Ann Hematol (1991) 6 2 : 7 6 - 8 0

/knn~ls of

Hematology 9 Springer-Verlag 1991

Case report Spontaneous interferonantibodies in a patient with pure red cell aplasia and recurrent cutaneous carcinomas O. Priimmer, N. Frickhofen, W. Digel, H. Heimpel, and E Porzsolt Department of Internal Medicine III, University of Ulm, Robert-Koch-Strasse 8, W-7900 Ulm, Federal Republic of Germany Received October 12, 1990/Accepted November 8, 1990

Summary. H i g h - t i t e r e d s p o n t a n e o u s i n t e r f e r o n ( I F N ) a n t i b o d i e s were detected in a p a t i e n t with pure red cell a p l a s i a ( P R C A ) , a b e n i g n m e d i a s t i n a l tumor, a n d recurrent c u t a n e o u s c a r c i n o m a s . T h e circulating I F N antib o d i e s reacted b r o a d l y with v a r i o u s h u m a n I F N - ~ subtypes ( 2 0 - 1 4 0 • 103 n e u t r a l i z i n g u n i t s / m l serum) b u t n o t with IFN-[3 or IFN-% a n d they n e u t r a l i z e d the antiviral activity o f the p a t i e n t ' s e n d o g e n o u s IFN-(x. T h e I F N - a b i n d i n g activity was restricted to the I g G l subclass in a n o n m o n o c l o n a l manner. W h e r e a s the P R C A r e p e a t e d l y r e s p o n d e d to i m m u n o s u p p r e s s i o n with h i g h - d o s e cyclos p o r i n A ( C S A ) a n d C S A plus p l a s m a p h e r e s i s , I F N antib o d y p r o d u c t i o n c o n t i n u e d d u r i n g t r e a t m e n t with cyclop h o s p h a m i d e a n d CSA. Serological analysis revealed p a s t infection with parvovirus B19 a n d persistent B19 I g M titers. A n t i b o d y - m e d i a t e d i m p a i r m e n t o f the IFN-(x system m i g h t have favored the d e v e l o p m e n t o f b o t h P R C A a n d t h e v a r i o u s c u t a n e o u s c a r c i n o m a s in this p a t i e n t .

Key words: I n t e r f e r o n - a a n t i b o d i e s - Pure red cell a p l a sia - Parvovirus B19 cancer

Cyclosporin A-

Cutaneous

Introduction S p o n t a n e o u s interferon ( I F N ) - a a n t i b o d i e s in m a n have been detected in a u t o i m m u n e disorders [16, 17, 19,21|, generalized viral infections [14], cancer [22, 25], a n d in n e w b o r n c o r d b l o o d [25]. Low-level p r o d u c t i o n o f I F N a n t i b o d i e s , which is n o t readily recognized by s t a n d a r d assay procedures, m a y even occur in healthy i n d i v i d u a l s [24]. Several i n b r e d m o u s e strains constitutively p r o d u c e IFN-ct a n d IFN-[3 a n t i b o d i e s [4]. T h e m e c h a n i s m s l e a d i n g to increased I F N a n t i b o d y p r o d u c t i o n in s o m e i n d i v i d u a l s a n d the p o t e n t i a l clinical i m p a c t o f these a n t i b o d i e s are u n k n o w n [19, 21]. Since

Offprint requests to: O. Priimmer

I F N - ~ is an active antiviral agent a n d has proved effective in the c o n t r o l o f s o m e m a l i g n a n t t u m o r s [6], it is conceivable t h a t n e u t r a l i z a t i o n o f e n d o g e n o u s IFN-(~ m a y predispose to s o m e viral infections a n d favor t u m o r development. We r e p o r t o n a p a t i e n t with high-titered s p o n t a n e ous I F N - a a n t i b o d i e s , p u r e red cell aplasia, persistent parvovirus B 19 I g M titers, a n d recurrent c u t a n e o u s carc i n o m a s , w h o might represent a n example o f the anticip a t e d h a r m f u l effect o f s p o n t a n e o u s I F N - a antibodies.

