CONCISE COMMUNICATIONS
247
JRA PP
Lanes 1-15
endemic area for Lyme disease thus far provide no evidence for B burgdorferi-related DNA sequences in synovial fluid of patients with juvenile rheumatoid arthritis. Our studies also underscore the fact that extreme care will be necessary to establish the proper role for PCR in pursuing the pathogenesis or improving the diagnosis of Lyme disease-in the choice of patient and control populations, the accession of specimens, and the containment of literally tens of billions of copies of the amplified material. Supported in part by grants from the USPHS (AR-40452 and AI-30548) and from the Arthritis Foundation. We thank Bing Lin, MD, and Paul Rhys for expert technical assistance.
JRA
Lyme
Other
16-20
21-26
27-30
Stephen E. Malawista, MD Robert T. Schoen, MD Yale University School of Medicine New Haven, CT Terry L. Moore, MD St. Louis University School of Medicine St. Louis, MO Deborah E. Dodge Thomas J. White, PhD Roche Diagnostics Research Alameda, CA David H. Persing, MD, PhD Mayo Clinic and Foundation Rochester, MN
pp
1 . Higuchi R: Simple and rapid preparation of samples for PCR,
2. 3.
0spAVar - - - - - + + + + + + - - - + + Figure 2. Results of studies in which synovial fluid specimens collected from 20 patients with juvenile rheumatoid arthritis (JRA), 6 different samples from a single patient with chronic Lyme arthritis, and 4 specimens from adult patients with other rheumatic diseases (Other) were DNA extracted (see text), amplified separately with three different primer pairs, and displayed following gel electrophoresis and Southern blotting, by hybridization with a 32P-labeled internal probe. In the run shown, primers OspA6 and OspA7 (see Figure I), directed against a conserved portion of the OspA gene, were employed. None of the JRA samples gave positive results. As indicated below each lane, these samples also gave negative results on amplifications from the OspA variable region (0spA"IU) or from 16s ribosomal DNA. P = positive controls (dilutions of Borrelia burgdorferi strain N40 [4]).
amplifications, adding a third primer pair targeting a sequence from 16s ribosomal DNA that is conserved in over 40 strains of Borrelia (6). Again, all were negative. A representative run for an OspA target is shown in Figure 2. The sensitivity of these procedures approaches 10 or fewer DNA copies. (The number of copies per spirochete is uncertain in virulent B burgdorferi; in B hermsii, there may be as many as 12 copies [Barbour A: personal communication].) Although the sample is small, our data from a non-
4.
5.
6.
7.
PCR Technology: Principles and Applications for DNA Amplification. Edited by HA Erlich. New York, Stockton Press, 1989 Nielsen SL, Peter JB: B. burgdorferi persistently detected by polymerase chain reaction in synovial fluid of a patient with lyme arthritis resistant to therapy (abstract). Clin Res 38:402A, 1990 Persing DH, Rys PN, van Blaricom G, Dodge DE, White TJ, Schoen RT, Rahn DW, Malawista SE:Multi-target detection of B. burgdorferi-associated DNA sequences in synovial fluids of patients with arthritis (abstract). Arthritis Rheum 33 (suppl 9):S36, 1990 Persing DH, Telford SR 111, Spielman A, Barthold SW: Detection of Borrelia burgdorferi infection in Ixodes dammini ticks with the polymerase chain reaction. J Clin Microbiol28:566-572, 1990 Cassidy JT, Levinson JE, Bass JC, Baum J, Brewer EJ Jr, Fink CW, Hanson V, Jacobs JC, Masi AT, Schaller JG, Fries JF, McShane D, Young D: A study of classification criteria for a diagnosis of juvenile rheumatoid arthritis. Arthritis Rheum 29: 274-281, 1986 Persing DH, Telford SR 111, Rys PN, Dodge DE, White TJ, Malawista SE, Spielman A: Detection of Borrelia burgdorferi DNA in museum specimens of Ixodes dammini ticks. Science 249:142&1423, 1990 Cimino GD, Mechette KC, Tessman JW, Hearst JE, Isaacs ST: Post-PCR sterilization: a method to control carryover contamination by the polymerase chain reaction. Nucleic Acids Res 1991 19:-107,
