- Email: [email protected]
Clinical association of autoantibodies to fibrillarin with diffuse scleroderma and disseminated telangiectasia
Journal of the American Academy of Dermatology
Vernon and Olsen may need to be continued more than 2 weeks, but perhaps a less potent steroid could be substituted for c1obetasol propionate ointment at that time. We thank Eldred E. Giefer of Glaxo, Inc., Research Triangle Park, North Carolina, for performing the statistical computations.
REFERENCES 1. Frank L, Stritzler C, Kaufman J. Hydrocortisone (com2.
pound F) free alcohOl and hydrocortisone acetate for topical uses. Arch Dermatol 1955;71:117-20. Kaidbey KH, Kligman AM. Assay oftopical corticosteroids, efficacy of suppression of experimental Rhus dermatitis in humans. Arch Dermatol 1976;112:808-13.
Clinical association of autoantibodies to fibrillarin with diffuse scleroderma and disseminated telangiectasia Gunter Kurzhals, MD,a Michael Meurer, MD,a Thomas Krieg, MD,a and Georg Reimer, MDb Munich and Erlangen, Federal Republic of Germany Circulating autoantibodies against a variety ofnuclear and nucleolar antigens are characteristic serologic findings in systemic scleroderma. Some of these antibodies correlate with clinical subsets of the disease. We describe three patients with systemic scleroderma and high autoantibody titers against U3 ribonucleoprotein-associated fibrillarin, a recently identified 34 leD nucleolar protein. These patients showed a progressive course with multiple organ and diffuse skin involvement with disseminated telangiectasia. (J AM ACAD DERMATOL 1990;23:832-6.)
Systemic scleroderma is a generalized disease of connective tissue that involves mainly the skin, the gastrointestinal tract, the lungs, the heart, and the kidneys. 1 Circulating antibodies against a variety of nuclear and nucleolar antigens can be detected in more than 95% ofpatients. 2-4 Some antibody specificities correlate with defined clinical subsets of the disease and have proved to be of diagnostic and prognostic significance. For example, anti-Sc1-70 antibodies directed against the nuclear/nucleolar enzyme DNA topoisomerase 1 are associated with diffuse scleroderma and multiple organ involvement. s On the other hand, anticentromere antibodies are characteristic of the CREST (calcinosis cutis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, and telangiectasia) syndrome From the Department of Dermatology, Ludwig-Maximilians-UniversHiit Munchen,' and the Department of Dermatology, Friedrich-ALexander-Universitat Erlangen-Nurnberg.b Accepted for publication Jan. 17,1990. Reprint requests: Michael Meurer, MD, DermatoLogische Klinik und Poliklinik, Ludwig-Maximilians-Universitat Miinchen, Frauenlobstr. 9-11, D-8000 Milnchen 2, FRG. 16/1/19464
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and of acroscleroderma with limited cutaneous and internal organ involvement. 6 More recently, a variety of nucleolar proteins within RNA-protein complexes have been identified as targets of autoantibodies in scleroderma patients. These nucleolar antigens include RNA polymerase 1,7 the Pm-Scl particle,8 U3 ribonucleoprotein CD3RNP)-associated fibrillarin, and 7-2 RNP.9 Antibodies directed against the Pm-Scl antigen identify a group of scleroderma patients with concomitant myositis. 10 The clinical significance of the other antinucleolar antibodies is less clear. We present evidence that antibodies against the D3-RNA-associated nucleolar protein fibrillarin may characterize a subset of patients with diffuse scleroderma and widespread telangiectasia. CASE REPORTS
Case 1 A 43-year-old white man had had Raynaud's disease for 8 years and diffuse scleroderma for 4 years. Recently, dysphagia and dyspnea on exertion developed. He had had psoriasis for 20 years. On examination, edema and swelling of the fingers and toes were present. Cutaneous
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sclerosis extended to the forearms and the upper part of the legs, the trunk, and the face. Disseminated telangiectasia with accentuation on the face, the neck, and the upper part of the trunk was noted. Psoriatic plaques were noted on the trunk, the elbows, and the knees. Chest roentgenogram showed bilateral pulmonary fibrosis. Pulmonary function tests revealed moderate impairment of diffusion capacity. Barium swallow roentgenography and manometric measurements indicated reduced esophageal motility. Except for a slightly increased erythrocyte sedimentation rate (10 to 30 mm/hr [Westergren]), a mild leukocytosis (12,000 cells/mm3), and elevated ')'-globulin level (20.5% relative) all routine laboratory tests were normal. Indirect immunofluorescence revealed IgG class antibodies directed against cell nucleoli. The staining pattern on HEp-2cells was clumped (titer> 1:10,000). Rheumatoid factor was negative. Autoantibodies to DNA and to extractable nuclear proteins, including Scl-70, UI-RNP, PM-Sel, and SS-B (La), were not detected.
