PART
VI.
P R O S P E C T I V E S O N P O S S I B L E THERAPY FOR P R E V E N T I O N OF JOINT DESTRUCTION IN RHEUMATOID ARTHRITIS
PENICILLAMINE TREATMENT OF RHEUMATOID ARTHRITIS: EFFECT ON IMMUNE COMPLEXES Israeli A. Jaffe Department oj Medicine New York Medical College New York, New York 10029
Penicillamine differs in structure from the naturally occurring amino acid cysteine by the presence of two methyl groups in the @-carbon position (FIGURE 1) . This structural change renders the compound relatively resistant to metabolic degradation and enables it to exert its various pharmacologic properties. It is a metabolite of penicillin that may be prepared from the parent molecule or synthetically, and the less toxic D isomer is the form used clinically. It is now more than 10 years since the first published report of the therapeutic efficacy of penicillamine in the treatment of rheumatoid arthritis ( R A ) Since that time, additional studies that confirmed this finding were conducted by investigators in England,z France,3 the Federal Republic of Germany,’ and the United States.s In 1973, the results of a controlled, double-blind, multicenter trial of penicillamine versus a placebo in severe rheumatoid disease were published. This trial, which was performed in England, demonstrated that by all clinical and laboratory criteria, penicillamine was an effective therapeutic agent in the treatment of RA. Based upon these findings, the Committee on Safety of Medicines in England approved penicillamine for the treatment of severe rheumatoid disease. At the present time, the drug is available on prescription in several European countries as an adjunct to the basic therapy of rheumatoid arthritis. T h e mechanism of action of penicillamine in RA is unknown; however, it appears to exert a very fundamental effect on the disease process and on its immunologic manifestations. There is a latent period of 12-20 weeks after therapy is instituted until clinical and laboratory response begins. Similarly, when the drug is discontinued, there is an interval of 8-10 weeks, and very often as long as 4-6 months, before clinical and laboratory exacerbation is observed. It therefore appears highly unlikely that the compound simply works as a n antiinflammatory agent. Furthermore, it has been shown to be without effect in influencing the established laboratory models of arthritis and inflammation.’ It is not the purpose of this presentation t o review the clinical pharmacologic features of penicillamine, dosage, side effects, and toxicity, because these subjects have been described adequately elsewhere.k.‘J In this presentation, emphasis will be directed toward the effect of penicillamine treatment on immune complexes and to the possible relevance of this effect to clinical improvement. The first evidence that penicillamine might be exerting a n effect on circulating complexes in a patient with R A was obtained by a comparison of the ultracentrifugal patterns of serum proteins before and after penicillamine 2 shows that after treatment, there therapy. A study of the patterns in FIGURE is a fall in the 22s complex and in the so-called intermediate complexes, which
330
Jaffe: Penicillamine Treatment
331
range from 9 to 17S, and also in the IgM peak. This reduction in the intermediate 7-globulin complexes is also demonstrated in the patient whose patterns are illustrated in FIGURE 3. A phenomenon that is probably identical can also be measured immunologically by the changes in the precipitin arc that occur when a rheumatoid serum is reacted with a monoclonal IgM rheumatoid factor, according to the technique of Winchester et al.Ir’ These changes, which occurred during and after the course of drug treatment, are depicted in FIGURE 4. There is a reduction in reactivity after 10 weeks of penicillamine therapy, and the reaction completely disappears at 6 months. The synovial fluid was not available for analysis after treatment, because the effusion had reabsorbed. Thus, the IgM rheumatoid factor measured by the latex fixation test, the r-globulin complexes seen in the ultracentrifuge, and the IgG rheumatoid factors (complexes) demonstrated by precipitation with a monoclonal IgM rheumatoid factor all decline after penicillamine treatment. The clinical relevance of this phenomenon is perhaps most readily apparent in the study of certain of the extraarticular manifestations of rheumatoid arthritis: rheumatoid lung disease, vasculitis, and Felty’s syndrome. The intermediate r-globulin complexes were shown by Tomasi et al. to bear a relationship to the presence of rheumatoid lung disease.” Lorber has described radiographic
t SH-C
I
I H
i
H
I - C --cooH I
NHe
SH-C
c?