Restriction fragment length polymorphism of the vitronectin gene in patients with rheumatic diseases Inherited complement deficiencies predispose to the development of autoimmune diseases, including systemic lupus erythematosus (SLE). The activation of complement is believed to play a prominent role in the pathogenesis of SLE and of rheumatoid arthritis (RA) with vasculitis. Activated
CONCISE COMMUNICATIONS
248 complement components have been observed in the synovial fluid of patients with RA (1). Vitronectin (VN), one of the glycoproteins in human plasma, interacts with the complement system, especially with the terminal complement complex C5b-9 (2). It circulates in a 75-kd single-chain form as well as in a proteolyzed form consisting of 2 chains of 65 kd and 10 kd, linked by a disulfide bond (3). A recent study revealed that a protein kinase in human plasma preferentially phosphorylates Ser378 of 75-kd VN in the presence of 75-kd and 65-kd VN (4). However, it remains to be determined whether the 2 forms of VN have different biologic activities or whether Ser-378 of VN can be phosphorylated in plasma under physiologic conditions. Human plasma and sera can be classified into 3 distinct categories according to the ratio of the 75-kd VN polypeptide to the 65-kd VN polypeptide. These VN blood types have been suggested to be due to 2 codominant VN alleles, 1 of which has C and the other T at nucleotide position 4056. A base change from C to T causes the amino acid change of Thr to Met at position 381. The VN Thr-381 allele encodes 75-kd VN, which is protease sensitive, while the VN Met-381 allele encodes protease-resistant VN (5). Using the polymerase chain reaction (PCR) and subsequent restriction enzyme digestion, we investigated the association between the VN genotypes and susceptibility to SLE or RA. Genomic DNA was isolated from 49 SLE patients, 76 RA patients, and 61 healthy controls. PCR and subsequent restriction enzyme digestion were performed as previously described (5). Pma CI, which distinguishes the base sequence causing the polymorphic change at nucleotide position 4056, was used as a restriction enzyme. The 2 primers used for amplification were homologous to nucleotides 3693-3712 and complementary to 4203-4222 of the vitronectin gene (6) and were kindly provided by Professor Y. Sakaki (Kyushu University). Table 1 shows the distribution of the 3 VN genotypes in each group. By chi-square analysis, no significant difference in the frequencies of VN genotypes was found between healthy controls and patients with either SLE or RA. VN binds to the nascent C 5 b 7 complex (7), inhibiting complex attachment to plasma membranes and thus functioning as a membrane-attack complex inhibitor to limit the lysis of bystander cells. It also may interact with the membrane-associated CSb-9 complex (8), and could inhibit
Table 1. Distribution of the genotypes of vitronectin at nucleotide position 4056 Genotype Group*
TiT
TIC
CIC
Total
Control SLE RA
28 27 46
30 17 29
3 5 1
61 49 76
* SLE = systemic lupus erythematosus; RA = rheumatoid arthritis.
