132 Immur~otogy T~May, rot. 3, No. 5, 1(.;82 Dextran hypersensitivity A. W. Richter and H. I. Hedin Department of Biomedical Research, Pharmacia AB, Uppsala, Sweden Dextran, a common plasma substitute, sometimes induces life-threatening hypersensitivity reactions, in this article Wolfgang Richter and Harriet Hedin discuss recent evidence that these Type III anap@lactic reactions, caused by natural antibodies, can be abolished by pretreatment of patients with monovalent h@ten dextran. The search for more efficient and safer plasma volume expanders led to the introduction of partially hydrolyzed and purified dextrans into medicine in 1947 (Ref. 1). Clinical dextrans have a molecular size range comparable to that of plasma proteins and are prepared from native dextrans which are polysaccharides of tool. wt 10-100 millions. The latter are produced ff'om sucrose by enzymes, mainly derived from bacteria of Leuconostoc spp (for review see Ref. 2). The dextran molecules consist of more or less branched chains of glucose units connected by ct 1-6 bonds. The first generation of clinical dextrans had a moderate degree of branching and frequently caused immediate-type allergic reactions involving the skin, respiratory tract and circulation. However, clinical dextrans from European and American manufacturers had varying incidence of reactions and it was found that the conditions of manufacture i.e. the Leuconostoc strain, degree of hydrolysis, and extent of removal of high molecular weight constituents were important in diminishing reactions 3. Since 1955, B 512 dextran- a linear dextran causing a minimum of allergic reactions and produced by Leuconostoc mesenteroides strain NRRL B 512 - has been in use worldwide. Until 1968 only a few cases of dextran-induced anaphylactoid/ana- phylactic reactions (ARs) were reported. However, the more widespread use of clinical dextrans, e.g. for improvement of blood flow ~ and prophylaxis of postoperative pulmonary embolism 4 led to an increase in the number of ARs reported 5. Since some of these reactions are lite-threatening, efforts to eliminate them were desirable. A collaborative study was carried out during 1968-1981 with the aim of elucidating the mech- anisms underlying ARs and finding ways to prevent them. This included research on the immunogenicity of dextran, serological studies on normal individuals, and on those people who reacted to dextran, develop- ment of animal models of dextran anaphylaxis, hapten inhibition studies, and clinical multicenter trials to determine the incidence of ARs and the efficacy of hapten prophylaxis. This work was done by W. Richter, H. Hedin, K Granath, B. Ingelman and II. Renck (Research Division, Pharmacia AB, Uppsala, Sweden), K. Messmer, J. Ring, C. Mendler and H. Laubenthal (Institute for Surgical Research Univer- e Elseviex Biomedical PEess 1982 0167-4919/82/(1000-0000/$2,75 sity of Munich, FRG), K.-G. Ljungstr6m (1)anderyds Hospital, Danderyd, Sweden), K. Peter U. Gruber (Kantonspital, Basel, Switzerland, Klinikum Gross- hadern, University of Munich, FRG), D. Kraft, O. Scheiner and H. Rumpold (Institute for General and Experimental Pathology, University of Vienna, Austria), M. Devey (Institute of Tropical Medicine, University of London), G. St~lenheim and J. Sj~Sqvist (Institute of Medical Chemistry, University of Uppsala, Sweden), H. J. M~ller-Eberhard (Scripps Clinic and Research Foundation, LaJolla, California) and others. Immunology of dextran The antigenicity of dextran was disclosed by its serological reactivity with antibodies to Leuconostoc and pneumococci of types lI and XX (for review see Ref. 6). Later the immunogenicity of native