Enhancement of AA-amyloid formation in mice by transthyretin amyloid fragments and polyethylene glycol

Biochimica et Biophysica Acta 1474 (2000) 331^336

www.elsevier.com/locate/bba

Enhancement of AA-amyloid formation in mice by transthyretin amyloid fragments and polyethylene glycol Charles Mambule a , Yukio Ando b;1 , Intissar Anan a , Go«sta Holmgren c , Ola Sandgren d , Torgny Stigbrandt e , Kazuhiro Tashima a , Ole Bernt Suhr a; * b

a Department of Medicine, Umea® University Hospital, Umea®, Sweden First Department of Medicine, Kumamoto University School of Medicine, Kumamoto, Japan c Department of Clinical Genetics, Umea® University Hospital, Umea®, Sweden d Department of Ophthalmology, Umea® University Hospital, Umea®, Sweden e Department of Immunology, Umea® University Hospital, Umea®, Sweden

Received 26 October 1999; received in revised form 3 February 2000; accepted 24 February 2000

Abstract The mechanism behind amyloid formation is unknown in all types of amyloidosis. Several substances can enhance amyloid formation in animal experiments. To induce secondary systemic amyloid (AA-type amyloid) formation, we injected silver nitrate into mice together with either amyloid fibrils obtained from patients with familial polyneuropathy (FAP) type I or polyethylene glycol (PEG). Mice injected with silver nitrate only served as controls. Amyloid deposits were detectable at day 3 in animals injected with amyloid fibrils and in those injected with PEG, whereas in control mice, deposits were not noted before day 12. Our results indicate that amyloid fibrils from FAP patients and even a non-sulfate containing polysaccharide (PEG) have the potential to act as amyloid-enhancing factors. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Amyloid formation; Amyloidosis ; Familial ; Secondary; Transthyretin ; Polyethylene glycol ; Amyloid A protein

1. Introduction In most types of amyloidosis, the proteins or protein precursors that form the amyloid ¢brils have been identi¢ed [1,2]. However, the mechanism leading to the L pleated sheet conformation of the proteins and the formation of amyloid ¢brils from the precursor proteins remains to be elucidated. Since the initial report of amyloid enhancement properties of spleen cells from amyloidotic mice [3], several substances have been identi¢ed, that possesses amyloid enAbbreviations : AA, amyloid A; AA-amyloidosis, secondary systemic amyloidosis; AEF, amyloid-enhancing factor; ELISA, enzyme-linked immunoassay; FAP, familial amyloidotic polyneuropathy ; PEG, polyethylene glycol; TTR, transthyretin ; ATTR V30M, transthyretin with the methionine for valine substitution at position 30 * Corresponding author. Section for Gastroenterology and Hepatology, Department of Medicine, Umea® University Hospital, S-901 85 Umea®, Sweden. Fax: +46-90-143-986; E-mail : [email protected] 1 Y.A. worked temporarily as a visiting professor at the Department of Medicine, Umea® University Hospital, Umea®, Sweden.

hancing factor (AEF) properties in susceptible animals. Apart from intact spleen cells, homogenated spleen cells also induce amyloid formation [4]. Even though studies have suggested that AEF is a protein of an approximately size of 10^16 kDa [5,6], AEF has never been identi¢ed. In addition, amyloid ¢brils, and also amyloid-like synthetic ¢brils constructed from transthyretin fragments have AEF properties as have several other non-¢bril substances, such as TNF-a and sulfated glycosaminoglycans [5,7^10], It has therefore been suggested, that AEF is not a single compound, and that several di¡erent molecules and structures have AEF properties [8]. There appears to be a lag phase before the onset of amyloid formation, but once the ¢rst amyloid ¢brils or pro-¢brils have been formed, the process accelerates [11]. This ¢nding indicates, that an AEF could be a substance that acts as a seed, triggering amyloid formation [8]. In secondary systemic amyloidosis (AA-amyloidosis) the precursor protein is an acute phase apolipoprotein, amyloid A (AA) [12], whereas in familial amyloidotic polyneuropathy (FAP) type I, the amyloidotic protein is mutated transthyretin (TTR) in which valine is exchanged for

0304-4165 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 0 ) 0 0 0 3 2 - 5