assembly of the complex, or block C9 polymerization, on cell membranes. Furthermore, recent evidence suggests that cells adhere to the VN-C5b-9 complex through interaction of the VN component with an integrin receptor, and cell attachment to terminal complement complexes could have a role in leukocyte adherence and migration during inflammation (9). Because the polymorphism of the VN gene at nucleotide position 4056 affects sensitivity to protease and thus determines the VN blood type, it may modify the role of VN as complement inhibitor and adhesion protein. These functional modifications may then alter the susceptibility to SLE or RA. Our results, however, demonstrated no association between VN polymorphism and susceptibility to SLE or RA. Conlan et al reported that the proportions of the 3 VN blood types among patients with disseminated intravascular coagulation were not different from those in a normal population (3). Although further study is required to reach a definite conclusion, no clinical significance can be assigned at present to the polymorphism of the VN gene or VN blood type. Satoshi Shiokawa, MD Masayuki Yasuda, MD Masashi Nobunaga, MD Kyushu University Beppu, Japan
1. Fearon DT: Complement mediators of inflammation, Arthritis and Allied Conditions. Eleventh edition. Edited by DJ McCarty. Philadelphia, Lea & Febiger, 1989 2. Podack ER, Muller-Eberhard HJ: Isolation of human S-protein, an inhibitor of the membrane attack complex of complement. J Biol Chem 254:9908-9914, 1979 3. Conlan MG, Tomasini BR, Schultz RL, Mosher DF: Plasma vitronectin polymorphism in normal subjects and patients with disseminated intravascular coagulation. Blood 72: 185-190, 1988 4. McGuire EA, Peacock ME, Inhorn RC, Siege1 NR, Tollefsen DM: Phosphorylation of vitronectin by a protein kinase in human plasma. J Biol Chem 263:1942-1945, 1988 5 . Kubota K, Hayashi M, Oishi N, Sakaki Y:Polymorphism of the human vitronectin gene causes vitronectin blood type. Biochem Biophys Res Commun 167:1355-1360, 1990 6. Jenne D, Stanley KK: Nucleotide sequence and organization of the human S-protein gene: repeating peptide motifs in the "pexin" family and a model for their evolution. Biochemistry 26:67354742, 1987 7. Podack ER, Kolb WP, Miiller-Eberhard HJ: The C5b-6 complex: formation, isolation, and inhibition of its activity by lipoprotein and the S-protein of human serum. J Immunol 1201841-1848, 1978 8. Bhakdi S, Kaeflein R, Halstensen TS, Hugo F, Preissner KT, Mollnes TE: Complement S-protein (vitronectin) is associated with cytolytic membrane-bound C5b-9 complexes. Clin Exp Immunol74:459-464, 1988 9. Biesecker G: The complement SC5b-9 complex mediates cell adhesion through a vitronectin receptor. J Immunol 145:209-214, 1990
247
JRA PP
Lanes 1-15
endemic area for Lyme disease thus far provide no evidence for B burgdorferi-related DNA sequences in synovial fluid of patients with juvenile rheumatoid arthritis. Our studies also underscore the fact that extreme care will be necessary to establish the proper role for PCR in pursuing the pathogenesis or improving the diagnosis of Lyme disease-in the choice of patient and control populations, the accession of specimens, and the containment of literally tens of billions of copies of the amplified material. Supported in part by grants from the USPHS (AR-40452 and AI-30548) and from the Arthritis Foundation. We thank Bing Lin, MD, and Paul Rhys for expert technical assistance.
JRA
Lyme
Other
16-20
21-26
27-30
Stephen E. Malawista, MD Robert T. Schoen, MD Yale University School of Medicine New Haven, CT Terry L. Moore, MD St. Louis University School of Medicine St. Louis, MO Deborah E. Dodge Thomas J. White, PhD Roche Diagnostics Research Alameda, CA David H. Persing, MD, PhD Mayo Clinic and Foundation Rochester, MN
pp
1 . Higuchi R: Simple and rapid preparation of samples for PCR,
2. 3.
0spAVar - - - - - + + + + + + - - - + + Figure 2. Results of studies in which synovial fluid specimens collected from 20 patients with juvenile rheumatoid arthritis (JRA), 6 different samples from a single patient with chronic Lyme arthritis, and 4 specimens from adult patients with other rheumatic diseases (Other) were DNA extracted (see text), amplified separately with three different primer pairs, and displayed following gel electrophoresis and Southern blotting, by hybridization with a 32P-labeled internal probe. In the run shown, primers OspA6 and OspA7 (see Figure I), directed against a conserved portion of the OspA gene, were employed. None of the JRA samples gave positive results. As indicated below each lane, these samples also gave negative results on amplifications from the OspA variable region (0spA"IU) or from 16s ribosomal DNA. P = positive controls (dilutions of Borrelia burgdorferi strain N40 [4]).
amplifications, adding a third primer pair targeting a sequence from 16s ribosomal DNA that is conserved in over 40 strains of Borrelia (6). Again, all were negative. A representative run for an OspA target is shown in Figure 2. The sensitivity of these procedures approaches 10 or fewer DNA copies. (The number of copies per spirochete is uncertain in virulent B burgdorferi; in B hermsii, there may be as many as 12 copies [Barbour A: personal communication].) Although the sample is small, our data from a non-
4.