I CH,
i
- C-COOH
I
NH2
FIGURE 1 . Penicillamine ( r i g h t ) is an analog of cysteine ( l e f t ) in which hydrogen atoms are replaced by methyl groups in the p-carbon position.
and functional improvement in rheumatoid lung disease after penicillamine treatment.l2 We have previously reported striking improvement in the vasculitis of rheumatoid arthritis during penicillamine therapy; l 3 , l1 relapse occurs after drug withdrawal. It is believed that the vasculitis that complicates RA may be analogous to experimentally induced immune complex vasculitis in anirnals.ls Recently, Hurd et at. demonstrated that in patients with Felty’s syndrome, the peripheral blood granulocytes contained large inclusions that exhibited properties of immunoglobulins, as assessed by immunofluorescent staining.16 They suggested that these inclusions (immune complexes) altered the morphologic status of peripheral blood granulocytes so as to predispose them to uptake by the spleen, a theory that would explain the leukopenia that is the hallmark of Felty’s syndrome. In two of their patients, penicillamine treatment produced a gradual disappearance of these inclusion-containing cells from the peripheral blood, which was accompanied by a rise in the neutrophil count to normal levels (FIGURE5). With respect to the articular manifestations of rheumatoid arthritis, there is considerable evidence to suggest that the sustained inflammatory response is due, at least in part, to the presence of complexes within the synovial space. A decrease in immune complexes in the synovial fluid during penicillamine treat-
332
Annals New York Academy of Sciences
Jaffe: Penicillamine Treatment
333
nient may be responsible for the improvement in the inflammatory articular process. As stated above, the mechanism of action of penicillamine in RA is not known. T h e effect upon the complexes could be caused by suppression of immunoglobulin synthesis per se, inhibition of complex formation, or alteration
FIGURE 3. Ultracentrifugal analysis of serum proteins in a penicillamine-responsive rheumatoid patient before and after 8 months of treatment. Sedimentation is at 52,640 rpm ( l e f t to right) at 64, 80, and 96 min. Serum in both determinations was diluted 1:2 with saline. Note the decline in 7s 7-globulin and in intermediate 7-globulin complexes.
of the antigenic stimulus to immunoglobulin production. With respect to the first possibility, it should be emphasized that penicillamine is not an immunosuppressant drug in experimental models.' Patients given penicillamine continuously for more than 10 years are not clinically immunosuppressed. They respond to antigenic stimuli i n the normal fashion and are not at increased risk for the development of opportunistic infections or neoplasia. Lymphocytes
334
Annals New Y o r k Academy of Sciences
FIGURE 4. This Figure illustrates the effect of penicillamine therapy on complexes, as measured by precipitation with purified IgM rheumatoid factor. Well 1 contains synovial fluid, and well 2 contains serum from the same patient prior to penicillamine therapy. Well 3 shows a decrease in the amount of complex after 6 weeks of penicillamine therapy. Wells 4 and 5 reveal the absence of demonstrable serum complexes at 3 and 6 months, respectively, after initiation of penicillamine therapy. Well C is a normal serum control. There is no posttreatment specimen of synovial fluid, because the synovial effusion had completely reabsorbed.
removed from patients during penicillamine therapy display a normal response to mitogens.J It seems unlikely that penicillamine affects the actual forination of coniplexes, because one of the most serious side effects of the drug is the development of an immune complex nephritis, which may be of sufficient severity to later result in nephrotic syndrome.” This immune complex nephritis occurs not only i n RA patients but may also develop in patients with Wilson’s disease and cystinuria who have been treated with the drug and whose basic immune mechanism is normal. I t is difficult to reconcile a drug-induced inhibition of complex formation to the fact that complexes may concomitantly be formed in response to the medication, which would give rise to nephropathy.