Case report The history of the patient, a 72-year-old man, was unremarkable until 1969, when an extrasphincteric perianal fistula was diagnosed in the absence of inflammatory bowel disease. Perianal abscesses were resected repeatedly until 1983. In 1981, a carcinoma spinocellulare of the left cheek was surgically removed, followed by local X-irradiation. Two years later, the patient was well, and there were no signs of regional or distant metastases. Hemoglobin (153g/1) and white blood cell (WBC) count (7.5• were normal. In April 1987, he underwent surgery for a squamous cell carcinoma of the right earlobe (cTiNoMo) and a morbus Bowen of the back. A solid mass in the anterior upper mediastinum (diameter 8 cm) had been known since 1981 and did not enlarge on repeated controls. Cytological or histological diagnosis study was refused by the patient, but the uneventful course suggested the presence of a benign thymoma or teratoma. In August 1987, the patient complained of progressive fatigue and persistent bronchitis. He was found anemic (hemoglobin 81 g/l) and failed to respond to folic acid and vitamin B12 treatment. One month later, the hemoglobin level was 71 g/l. Based on low reticulocyte counts (2.3 x 109/t) and severely depressed bone marrow erythropoiesis in the presence of an adequate white blood cell count (10.3x109/1), differential count, and platelet count (459x 109/1), the diagnosis of hypoproliferative anemia was made. Serum iron (37 ~tmol/1) and lactate dehydrogenase levels (472 U/l) were increased. Immunoglobulin levels were normal. Nuclear- and mitochondrial-specific antibodies were absent, and an extensive search for a rheumatoid or a malignant disorder remained negative. Supportive care with repeated red blood cell (RBC) transfusions was initiated (Fig. 1). In January 1988, the patient was referred to the Department of Internal Medicine III, University of Ulm. He complained of a productive cough but was otherwise well. There were no malignancyassociated symptoms and no history of allergies. Physical examina-

77

RBC

N.

I[11II II IIII

IIIII [Ill IIII nil

II

IIII

IIII II p v ,~v

1~176 T Fig. 1. Course of blood hemoglobulin levels, reticulocyte count, and serum interferon (IFN-c0 antibody levels (BU, binding units; NU, neutralizing units). Treatment is indicated at the top of the figure: CP, cyclophosphamide; CSA, cyclosporin A; RBC, red blood cell transfusion; P, plasmapheresis

~"~o~ 2oo I D

[]

ol 83

87

88

Time

89

tion revealed a grade 2/6 holosystolic murmur and the known perianal fistula but was otherwise noncontributory. The hypoproliferative anemia (hemoglobin 75 g/l, reticulocyte count below 3.1• was confirmed; leucocytes (11.9x109/1) and platelets (485 x 109/1) were marginally increased, and there was a moderate toxic granulation of the neutrophils. Serum iron (33 pmol/1) and ferritin levels (1,170 ng/ml) were increased. Serum immunoglobulin levels were normal (IgG 9.6 g/l) or moderately decreased (IgA 0.62 g/1, IgM 0.31 g/l); there was no detectable M-protein. Nuclear-specific antibodies were weakly positive (titer 1:160, speckled pattern), but a search for additional autoantibodies and rheumatoid factor remained negative. Erythropoietic cells were almost absent from bone marrow smears and biopsies (less than 1 per 1,000 nuclear cells) in the presence of regular maturation of granulopoiesis and megakaryopoiesis and a moderate elevation of eosinophils. Prominent focal lymphocyte infiltrates were primarily composed of T cells (CD4/CD8 ratio, 2.0) with few accompanying B cells. Erythroid progenitor cell numbers were low in bone marrow [burst-forming unit-erythroid (BFU-E) 27/105 mononuclear cells (MNC), colony-forming uniterythroid (CFU-E) 82/105 MNC; normal values: BFU-E 148 +_ 99/105 MNC, CFU-E 326 + 144/105 MNC] and peripheral blood (BFU-E 0-15/106 MNC; normal values: 6 _+ 4/106 MNC). Serum levels of immunoreactive erythropoietin were tenfold elevated (262 mU/ml). T cells predominated in the blood (87~ CD3-positive, 1% CD20-positive lymphocytes) with an inverse CD4/CD8 ratio of 0.57; there were no signs of T-cell activation (1% CD25-positive, 3% HLA-DR-positive lymphocytes). The natural killer cell-associated antigen CD56 was expressed on 0.1% of blood MNC. A computed tomography (CT) scan of the chest revealed a mediastinal mass with a diameter of 8.5 cm. A diagnosis of pure red cell aplasia (PRCA) was made, and treatment was initiated as indicated below.