Case 2 A 60-year-old white man had a 4-year history of progressive thickening of the skin preceded by Raynaud's phenomenon for 1 year. He also had dyspnea, dysphagia, and renal hypertension. Physical examination showed scleroderma ofthe upper and lower extremities and of the upper part of the trunk. He had a beaked nose, narrow lips, small mouth, and perioral radial furrowing. Sclerotic skin areas, especially on the face and trunk, contained numerous telangiectases (Fig. 1). Telangiectases were also present on normal perilesional skin. Assessment of esophageal function revealed reduced motility, delayed emptying, and gastroesophageal reflux. Blood pressure, controlled with treatment with angiotensin-converting enzyme inhibitor, was elevated (160/100 mm Hg). Electrocardiogram, echocardiogram, chest roentgenogram, and pulmonary function were normal. The patient had proteinuria (1650 mg/24 hr), reduced renal clearance, and an elevated serum creatinine level (1.5 mg/ 100 mI). With the exception ofa slightly elevated erythrocyte sedimentation rate (15 to 25 mm [Westergren)) and a test result positive for rheumatoid factor, other routine laboratory findings were within normal limits. By indirect immunofluorescence on HEp-2 cells, antinucleolar antibodies of the IgG type with a titer of more than 1:I0,000 and clumped nucleolar staining were detected. Antibodies against Scl-70, UI-RNP, Pm-Scl, and SS-B (La), and anti-DNA antibodies were absent.
Case 3 A 48-year-old white woman first noted Raynaud's phenomenon in 1982 and was referred to the hospital in 1984 because of sclerodactyly, dysphagia, heartburn, and dyspnea on exertion. In addition, the patient had had
Fig. 1. Case 2. Multiple telangiectases on face. psoriasis for more than 30 years. Sclerodermatous involvement of the fingers and face was marked but less pronounced on the feet and forearms. Sclerodactyly was prominent, with multiple ulcers and pitting scars on the fingertips. Microcheilia, microstomia, and radial perioral furrowing were present. This patient also had disseminated telangiectases that involved mainly the face and upper part of the chest but also appeared on apparently normal skin. In addition, psoriatic plaques were present on the elbows, knees, and trunk. Scintigraphy showed reduced esophageal motility, delayed emptying, and a gastroesophageal reflux. Arteriography showed marked obliterative disease of almost all digital arteries. Chest roentgenogram and pulmonary function were normal. Echocardiography showed signs of endocardial and myocardial fibrosis. No proteinuria was found, and renal function was normal. Besides a positive rheumatoid factor test result, all routine laboratory data were within normal limits. Indirect immunofluorescence of HEp-2 cells showed antinucleolar antibodies with clumped staining and a titer of more than 1: 10,000.
Immunologic studies All three sera displayed clumped nucleolar staining on HEp-2 cells (Fig. 2). Antigens were further characterized by immunoblotting; HeLa cell nucleoli were used as the source of antigen.?' 10 Nucleoli were isolated by sonication. lI Nucleolar proteins were separated by sodium dodecylsulfate--polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose paper. 12 After incubation with antinucleolar scleroderma sera a,nd control sera, diluted at 1: 100, specific IgG was detected by a second incubation with protein A (Du Pont New
834 Kurzhals et al.
Journal of the American Academy of Dermatology
Fig. 2. Indirect immunofluorescence of serum from case 1: Sera were diluted 1:40 in phosphate-buffered saline solution (pH 7.4) and evaluated by indirect immunofluorescence for presence of ANA with use of HEp-2 cells as substrate. Fluoresceinated goat antihuman immunoglobulin conjugate specific for human IgG (Behring Diagnostics, La Jolla, Calif.) was used as detecting reagent. Bright nucleolar staining in a clumped pattern is produced by antibodies from all sera. In dividing cells both chromosomal and nucleoplasmic staining are observed (bright cells at left). Nucleolus has disintegrated in these cells and is no longer present.
England Nuclear Research Products, Boston, Mass.) followed by autoradiography. I I Serum from patient 1 was also reacted with purified fibrilIarin (see legend to Fig. 3). Antibodies from the sera of the three patients reacted with a 34 kD protein present in the nucleolar extract from HeLa cells. By use of the reference serum from patient 1 (same serum as that used by Reimer et al. l !), the 34 kD protein was identified as fibrillarin (see Fig. 3). DISCUSSION
Antinucleolar antibodies are part ofthe autoantibody spectWm present in scleroderma sera. 13 By indirect immunofluorescence antinucleolar antibodies produce distinct staining patterns on tissue culture cells. Among these are speckled, homogeneous, and clumped patterns. 14 Several reports have correlated the speckled staining pattern with anti-RNA polymerase I antibodies, 7 the homogeneous staining with anti-Pm-Scl specificity, 10 and the clumped staining pattern with antifibrillarin antibodies. 11 Fibrillarin is an RNP particle that contains U3-RNA and is located in the fibrillar regions of nucleoli. IS The protein of the U3-RNP particle that contains the antigenic determinants is a 34 kD nucleolar protein. I6 Antibodies to U3-RNP-associated fibrillarin have been found in 22 of 646 patients with systemic scleroderma 13 and appear to be highly specific for
this disease. In this study patients with antifibrillarin antibodies were found to have significantly less joint involvement but otherwise were not different from patients without antinucleolar antibodies, including randomly selected patients with the CREST syndrome and diffuse scleroderma. Our three patients with this antibody specificity shared strikingly similar clinical features of systemic scleroderma, including diffuse skin involvement, widespread telangiectasia, and multiple internal manifestations. The vascular changes were even more pronounced than in patients with CREST syndrome. Cutaneous calcinosis, however, was absent. In contrast to most patients with CREST syndrome, our patients showed a progressive course with the development of sclerotic cutaneous and extracutaneous manifestations within 1 to 2 years after the onset of Raynaud's phenomenon. Therefore patients with antifibrillarin antibodies are different from patients with the CREST syndrome, which is serologically characterized by anticentromere antibodies 6, 17 and which usually follows a more benign course. IS•20 In scleroderma patients with widespread telangiectasia, these two antibody specificities may thus be helpful for determining prognosis. Two of our three patients had psoriasis in associ-
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patients in this study is small, a specific definition of a new clinical subset of scleroderma would be premature and would require a multicenter study of a larger number of well-characterized patients. We thank Dr. Michael Lischwe (Baylor College of Medicine, Department ofPharmacology, Houston, Tex.) for providing the data of the experiment shown in Fig. 3.