Jaff c : Penicillaminc ‘Treatment
335
Another possibility is that penicillamine acts upon the primary stimulus for abnormal immunoglobulin production, perhaps an infectious (viral) agent. It is of interest in this regard that penicillamine has potent and selective antiviral properties in vilro (FIGURE 6). Its entire antiviral spectrum has not been elucidated, and whether these properties exist in vivo has not been determined. Alternatively, a role for penicillamine in inhibiting the denaturation of proteins, including IgG, has been demonstrated in vitr.o.’O This inhibition could be a mechanism, if it occurred in vivo, by which a noninfectious protein, IgG, might be rendered less antigenic.
SUMMARY A N D CONCLUSIONS The prolonged administration of pcnicillamine to patients with KA not only produces clinical improvement with respect to both articular and extraarticular manifestations but also yields improvement in immunologic parameters, as is evidenced by a decline in IgM and IgG rheumatoid factors and in circulating complexes. It is not known whether this effect is mediated by a selective inhibition of immunoglobulin synthesis or is secondary to a basic effect of the drug on the presumed primary antigenic stimulus responsible for both the clinical and the laboratory manifestations of the disease. It is believed that by further study of this compound, more fundamental insights can be achieved into the basic pathogenetic mechanisms in rheumatoid arthritis.
+ 4 -0,
Total Neutrophiles
-14,000
fi
E
-12,000 E Z
-10,000
0
;
- 8,000 W 4
2.2-
2
J
-
6,000 0 4,000 - 2,000
2
z
TIME IN WEEKS -PENlClLLAMlNE
FIGURE 5 . This Figure illustrates the changes in neutrophil inclusions and total neutrophil count in a patient with Felty’s syndrome during penicillamine therapy (direct
method). The IgG inclusions gradually disappeared, and the granulocyte count increased to normal.
336
Annals New York Academy of Sciences
Jaffe: Penicillamine Treatment
337
REFERENCES I . JAFFE,I. A. 1963. Comparison of the effect of plasmapheresis and penicillamine on the level of circulating rheumatoid factor. Ann. Rheumat. Diseases 22: 71. 2. GOLDING, J . R., J. V. WILSON& T. G. PLUNKETT.1968. Laboratory observations on the use of penicillamine in rheumatoid arthritis. Postgrad. Med. I. (London) (October suppl.) :40. 3. CAMUS,J. P., C. BENICHOU, P. GUILLIEN,J. CROUZET& J. A. LIEVRE. 1971. Traitement de la polyarthrite ruhmatoide commune par la D-penicillamine. Rev. Rhumat. 3 8 809. 4. MIEHLKE,K. & I. KOHLHARDT. 1966. Uber die immuno-depressorische Wirkung von D-penicillamin unter der Behandlung der progredient chronischen Polyarthritis. Z. Rheumaforsch. 26: 56. 5. ZUCKNER, J., R. H. RAMSEY,R. W. DORMER& G. E. GANTNER.1970. Dpenicillamine in rheumatoid arthritis. Arthritis Rheumat. 13: 131. 6. MULTICENTRE TRIAL GROUP. 1973. Controlled trial of D ( - ) penicillamine in severe rheumatoid arthritis. Lancet 1: 275. 7. LIYANAGE, S. P. & H. L. F. CURREY.1972. Failure of oral D-penicillamine to modify adjuvant arthritis or immune response in the rat. Ann. Rheumat. Diseases 31: 521. 8. JAFFE,I. A. 1970. The treatment of rheumatoid arthritis and necrotizing vasculitis with penicillamine. Arthritis Rheumat. 13: 436. 9. DAY,A. T., J. R. GOLDING,P. N. LEE & A. D. BUTTERWORTH. 1974. Penicillamine in rheumatoid disease: a long-term study. Brit. Med. J. 1: 180. 10. WINCHESTER, R. J., H. G. KUNKEL & V. AGNELLO.1971. Occurrence of gammaglobulin complexes in serum and joint fluid of rheumatoid arthritis patients: use of monoclonal rheumatoid factors as reagents for their demonstration. J. Exp. Med. 134: 2863. 1 1 . TOMASI,T. B., JR., H. H. FUDENBERG & N. FINBY.1962. Possible relationship of rheumatoid factors and pulmonary disease. Amer. I. Med. 33: 243. 12. LORBER,A. 1966. Penicillamine therapy for rheumatoid lung disease: effects on protein sulphydryl groups. Nature (London) 210: 1235. 13. JAFFE, I. A. 1964. Rheumatoid arthritis with arteritis. Ann. Intern. Med. 61: 556. 14. JAFFE,I. A. & R. W. SMITH.1968. Rheumatoid vasculitis-report of a second case treated with penicillamine. Arthritis Rheumat. 