5.
6.
7.
PCR Technology: Principles and Applications for DNA Amplification. Edited by HA Erlich. New York, Stockton Press, 1989 Nielsen SL, Peter JB: B. burgdorferi persistently detected by polymerase chain reaction in synovial fluid of a patient with lyme arthritis resistant to therapy (abstract). Clin Res 38:402A, 1990 Persing DH, Rys PN, van Blaricom G, Dodge DE, White TJ, Schoen RT, Rahn DW, Malawista SE:Multi-target detection of B. burgdorferi-associated DNA sequences in synovial fluids of patients with arthritis (abstract). Arthritis Rheum 33 (suppl 9):S36, 1990 Persing DH, Telford SR 111, Spielman A, Barthold SW: Detection of Borrelia burgdorferi infection in Ixodes dammini ticks with the polymerase chain reaction. J Clin Microbiol28:566-572, 1990 Cassidy JT, Levinson JE, Bass JC, Baum J, Brewer EJ Jr, Fink CW, Hanson V, Jacobs JC, Masi AT, Schaller JG, Fries JF, McShane D, Young D: A study of classification criteria for a diagnosis of juvenile rheumatoid arthritis. Arthritis Rheum 29: 274-281, 1986 Persing DH, Telford SR 111, Rys PN, Dodge DE, White TJ, Malawista SE, Spielman A: Detection of Borrelia burgdorferi DNA in museum specimens of Ixodes dammini ticks. Science 249:142&1423, 1990 Cimino GD, Mechette KC, Tessman JW, Hearst JE, Isaacs ST: Post-PCR sterilization: a method to control carryover contamination by the polymerase chain reaction. Nucleic Acids Res 1991 19:-107,
Restriction fragment length polymorphism of the vitronectin gene in patients with rheumatic diseases Inherited complement deficiencies predispose to the development of autoimmune diseases, including systemic lupus erythematosus (SLE). The activation of complement is believed to play a prominent role in the pathogenesis of SLE and of rheumatoid arthritis (RA) with vasculitis. Activated
CONCISE COMMUNICATIONS
248 complement components have been observed in the synovial fluid of patients with RA (1). Vitronectin (VN), one of the glycoproteins in human plasma, interacts with the complement system, especially with the terminal complement complex C5b-9 (2). It circulates in a 75-kd single-chain form as well as in a proteolyzed form consisting of 2 chains of 65 kd and 10 kd, linked by a disulfide bond (3). A recent study revealed that a protein kinase in human plasma preferentially phosphorylates Ser378 of 75-kd VN in the presence of 75-kd and 65-kd VN (4). However, it remains to be determined whether the 2 forms of VN have different biologic activities or whether Ser-378 of VN can be phosphorylated in plasma under physiologic conditions. Human plasma and sera can be classified into 3 distinct categories according to the ratio of the 75-kd VN polypeptide to the 65-kd VN polypeptide. These VN blood types have been suggested to be due to 2 codominant VN alleles, 1 of which has C and the other T at nucleotide position 4056. A base change from C to T causes the amino acid change of Thr to Met at position 381. The VN Thr-381 allele encodes 75-kd VN, which is protease sensitive, while the VN Met-381 allele encodes protease-resistant VN (5). Using the polymerase chain reaction (PCR) and subsequent restriction enzyme digestion, we investigated the association between the VN genotypes and susceptibility to SLE or RA. Genomic DNA was isolated from 49 SLE patients, 76 RA patients, and 61 healthy controls. PCR and subsequent restriction enzyme digestion were performed as previously described (5). Pma CI, which distinguishes the base sequence causing the polymorphic change at nucleotide position 4056, was used as a restriction enzyme. The 2 primers used for amplification were homologous to nucleotides 3693-3712 and complementary to 4203-4222 of the vitronectin gene (6) and were kindly provided by Professor Y. Sakaki (Kyushu University). Table 1 shows the distribution of the 3 VN genotypes in each group. By chi-square analysis, no significant difference in the frequencies of VN genotypes was found between healthy controls and patients with either SLE or RA. VN binds to the nascent C 5 b 7 complex (7), inhibiting complex attachment to plasma membranes and thus functioning as a membrane-attack complex inhibitor to limit the lysis of bystander cells. It also may interact with the membrane-associated CSb-9 complex (8), and could inhibit
Table 1. Distribution of the genotypes of vitronectin at nucleotide position 4056 Genotype Group*
TiT
TIC
CIC
Total
Control SLE RA
28 27 46
30 17 29
3 5 1
61 49 76
* SLE = systemic lupus erythematosus; RA = rheumatoid arthritis.