94-
68-
REFERENCES
-34
30-
2114Fig. 3. Immunoblot obtained with serum from case 1: HeLa nucleolar proteins and purified 34 leD protein fibrillarin 7 were separated by electrophoresis in a 11%
polyacrylamide Laernmli gel21 and electrophoretically transferred to nitrocellulose paper incubated with antibodies from the serum of patient presented as case 1. The antigen-antibody complexes were detected with 1251_protein A. Lane A, Amido black-stained HeLa nucleolar proteins. Lane B, Amido black-stained purified 34 leD fibrillarin. Lane C, Autoradiograph of the immunoblot with scleroderma serum (case 1) against total HeLa nucleolar proteins. Lane D, autoradiograph of immunoblot with this serum with purified fibrillarin as antigen. 15, 16 Molecular weight markers are phosphorylase B (94,000 leD), bovine serum albumin (68,000 leD), ovalbumin (43,000 leD), carbonic anhydrase (30,000 leD), and soybean trypsin inhibitor (21,000 leD).
ation with systemic scleroderma. The frequency of psoriasis in systemic scleroderma is not higher than in the normal population. Autoantibodies against nuclear antigens, particularly against denatured DNA, have been reported in patients with psoriasis after ultraviolet light therapy.22,23 To our knowledge nucleolar staining by psoriatic sera has not been reported, although antibodies to UI-RNP and U2-RNP have been described in two patients with severe Raynaud's phenomenon and concomitant psoriasis. 24 The association of antifibrillarin antibodies and psoriasis in two of our three patients needs further investigation. Because the number of
I. Krieg T, Meurer M. Systemic scleroderma: clinical and pathophysiologic aspects. J AM ACAD DERMATOL 1988; 18:457-81. 2. Tan EM. Autoantibodies to nuclear antigens (ANA): their immunobio10gy and medicine. Adv TmmunoI1982;33: 167240. 3. Tan EM, Rodnan GP, Garcia I, et aL Diversity of antinuclear antibodies in progressive systemic sclerosis: antieentromere antibody and its association to CREST syndrome. Arthritis Rheum 1980;23:617-25. 4. Bernstein RM, Steigerwald IC, Tan EM. Association of antinuclear and antinucleolar antibodies in progressivesystemie sclerosis. Clin Exp Immunol 1982;48:43·57. 5. Shero JH, Bordwell B, Rothfield NF, et al. High titers of autoantibodies to topoisomerase I (Scl·70) in sera from scleroderma patients. Science 1986;231:737-40. 6. Moroi Y, Peebles C, Fritzler MJ, et al. Autoantibody to centromere (kinetochore) in scleroderma sera. Proc Natl Acad Sci USA 1980;77:1627-31. 7. Reimer G, Rose KM, Scheer U, et al. Autoantibodies to RNA polymerase I in scleroderma sera. J Clin Invest 1987; 79;65-72. 8. Reichlin M, Maddison PS, Targolf J, et al. Antibodies to nuclear/nucleolar antigen in patients with polymyositis overlap syndromes. J Clin Immunol 1984;42:40-4. 9. Reddy R, Tan EM, Henning D, et al. Detection of a nucleolar 7-2 ribonucleoprotein and cytoplasmic 8-2 ribonucleoprotein with autoantibodies from patients with scleroderma. J Bioi Chem 1983;258:1383-6. 10. Reimer G, Scheer U, Peters JM, ct al. Immunolocalization and partial characterization of an nucleolar autoantigen (PM-Scl) associated with polymyositis/scleroderma overlap syndromes. J Immunol 1986;137:3802-8. II. Reimer G, Pollard KM, Penning CA, et al. Monoclonal autoantibodies and some human scleroderma sera traget a Mr 34,000 nucleolar protein of the U3-ribonucleoprotein particle. Arthritis Rheum 1987;30:793·800. 12. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979;76:4350-4. 13. Reimer G, Steen VD, Penning CA, et al. Correlates between autoantibodies to nucleolar antigens and clinical features in patients with systemic sclerosis (scleroderma). Arthritis Rheum 1988;31:525-32. 14. Bernstein RM, Steigerwand IC, Tan EM. Association of antinuclear and antinucleolar antibodies in progressive systemic sclerosis. Clin Exp Immunol 1982;48:43-51. 15. Oehs RL, Lischwe MA, Spohn WH, et al. Fibrillarin: a new protein ofthe nucleolus identified by autoimmune sera. Bioi Cell 1985;54:123-34. 16. Lischwe MA, Reddy R, Dchs RL, et al. Purification of a nucleolar scleroderma antigen (Mr = 34,000; pI 8.5) rich in NO, NO-dimethyl-arginine. J Bioi Chem 1985;260:1430410.