BBAGEN 24997 10-4-00

332

C. Mambule et al. / Biochimica et Biophysica Acta 1474 (2000) 331^336

methionine at position 30 (ATTR V30M) [13]. It is suggested that AA precursor proteins can aggregate with sonicated FAP amyloid ¢brils and act as a seed for the enhancement of AA amyloidosis thus shortening the lag phase before the onset of AA amyloid deposition [7]. AAamyloidosis can be induced experimentally in animals by induction of a long-lasting in£ammation, e.g. by subcutaneous injection of silver nitrate (AgNO3 ) [7]. By this method, amyloid is formed in the animal within 2 weeks, making this model valuable for studying amyloid formation. The aim of the present study was to investigate the amyloid enhancing properties of sonicated ATTR V30M amyloid ¢brils and a hydrophobic 10-kDa non-sulfated polysaccharide (PEG) in AgNO3 -treated mice. 2. Materials and methods 2.1. Animals Forty-eight outbred male NMRI mice aged 11^12 weeks and with a weight of 30 g at the beginning of the experiments were obtained from Alab, So«derta«lje, Sweden. The animals were fed on ordinary pellets and water. 2.2. Preparation of amyloid ¢brils Vitreous samples were obtained from FAP patients who underwent vitrectomy for vitreous opacity, caused by amyloid deposits. During the operation, 50 ml of vitrectomized corpus vitrium from a heterozygous 54-year-old male and a homozygous, 64-year-old male FAP type I patient, were collected. The FAP type I patient's diagnosis was based upon typical clinical ¢ndings, the presence of amyloid deposits, and a positive test for the ATTR V30M mutation [14]. The insoluble precipitates of the amyloid ¢brils were consecutively washed 5 times with saline and sonicated. The ¢bril suspensions from both homozygotic and heterozygotic FAP patients were diluted in saline to a concentration of 1 mg/ml in dry weight. Samples of sonicated vitreous amyloid from FAP patients were analyzed by gel analysis and immunoblotting, before being injected into the experimental animals. 2.3. Gel analysis and immunoblotting The vitreous amyloid samples from FAP patients were dissolved in 20% volume of sample bu¡er consisting ; 3 M urea, 2.5% sodium dodecylsulfate sulfate (SDS), 0.1 M DTT, 0.05 M Tris pH 6.8 and 0.05% bromophenol blue and boiled at 95³C for 10 min. Thereafter the solution was run through a 20% sodium dodecylsulfate^polyacrylamide gel (SDS^PAGE). For immunoblotting, the primary antibody was rabbit anti-human TTR antibody (Dako, Dakopatts, Glostrup, Denmark) and the secondary antibody

was goat anti-rabbit IgG horseradish peroxidase (BioRad, Richmond, CA). 2.4. Induction of amyloid deposits Forty-eight animals were divided according to the intravenous injections they received into groups I^IV, each consisting of 12 animals : group I, 0.1 ml isotonic saline solution; group II, 0.1 ml homozygous amyloid ¢bril suspension; group III, 0.1 ml heterozygous amyloid ¢bril suspension; and group IV, 0.2 ml of a 2% (w/v), 10 000 Da polyethylene glycol solution in water (Sigma, St. Louis). Simultaneously all mice received a subcutaneous injection of 0.5 ml 1% (w/v) AgNO3 . Three animals from each of the four groups were exsanguinated by bleeding and killed on days 3, 6, 9 and 12. Specimens from the liver, spleen and kidney were ¢xed in 4% bu¡ered formalin, embedded in para¤n and serial cut at 5 mm. 2.5. Enzyme-linked immuno assay (ELISA) for AA protein AA levels in mice blood were determined by a simpli¢ed micro-ELISA as described by Zuckerman and Surprenant [15]. Serum samples were diluted by a bicarbonate bu¡er without prior denaturation and coated overnight onto microtiter plates. A rabbit antiserum to mouse protein AA (Per Westermark, Lindko«ping University Hospital, Sweden), was used as the primary antibody followed by a (HRP) conjugated goat anti-rat IgG serum (Dako, Denmark). For each of the three animals in the groups, the test was performed three times and the average titer in each group was calculated. 2.6. In vivo radioisotope experiment The vitreous ATTR V30M amyloid samples were labeled by 125 I-Bolton^Hunter reagent (2200 Ci/nmol) (New England Nuclear, Boston, USA) [16]. After 12 h of fasting, six mice were injected with the 125 I-labeled amyloid between 09.00 and 12.00 h. Under anesthesia, 125 Ilabeled amyloid (0.1 mg/30 g) was intravenously injected into the mice. Three mice were killed 3 h after the injection, and additional three mice after 24 h. Under anesthesia, the animals were exsanguinated by bleeding from the lower part of the abdominal aorta. The remaining blood in the tissues was removed by perfusion with 5 ml ice-cold saline through the abdominal artery. Then, the liver, kidney, spleen, heart, lung, stomach, intestines, urine and muscles were removed. Radioactivity was determined in the tissues, blood and urine by a Packard Model 5130 auto gamma-scintillation spectrometer, and expressed as percentage of given dose per g tissue. 2.7. Congo red staining and immunohistochemistry Consecutive coded sections were stained with hematox-