dextrans of varying structure was demonstrated in man 3. The immunogenicity of B 512 dextran has been shown to be dependent on its molecular weight, in man 7 and mouse s. The choice of a non-immunogenic mol. wt range of 40,000-75,000 for clinical dextrans has been influenced by these findings. It must he pointed out here that the amount of clinical dextran given to patients per infusion (30-100 grams) represents an 'overwhelming' dose, which most probably leads to immunological unresponsiveness as it is known to be induced by large doses of purified polysaccharides (cited in Ref. 9). Like other polysaccharides of repeti- tive structure, dextran induces a thymus-independent humoral IgM class response in mice 1° and is a polyclonal B-cell activator ~. However, dextran can be converted to a thymus-dependent antigen by covalent coupling to protein 9. Such conjugates elicit a strong IgG antidextran response upon immunization. Clinical dextran does not induce mitogenic stimula- tion of human lymphocytes in vitro ~2,13. The antigenic determinants of different dextrans reflect their simple structure. They are constituted by oligosaccharides containing ctl-6, 1-2, 1-3 and 1-4 linked glucosyl residues in varying proportions 2. The clinically used B 512 dextran contains about 95% al-6 linkages 1. Inhibition studies indicated that combining sites of antidextrans are complementary to sequences
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`I,~,~u,oh;gy T~Jday, wd. d, .M~. 5, 1~82 t33 of 2-7 glucose residues, also suggesting the size range of its antigenic determinants t4. Antidextran combin- ing sites may react with terminal or non-terminal determinants% The minimal size of the imrnun<;genic determinants' of B 512 dextran corresponds to two, and exceptionally single, d-6 linked glucosyl residuesL In mice, the immune responses to the al-3 and cd-6 determinants of dextran ~(',lv have been shown to be under separate genetic control. Thus, high responder strains to the former may be low responders to the latter, and vice versa. In man, high and low responders to B 512 dextran can also be dist- inguished 18 but the genetic background has not been studied. Antidextrans in humans may belong to the lgG, lgA and lgM classes. Within the IgG class, the IgG 2 subgroup has been found to be predominant 19 as an expression of a restricted response. Most people have natural dextran-reactive anti- bodies (DRA) 3,1u,2°,21,22,23. They may be induced by dextran itself or by cross-reactive microbial poly- saccharides..Native dextran is ingested as a regular contaminant of sucrose <'. Dextran is also a componenl of dental plaque, may occur in food, and is produced by microorganisms of the gastrointestinal tract (cited in Ref. 29). Recently, a ubiquitous antigen found in the tissues and sera of human beings was identified as native dextran. In patients with gastrointestinal disease, high levels of 40-260 ng/ml serum were demonstrable 23. It is also of interest that in guinea pigs the permeability of the gastric mucosa to ingested dextran is increased by acetylsalicylic acid in thera- peutic doses; the influx of dextran into the circulation was sufficient to induce passive cutaneous ana- phylaxis 24. Thus, ample opportunity exists for dextran to induce I)RA in random human populations. On the other hand, hyperimmune animal sera to pneumo- cocci, streptococci, Salmonella, and teichoic acids cross- react with dextran (cited in Ref. 20), so theoretically 1)RA could be induced by cross-immunization. In the case of cross-reactive pneumococcal polysaccharides, results of absorption tests on human sera did not support such a view 22 (Table I).