Kurzhals et al. 17. Fritzler MJ, Kinsella TD, Garbutt E. The CREST syndrome: a distinct serological entity with anticentromere antibodies. Am J Moo 1980;69:520-6. 18. Chorzelski TP, Jablonska S, Beutner EH, et al. Anticentromere antibody: an immunological marker of a subset of systemic sclerosis. Br J DermatoI1985;114:381-9. 19. Meurer M, Scharf A, Luderschmidt CH, et al. Zentromerantikorper und Antikorper gegen Scl-70-Nucleoprotein bei progressiver systemischer Sklerodermie. Dtsch Med Wochenschr 1985;110:8-14. 20. Steen YD, Ziegler GL, Rodnan GP, et al. Clinical and laboratory association of anticentromere antibodies in patients with progressive systemic sclerosis. Arthritis Rheum 1984; 27:125-31.
Journal of the American Academy of Dermatology 21. Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970; .227:680-5. 22. Leipold B, Grimm H, Yogt HJ, et al. Effect of selective ultraviolet phototherapy on DNA and antinuclear antibody titers in psoriatic patients. Arch Dermatol Res 1984; 276:297-302. 23. Chetkov NB, Bozhkov B, Nikolov K, et al. Anti-DNA antibodies in blood serum of psoriatic patients. Dermatologica 1984;169:121-4. 24. Reeves WH, Fisher DE, Wisniewski R, et al. Psoriasis and Raynaud's phenomenon associated with autoantibodies to Uland U2 small nuclear ribonucleoproteins. N Engl J Moo 1986;315:105-11.
Occurrence of human papillomavirus type 16 DNA in cutaneous squamous and basal cell neoplasms Yehuda D. Eliezri, MD,a Saul J. Silverstein, PhD,b,d and Gerard J. Nuovo, MDc,d New York, New York Sixty-eight cutaneous squamous cell neoplasms (in situ and invasive) and 26 basal cell carcinomas from 89 patients were analyzed for DNA sequences homologous to the human papillomavirus (HPV) types found predominantly in the genital tract. Thirty-six (53%) of the squamous cell neoplasms contained HPV DNA as detected by filter or in situ hybridization analysis. The frequency of detection of HPV DNA was dependent on the site of the lesion. Of 40 genital squamous cell neoplasms (penile, vulvar, and perianal), 27 (68%) had detectable HPV DNA. In 25 of these, the HPV type was 16 or HPV-16-re1ated, which was similar to the results for the squamous cell neoplasms of the finger (HPV DNA in 9 of 11 tumors with HPV-16 in seven). None of 16 squamous cell neoplasms from sites other than the genital tract or the finger had detectable HPV DNA. HPV DNA was detected in one of the 26 basal cell carcinomas (4%). We conclude that, for cutaneous epithelial malignancies, HPV16 is restricted to squamous cell neoplasms of the genital tract and finger. These data are consistent with venereal transmission ofHPV-16 to the periungual region and suggests a role for this virus in the evolution of squamous cell carcinoma at this site. (J AM ACAD DERMATOL 1990;23:836-42.)
Cutaneous squamous and basal cell carcinomas (SCCs and BCCs, respectively) are the most common tumors in human beings. Their incidence corFrom the Departments of Dermatology,· Microbiology,b Pathology,C and The Cancer Research Center,d Columbia University. Supported by a grant from the National Institutes of Health (CA 23767) to Dr, Silverstein and a grant from the Lewis Foundation to Dr. Nuovo, Accepted for publication Feb. 1, 1990. Reprint requests: Gerard J. Nuovo, MD, Department of Pathology, State University of New York at Stony Brook, Stony Brook, NY
11794-8691.
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relates with exposure to the sun. 1 Other less common risk factors include exposure to arsenic and coal tar derivatives, chronic inflammation, scars related to burns or x-radiation2,3 decreased immunocompetence,4,5 and certain congenital conditions, such as xeroderma pigmentosa6 and epidermodysplasia verruciformis (EDV).7-11 Evidence for an etiologic role for certain human papillomavirus (HPV) types in cutaneous SCC at various sites in accumulating. At nongenital sites this association is best characterized in patients with EDV. Patients with EDV are at much higher risk to develop in situ and invasive SCCs in sun-exposed
Vernon and Olsen may need to be continued more than 2 weeks, but perhaps a less potent steroid could be substituted for c1obetasol propionate ointment at that time. We thank Eldred E. Giefer of Glaxo, Inc., Research Triangle Park, North Carolina, for performing the statistical computations.