BBAGEN 24997 10-4-00

C. Mambule et al. / Biochimica et Biophysica Acta 1474 (2000) 331^336

ylin/alkaline Congo red and by immunohistochemistry. The Congo red-stained specimens were examined in polarized light. A ¢nding of a green birefringence was taken as evidence for amyloid deposit. The amount of amyloid in the organs was graded as follows : 0, no detectable amyloid; 1, small amounts of amyloid ; 2, moderate amounts of amyloid; and 3, extensive amyloid deposits. The sections were examined without the knowledge of treatment given to the animals. The avidin^biotin complex (ABC) method was used (Dakopatts, Glostrup, Denmark) to identify the type of amyloid deposits in the samples. The sections were immersed in 0.1% hydrogen peroxide and Tris bu¡er, pH 7.4, for 10 min to inhibit the endogenous peroxidase activity and treated with 1% bovine serum to occupy nonspeci¢c binding sites. The anti-AA antibody (Per Westermark, Lindko«ping University Hospital, Sweden) was diluted 1:1000, and the sections were incubated with the antibody overnight at room temperature. Secondary antibodies (biotinylated anti-rabbit IgG) were added the next day followed by avidin^biotin^peroxidase complex. Thereafter, the sections were stained with 3,3P-diaminobenzidine. As negative controls, the primary antibodies were replaced by non-immune rabbit serum or 1% albumin. 2.8. Statistics Fisher's exact probability test was used to calculate the di¡erence between groups. 2.9. Ethics This study was approved by the Ethical Committee for Animals Experiments, Umea® University. 3. Results 3.1. Gel analysis and immunoblotting The SDS^PAGE electrophoresis showed that the injected material contained TTR fragments, and aggregates of amyloid ¢brils in addition to monomeric and dimeric forms of TTR (data not shown).

333

Fig. 1. AA-protein concentration in serum during the experiment. The data are expressed as the average of the optical density titers obtained from the serum of three animals.

had accumulated especially in the lungs. Smaller amounts were noted in the liver, stomach, and small intestine. Twenty-four hours after the injection, a similar pattern of distribution was observed though the levels of radioisotope activity were diminished. 3.4. Congo red staining and histochemical analysis of the tissues A summary of the results is given in Table 1. On day 12 after AgNO3 injection, a small amount of amyloid was found in group I mice. It was surrounding the splenic follicles and in the wall of the blood vessels. In the liver, deposits were found in the perivascular areas of the central veins, and in the kidney, deposits were noted around the papillae and along the tubules of the kidney (Fig. 3). For groups II and III, amyloid deposits were found in the liver, kidney, and spleen from day 3: the deposits were surrounding the splenic follicles and vessels, the perivascular areas of the central veins of the liver, around the papillae and along the tubules of the kidney. The amount of amyloid increased in the animals until day 9, thereafter,

3.2. ELISA for AA protein AA protein reached a peak level in the blood at day 3 in all groups. In the animals injected with amyloid or polyethylene glycol, the levels of SAA decreased rapidly after day 3, whereas those of control animals continued to be high until day 9, thereafter the levels diminished (Fig. 1). 3.3. Distribution of the injected amyloid in mice As shown in Fig. 2, after 3 h, the labeled TTR amyloid

Fig. 2. Distribution of injected radioisotope-labelled amyloid in various tissues. (mean þ S.D.; n = 4)

BBAGEN 24997 10-4-00

334

C. Mambule et al. / Biochimica et Biophysica Acta 1474 (2000) 331^336

Fig. 3. Kidney tissue from polyethylene glycol-injected mice killed on day 9. The slides are stained with Congo red and viewed in polarized light. Arrow indicates area with greenish birefringe, indicative of amyloid deposits.