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`134 Immunology Today, yd. 3, .Nb. 5, 1982 TABLE I. Micrograms antibody N precipitated per ml of' serum from medical volunteer's receiving subcutaneous injections of 1 mg of various native dextrans. Anti- Antidextran Antidextran Subject Native dextran after absorption before absorption No. Code Bleeding CI a SII SXII SXX with pneumococcal with pneumococcal polysaccharides polysaccharides 21 CSC-N 279 Pre-immunization 0.3 1.1 0.9 1.0 0.2 1.8 Post- 1.2 1.4 0.7 1.0 5.9 6.0 34 Swed. 3079 Pre- 0.9 0.5 0.9 0.5 1.1 1.5 Post- 1.0 1.3 0.4 0.6 14.9 16.2 44 NRRL-B 742 Pre- 1.5 0.0 0.6 0.6 3.0 5.1 Post- 1.3 0.0 1.6 1.2 6.5 6.3 49 NRRL-B 1254 Pre- 0.7 2.8 0.0 0.0 0.8 0.5 Post- 0.5 2.9 0.4 0.7 4.1 4.1 51 NRRL-B 512 F Pre- 1.6 0.0 1.2 0.3 2.7 3.1 Post- 1.2 0.0 0.0 0.5 19.4 17.1 a CI C polysaccharide from type I pneumococcus SII = pneumococcal polysaccharide specific for type 1I Pathogenetic mechanisms Impurities In adverse drug reactions, the drug itself, its metabolites, or impurities may be elicitors. With regard to dextran, the molecule itself was incriminated in severe ARs 3.20,30 rather than impurities 31-33 but the role of macromolecular contaminants in provoking mild reactions cannot be ruled out since traces of such material are demonstrable in the majority of clinical dextran preparations 33. The removal of impurities is therefore desirable and one manufacturer has recently achieved this 33. Benefits may be expected but demons- trating them will be difficult, for technical and bio- metric reasons. Dextran reactive antibodies ( DRA ) The theoretical risks of dextran infusion in individuals with high titers of circulating DRA were pointed out in 1950 (Ref. 34). A few years later, Kabat el al) demonstrated a positive relationship between skin tests to native dextran, high spec!fic precipitin levels, and a propensity to systemic allergic reactions upon infusion of non-B 512 and B 512 clinical dextrans into 101 volunteers. The results indicated that DRA participate in elicitation of such reactions. However, Jacobsson 2~, concluded that DRA have no pathogenic importance, since the great majority of volunteers with high DRA levels tolerate B 512 dextran infusion. Despite the many published case reports of ARs immunological observations are rare and give no clear picture of the underlying mechanisms. We therefore studied the levels and composition of DRA in sera from many patients who had experienced ARs of varying severity and from nor- mal individuals. Sera were collected over 12 years from different countries. In anaphylactic shock in humans, specific IgE-class antibodies are often considered responsible for the SXII - pneumococcal polysaccharide speeific for type Xll SXX = pneumococcal polysaccharide specific for type XX From Ref. 22 development of clinical signs, but IgG-mediated ana- phylaxis has also been documented 35. No DRA of IgE- class could be found before or after ARs by passive cutaneous anaphylaxis in monkeys, the radioallergo- sorbent technique, and the modified radioactive red- cell-linked antigen-antiglobulin reaction 2°,3°. Thus, ARs do not conform to the concept of IgE-mediated anaphylaxis. Sera of dextran reactors were also examined for DRA by passive hemagglutination 2°. In this way, total activity of the Ig-classes G, A, M and D is determined. It is apparent from Fig. 1 that the titer of DRA is positively related to the degree of severity of ARs. All patients with severe reactions have high titers. It should be emphasized that these results have been obtained from serum samples fortuitously collected from patients shortly before ARs occurred. If sera drawn soon after ARs are examined, erroneously low titers are found, due to neutralization of DRA by the infused dextran. A comparison between DRA-titer distributions in patients with severe ARs and in a random population shows that 76% of the dextran reactors and only 4% of normals had high titers of 512 or more (Fig. 2). Clearly, the group at risk is a small subpopulation of high responders to dextran. However, comparison of the incidence of severe ARs (0.05%) and the frequency of high titers of I)RA in normal people shows that only a small proportion of those with high titers develop ARs. This is in accord with Jacobsson's results 2~ and may be explained by several sets of factors TM. Some of these are: Ig-class and subgroup composition, affinity and concentration of DRA, platelet fragility and responsiveness of the patient's vascular system. For further analysis of DRA, the modified red-cell- linked antigen-antiglobulin reaction was employed to determine Ig-class and subgroup composition 3°.