REFERENCES 1. Frank L, Stritzler C, Kaufman J. Hydrocortisone (com2.
pound F) free alcohOl and hydrocortisone acetate for topical uses. Arch Dermatol 1955;71:117-20. Kaidbey KH, Kligman AM. Assay oftopical corticosteroids, efficacy of suppression of experimental Rhus dermatitis in humans. Arch Dermatol 1976;112:808-13.
Clinical association of autoantibodies to fibrillarin with diffuse scleroderma and disseminated telangiectasia Gunter Kurzhals, MD,a Michael Meurer, MD,a Thomas Krieg, MD,a and Georg Reimer, MDb Munich and Erlangen, Federal Republic of Germany Circulating autoantibodies against a variety ofnuclear and nucleolar antigens are characteristic serologic findings in systemic scleroderma. Some of these antibodies correlate with clinical subsets of the disease. We describe three patients with systemic scleroderma and high autoantibody titers against U3 ribonucleoprotein-associated fibrillarin, a recently identified 34 leD nucleolar protein. These patients showed a progressive course with multiple organ and diffuse skin involvement with disseminated telangiectasia. (J AM ACAD DERMATOL 1990;23:832-6.)
Systemic scleroderma is a generalized disease of connective tissue that involves mainly the skin, the gastrointestinal tract, the lungs, the heart, and the kidneys. 1 Circulating antibodies against a variety of nuclear and nucleolar antigens can be detected in more than 95% ofpatients. 2-4 Some antibody specificities correlate with defined clinical subsets of the disease and have proved to be of diagnostic and prognostic significance. For example, anti-Sc1-70 antibodies directed against the nuclear/nucleolar enzyme DNA topoisomerase 1 are associated with diffuse scleroderma and multiple organ involvement. s On the other hand, anticentromere antibodies are characteristic of the CREST (calcinosis cutis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, and telangiectasia) syndrome From the Department of Dermatology, Ludwig-Maximilians-UniversHiit Munchen,' and the Department of Dermatology, Friedrich-ALexander-Universitat Erlangen-Nurnberg.b Accepted for publication Jan. 17,1990. Reprint requests: Michael Meurer, MD, DermatoLogische Klinik und Poliklinik, Ludwig-Maximilians-Universitat Miinchen, Frauenlobstr. 9-11, D-8000 Milnchen 2, FRG. 16/1/19464
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and of acroscleroderma with limited cutaneous and internal organ involvement. 6 More recently, a variety of nucleolar proteins within RNA-protein complexes have been identified as targets of autoantibodies in scleroderma patients. These nucleolar antigens include RNA polymerase 1,7 the Pm-Scl particle,8 U3 ribonucleoprotein CD3RNP)-associated fibrillarin, and 7-2 RNP.9 Antibodies directed against the Pm-Scl antigen identify a group of scleroderma patients with concomitant myositis. 10 The clinical significance of the other antinucleolar antibodies is less clear. We present evidence that antibodies against the D3-RNA-associated nucleolar protein fibrillarin may characterize a subset of patients with diffuse scleroderma and widespread telangiectasia. CASE REPORTS
Case 1 A 43-year-old white man had had Raynaud's disease for 8 years and diffuse scleroderma for 4 years. Recently, dysphagia and dyspnea on exertion developed. He had had psoriasis for 20 years. On examination, edema and swelling of the fingers and toes were present. Cutaneous
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sclerosis extended to the forearms and the upper part of the legs, the trunk, and the face. Disseminated telangiectasia with accentuation on the face, the neck, and the upper part of the trunk was noted. Psoriatic plaques were noted on the trunk, the elbows, and the knees. Chest roentgenogram showed bilateral pulmonary fibrosis. Pulmonary function tests revealed moderate impairment of diffusion capacity. Barium swallow roentgenography and manometric measurements indicated reduced esophageal motility. Except for a slightly increased erythrocyte sedimentation rate (10 to 30 mm/hr [Westergren]), a mild leukocytosis (12,000 cells/mm3), and elevated ')'-globulin level (20.5% relative) all routine laboratory tests were normal. Indirect immunofluorescence revealed IgG class antibodies directed against cell nucleoli. The staining pattern on HEp-2cells was clumped (titer> 1:10,000). Rheumatoid factor was negative. Autoantibodies to DNA and to extractable nuclear proteins, including Scl-70, UI-RNP, PM-Sel, and SS-B (La), were not detected.