11: 585. 15. EPSTEIN,W. 1957. F I1 precipitation test. In Serological Reactions of Rheumatoid Arthritis. R. W. Lamont-Havers, Ed. : 22. Arthritis and Rheumatism Foundation. New York, N.Y. 16. HURD,E. R.,J . LOSPALLUTO & M. ZIFF. 1974. The role of immune complexes in production of the neutropenia of Felty’s syndrome. In VIth Pan-American Congress on Rheumatic Diseases. : 194(abs.). 17. JAFFE,I. A., G. TRESER,Y.SUZUKI& T. EHRENREICH. 1968. Nephropathy induced by D-penicillamine, Ann. Intern. Med. 69: 549. 18. GESSA,G. L., B. LODDO,G. BROTZU,M. L. SCHIVO,A. TAGLIAMONTE, A. SPANEDDA, G. Bo & W. FERRARI.1966. Selective inhibition of poliovirus growth by D-penicillamine in vitro. Virology 30: 618. 19. MERRYMAN, P.,I. A. JAFFE& E. EHRENFELD.1974. Effect of D-penicillamine on poliovirus replication in HeLa cells. J. Virol. 13: 881. 20. GERBER,D. 1974. Copper-catalyzed thermal aggregation of human gammaglobulin. Inhibition by histidine. gold thiomalate and penicillamine. Arthritis Rheumat. 17: 85.
VI.
P R O S P E C T I V E S O N P O S S I B L E THERAPY FOR P R E V E N T I O N OF JOINT DESTRUCTION IN RHEUMATOID ARTHRITIS
PENICILLAMINE TREATMENT OF RHEUMATOID ARTHRITIS: EFFECT ON IMMUNE COMPLEXES Israeli A. Jaffe Department oj Medicine New York Medical College New York, New York 10029
Penicillamine differs in structure from the naturally occurring amino acid cysteine by the presence of two methyl groups in the @-carbon position (FIGURE 1) . This structural change renders the compound relatively resistant to metabolic degradation and enables it to exert its various pharmacologic properties. It is a metabolite of penicillin that may be prepared from the parent molecule or synthetically, and the less toxic D isomer is the form used clinically. It is now more than 10 years since the first published report of the therapeutic efficacy of penicillamine in the treatment of rheumatoid arthritis ( R A ) Since that time, additional studies that confirmed this finding were conducted by investigators in England,z France,3 the Federal Republic of Germany,’ and the United States.s In 1973, the results of a controlled, double-blind, multicenter trial of penicillamine versus a placebo in severe rheumatoid disease were published. This trial, which was performed in England, demonstrated that by all clinical and laboratory criteria, penicillamine was an effective therapeutic agent in the treatment of RA. Based upon these findings, the Committee on Safety of Medicines in England approved penicillamine for the treatment of severe rheumatoid disease. At the present time, the drug is available on prescription in several European countries as an adjunct to the basic therapy of rheumatoid arthritis. T h e mechanism of action of penicillamine in RA is unknown; however, it appears to exert a very fundamental effect on the disease process and on its immunologic manifestations. There is a latent period of 12-20 weeks after therapy is instituted until clinical and laboratory response begins. Similarly, when the drug is discontinued, there is an interval of 8-10 weeks, and very often as long as 4-6 months, before clinical and laboratory exacerbation is observed. It therefore appears highly unlikely that the compound simply works as a n antiinflammatory agent. Furthermore, it has been shown to be without effect in influencing the established laboratory models of arthritis and inflammation.’ It is not the purpose of this presentation t o review the clinical pharmacologic features of penicillamine, dosage, side effects, and toxicity, because these subjects have been described adequately elsewhere.k.‘J In this presentation, emphasis will be directed toward the effect of penicillamine treatment on immune complexes and to the possible relevance of this effect to clinical improvement. The first evidence that penicillamine might be exerting a n effect on circulating complexes in a patient with R A was obtained by a comparison of the ultracentrifugal patterns of serum proteins before and after penicillamine 2 shows that after treatment, there therapy. A study of the patterns in FIGURE is a fall in the 22s complex and in the so-called intermediate complexes, which