Materials

and methods

Binding IFN-a antibodies. Serum antibodies directed at human recombinant IFN-c~2a (rIFN-ct2a) were detected with a sandwich enzyme immunoassay (EIA [8]; reagents kindly donated by Hoff-

mann-La Roche, Basel, Switzerland). Binding units were expressed according to a polyclonal rabbit standard serum raised against rIFN-a2a. Specificity of antibody binding was confirmed by the addition of rIFN-c~2a (Roferon; Hoffmann-La Roche), rIFN-a2b (Intron A; Essex-Pharma, Munich, FRG), lymphoblastoid IFNanl (Wellferon; Wellcome, Beckenham, UK), IFN-13 (Fiblaferon; Bioferon, Laupheim, FRG), and rIFN-7 (a gift of Dr. A. Wolf, Bioferon) to the test sample.

Immunoglobulin class of IFN-c~-binding antibodies. An enzymelabelled immunosorbent assay (ELISA) was used for the demonstration of immunoglobulin class and subclass of IFN-a-binding antibodies. Recombinant IFN-a2a was coupled to the solid phase (Nunc Immunoplate II; Nunc, Roskilde, Denmark) with the aid of bovine antihuman IFN-a antibodies (Ig fraction of serum AS94 at 1 : 5,000; kindly donated by Dr. G. Aim, Uppsala, Sweden) as described [23]. Free protein-binding sites were blocked with PBS containing 5% nonfat dry milk (BLOTTO 5), and 0.1 ml serum samples diluted 1 : 1,000 in BLOTTO 5 were added to triplicate wells for 1 h at 37~ After washing (PBS plus 0.05% Tween 20), 0.1 ml of appropriately diluted (BLOTTO 5), peroxidase-conjugated, goat antihuman IgA, IgG, and IgM antibodies (Jackson, West Grove, USA), peroxidase-conjugated, rabbit antihuman K- and X-light chain antibodies (Dako, Glostrup, Denmark) or subclass-specific monoclonal mouse antihuman IgGl_4 antibodies (Immunotech, Marseille, France) were added for 1 h at 37~ Mouse antibodies were visualized with peroxidase-conjugated, goat antimouse IgG ( H + L ) antibodies (Jackson). Plates were washed, 0.1 ml of Ophenylenediamine H20 z substrate solution was added per well, and absorbance was read at 490 nm after 10 min. Control wells contained no rIFN-a2a.

Bioassay for IFN. An antiviral cytopathic effect (CPE) reduction assay (Madin-Darby bovine kidney ceils plus vesicular stomatitis virus; both kindly donated by Dr. W. Jucker, Hoffmann-La Roche) was used for the quantitation of IFN-a in serum samples and culture supernatants [12]. MDBK cells were propagated in RPMI-1640 medium containing 10% fetal calf serum, 2 m M L-glutamine, penicillin-streptomycin, and 25 m M H~PES buffer. After monolayer formation, cells were exposed to IFN-c~ for 24 h and challenged

78 with virus for 18-24 h. IFN-[3 and IFN-y was quantitated on Wish cells challenged with encephalomyocarditis virus [181. Laboratory standards calibrated with international reference preparations (NIH Ga 23-902-50 and NIH Gg 23-901-530) were incorporated in each assay.

Neutralizing lFN antibodies, Neutralization by the patient's serum of tlae antiviral IFN-cq IFN-~3, and IFN-y activity was carried out according to published recommendations [7]. Serial twofold serum dilutions were incubaled with final concentrations of 5-20 IU/ml of various IFN-ct preparations (rlFN-ct2c, kindly donated by Dr. G. R. Adolf, Ernst Boehringer Institute, Vienna, Austria; leukocyte IFN-ctLe, donated by Dr. Myllyla, Finnish Red Cross Blood Transfusion Service, Helsinki) with IFN-B and rIFN-~ for I h at 37~ and the mixture was tested for residual IFN activity as described above. Besides standard virus and cell controls, serum controls with and without virus challenge as well as titrations of the IFNs used and their respective reference standards were run simultaneously. Serum dilutions yielding a 50070 CPE were taken as the reciprocal titer and converted to neutralizing units (NU)/ml by multiplying by the IFN concentration neutralized. Interferon production. Mononuclear blood cells of the patient were stimulated with Sendal virus for IFN-c~ production and with phytohemagglutinin for IFN-y production as described [18]. Supernatants were removed after 20 h and stored frozen until analysis. Parvovirus studies. Serum antibodies to parvovirus B19 were measured by capture immunoassay [3]_ A search for viral DNA in serum and bone marrow sections was done by standard dot hybridization or polymerase chain reaction [2, 10]. Treatment and experimental analyses were carried out with the patient's informed consent,