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`Immunology Today, vol. 3, No. 5, 1982 135 262144. 131072- 65536- 32768- 16384- 8102- 4096- 2048- 1024- < 512- 256- 128- '~ 64- ~ 32- 16- E 8- -~ 4- ~8 2- ~- 0 ill lllll ll Ill I it lit mm • • • • mm mmmm n• • •magi •••••t mn •m•nmn mum mum emiR iit lib Hi n, mnmu • • ii Ill II ill Grade of severity of AR Fig. ! Relationship between serum titers of hemagglutlnating DRA and grade of severity in patients with ARs. Samples were collected prior to the dextran infusion (n = 84). ] = fatal outcome. (From Hedin and Richter, in press) Dextran-reactive IgG antibodies of high titer were regularly found in patients with severe reactions. IgG- titer strongly correlated with grade of severity; four lethal cases all belonged to the highest titer range of 16,384 to 32,768. This corresponds to 0.4 mg DRA per ml serum. In addition, high IgA-titers were sometimes found, whereas IgM levels were low and IgD anti- bodies were absent. All four subgroups of IgG were demonstrable but the contribution of IgG 2 was con- sidered the most important. Skin tests with clinical B 512 dextran were positive in 32% of dextran reactors and often correlated with high titers of hemagglutinating DRA 2°. The best predictive diagnostic test for dextran hypersensitivity is determination of dextran reactive IgG. However, such a test can only delineate a risk group of a few per cent in a random population, and cannot predict individual predisposition. CompLement Since native dextran can activate the alternative pathway of complement in vitro, it was suggested that ARs might be triggered in a similar way (for review see Ref. 31). Complement profiles were established for dextran reactors to study this possibility. In addition, serum carboxypeptidase B levels were determined, since low levels might lead to high concentrations of anaphylatoxin with potential shock producing activity. The most important finding was a significant decrease in tile levels of Clq in severe ARs ~t. Con- centrations of the other complement proteins and of serum carboxypeptidase B were normal. These results indicate that the classical pathway is being activated in dextran reactors by immune complexes and accord with the presence of high titers of DRA in patients with severe reactions. Available evidence does not favour the activation of the alternative pathway by infusion of clinical dextran. Lung histopathology Histological examination of sections of lung tissue from patients with fatal ARs showed occlusion of pulmonary vessels (Ref. 36 and unpublished data). The occluding material consisted of platelets, leuko- cytes and hyaline globuli with the staining properties of fibrin. These findings are interpreted to suggest that % 22- 20- 18- 16- 14- 12- 10- 8- ,-]- 2- 0 %J 22- 20- 18- 16- 14- 12- lO. 8. 6"- k- 2- 0 16 64 256 1024 4086 16384 65536 262144 8 32 128 512 2848 8192 32768 131072 Titer of hemagglutinating DRA Fig. 2 Distribution of serum titers of hemagglutinating DRA in individuals with no history of ARs, n = 1,408 (upper graph) and in patients with ARs of grades III to IV, n - 46 (lower graph). (From Hedin and Richter, in prcss)