330
Jaffe: Penicillamine Treatment
331
range from 9 to 17S, and also in the IgM peak. This reduction in the intermediate 7-globulin complexes is also demonstrated in the patient whose patterns are illustrated in FIGURE 3. A phenomenon that is probably identical can also be measured immunologically by the changes in the precipitin arc that occur when a rheumatoid serum is reacted with a monoclonal IgM rheumatoid factor, according to the technique of Winchester et al.Ir’ These changes, which occurred during and after the course of drug treatment, are depicted in FIGURE 4. There is a reduction in reactivity after 10 weeks of penicillamine therapy, and the reaction completely disappears at 6 months. The synovial fluid was not available for analysis after treatment, because the effusion had reabsorbed. Thus, the IgM rheumatoid factor measured by the latex fixation test, the r-globulin complexes seen in the ultracentrifuge, and the IgG rheumatoid factors (complexes) demonstrated by precipitation with a monoclonal IgM rheumatoid factor all decline after penicillamine treatment. The clinical relevance of this phenomenon is perhaps most readily apparent in the study of certain of the extraarticular manifestations of rheumatoid arthritis: rheumatoid lung disease, vasculitis, and Felty’s syndrome. The intermediate r-globulin complexes were shown by Tomasi et al. to bear a relationship to the presence of rheumatoid lung disease.” Lorber has described radiographic
t SH-C
I
I H
i
H
I - C --cooH I
NHe
SH-C
c?
I CH,
i
- C-COOH
I
NH2
FIGURE 1 . Penicillamine ( r i g h t ) is an analog of cysteine ( l e f t ) in which hydrogen atoms are replaced by methyl groups in the p-carbon position.
and functional improvement in rheumatoid lung disease after penicillamine treatment.l2 We have previously reported striking improvement in the vasculitis of rheumatoid arthritis during penicillamine therapy; l 3 , l1 relapse occurs after drug withdrawal. It is believed that the vasculitis that complicates RA may be analogous to experimentally induced immune complex vasculitis in anirnals.ls Recently, Hurd et at. demonstrated that in patients with Felty’s syndrome, the peripheral blood granulocytes contained large inclusions that exhibited properties of immunoglobulins, as assessed by immunofluorescent staining.16 They suggested that these inclusions (immune complexes) altered the morphologic status of peripheral blood granulocytes so as to predispose them to uptake by the spleen, a theory that would explain the leukopenia that is the hallmark of Felty’s syndrome. In two of their patients, penicillamine treatment produced a gradual disappearance of these inclusion-containing cells from the peripheral blood, which was accompanied by a rise in the neutrophil count to normal levels (FIGURE5). With respect to the articular manifestations of rheumatoid arthritis, there is considerable evidence to suggest that the sustained inflammatory response is due, at least in part, to the presence of complexes within the synovial space. A decrease in immune complexes in the synovial fluid during penicillamine treat-
332
Annals New York Academy of Sciences
Jaffe: Penicillamine Treatment
333
nient may be responsible for the improvement in the inflammatory articular process. As stated above, the mechanism of action of penicillamine in RA is not known. T h e effect upon the complexes could be caused by suppression of immunoglobulin synthesis per se, inhibition of complex formation, or alteration
FIGURE 3. Ultracentrifugal analysis of serum proteins in a penicillamine-responsive rheumatoid patient before and after 8 months of treatment. Sedimentation is at 52,640 rpm ( l e f t to right) at 64, 80, and 96 min. Serum in both determinations was diluted 1:2 with saline. Note the decline in 7s 7-globulin and in intermediate 7-globulin complexes.