Case 2 A 60-year-old white man had a 4-year history of progressive thickening of the skin preceded by Raynaud's phenomenon for 1 year. He also had dyspnea, dysphagia, and renal hypertension. Physical examination showed scleroderma ofthe upper and lower extremities and of the upper part of the trunk. He had a beaked nose, narrow lips, small mouth, and perioral radial furrowing. Sclerotic skin areas, especially on the face and trunk, contained numerous telangiectases (Fig. 1). Telangiectases were also present on normal perilesional skin. Assessment of esophageal function revealed reduced motility, delayed emptying, and gastroesophageal reflux. Blood pressure, controlled with treatment with angiotensin-converting enzyme inhibitor, was elevated (160/100 mm Hg). Electrocardiogram, echocardiogram, chest roentgenogram, and pulmonary function were normal. The patient had proteinuria (1650 mg/24 hr), reduced renal clearance, and an elevated serum creatinine level (1.5 mg/ 100 mI). With the exception ofa slightly elevated erythrocyte sedimentation rate (15 to 25 mm [Westergren)) and a test result positive for rheumatoid factor, other routine laboratory findings were within normal limits. By indirect immunofluorescence on HEp-2 cells, antinucleolar antibodies of the IgG type with a titer of more than 1:I0,000 and clumped nucleolar staining were detected. Antibodies against Scl-70, UI-RNP, Pm-Scl, and SS-B (La), and anti-DNA antibodies were absent.
Case 3 A 48-year-old white woman first noted Raynaud's phenomenon in 1982 and was referred to the hospital in 1984 because of sclerodactyly, dysphagia, heartburn, and dyspnea on exertion. In addition, the patient had had
Fig. 1. Case 2. Multiple telangiectases on face. psoriasis for more than 30 years. Sclerodermatous involvement of the fingers and face was marked but less pronounced on the feet and forearms. Sclerodactyly was prominent, with multiple ulcers and pitting scars on the fingertips. Microcheilia, microstomia, and radial perioral furrowing were present. This patient also had disseminated telangiectases that involved mainly the face and upper part of the chest but also appeared on apparently normal skin. In addition, psoriatic plaques were present on the elbows, knees, and trunk. Scintigraphy showed reduced esophageal motility, delayed emptying, and a gastroesophageal reflux. Arteriography showed marked obliterative disease of almost all digital arteries. Chest roentgenogram and pulmonary function were normal. Echocardiography showed signs of endocardial and myocardial fibrosis. No proteinuria was found, and renal function was normal. Besides a positive rheumatoid factor test result, all routine laboratory data were within normal limits. Indirect immunofluorescence of HEp-2 cells showed antinucleolar antibodies with clumped staining and a titer of more than 1: 10,000.
Immunologic studies All three sera displayed clumped nucleolar staining on HEp-2 cells (Fig. 2). Antigens were further characterized by immunoblotting; HeLa cell nucleoli were used as the source of antigen.?' 10 Nucleoli were isolated by sonication. lI Nucleolar proteins were separated by sodium dodecylsulfate--polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose paper. 12 After incubation with antinucleolar scleroderma sera a,nd control sera, diluted at 1: 100, specific IgG was detected by a second incubation with protein A (Du Pont New
834 Kurzhals et al.
Journal of the American Academy of Dermatology
Fig. 2. Indirect immunofluorescence of serum from case 1: Sera were diluted 1:40 in phosphate-buffered saline solution (pH 7.4) and evaluated by indirect immunofluorescence for presence of ANA with use of HEp-2 cells as substrate. Fluoresceinated goat antihuman immunoglobulin conjugate specific for human IgG (Behring Diagnostics, La Jolla, Calif.) was used as detecting reagent. Bright nucleolar staining in a clumped pattern is produced by antibodies from all sera. In dividing cells both chromosomal and nucleoplasmic staining are observed (bright cells at left). Nucleolus has disintegrated in these cells and is no longer present.
England Nuclear Research Products, Boston, Mass.) followed by autoradiography. I I Serum from patient 1 was also reacted with purified fibrilIarin (see legend to Fig. 3). Antibodies from the sera of the three patients reacted with a 34 kD protein present in the nucleolar extract from HeLa cells. By use of the reference serum from patient 1 (same serum as that used by Reimer et al. l !), the 34 kD protein was identified as fibrillarin (see Fig. 3). DISCUSSION
Antinucleolar antibodies are part ofthe autoantibody spectWm present in scleroderma sera. 13 By indirect immunofluorescence antinucleolar antibodies produce distinct staining patterns on tissue culture cells. Among these are speckled, homogeneous, and clumped patterns. 14 Several reports have correlated the speckled staining pattern with anti-RNA polymerase I antibodies, 7 the homogeneous staining with anti-Pm-Scl specificity, 10 and the clumped staining pattern with antifibrillarin antibodies. 11 Fibrillarin is an RNP particle that contains U3-RNA and is located in the fibrillar regions of nucleoli. IS The protein of the U3-RNP particle that contains the antigenic determinants is a 34 kD nucleolar protein. I6 Antibodies to U3-RNP-associated fibrillarin have been found in 22 of 646 patients with systemic scleroderma 13 and appear to be highly specific for
this disease. In this study patients with antifibrillarin antibodies were found to have significantly less joint involvement but otherwise were not different from patients without antinucleolar antibodies, including randomly selected patients with the CREST syndrome and diffuse scleroderma. Our three patients with this antibody specificity shared strikingly similar clinical features of systemic scleroderma, including diffuse skin involvement, widespread telangiectasia, and multiple internal manifestations. The vascular changes were even more pronounced than in patients with CREST syndrome. Cutaneous calcinosis, however, was absent. In contrast to most patients with CREST syndrome, our patients showed a progressive course with the development of sclerotic cutaneous and extracutaneous manifestations within 1 to 2 years after the onset of Raynaud's phenomenon. Therefore patients with antifibrillarin antibodies are different from patients with the CREST syndrome, which is serologically characterized by anticentromere antibodies 6, 17 and which usually follows a more benign course. IS•20 In scleroderma patients with widespread telangiectasia, these two antibody specificities may thus be helpful for determining prognosis. Two of our three patients had psoriasis in associ-
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patients in this study is small, a specific definition of a new clinical subset of scleroderma would be premature and would require a multicenter study of a larger number of well-characterized patients. We thank Dr. Michael Lischwe (Baylor College of Medicine, Department ofPharmacology, Houston, Tex.) for providing the data of the experiment shown in Fig. 3.