the deposits tended to be diminished. In group IV, a similar pattern of distribution was observed from day 3, though the amount of amyloid was smaller compared with groups II and III. In all organs examined, amyloid deposits were found in groups II, III, and IV from days 3 to 9. In all groups mentioned above, day 6 and 9 sections Table 1 Amyloid deposits in the examined organs Organ Day 0

Day 3

Day 6

Day 9

Day 12

liver kidney spleen liver kidney spleen liver kidney spleen liver kidney spleen liver kidney spleen

Group

contained the most amyloid (Table 1). For controls, no amyloid was detected from days 3 to 9, slight amyloid deposition were observed at day 12. (P 6 0.001 for groups II and III and P = 0.005 for group IV, compared with group I). The amyloid deposits were typed immunohistochemically as AA proteins. No reactivity was found for TTR. 4. Discussion

I

II

III

IV

0 0 0 0 0 0 0 0 0 0 0 0 0 1 1

N.D. N.D. N.D. 1 2 1 1 2 1 2 2 2 1 1 1

N.D. N.D. N.D. 2 1 1 1 2 1 2 3 2 0 2 1

N.D. N.D. N.D. 0 1 0 1 1 0 1 2 1 0 1 0

Group I, controls; group II, mice injected with heterozygous TTR amyloid ; group III, mice injected with homozygous TTR Met30 amyloid; group IV, polyethylene glycol-injected mice. The amount of amyloid was graded from 0 to 3, where 0 denotes no amyloid detected, and 3 heavy amyloid deposits. The ¢gures shown represent median values from three mice. N.D., not done.

The injection of AgNO3 induces an in£ammatory response in the animals with a corresponding rise in AA protein (the precursor of AA amyloid) levels. The di¡erences in serum concentrations of AA protein between amyloid-/PEG-injected animals and controls (group I) may re£ect a consumption of AA protein caused by amyloid formation, though the decline for PEG was not so pronounced as that of TTR amyloid (Fig. 1). The labeled amyloid fragments appeared to accumulate predominantly in the lungs. Since the lung is the ¢rst organ the injected amyloid passes through, this is not unexpected. It has been suggested, that AA amyloid is formed via a reaction carried out by macrophages [17]. Since macrophages are abundantly present in the alveoli of the lung, it is tempting to suggest that the injected amyloid ¢brils are cleared from the blood stream by macrophages, and that these cells may participate in amyloid formation in other organs.

BBAGEN 24997 10-4-00

C. Mambule et al. / Biochimica et Biophysica Acta 1474 (2000) 331^336

TTR was not detected by immunohistochemistry in the amyloid deposits, where reactivity was noted for AA amyloid only. However, the injected amount of amyloid of 0.1 mg/mouse appears to be too small for immunohistochemical detection, since Johan et al. were able to disclose the injected amyloid ¢brils in the amyloid deposits by radiolabeling [9]. Amyloid deposits were found in all group II and III mice (amyloid injected) on day 3, whereas no deposits were noted in controls. This ¢nding corresponds to an earlier report in which synthetic heterologous amyloidlike ¢brils derived from four di¡erent short amino acid sequences of TTR induced amyloid formation, probably by acting as a seed [9]. Further support for the seeding theory was derived from Tamaoka et al. who used a mouse model of Alzheimer's disease, where the long-tail from AL 1^42/43 of the L amyloid proteins was shown to act as a seed molecule for cerebral amyloid deposits [18]. It therefore appears reasonable to suggest that the injected amyloid fragments act as a seed upon which amyloidogenic proteins can assemble into amyloid ¢brils. There was no di¡erence between the amyloid deposits observed in sections from homozygous or heterozygous injected animals. This could mean that the composition of amyloid fragments in both cases is very similar, at least in their AEF e¡ect on mouse AA amyloidosis. To our surprise, we found, by day 3, small amounts of amyloid deposits in the liver and kidney and by day 9, amyloid in all investigated organs of animals who had received PEG, whereas none was found in controls. This indicates that a foreign substance, like a 10-kDa non-protein hydrophobic molecule, may act as an AEF in mice. However, the deposits in this group of animals, were less pronounced than those in groups II and III, but the decline in AA concentration was also lower than that observed for TTR amyloid ¢bril injected mice. Our ¢nding indicates that PEG could have amyloid-enhancing abilities. PEG is a substance that is widely used clinically to investigate permeability over biological membranes, such as intestines. It is regarded as an inert substance, that appears not to induce an in£ammatory or immunological response; and in the present investigation, AA protein levels decreased after the injection of PEG, so amyloid enhancement in the PEG-injected animals is not related to an increased in£ammatory response. However, PEG a¡ects the function of macrophages and mast cells. For mast cells, an e¡ect has been noted on histamine release, and IgE antibody titer in PEG-treated sensitized mice has been noted to be approximately 10-fold lower than in saline-treated animals, and in that experiment, histamine response was also markedly reduced [19,20]. Furthermore, PEG appears to be able to modify the macrophage lysosomas' handling of particles and the lysosome membranes' accessibility for endocytic tracers [21]. These e¡ects on macrophages and mast cells, may have an impact on the