of the antigenic stimulus to immunoglobulin production. With respect to the first possibility, it should be emphasized that penicillamine is not an immunosuppressant drug in experimental models.' Patients given penicillamine continuously for more than 10 years are not clinically immunosuppressed. They respond to antigenic stimuli i n the normal fashion and are not at increased risk for the development of opportunistic infections or neoplasia. Lymphocytes
334
Annals New Y o r k Academy of Sciences
FIGURE 4. This Figure illustrates the effect of penicillamine therapy on complexes, as measured by precipitation with purified IgM rheumatoid factor. Well 1 contains synovial fluid, and well 2 contains serum from the same patient prior to penicillamine therapy. Well 3 shows a decrease in the amount of complex after 6 weeks of penicillamine therapy. Wells 4 and 5 reveal the absence of demonstrable serum complexes at 3 and 6 months, respectively, after initiation of penicillamine therapy. Well C is a normal serum control. There is no posttreatment specimen of synovial fluid, because the synovial effusion had completely reabsorbed.
removed from patients during penicillamine therapy display a normal response to mitogens.J It seems unlikely that penicillamine affects the actual forination of coniplexes, because one of the most serious side effects of the drug is the development of an immune complex nephritis, which may be of sufficient severity to later result in nephrotic syndrome.” This immune complex nephritis occurs not only i n RA patients but may also develop in patients with Wilson’s disease and cystinuria who have been treated with the drug and whose basic immune mechanism is normal. I t is difficult to reconcile a drug-induced inhibition of complex formation to the fact that complexes may concomitantly be formed in response to the medication, which would give rise to nephropathy.
Jaff c : Penicillaminc ‘Treatment
335
Another possibility is that penicillamine acts upon the primary stimulus for abnormal immunoglobulin production, perhaps an infectious (viral) agent. It is of interest in this regard that penicillamine has potent and selective antiviral properties in vilro (FIGURE 6). Its entire antiviral spectrum has not been elucidated, and whether these properties exist in vivo has not been determined. Alternatively, a role for penicillamine in inhibiting the denaturation of proteins, including IgG, has been demonstrated in vitr.o.’O This inhibition could be a mechanism, if it occurred in vivo, by which a noninfectious protein, IgG, might be rendered less antigenic.
SUMMARY A N D CONCLUSIONS The prolonged administration of pcnicillamine to patients with KA not only produces clinical improvement with respect to both articular and extraarticular manifestations but also yields improvement in immunologic parameters, as is evidenced by a decline in IgM and IgG rheumatoid factors and in circulating complexes. It is not known whether this effect is mediated by a selective inhibition of immunoglobulin synthesis or is secondary to a basic effect of the drug on the presumed primary antigenic stimulus responsible for both the clinical and the laboratory manifestations of the disease. It is believed that by further study of this compound, more fundamental insights can be achieved into the basic pathogenetic mechanisms in rheumatoid arthritis.
+ 4 -0,
Total Neutrophiles
-14,000
fi
E
-12,000 E Z
-10,000
0
;
- 8,000 W 4
2.2-
2
J
-
6,000 0 4,000 - 2,000
2
z
TIME IN WEEKS -PENlClLLAMlNE
FIGURE 5 . This Figure illustrates the changes in neutrophil inclusions and total neutrophil count in a patient with Felty’s syndrome during penicillamine therapy (direct
method). The IgG inclusions gradually disappeared, and the granulocyte count increased to normal.