Results

Interferon antibodies Retrospective analysis by E I A o f a s e r u m s a m p l e o b t a i n ed o n a d m i s s i o n o f the p a t i e n t revealed the presence o f a high-titered activity b i n d i n g to i m m o b i l i z e d r I F N - a 2 a (240,000 B U / m l ) . Despite c o n t i n u e d i m m u n o s u p p r e s s i v e t h e r a p y with c y c l o p h o s p h a m i d e a n d cyclosporin A (CSA) the activity persisted for at least 18 m o n t h s , a l t h o u g h at r e d u c e d levels for s o m e t i m e (Fig. 1). O n analysis by E L I S A , the I F N - a 2 a - b i n d i n g molecules t u r n e d o u t to be Table 1. Immunoglobulin class of spontaneous interferon (IFNbinding antibodies Antigen detected

IgA IgG IgM IgG 1 IgG2 IgG3 lgG4 Kappa Lambda

Absorbance 490 - IFN-e2a

+ IFN-a2a

0,053 0,051 0,056 0,056 0,018 0.020 0.028 0.055 0.055

0,045 0.790 0.046 0.417 0.010 0.015 0.015 0.229 0.223

+_ 0.012 + 0.004 _+ 0_006 _+ 0.018 +_ 0.003 _+0.009 _+ 0.003 _+ 0.049 +_ 0.018

_+ 0.002 _+ 0.028 _+ 0,006 _+ 0.029 + 0,001 _+ 0.006 _+ 0.003 +_ 0.016 +_ 0.009

Immunoglobulin class and subclass of IFN-a2a-binding antibodies were characterized by ELISA. Control wells (-IFN-a2a) were run without IFN-ct2a

600 500E

4003000 ..Q

m!FN-~nl

"~ 200-

~

\

..Q


100 > 100 62 69 > 100 > 100

a Polymerase chain reaction b Arbitrary units; titers > 2 are significant

A squamous cell carcinoma at the tip of the nose was resected in April 1989. IFN-~ antibodies, which had been lowered by the plasmaphereses, were present again at high levels 4 months after the procedure. Since then, the patient has been treated by his family doctor. Because of recurrent bronchitis and pneumonia, the CSA dosage was reduced and finally discontinued. This was associated with recurrence of the PRCA, and by June 1990 the patient again needed RBC support.

Discussion

This is the first report of a coincidence of high-titered spontaneous IFN-a antibodies, PRCA, and recurrent cutaneous carcinomas. Similar to previous reports [17, 19, 21, 25], the IFN antibodies in our patient were of the IgG type, but their restriction to IgGl distinguished them from IFN-a antibodies in a patient with zoster infection, which were composed of all IgG subclasses [20]. The presence of both kappa and lambda light chains excluded a monoclonal origin. The IFN antibodies reacted strongly with several recombinant human IFN-a subtypes and the major components of lymphoblastoid and leukocyte IFN-a, both by binding and neutralization of the antiviral and antiproliferative (data not shown) IFN-a effect. Similar broad specificities have been reported for spontaneous IFN-c~ antibodies [20,25], whereas treatment-induced IFN-a antibodies predominantly recognize the administered rIFN-a and related subtypes ([251, own observation). Natural IFN antibodies in mice react with both IFN-a and IFN-[3 [41. The mechanisms leading to spontaneous IFN antibody formation are unknown [21]. In our patient, IFN-a antibody production was remarkably stable and resistant to immunosuppression with cyclophosphamide and CSA. Lack of the respective antigen presumably did not cause IFN antibody formation, since the patient's blood leukocytes readily produced substantial amounts of IFN-(x upon viral stimulation. The likely presence of thymoma might have been relevant, although the pathogenetic link between thymoma and autoantibody formation is not understood. Except for a temporary elevation of specific antibodies, no additional autoantibodies could be disclosed. What is the clinical significance of IFN-a antibodies in our patient? It is possible that high-level production of IFN antibodies simply reflected an underlying, as yet unidentified immune derangement which was also relevant for the pathogenesis of PRCA and cancer development. However, there were sufficient antibodies to neutralize even high local concentrations of endogenous IFN-a I1, 15]. Thus, impairment of in vivo processes depending on an intact IFN-a system was a likely consequence. Acquired PRCA in adults is associated with the presence of thymoma, autoimmune disorders, malignancies, and infections [5, 26]. Antibody- or cell-mediated suppression of erythropoiesis is probably involved in most patients [5, 11, 13], but persistent parvovirus B19 infection has been described rarely [9, 10, 26]. The continued