335

cellular handling of amyloid proto¢brils within the cells and in the extracellular space where amyloid ¢brils are deposited. PEG contains no sulfates, a component which has been proposed to be crucial for glycosaminoglycans' functions as an AEF [10], so it is unlikely that its action as an AEF is similar to that of glycosaminoglycans. It is worthy of note, that the molecular weight of the PEG used in the study is close to that previously proposed for AEF [5,6]. Lately, in experiments with organic osmolytes as chemical chaperons, glycerol exhibits an ability to rapidly accelerate the AL random coil-to-L-sheet conformational changes necessary for ¢bril formation. This was accompanied by a conversion of amorphous unstructured aggregates into uniform globular and possibly nucleating structures. Even an accelerated transmission from proto¢brils into mature ¢brils was noted [22]. It is therefore possible, that PEG could have a similar e¡ect on AA protein and accelerate conformational changes into L-sheet, and thereby facilitate amyloid formation. In summary, TTR-amyloid ¢brils act as an AEF in silver nitrate-injected mice. Even a hydrophobic polysaccharide, polyethylene glycol with a molecular weight of 10 kDa, that generally is considered to be inert, appears to be able to enhance amyloid formation. The mechanism for this is unknown. It may be mediated through its e¡ect on macrophages and mast cells and/or by its newly discovered ability to accelerate transformational changes of an amyloidogenic protein into a L-sheet structure. Acknowledgements This study was supported by grants from the patients association (FAMY), the Medical Faculty, Umea® University, Va«sterbottens Health District, the Swedish Cancer Foundation (CF), The Swedish Council for Planning and Co-ordination of Research (FRN) and the Swedish Research Council (Grant 14X-013045-01A). The authors are indebted to Prof. Per Westermark, Linko«ping University Hospital, Sweden, for supplying the anti-AA antibody. References [1] M.D. Benson, T. Uemichi, Transthyretin amyloidosis, Amyloid 3 (1996) 44^56. [2] R.H. Falk, R.L. Comenzo, M. Skinner, The systemic amyloidoses [see comments], New Engl. J. Med. 337 (1997) 898^909. [3] O. Werdelin, P. Ranlov, Amyloidosis in mice produced by transplantation of spleen cells from casein-treated mice, Acta. Pathol. Microbiol. Scand. 68 (1966) 1^18. [4] F. Hardt, Transfer amyloidosis. I. Studies on the transfer of various lymphoid cells from amyloidotic mice to syngeneic nonamyloidotic recipients. II. Induction of amyloidosis in mice with spleen, thymus and lymph node tissue from casein-sensitized syngeneic donors, Am. J. Pathol. 65 (1971) 411^424.