336
Annals New York Academy of Sciences
Jaffe: Penicillamine Treatment
337
REFERENCES I . JAFFE,I. A. 1963. Comparison of the effect of plasmapheresis and penicillamine on the level of circulating rheumatoid factor. Ann. Rheumat. Diseases 22: 71. 2. GOLDING, J . R., J. V. WILSON& T. G. PLUNKETT.1968. Laboratory observations on the use of penicillamine in rheumatoid arthritis. Postgrad. Med. I. (London) (October suppl.) :40. 3. CAMUS,J. P., C. BENICHOU, P. GUILLIEN,J. CROUZET& J. A. LIEVRE. 1971. Traitement de la polyarthrite ruhmatoide commune par la D-penicillamine. Rev. Rhumat. 3 8 809. 4. MIEHLKE,K. & I. KOHLHARDT. 1966. Uber die immuno-depressorische Wirkung von D-penicillamin unter der Behandlung der progredient chronischen Polyarthritis. Z. Rheumaforsch. 26: 56. 5. ZUCKNER, J., R. H. RAMSEY,R. W. DORMER& G. E. GANTNER.1970. Dpenicillamine in rheumatoid arthritis. Arthritis Rheumat. 13: 131. 6. MULTICENTRE TRIAL GROUP. 1973. Controlled trial of D ( - ) penicillamine in severe rheumatoid arthritis. Lancet 1: 275. 7. LIYANAGE, S. P. & H. L. F. CURREY.1972. Failure of oral D-penicillamine to modify adjuvant arthritis or immune response in the rat. Ann. Rheumat. Diseases 31: 521. 8. JAFFE,I. A. 1970. The treatment of rheumatoid arthritis and necrotizing vasculitis with penicillamine. Arthritis Rheumat. 13: 436. 9. DAY,A. T., J. R. GOLDING,P. N. LEE & A. D. BUTTERWORTH. 1974. Penicillamine in rheumatoid disease: a long-term study. Brit. Med. J. 1: 180. 10. WINCHESTER, R. J., H. G. KUNKEL & V. AGNELLO.1971. Occurrence of gammaglobulin complexes in serum and joint fluid of rheumatoid arthritis patients: use of monoclonal rheumatoid factors as reagents for their demonstration. J. Exp. Med. 134: 2863. 1 1 . TOMASI,T. B., JR., H. H. FUDENBERG & N. FINBY.1962. Possible relationship of rheumatoid factors and pulmonary disease. Amer. I. Med. 33: 243. 12. LORBER,A. 1966. Penicillamine therapy for rheumatoid lung disease: effects on protein sulphydryl groups. Nature (London) 210: 1235. 13. JAFFE, I. A. 1964. Rheumatoid arthritis with arteritis. Ann. Intern. Med. 61: 556. 14. JAFFE,I. A. & R. W. SMITH.1968. Rheumatoid vasculitis-report of a second case treated with penicillamine. Arthritis Rheumat. 11: 585. 15. EPSTEIN,W. 1957. F I1 precipitation test. In Serological Reactions of Rheumatoid Arthritis. R. W. Lamont-Havers, Ed. : 22. Arthritis and Rheumatism Foundation. New York, N.Y. 16. HURD,E. R.,J . LOSPALLUTO & M. ZIFF. 1974. The role of immune complexes in production of the neutropenia of Felty’s syndrome. In VIth Pan-American Congress on Rheumatic Diseases. : 194(abs.). 17. JAFFE,I. A., G. TRESER,Y.SUZUKI& T. EHRENREICH. 1968. Nephropathy induced by D-penicillamine, Ann. Intern. Med. 69: 549. 18. GESSA,G. L., B. LODDO,G. BROTZU,M. L. SCHIVO,A. TAGLIAMONTE, A. SPANEDDA, G. Bo & W. FERRARI.1966. Selective inhibition of poliovirus growth by D-penicillamine in vitro. Virology 30: 618. 19. MERRYMAN, P.,I. A. JAFFE& E. EHRENFELD.1974. Effect of D-penicillamine on poliovirus replication in HeLa cells. J. Virol. 13: 881. 20. GERBER,D. 1974. Copper-catalyzed thermal aggregation of human gammaglobulin. Inhibition by histidine. gold thiomalate and penicillamine. Arthritis Rheumat. 17: 85.