94-
68-
REFERENCES
-34
30-
2114Fig. 3. Immunoblot obtained with serum from case 1: HeLa nucleolar proteins and purified 34 leD protein fibrillarin 7 were separated by electrophoresis in a 11%
polyacrylamide Laernmli gel21 and electrophoretically transferred to nitrocellulose paper incubated with antibodies from the serum of patient presented as case 1. The antigen-antibody complexes were detected with 1251_protein A. Lane A, Amido black-stained HeLa nucleolar proteins. Lane B, Amido black-stained purified 34 leD fibrillarin. Lane C, Autoradiograph of the immunoblot with scleroderma serum (case 1) against total HeLa nucleolar proteins. Lane D, autoradiograph of immunoblot with this serum with purified fibrillarin as antigen. 15, 16 Molecular weight markers are phosphorylase B (94,000 leD), bovine serum albumin (68,000 leD), ovalbumin (43,000 leD), carbonic anhydrase (30,000 leD), and soybean trypsin inhibitor (21,000 leD).
ation with systemic scleroderma. The frequency of psoriasis in systemic scleroderma is not higher than in the normal population. Autoantibodies against nuclear antigens, particularly against denatured DNA, have been reported in patients with psoriasis after ultraviolet light therapy.22,23 To our knowledge nucleolar staining by psoriatic sera has not been reported, although antibodies to UI-RNP and U2-RNP have been described in two patients with severe Raynaud's phenomenon and concomitant psoriasis. 24 The association of antifibrillarin antibodies and psoriasis in two of our three patients needs further investigation. Because the number of
I. Krieg T, Meurer M. Systemic scleroderma: clinical and pathophysiologic aspects. J AM ACAD DERMATOL 1988; 18:457-81. 2. Tan EM. Autoantibodies to nuclear antigens (ANA): their immunobio10gy and medicine. Adv TmmunoI1982;33: 167240. 3. Tan EM, Rodnan GP, Garcia I, et aL Diversity of antinuclear antibodies in progressive systemic sclerosis: antieentromere antibody and its association to CREST syndrome. Arthritis Rheum 1980;23:617-25. 4. Bernstein RM, Steigerwald IC, Tan EM. Association of antinuclear and antinucleolar antibodies in progressivesystemie sclerosis. Clin Exp Immunol 1982;48:43·57. 5. Shero JH, Bordwell B, Rothfield NF, et al. High titers of autoantibodies to topoisomerase I (Scl·70) in sera from scleroderma patients. Science 1986;231:737-40. 6. Moroi Y, Peebles C, Fritzler MJ, et al. Autoantibody to centromere (kinetochore) in scleroderma sera. Proc Natl Acad Sci USA 1980;77:1627-31. 7. Reimer G, Rose KM, Scheer U, et al. Autoantibodies to RNA polymerase I in scleroderma sera. J Clin Invest 1987; 79;65-72. 8. Reichlin M, Maddison PS, Targolf J, et al. Antibodies to nuclear/nucleolar antigen in patients with polymyositis overlap syndromes. J Clin Immunol 1984;42:40-4. 9. Reddy R, Tan EM, Henning D, et al. Detection of a nucleolar 7-2 ribonucleoprotein and cytoplasmic 8-2 ribonucleoprotein with autoantibodies from patients with scleroderma. J Bioi Chem 1983;258:1383-6. 10. Reimer G, Scheer U, Peters JM, ct al. Immunolocalization and partial characterization of an nucleolar autoantigen (PM-Scl) associated with polymyositis/scleroderma overlap syndromes. J Immunol 1986;137:3802-8. II. Reimer G, Pollard KM, Penning CA, et al. Monoclonal autoantibodies and some human scleroderma sera traget a Mr 34,000 nucleolar protein of the U3-ribonucleoprotein particle. Arthritis Rheum 1987;30:793·800. 12. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979;76:4350-4. 13. Reimer G, Steen VD, Penning CA, et al. Correlates between autoantibodies to nucleolar antigens and clinical features in patients with systemic sclerosis (scleroderma). Arthritis Rheum 1988;31:525-32. 14. Bernstein RM, Steigerwand IC, Tan EM. Association of antinuclear and antinucleolar antibodies in progressive systemic sclerosis. Clin Exp Immunol 1982;48:43-51. 15. Oehs RL, Lischwe MA, Spohn WH, et al. Fibrillarin: a new protein ofthe nucleolus identified by autoimmune sera. Bioi Cell 1985;54:123-34. 16. Lischwe MA, Reddy R, Dchs RL, et al. Purification of a nucleolar scleroderma antigen (Mr = 34,000; pI 8.5) rich in NO, NO-dimethyl-arginine. J Bioi Chem 1985;260:1430410.