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`136 Immunology Today, ~o{. ,3, ,No..5, 7~)~2 insoluble antigen-antibody complexes have been deposited in the lungs and bound ieukocytes and platelets by an interaction between Fc-receptors on cells and Fc-pieces of DRA. The fibrin deposits appearing as globular microthrombi are thought to be due to activation of the coagulation system via its con- nection with other plasma enzyme systems, e.g. the complement system. Conclusion All these findings taken together strongly support immune complex (Type III) anaphylaxis as the mechanism of severe ARs. Mild reactions may be anti- body-dependent or not. The sequence of events leading to antibody-dependent reactions may be envisaged as follows. In a small subpnpulation of individuals with high titers of preformed DRA of IgG- class, infusion of clinical dextran generates harmful immune complexes. They activate complement and aggregation of leukocytes and platelets occurs. The aggregated material is sequestered in the lung and release of vasoactive mediators leads to the ana- phylaetic symptoms. Since antigen-antibody com- plexes are of crucial pathogenic importance, inter- ference with their formation by hapten inhibition was proposed as a means of preventing ARs 2°. ,.... S ~*'°~ ." i • • o d~ • Ioe•ea eeeeee Z 5.... % e 7°* j i / ee eo eo / %.z eo%~ °eee°*~ • • "" "-.......-......t Fig. 3 Illustration of the hapten inhibition principle Monovalent haptendextran, i.e. Dextran 1 (3) binds competitively to combining sites (4) of DRA (I) thus preventing formation of large complexes between polyvalent clinical dextran (2) and DRA (1). Hapten inhibition A hapten is a substance capable of binding to anti- bodies with corresponding specificity but which cannot induce antibody tbrmation. Haptens may be polyvalent or monovalent with regard to the number of antigenic determinants. A polyvalent hapten may form complexes with antibodies, as an antigen does, but a monovalent hapten can bind only to individual combining sites of antibodies. Fig. 3 illustrates the inhibition of immune complex formation by a monovalent hapten. ln-vilro studies Hapten inhibition of precipitation in the dextran- antidextran system has been extensively investigated by Kabat and co-workers (for review see Ref. 14). Only inhibition studies with B 512 dextran are con- sidered here. Isomalto-oligosaccharides consisting of al-6 linked glucosyl residues were found most effec- tive. Their inhibitory power increased strikingly from isomaltose to isomaltopentaose with little further increase for isomalto-hexaose and -heptaose 3v (Fig. 4). These results were confirmed by the technique of 'indirect' single radial immunodiffusion ~*. In this way a dextran fragment of 6 glucose units (mol. wt 990) was found suitable as a monovalent hapten prepara- tion for in-vivo experiments. Animal models Hapten inhibition has been shown to prevent lethal cytotropic dextran anapbylaxis in guinea pigs. Dextran cross-reactive rabbit antipneumococcal type II sera 3v and rabbit antisera to B 512 dextran 4° were used for sensitization. The following information on the molecular mechanism was obtained, using B 512 dextran fractions of varying size tot challenge. The amount of sensitizing antibody is critical: polyvalent dextran may elicit anaphylaxis in strongly sensitized animals, but not in weakly sensitized ones. Non- eliciting dextran fractions always exerted inhibition, when given together with eliciting fractions. The molecular weight of such protective isomalto-oligo- saccharides or dextran fi'actions ranged from about 1,000-10,000 (Ref. 40,41). Inhibition of anaphylaxis was achieved by a moderate molar excess of non- eliciting/eliciting fractions (1.9-7.6). With maximal sensitization, the smallest dextran fragment with elicitor action in passive cutaneous anaphylaxis proved to be isomaltodecaose (mol. wt 1,600) 41, see Table I1. In contrast, isomaltohexaose (mol. wt 990) never elicited anaphylaxis, showing that it was truly a monovalent hapten. It was also considered important to study immune complex anaphylaxis in animals, since ARs in man conform to this type. For this purpose, a special low molecular dextran product (Dextran 1, i5% solution) comprising a selected mixture of isomalto- oligosaccharides of mol. wt (~w) 1,000 was developed (Ingelman el al., unpublished); factors other than inhibitory power were also taken into consideration. A model was established in dogs made hypersensitive to