80 presence in o u r p a t i e n t o f significant I g M titers to p a r v o virus B 19 suggested persistent infection, b u t viral D N A c o u l d never be detected in his serum. Since p e r i p h e r a l b l o o d or b o n e m a r r o w cells were n o t available for m o r e sensitive viral assays [9], a low-level p a r v o v i r u s infection c a n n o t definitely be excluded. Alternatively, the a n t i b o d y p a t t e r n m i g h t indicate p a s t parvovirus infection with persistent I g M titers owing to a t y p i c a l i m m u n e regulation. H i g h serum e r y t h r o p o i e t i n levels excluded the presence o f p a t h o g e n e t i c a l l y relevant e r y t h r o p o i e t i n a n t i b o d i e s [5]. H i g h - d o s e C S A t r e a t m e n t reversed the P R C A for s o m e t i m e in o u r p a t i e n t despite the c o n t i n u e d presence o f I F N - a - s p e c i f i c a n d p a r v o v i r u s B19-specific I g M antibodies. Thus, a solely viral e t i o l o g y o f the P R C A a p p e a r ed unlikely. I f I F N - ~ a n t i b o d i e s were relevant for the p a t h o g e n e s i s o f the disorder, their a c t i o n m u s t have been c o u n t e r a c t e d by C S A . It is u n k n o w n w h e t h e r the prolonged s e c o n d a r y response o f the P R C A was merely due to elevated C S A levels or w h e t h e r the p l a s m a p h e r e s i s i n d u c e d t e m p o r a r y decrease o f I F N a n t i b o d y levels a n d the a c c o m p a n y i n g p l a s m a exchange [10, 12, 13] were critical d e t e r m i n a n t s . Recurrence o f c u t a n e o u s c a r c i n o m a s at different sites a n d with various histologies raised the q u e s t i o n as to the n a t u r e o f the u n d e r l y i n g defect in o u r patient. Since I F N - a t r e a t m e n t has proved effective in several m a l i g n a n cies, in p a r t i c u l a r at early stages o f the disease [6], a n d I F N - ~ a n t i b o d i e s were present in s o m e c a n c e r - b e a r i n g patients [22, 25], a n t i b o d y - m e d i a t e d i m p a i r m e n t o f I F N - ~ a c t i o n m i g h t have favored t u m o r d e v e l o p m e n t a n d p r o gression in this patient.

Acknowledgments. The technical assistance of Helga Dietrich, Sybille Rink, and B~rbel Neuh~user is gratefully acknowledged. Dr. B. J. Cohen (Virus Reference Laboratory, Central Public Health Laboratory, London) determined parvovirus B19 antibodies. U. Hennes and Dr. W. Jucker (Hoffmann-La Roche, Basel) kindly donated EIA reagents, MDBK cells, and vesicular stomatitis virus. Serum AS94 was a kind gift of Dr. G. Alto, (Interferon Laboratory, University of Uppsala). Interferons were donated by Dr. G.R. Adolf (rIFN-c~2c; Ernst-Boehringer-Institut far Arzneimittelforschung, Vienna) and Dr. A. Wolf, (rIFN-7; Bioferon, Laupheim). This work was supported by a research development grant of the University of Ulm.