Kurzhals et al. 17. Fritzler MJ, Kinsella TD, Garbutt E. The CREST syndrome: a distinct serological entity with anticentromere antibodies. Am J Moo 1980;69:520-6. 18. Chorzelski TP, Jablonska S, Beutner EH, et al. Anticentromere antibody: an immunological marker of a subset of systemic sclerosis. Br J DermatoI1985;114:381-9. 19. Meurer M, Scharf A, Luderschmidt CH, et al. Zentromerantikorper und Antikorper gegen Scl-70-Nucleoprotein bei progressiver systemischer Sklerodermie. Dtsch Med Wochenschr 1985;110:8-14. 20. Steen YD, Ziegler GL, Rodnan GP, et al. Clinical and laboratory association of anticentromere antibodies in patients with progressive systemic sclerosis. Arthritis Rheum 1984; 27:125-31.
Journal of the American Academy of Dermatology 21. Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970; .227:680-5. 22. Leipold B, Grimm H, Yogt HJ, et al. Effect of selective ultraviolet phototherapy on DNA and antinuclear antibody titers in psoriatic patients. Arch Dermatol Res 1984; 276:297-302. 23. Chetkov NB, Bozhkov B, Nikolov K, et al. Anti-DNA antibodies in blood serum of psoriatic patients. Dermatologica 1984;169:121-4. 24. Reeves WH, Fisher DE, Wisniewski R, et al. Psoriasis and Raynaud's phenomenon associated with autoantibodies to Uland U2 small nuclear ribonucleoproteins. N Engl J Moo 1986;315:105-11.
Occurrence of human papillomavirus type 16 DNA in cutaneous squamous and basal cell neoplasms Yehuda D. Eliezri, MD,a Saul J. Silverstein, PhD,b,d and Gerard J. Nuovo, MDc,d New York, New York Sixty-eight cutaneous squamous cell neoplasms (in situ and invasive) and 26 basal cell carcinomas from 89 patients were analyzed for DNA sequences homologous to the human papillomavirus (HPV) types found predominantly in the genital tract. Thirty-six (53%) of the squamous cell neoplasms contained HPV DNA as detected by filter or in situ hybridization analysis. The frequency of detection of HPV DNA was dependent on the site of the lesion. Of 40 genital squamous cell neoplasms (penile, vulvar, and perianal), 27 (68%) had detectable HPV DNA. In 25 of these, the HPV type was 16 or HPV-16-re1ated, which was similar to the results for the squamous cell neoplasms of the finger (HPV DNA in 9 of 11 tumors with HPV-16 in seven). None of 16 squamous cell neoplasms from sites other than the genital tract or the finger had detectable HPV DNA. HPV DNA was detected in one of the 26 basal cell carcinomas (4%). We conclude that, for cutaneous epithelial malignancies, HPV16 is restricted to squamous cell neoplasms of the genital tract and finger. These data are consistent with venereal transmission ofHPV-16 to the periungual region and suggests a role for this virus in the evolution of squamous cell carcinoma at this site. (J AM ACAD DERMATOL 1990;23:836-42.)
Cutaneous squamous and basal cell carcinomas (SCCs and BCCs, respectively) are the most common tumors in human beings. Their incidence corFrom the Departments of Dermatology,· Microbiology,b Pathology,C and The Cancer Research Center,d Columbia University. Supported by a grant from the National Institutes of Health (CA 23767) to Dr, Silverstein and a grant from the Lewis Foundation to Dr. Nuovo, Accepted for publication Feb. 1, 1990. Reprint requests: Gerard J. Nuovo, MD, Department of Pathology, State University of New York at Stony Brook, Stony Brook, NY
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relates with exposure to the sun. 1 Other less common risk factors include exposure to arsenic and coal tar derivatives, chronic inflammation, scars related to burns or x-radiation2,3 decreased immunocompetence,4,5 and certain congenital conditions, such as xeroderma pigmentosa6 and epidermodysplasia verruciformis (EDV).7-11 Evidence for an etiologic role for certain human papillomavirus (HPV) types in cutaneous SCC at various sites in accumulating. At nongenital sites this association is best characterized in patients with EDV. Patients with EDV are at much higher risk to develop in situ and invasive SCCs in sun-exposed