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`Immunology To&O, , voL 3, No..5, 1982 137 so A' ¢/'~. / 0.5ml 30 D4 ,o /~ ~'/" ~/ 0 , i i ~l,nT " J i i i i,,g° , ....... 1 i J i , i ,o~ 1.0ml20D,o '4 ,1/./ /S / ,o1- /7/ /P" ...- ....... ....... ,,,o ,,,, MICRflMOLES OLIGOSACCHAHIOE AODED ISOMALTOHEIAOSE , METR~'L-a-ISOMALTOSIDE • ISOMALTOPENTAQSE • ISOMALTOSE o tSOMALTOTETRAOSE * PAAOSE • METHYL-a-ISDMALTOTRIOSIDE • 4~ISOMALTOTRIOSYL-D-GLUCOSE • ISOMALTOTfllOSE Fig. 4 Inhibition by various mono- and oligo-saccharides of precipitation of antidextran by dextran. (From Ref. 3) B512 dextran by immunization with a protein- dextran corljugate 42. Upon challenge of these dogs under anaesthesia with 1 ml of 'Macrodex' (mol. wt 60,000) 59% of animals showed anaphylactic reactions of varying severity. Among reactors, severe reactions occurred in 37%. They were characterized by decrease in cardiac output and of mean arterial pressure, whereas pulmonary arterial pressure and pulmonary resistance increased. Further, the number of circulat- ing leukocytes and platelets fell, and a decrease in the titer of DRA was noted. The administration of Dextran 1 either as preinjection or as admixture to the challenging anaphylactogenic dextran, significantly reduced both the incidence and the severity of ana- phylaxis. In the combined hapten-treated groups, only 16% of the dogs showed reactions. Among the reactors, 7% developed severe anaphylaxis4L Similar results were obtained in non-anaesthetized dogs by preinjection of Dextranl (Ref. 43). Preventior7 of a&erse reaclions z'r~ man Clinical trials with Dextran 1 (Promit ®, 15°70, Phar- macia AB) were now considered. Theoretically, a given dose should be more effective when administered as preinjection than when mixed with clinical dextran. Suitable doses, representing a molar excess of 50-200 times, were calculated from the number of antibody combining sites of DRA of lgG-class in sera of patients with severe DIAR. Clinical studies were initiated in Sweden and Germany in 1978. In each patient, a preinjection of 10 or 20 ml of Dextran 1 or an admixture of 20 ml of Dextran 1-500 ml of clinical dextran is given. For each patient, a protocol containing information on sex, age, main diagnosis, type of operation performed etc., is completed. When an adverse reaction is observed or suspected, details about the reaction and the patient's history are obtained. Further, serum samples drawn before and after the reaction are analyzed for DRA. To achieve a uniform classification of reactions, clinical and immunological data are continuously TABLE 11. Monovalency of dextran fragments ot six glucose residues indicated by incapability to elicit passive cutaneous anaphylaxis (PCA) in guinea pigs. gg antidextran Challenging Mo[. wt PCA-lesion per skin site agent (X'iw) area (mm 2) 0.3 IM-6 990 0 0.3 IM-10 1,638 0 0.3 Dx-fr. 3,100 121 0.3 Dx-fr. 10,500 121 0.3 Dx-fr. 71,000 289 0.9 IM-6 990 0 0.9 [M-10 1,638 225 0.9 I)x-fr. 3,100 196 0.9 Dx-fr. 71,000 361 From Ref. 41 TABLE Ill. Statistical comparison (Fisher's one-sided test) of the incidences of mild and severe ARs in patients receiving 10 or 20 ml of Dextran 1 before infusion of clinical dextran. Status: September, 1981. Incidence of Dose Mild reactions Severe reactions (Grades I + 11) (Grades Ill + IV) 10ml 65/35346=0.184% 9/35346 = 0.025% 20ml 92/65048=0.141% 3/65048=0.005% p = 0.06 p = 0.006 Note: The reported incidencc per patient of severe ARs without hapten prophylaxis is 0.037%-0.080% (Ljungstr6m, Renck el at., to be published). discussed between the physicians responsible arm the study co-ordination centre. At present, trials are going on in six countries and more than 100,000 patients have been i~westigated. Interim results from Scandinavia (Co-ordinators: H. Renck and K.