperitoneal inoculation with Listeria monocytogenes EGD, increases the acute phase response as determined by measurements of serum amyloid protein-P (SAP), and decreases the severity of the infection. Conversely, when T-2 toxin is given simultaneously with L. monocytogenes the mice become more susceptible to the infection, and the SAP levels attained are diminished relative to the non-toxin-treated Listeria-infected controls. T-2 toxin given 4 days prior to intraperitoneal inoculation with Salmonella typhimurium had no effect on either the resultant infection or SAP levels. These results indicate that T-2 toxin modulates the acute phase response to infection, and are consistent with an in vivo role for SAP as a nonspecific host resistance factor.

Serum amyloid P component is the major calcium-dependent specific DNA binding protein of the serum.

Serum amyloid P component (SAP), a member of the highly conserved pentraxin family of plasma proteins, was found to be the only protein in whole normal or acute phase serum which underwent specific calcium-dependent binding to either single or double-stranded DNA immobilised on gel. Isolated purified SAP also bound to long chromatin, to H1-stripped chromatin and to native DNA in solution at physiological ionic strength. Pure SAP which had been immobilised on gel, specifically bound nucleosome core particles from solution. These observations strongly suggest that SAP may bind to extracellular chromatin and DNA in vivo and that this may be its physiological role.

Levels of serum amyloid P-component associated with pregnancy and collagen-induced arthritis in DBA/1 (H-2q) mice.

The levels of the acute-phase reactant serum amyloid P-component (SAP) were measured by quantitative rocket immunoelectrophoresis in pregnant and non-pregnant DBA/1 female mice with or without collagen-induced rheumatoid arthritis (CIA). Non-pregnant animals with CIA showed elevated SAP titres related to the severity of the disease. Pregnancy alone also caused increased SAP levels equivalent to those found in animals with established CIA but which were virgin. The clinical remission seen in arthritic animals during pregnancy was not associated with reductions in circulating SAP levels. Increasing parity, however, caused a lowering of SAP levels in animals with or without CIA compared to the primiparous individuals. Pregnancy causes a strain-dependent elevation of serum SAP which is not further elevated by CIA, thus limiting the usefulness of SAP measurements in assessment of disease progression or remission during gestation.

Identification and isolation of two pentraxins from bovine serum.

Two distinct pentraxin proteins were isolated from bovine serum by calcium-dependent affinity chromatography on high pyruvate agarose gel. One of these proteins cross-reacted specifically with certain rabbit anti-human CRP antisera and was therefore designated as bovine CRP. The other cross-reacted specifically with a sheep anti-human SAP antiserum and was therefore designated as bovine SAP. Although the mixture of these two pentraxins was not resolved by gel filtration chromatography, they were separated by solid phase absorption of the CRP with immobilized rabbit anti-human CRP antibodies. Their identity as pentraxins was confirmed by their electron microscopic appearance. Bovine CRP was composed of a single type of non-glycosylated subunit whilst bovine SAP contained two major types of glycosylated subunits and a minor polypeptide, the glycosylation of which was not determined. Serum concentrations were in the range of 5-40 mg/l and neither protein behaved as an acute phase reactant. No bovine serum protein undergoing calcium-dependent binding to phosphoryl choline or pneumococcal C-polysaccharide was obtained.