5. Duermeyer, W., F. Wieland, andJ. van der Veen. 1979. A newprinciple for the detection of specificIgM antibodies applied in an ELISAfor hepatitis A. J. Med. Virol. 4:25-32.

6. Field, P. R., S. Shanker, and A. M.Murphy. 1980. The use of proteinA-Sepharose affinity chromatog­raphy for separation and detection ofspecific IgM antibody in acquiredrubella infection: A comparisonwith absorption by staphylococcicontaining protein A and densitygradient ultracentrifugation. J.Immunol. Methods 32:59-70.

7. Gerlich, W. H., and W. Liier, 1979.Selective detection of IgM antibodyagainst core antigen of the hepatitisB virus by a modified enzymeimmune assay. J. Med. Virol. 4:227-238.

8. Halonen, P., H. Bennich, E.Torfason, J. Karlsson, B. Zlola,M. Matikainen, E. Hjertsson, andT. Wesslen. 1979. Solid-phase radio-immunoassay of serum immuno­globulin A antibodies to respiratorysyncytial virus and adenovirus. J.Clin. Microbiol. 10:192-197.

9. Halonen, P., O. Meurman, and

M. Matikainen. 1979. IgA antibodyresponse in acute rubella determinedby solid-phase radioimmunoassay.J. Hyg. (Lond.) 83:69-75.

10. Henle, G., and W. Henle. 1976.Epstein-Barr virus-specific IgAserum antibodies as an outstandingfeature of nasopharyngeal car­cinoma. Int. J. Cancer 17:1-7.

11. Horwitz, C. A., W. Henle, andG. Henle. 1979.Diagnostic aspects ofthe cytomegalovirus mononucleosissyndrome in previously healthypersons. Postgrad. Med. 66:153-158.

12. Joncas, J. H., M. Granger-Julien,and F. Gervais. 1975. ImprovedEpstein-Barr virus immunoglobulinM antibody test. J. Clin. Microbiol.1:192-195.

13. Leinikki, P.O., I. Shekarchi, P.Dorsett, and J. L. Sever. 1978.Determination of virus-specific IgMantibodies by using ELISA: elimina­tion of false-positive results withprotein A-Sepharose absorption andsubsequent IgM antibody assay.J. Lab. Clin. Med. 92:849-857.

14. Meurman, O. H., M. K. Viljanen,and K. Granfors. 1977. Solid-phaseradioimmunoassay of rubella virusimmunoglobulin M antibodies:

Comparison with sucrose densitygradient centrifugation test. J. Clin.Microbiol. 5:257-262.

15. Pattison, J. R., C. M. Jackson,J. A. Hiscock, J. E. Cradock­Watson, and M. K. S. Ridehalgh.1978. Comparison of methods fordetecting specific IgM antibody ininfants with congenital rubella. J.Med. Microbiol. 11:411-418.

16. Schmitz, H., D. Kampa, H. W.Doerr, T. Luthardt, H. G. Hille­manns, and A. Wiirtele. 1977. IgMantibodies to cytomegalovirus duringpregnancy. Arch. Virol. 53:177-184.

17. Vesikari, T., and A. Vaheri. 1968.Rubella. A method for rapid diag­nosis of a recent infection by demon­stration of the IgM antibodies. Br.Med. J. i:221-223.

18. Yolken, R. H., R. G. Wyatt, H. W.Kim, A. Z. Kapikian, and R. M.Chanock. 1978. Immunologicalresponse to infection with humanreovirus-like agent: Measurement ofanti-human reovirus-like agentimmunoglobulin G and M levels bythe method of enzyme-linkedimmunosorbent assay. Infect.Immun. 19:540-546.

Immune Complex Disease: Techniques for the Detection of Circulating Immune Complexes

L. R. Espinoza, M.D.Frank B. Vasey, M.D.Bernard F. Germain, M.D.Division of RheumatologyCollege of MedicineUniversity of South Florida.Tampa. Florida 336/2

The role played by circulatingimmune complexes (CIC) in themediation of tissue injury in a widerange of human disorders has beenclearly established (1, 4, 8). Theproposed sequence of events leadingto immune complex tissue injury is asfollows: After antigen exposure,certain types of soluble antigen-anti­body complexes that are formed inantigen excess freely circulate and, ifnot cleared from the circulation bythe reticuloendothelial system, willbe deposited in the wall of bloodvessels.This immune complex deposi­tion induces an increase in vascularpermeability caused, at least in part,by the action of vasoactive aminesreleased from platelets and immuno-

globulin E-triggered basophils.Following immune complex deposi­tion in vessel walls, the complementsystem is activated, resulting in theformation of chemotactic factors forpolymorphonuclear leukocytes.These cells infiltrate the vessel wallsand release their lysosomal enzymes,particularly collagenase and elastase,causing necrosis of the vessel wall.The concentration of immune com­plexes in the circulation, the cir­culating half-life, and the physicalcharacteristics of the antibodymolecules forming the complexinfluence the nature of diseaseactivity.