-G. Ljungstr6m) and Bavaria (Co- ordinators: K. Messmer, K. Peter and H. Laubentha[) are reported in Ref. 44 and in "Fable II1. A dose- dependent prophylactic effect of preinjection of Dextran 1 is evident. The incidence of severe ARs is decreased by 30-50% with the ]0 ml dose and by 90% with the 20 ml dose in comparison with controls (I~jungstr/Sm, Renck et aL, to be published). The pro- phylactic effect of admixture of Dextran 1 to clinical dextran on severe ARs is insignificant. The clinical results are supported by immunological findings (DRA titer analysis), indicating attenuation of severe reactions into mild ones. The incidence of mild reac- tions is affected only slightly by hapten prophylaxis. The series of investigations described here demon- strates the applicability of hapten inhibition to the elimination of adverse drug reactions - a principle that has also been applied to penicillin allergy 4s. References 1 lngelman, B., Gr6nwall, A., Gelin, L. E. and Eliasson, R. (1969) Properties a~d App/icalion oj De.~harT.~, Ahnqvist and Wiksell, Stockholm 2 Jeanes, A., Haynes, W. C., Wilham, C. A. e/a/. (1954)J. Am Chem. Soc. 76, 5041-5052
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`138 3 Kabat, E. A., Turino, G. M., Tarrow, A. B. and Maurer, P. H. (t957) J. Clin. lnvest. 36, 1160-1170 4 Bergentz, S. E. (1978) WorldJ. Surg. 2, 19-24 5 Furhoff, A. K. (1977) Acla Anaesthesinl. Seand. 21,161-167 6 Hehre, E. J., Sugg, J. Y. and Neill, J. M. (1952) Ann. N. Y. Acad. &'i. 55,467-470 7 Kabat, E. A. and Bezer, A. E. (1958) Arch. Biochem. Biophys. 78, 306-318 8 Howard, J. G., Vicari, G. and Courtenay, B. (1975) immunology 29, 585-597 9 Richter, W. (1981) in Clin. Immunol. (Steften, C. and Ludwig, H., eds) Vol. 14, pp. 235-246, Elsevier/North Holland Biomedical Press, Amsterdam 10 Chen, J. C. and Leon, M. A. (1976) J. lmmuuol. 116,416-422 II Coutinho, A., M611er, G. and Richter, W. (1974) Scan& J. Immunol. 3, 321-338 12 Ring, J. (1978) Anaphylaktoide Reaklior~en (Diss) Anaeslhe.siologie und Intenriv-medizin Vol. tit, Springer Verlag, Berlin 13 Cunnington, P. G., Blackshaw, R. M. and Sykes, 1. K. (1980) Int. Arch. Allergy Appl. Immunol. 63, 195-200 14 Kabat, E. A. (1961) in Experimental Irumunochemistry (Kabat, E. A. and Mayer, M. M., eds) 2nd edn, pp. 241-267, Thomas, U.S.A. 15 Cisar, J., Kabat, E. A., Dorner, M. E. and Liao, J. (1975) J. Exp. Med. 142, 435-459 16 Blomberg, B. S., Geckeler, W. R. and Weigert, M. (1972) &ience 177, 178-180 17 Fernandez, C., Liebermann, R. and M611er, G. (1979) &and. J. Immunol. 10, 77-80 t8 Hedin, H. and Richter, W. (1982) [rTt. Arch. Allergy Appl. Immunol. in press 19 Yount, W.J., Dorner, M. M., Kunkel, H. G. and Kabat, E. A. (1968) J. Exp. Med. i27,633-646 20 Hedin, H., Richter, W. and Ring, J. (1976) Int. Arch. Allergy Appl. Immunol. 52, 145-159 2l Jacobsson, L. (1959) &udies on Partially ttydrolyzed Dextran with Special Reference to its use fi)r Plasma Vdume Determination in Man (Diss), Uppsala, Sweden Immurmlogy Today, vol. 3, No. 5, 1982 22 Maurer, P. H. (1953) Proc. Soc. t'2xp. Biol. Med. 83,879-884 23 Palosuo, T. and Milgrom, F. (1980) Int. Arch. Alle*igy Appl. Immunol. 65, 153-161 24 Flemstr6m, G., Marsden, N. V. B. and Richter, W. (1976) Int. Arch. Allergy Appl. ImmunoI. 51,627-636 25 B6ttiger, L. E. (1979) Acla Med. &'and. 205,451-456 26 Ring, J. and Messmer, K. (I977) Lancet i, 466-469 27 Bauer, ,~. and Ostling, G (I970) Acta Anaestheszol. Stand. Suppl. 