assembly of the complex, or block C9 polymerization, on cell membranes. Furthermore, recent evidence suggests that cells adhere to the VN-C5b-9 complex through interaction of the VN component with an integrin receptor, and cell attachment to terminal complement complexes could have a role in leukocyte adherence and migration during inflammation (9). Because the polymorphism of the VN gene at nucleotide position 4056 affects sensitivity to protease and thus determines the VN blood type, it may modify the role of VN as complement inhibitor and adhesion protein. These functional modifications may then alter the susceptibility to SLE or RA. Our results, however, demonstrated no association between VN polymorphism and susceptibility to SLE or RA. Conlan et al reported that the proportions of the 3 VN blood types among patients with disseminated intravascular coagulation were not different from those in a normal population (3). Although further study is required to reach a definite conclusion, no clinical significance can be assigned at present to the polymorphism of the VN gene or VN blood type. Satoshi Shiokawa, MD Masayuki Yasuda, MD Masashi Nobunaga, MD Kyushu University Beppu, Japan
1. Fearon DT: Complement mediators of inflammation, Arthritis and Allied Conditions. Eleventh edition. Edited by DJ McCarty. Philadelphia, Lea & Febiger, 1989 2. Podack ER, Muller-Eberhard HJ: Isolation of human S-protein, an inhibitor of the membrane attack complex of complement. J Biol Chem 254:9908-9914, 1979 3. Conlan MG, Tomasini BR, Schultz RL, Mosher DF: Plasma vitronectin polymorphism in normal subjects and patients with disseminated intravascular coagulation. Blood 72: 185-190, 1988 4. McGuire EA, Peacock ME, Inhorn RC, Siege1 NR, Tollefsen DM: Phosphorylation of vitronectin by a protein kinase in human plasma. J Biol Chem 263:1942-1945, 1988 5 . Kubota K, Hayashi M, Oishi N, Sakaki Y:Polymorphism of the human vitronectin gene causes vitronectin blood type. Biochem Biophys Res Commun 167:1355-1360, 1990 6. Jenne D, Stanley KK: Nucleotide sequence and organization of the human S-protein gene: repeating peptide motifs in the "pexin" family and a model for their evolution. Biochemistry 26:67354742, 1987 7. Podack ER, Kolb WP, Miiller-Eberhard HJ: The C5b-6 complex: formation, isolation, and inhibition of its activity by lipoprotein and the S-protein of human serum. J Immunol 1201841-1848, 1978 8. Bhakdi S, Kaeflein R, Halstensen TS, Hugo F, Preissner KT, Mollnes TE: Complement S-protein (vitronectin) is associated with cytolytic membrane-bound C5b-9 complexes. Clin Exp Immunol74:459-464, 1988 9. Biesecker G: The complement SC5b-9 complex mediates cell adhesion through a vitronectin receptor. J Immunol 145:209-214, 1990