The tissue injury induced byimmune complexes has been shown tobe mediated by cellular and humoralfactors. In particular, the comple­ment system activates neutrophils,platelets, basophils, and macro­phages. Polymorphonuclear leuko­cytes, with their intracytoplasmicenzymes, play a fundamental role in

the mediation of tissue injury byimmune complexes. The classicanimal serum sickness model ofimmune complex disease developedby Germuth (6) and Dixon et al (2)remains the prototype for the study ofhuman diseases in which CIC par­ticipates. It may be produced inrabbits by repeated injections of largeamounts of an appropriate antigen,e.g., heterologous serum proteins.After 10-14 days some animals willdemonstrate signs of systemic toxicitywith inflammatory lesions beingpresent in the heart, arteries, kidneys,and joints. The onset of these clinicalmanifestations correlates with theappearance of CIC. Furthermore,localization of antigen-antibody andcomplement can be demonstrated inthe heart, arteries, kidney and sy­novial membrane. The pathogenesisof serum sickness is thought to havethe same mechanism as that of theexperimental model just described. Atpresent, this entity is much less

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frequently seen than in past yearswhen the use of heterologous serum,such as equine tetanus antitoxin, wasmore prevalent in clinical practice.Penicillin allergy and other drugallergies are the most common causesof serum sickness in 1980.

Many of the clinical manifestationsseen in patients with connective tissuedisorders are thought to be secondaryto tissue injury induced by CIC. Oneof the major entities in this group issystemic lupus erythematosus (SLE).This condition characteristicallyinvolves multiple systems in the body.Nephritis, arthritis, serositis, dermati­tis, cerebritis and blood cytopeniasare common occurrences. Otherconditions in this group, i.e., rheuma­toid arthritis, polyarteritis nodosa,temporal arteritis, are also charac­terized by the deposition of immunecomplex material in several tissuesthroughout the body.

Better understanding of the inter­action between biological systems andCIC has facilitated the developmentof sensitive and reproducible tech­niques for complex detection. Mostof the techniques for detecting CICare antigen nonspecific as they dealwith complexes of unknown speci­ficity. So far, more than 40 differentprocedures have been described.Antigen-specific techniques onlyapply to those unusual clinical cir­cumstances where the complex'santigen has been identified. (10, 11)

General techniques in use to detectCIC can be divided into those usingphysicochemical methods and thoseusing biological or immunologicmethods. The main advantage ofphysicochemical methods is that theycan be preparative, permitting furthercharacterization of the complexes.They are based on size separation,differential solubility in solutionssuch as polyethylene glycol , anddiminished solubility at 4°C. A searchfor cryoglobulins is often used as ascreening technique for the presenceof immune complexes. Unfortu­nately, it lacks specificity. Composi­tion of the cryoglobulin should bedetermined by the demonstration ofimmunoglobulin classes (mixedcryoglobulinemia), complement

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components, or rheumatoid factor(RF) activity within the cryoprecipi­tate. Polyethylene glycol (pEG) isknown to enhance precipitation ofantigen-antibody complexes and tofractionate plasma proteins accordingto the size of the molecules and theirelectric charge. Demonstration ofimmunoglobulin or C3 in highmolecular weight fractions fromcentrifuged or chrornatographed serais suggestive of their complexed, orcomplex-bound nature. Biological orimmunologic methods are based onthe ability of antigen-antibody com­plexes to interact with humoral or cellreceptor systems. Cl q, rheumatoidfactors, and Fe receptors on B lym­phocytes, macrophages and plateletsall recognize the Fe part of aggregated(or complexed) IgG. Other receptorssuch as conglutinin or C3 receptorson B lymphocytes recognize C3bound to complexes.