37, 182 185 28 Schaning, B. and Koch, H. (1975) Anaesthesirt 24, 507-516 29 Gruber, U. F., Saldeen, T., Brokop, T., Ekl6f, B. ET AL. (1980) Br. Med. J. 280, 69-77 30 Hedin, H., Kraft, D., Richter, W., Scheiner, O. and Devey, M. (1979) lmmunobiology 156,289 31 Hedin, H. (1977) Dextran-InducedAr~aphylactoidReactim~s in Man. Immunological in vitro and in vivo Studies (Diss), Uppsala, Abs- tract Ups. Diss. Fac. Sci. No. 432 32 Richter, W. (1973) Immunological in vivo and in vitro Studies q[ the 1)extran Antidexlran System (Diss), Uppsala 33 Richter, W. (1980) Int. Arch. Allergy Appl. lmmanol. 61,457-466 34 Hehre, E.J. and Sugg, J. Y. (1950) Fed. Proc. 9,383 35 Leikola, J., Koistinen, J., Lehtinen, M. and Virolainen, M. (I973) Blood42, 111-119 36 Ziegler, H. K. (1978) Med. Klin. 73, 1089-1090 37 Kabat, E. A. (1957) J. CellComp. Phy.~iol. 50, 79-102 38 Richter, W. (1971)J. Immunology 107, 948 952 39 Hoene, R., Swineford, O. and Quelch, S. (1961)J. Allergy 32, 381-391 40 Richter, W. (1971) Int. Azch. Allezgy Appl. lmmunol. 41,826-844 41 Richter, W. (1972) Inl. Arch. Allergy Appl. fmmunology 43, 252-268 42 Mendler, C. (1980) Hapten-Hemmung der Dexlran-lrdnzierlen Ana- phylaklischen Reaktion beim Hund (Diss), University Munich 43 Schwarz, J. A. and Raschak, M. (1978) Allergologie 1,184 44 Hedin, H., Richter, W., Messmer, K., Renck, H., Ljungstr6m, K. G. and Laubenthal, H. (1981) Dev. Biol. &and. 48, 179-189 45 Week, de, A. L. and Girard, J. P. (1972) Int. Arch. AllergyAppl. lmmunol. 42, 798-815 T-cell membrane antigens associated with cytotoxic function Benjamin Bonavida, John Fan and John C. Hiserodt Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90024, U.S.A. In the pas! .few years the.first steps have been taken towards an understanding of the molecular basis o[ %ce//- medialed eyloloxicity. Parlicular alten~ion has been paid to the par! played in cyloloxicily by idiotype-bearing antigen-receptors arid other molecules on lhe T-eel! .r1~rface. This arlicle reveiws lhese .rludies and.focuses on lhe information obtained from studies on the inhibition (~f cylotoxic T cells with anlisera or monoclonal antibodies directed against ran/codes on their szlr[ace. Cytotoxic T lymphocytes (CTLs) are involved in allo- graft rejection in vivo I and the lysis of allogeneic 2 and virus- or hapten-modified syngeneic target cells in /3itro 3,4. Lysis takes place in three discrete stepsS: (i) the binding of CTLs to target cells (a process probably involving T-ceil receptor and target cell antigens); (ii) 'programming for lysis' (a Ca2+ and temperature- dependent process) and; (iii) the disintegration of the target cell (a step independent of the effector cell). In the initial event of CTL-mediated target cell lysis, eflector lymphocytes interact with and bind to target cells. Elsevier Biomedical Piess 1982 0167 4919/82/0000 0000/$275 Despite the recent advances in our" understanding of the physiological requirements and cellular processes in CTL-mediated cytolysis, the molecular mechanism involved in target cell lysis remains to be resolved. Several models have been proposed. These include (l) interactions involving membrane-associated molecules of CTL other than the antigen-binding receptor; (2) the release from CTI.s of soluble mediators and; (3) the role of the perturbation of the target cell membrane following an interaction between the CTL receptor and target-cell antigen - which perturbs the target cell's membrane and so results in its lysis. There is
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`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1072 - Page 7
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