References 1. Armstrong RW, Gurwith M J, Waddel D, Merigan TC (1970) Cutaneous interferon production in patients with Hodgkin's disease and other cancers infected with varicella and vaccinia. N Engl J Med 283:1182-1187 2. Clewely JP (1985) Detection of human parvovirus using a molecularly cloned probe. J Med Virol 15:173-181 3. Cohen BJ, Mortimer PP, Pereira MS (1983) Diagnostic assays with monoclonal antibodies for the human serum parvoviruslike virus (SPLV). J Hyg (Lond) 91:113-130 4. De Maeyer-Guignard J, de Maeyer E (1986) Natural antibodies to interferon-a and interferon-~ are a common feature of inbred mouse strains. J Immunol 136:1708-1711

5. Dessypris EN (1988) Pure red cell aplasia. Johns Hopkins University Press, Baltimore 6. Figlin RA (1988) Biotherapy with interferon 1988. Semin Oncol 15:3-9 7. Grossberg SE (1988) Human antibodies to interferons: report of a National Institute of Health Workshop held 25-26 July 1988 in Bethesda, MD. J Interferon Res 8 (6): V-VII 8. Hennes U, Jucker W, Fischer EA, Krummenacher T, Palleroni AV, Trown PW, Linder-Ciccolunghi S, Rainisio M (1987) The detection of antibodies to recombinant interferon alfa-2a in human serum. J Biol Stand 15:231-244 9. Kurtzmann GJ, Gascon P, Caras M, Cohen B, Young NS (1988) B 19 parvovirus replicates in circulating cells of acutely infected patients. Blood 71:1448-1454 10. Kurtzman G, Frickhofen N, Kimball J, Jenkins DW, Nienhuis AW, Young NS (1989) Pure red cell aplasia of 10 years' duration due to persistent parvovirus B 19 infection and its cure with immunoglobulin therapy. N Engl J Med 321:519-523 11. McGuire WA, Yang HH, Bruno E et al. (1987) Treatment of antibody-mediated pure red-cell aplasia with high-dose intravenous gamma globulin. N Engl J Med 317:1004-1008 12. Meager A (1987) Quantification of interferons by anti-viral assays and their standardization. In: Clemens M J, Morris AG, Gearing AJH (eds) Lymphokines and interferons: a practical approach. IRL Press, Oxford, pp 129-147 13. Messner HA, Fauser AA, Curtis JE, Dotten D (1981) Control of antibody-mediated pure red-cell aplasia by plasmapheresis. N Engl J Med 304:1334-1338 14. Mogensen KE, Daubas PH, Gresser I, Sereni D, Varet B (1981) Patient with circulating antibodies to c~-interferon. Lancet II: 1227-1228 15. Overall JC, Spruance SL, Green JA (1981) Viral-induced leukocyte interferon in vesicle fluid from lesions of recurrent herpes labialis. J Infect Dis 143:543-547 16. Panem S (1984) Antibodies to interferon in man. In: Vilcek J, de Maeyer E (eds) Interferons and the immune system. Elsevier, Amsterdam, pp 175-183 17. Panem S, Check I J, Henriksen D, Vilcek J (1982) Antibodies to ct-interferon in a patient with systemic lupus erythematosus. J Immunol 129:1-3 18. Porzsolt F, Raghavachar A, Digel W, Raghavachar A, Bartram CR, Heimpel H (1987) Strategy for the treatment of hairy cell leukemia. Leukemia 1:334-337 19. Pozzetto B, Mogensen KE (1986) Immunisation anti-interf6ron chez l'homme. Pathol Biol (Paris) 34:1097-1103 20. Pozzetto B, Mogensen KE, Tovey MG, Gresser I (1984) Characteristics of antibodies to human interferon in a patient with varicella-zoster disease. J Infect Dis 150:707-713 21. Prtimmer O, Seyfarth C, Scherbaum WA, Drees N, Porzsolt F (1989) Interferon-ct antibodies in autoimmune diseases. J Interferon Res 9 (Suppl 1): 67-74 22. Prt~mmer O, Trauschel B, Drees N et al. (1989) Antibodies to interferon (IFN)-e~ as a consequence of adjuvant IFN-c~2a treatment of renal cell carcinoma. J Interferon Res 9 (Suppl 2): 114 23. Pr/immer O, Cederblad B, Alto G, Drees N, Porzsolt F (1990) Filter spot-ELISA for the enumeration of interferon-e~ antibody-secreting cells. J Immunol Methods 130:187-193 24. Ross C, Hansen MB, Schuberg T, Berg K (1989) Autoantibodies to the human interferon system. J Interferon Res 9 (Suppl 2) 25. Trown PW, Kramer M J, Dennin RA Jr, Connell EV, Palleroni AV, Quesada J, Gutterman FU (1983) Antibodies to human leukocyte interferons in cancer patients. Lancet I: 81-84 26. Young N (1988) Hematologic and hematopoietic consequences of B 19 parvovirus infection. Semin Hematol 25:159-172