BBAGEN 24997 10-4-00

336

C. Mambule et al. / Biochimica et Biophysica Acta 1474 (2000) 331^336

[5] T.A. Niewold, P.R. Hol, A.A. van, E.T. Lutz, E. Gruys, Enhancement of amyloid induction by amyloid ¢bril fragments in hamster, Lab. Invest. 56 (1987) 544^549. [6] E. Gruys, F.W. Snel, Animal models for reactive amyloidosis, Baillieres Clin. Rheumatol. 8 (1994) 599^611. [7] K. Ganowiak, P. Hultman, U. Engstrom, A. Gustavsson, P. Westermark, Fibrils from synthetic amyloid-related peptides enhance development of experimental AA-amyloidosis in mice, Biochem. Biophys. Res. Commun. 199 (1994) 306^312. [8] R. Kisilevsky, E. Gruys, T. Shirahama, Does amyloid enhancing factor (AEF) exist ? Is AEF a single biological entity?, Amyloid 2 (1995) 128^133. [9] K. Johan, G. Westermark, U. Engstrom, A. Gustavsson, P. Hultman, P. Westermark, Acceleration of amyloid protein A amyloidosis by amyloid-like synthetic ¢brils, Proc. Natl. Acad. Sci. USA 95 (1998) 2558^2563. [10] A.D. Snow, W. Lukito, G.M. Castillo, VIII International Symposium on Amyloidosis : The Sulfate Moieties of Glycosaminoglycans are Critical for the Enhancement of beta-Amyloid Protein Fibril Formation, Mayo Press, Rochester, 1998, p. 171. [11] C.J. Barrow, A. Yasuda, P.T. Kenny, M.G. Zagorski, Solution conformations and aggregational properties of synthetic amyloid betapeptides of Alzheimer's disease. Analysis of circular dichroism spectra, J. Mol. Biol. 225 (1992) 1075^1093. [12] P. Westermark, K.H. Johnson, K. Sletten, D.W. Hayden, AA-amyloidosis in dogs: partial amino acid sequence of protein AA and immunohistochemical cross-reactivity with human and cow AA-amyloid, Comp. Biochem. Physiol. 82 (1985) 211^215. [13] S. Tawara, M. Nakazato, K. Kangawa, H. Matsuo, S. Araki, Identi¢cation of amyloid prealbumin variant in familial amyloidotic polyneuropathy (Japanese type), Biochem. Biophys. Res. Commun. 116 (1983) 880^888. [14] G. Holmgren, E. Holmberg, A. Lindstrom, E. Lindstrom, I. Norden-

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

son, O. Sandgren, L. Steen, B. Svensson, E. Lundgren, G.A. Von, Diagnosis of familial amyloidotic polyneuropathy in Sweden by RFLP analysis, Clin. Genet. 33 (1988) 176^180. S.H. Zuckerman, Y.M. Surprenant, Simpli¢ed microELISA for the quantitation of murine serum amyloid A protein, J. Immunol. Methods 92 (1986) 37^43. Y. Ando, S. Ikegawa, A. Miyazaki, M. Inoue, Y. Morino, S. Araki, Role of variant prealbumin in the pathogenesis of familial amyloidotic polyneuropathy: fate of normal and variant prealbumin in the circulation, Arch. Biochem. Biophys. 274 (1989) 87^93. T. Shirahama, K. Miura, S.T. Ju, R. Kisilevsky, E. Gruys, A.S. Cohen, Amyloid enhancing factor-loaded macrophages in amyloid ¢bril formation [see comments], Lab. Invest. 62 (1990) 61^68. A. Tamaoka, T. Kondo, A. Odaka, N. Sahara, N. Sawamura, K. Ozawa, N. Suzuki, S. Shoji, H. Mori, Biochemical evidence for the long-tail form (A beta 1^42/43) of amyloid beta protein as a seed molecule in cerebral deposits of Alzheimer's disease, Biochem. Biophys. Res. Commun. 205 (1994) 834^842. V. Holford-Strevens, W.Y. Lee, K.A. Kelly, A.H. Sehon, Suppression of IgE antibody production in sensitized mice and rats by tolerogenic conjugates of synthetic hydrophilic polymers with antigen or hapten: e¡ect on antigen-induced histamine release from peritoneal mast cells, Int. Arch. Allergy Appl. Immunol. 67 (1982) 109^116. G. Decorti, F.B. Klugmann, L. Candussio, M. Basa, F. Mallardi, V. Grill, L. Baldini, E¡ect of polyethylene glycol 400 on adriamycin induced histamine release, Eur. J. Cancer Clin. Oncol. 22 (1986) 793^799. Y.K. Oh, J.A. Swanson, Di¡erent fates of phagocytosed particles after delivery into macrophage lysosomes, J. Cell Biol. 132 (1996) 585^593. D.-S. Yang, C.M. Yip, T.H. Jackson Huang, A. Chakrabartty, P.E. Fraser, Manipulating the amyloid-L aggregation pathway with chemical charerons, J. Biol. Chem. 274 (1999) 32970^32974.

BBAGEN 24997 10-4-00