In general the Clq tests and theRaji cell assay are among the mostwidely used. Each assay has itsadvantages as well as its limitations.In the Clq-deviation test complexes,if present in the test serum, will bindradiolabeled Clq and decrease orprevent its binding to sensitized sheeperythrocytes. The fluid-phase Clqbinding test is quantitative and moresensitive. In this test, the serum con­taining immune complexes is in­cubated with radiolabeIed Clq andPEG; separation of the complex­bound radiolabeled Clq from the freeradiolabeled C1q is achieved byprecipitation with PEO (2.50/0 finalconcentration) and centrifugation,leaving free radiolabeled Clq in thesupernate. The solid-phase Clqbinding assay is performed by in­cubating the test serum in polystyrenetubes coated with Clq; the tubes arethen washed, and the complexesbound to the insolubilized Clq aredetected by adding radiolabeledanti-IgG antibodies or radiolabeledstaphylococcal protein A, both ofwhich bind to the Fe region of IgG.The radiolabeled probe detects onlymacromolecules that contain IgG,and will not detect other immunecomplex material that fails to bind toClq.

Conglutinin is a bovine plasmaprotein that specificaIIy recognizes aC3 fragment when it is bound toimmune complexes. It can be in­solubilized by coupling to Sepharoseor by coating polystyrene tubes sothat it can bind complexes that can bespecifically detected with radio­labeled anti-IgG antibodies. Raji cellassay is based on CIC binding to theC3b receptor of an in-vitro-trans­formed Iymphoblastoid cell line, withsubsequent detection of the immunecomplexes by radiolabeled anti-IgQantibodies. Platelets and monocYtesexhibit Fe receptors; this property isused to detect CIC in a variety oftests. The erythrocyte antibody COIl1_

plement (EAC) rosette inhibition testmeasures the inhibition by immunecomplexes of rosette formationbetween a subpopulation of periph_eral B lymphocytes and sensitizedsheep erythrocytes carrying C3.

Most tests used for detecting CICare semiquantitative. Sensitivity isusually expressed as an equivalent of~g/ml detected in in-vitro-aggregatedhuman IgO. In a collaborative WorldHealth Organization study (7), inwhich 18 techniques were compared,the most sensitive techniques for thedifferentiation of sera containingimmunoglobulin aggregates fromnormal sera were the conglutinin­binding test and the Raji cell assay,followed by the rheumatoid factorinhibition assay, the solid-phaseClq-binding test, and the Clq devia,tion test. Reproducibility is some­times difficult to achieve, and, ingeneral, better reproducibility isobtained in humoral than in cellularsystems, except in the Raji cell as­say in which a constant source ofcellular receptor is available. Inregard to specificity, it is important toremember that any given test onlydetects a restricted population ofcomplexes, so that a negative result inone assay system does not necessarilyrule out the presence of immunecomplexes. In assays using Clq orreceptor for C3, only complement­fixing immune complexes are de­tected, such as complexes containingIgOl and Ig03, or complexes con­taining complement-fixing IgM

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antibodies. Tests that detect immunecomplex-bound C3 will also revealthose complexes that can activatecomplement by the alternative path­way. False-positive results can beinduced in a number of differentways. First of all, IgG aggregated byrepeated freezing and thawing, byheat inactivation, and by storage mayinteract in most of the tests described;endotoxin, DNA, and low-molecular­weight polyanions can react withClq. In addition, immune complexesnot binding Clq, or of insufficientsize to precipitate will remain unde­tected. Lymphocytotoxic antibodiesmay bind to Raji cells and should beadsorbed from the serum prior totesting.

It has been shown that assays donot necessarily correlate with oneanother. For example, using the Rajicell assay, higher levels of immunecomplexes are found in SLE than inrheumatoid arthritis, while theopposite is observed using the Clqbinding assay or the monoclonal RFinhibition assay.

At the present time, there are nosimple techniques for detecting cir­culating complexed 19A and IgE anti­bodies, but it is anticipated they will

Case Report

Polymyositis

Mahadevappa Vagesh, M.D.Frank B. Vasey, M.D.Luis R. Espinoza, M.D.Bernard F. Germain, M.D.Department of Internal MedicineCollege of MedicineUniversity of South FloridaTampa. Florida 33612

Polymyositis (PM) is a disorder ofstriated muscle characterized bydiffuse inflammatory and degenera­tive changes resulting in symmetricalweakness and, to a lesser degree,muscle atrophy. The term derma­tomyositis (OM) is applied in thepresence of typical skin lesions.

The first recorded case of PM wasdescribed by Wagner in 1863, butuntil recently this condition hasreceived comparatively little atten-

be developed in the near future.The clinical usefulness of the

detection of CIC is currently understudy. It appears that their detectionmay be useful in management ofSLE, rheumatoid arthritis and bac­terial endocarditis (3, 5, 9), yet nouniformity of opinion has beenreached. It is hoped that these testswill provide new insights into thepathogenesis, diagnosis and manage­ment of immune complex disease.

References1. Cochrane, C. G., and F. J. Dixon.

1976. Antigen-antibody complexinduced disease, p. 137. In P. A.Meischer, and H. J. Muller-Eberhard(eds.), Textbook of immunopa­thology, 2nd ed., vo\. 1. Grune &Stratton, New York.

2. Dixon, F. J., J. J. Vasquez,W. O. Weigle, and C. G. Cochrane.1958. Pathogenesisof serumsickness.Arch. Patho!. Lab. Med. 65:18-27.

3. Espinoza, L. R., S. W. Gaylord.F. B. Vasey, B. F. Germain, andC. K. Osterland, 1980. The "active"rosette test in rheumatoid arthritis:Correlation with disease activity.Clint Immuno\. lmrnunopathol.17:110-116.

4. Fauci, A. S. 1978. Pathophysiologyand immunologic mechanisms,pp. 660-662. In A. S. Fauci (mod­erator), The spectrum of vasculitis.

tion. The more dramatic subgroup,OM, was first outlined by Unverrichtin 1887. OM held the spotlight formore than fifty years as the pre­dominant inflammatory myopathy.For the past two decades it has beenrecognized that patients with myositismay lack the typical skin lesions.

A 62-year-old male developedproximal muscle weakness six weeksprior to admission to the hospital inApril 1980. The patient initiallydeveloped difficulty in climbing stairsand rising from a chair. This pelvicgirdle muscle weakness was followedby weakness of the shoulder girdlemuscle, which caused difficultycombing his hair and lifting objects.The muscle weakness graduallyprogressed over the next few weeks,causing the patient to become non­ambulatory. He also complained of

Clinical, pathologic, immunologic,and therapeutic considerations.Ann. Intern. Med. 89(Part 1):660­676.

5. Frank, M. M., M. I. Hamburger,T. J. Lawley, R. P. Kimberly, andP. H. Plotz. 1979. Defective reticu­loendothelial system Fc-receprorfunction in systemic lupus ery­thematosus. N. Eng!. J. Med. 300:518-523.

6. Germuth, F. G. 1953. A comparativehistologic and immunologic study inrabbits of induced hypersensitivity ofthe serum sickness type. J. Exp.Med. 97:257-268.

7. Lambert, P. H., et al, 1978. A WHOcollaborativestudy for the evaluationof eighteen methods for detectingimmune complexes in serum. J. ClintLab. Immuno\. 1:1-15.

8. McDuffie, F. C. 1978. Immune com­plexes in the rheumatic diseases. J.Allergy Clin. Immunol. 62:37-43.

9. Pussell, B. A., C. M. Lockwood,D. M. Scott, A. J. Pinching, andD. K. Peters. 1978. Valueof immune­complex assays in diagnosis andmanagement. Lancet 11:359-364.

10. Theofilopoulos, A. N., C. B. Wilson,and F. J . Dixon. 1976. The Raji cellradioimmunoassay for detectingimmune complexes in human sera.J. C1in. Invest. 57:169-182 .

11. Zubler, R. H., and P. H. Lambert.1978. Detection of immune com­plexes in human diseases. Prog.Allergy 24:1-48.

proximal myalgia, malaise, anorexiaand a 15-lb weight loss during the sixweeks prior to admission. He did nothave Raynaud's phenomenon, oralUlcers, arthralgia, arthritis, sclero­dactyly or skin rashes.

Examination revealed severe weak­ness and atrophy of the proximalmuscles of the upper and lowerextremities. Laboratory studiesrevealed markedly elevated muscleenzymes (creatine phosphokinase(CPK), aldolase, transaminases),negative antinuclear antibody test(ANA), and an abnormal electro­myogram that showed a myopathicpattern. A muscle biopsy revealedmarked infiltration of acute andchronic inflammatory cells, necrosis,noticeable variation of muscle fibersize and atrophy. The erythrocytesedimentation rate was 49 mm/hr,

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