- Email: [email protected]
New diagnostic test for lupus
N
e
w
s
Defects in the DNA NER pathway cause three separate clinical syndromes: xeroderma pigmentosa (XP), Cockayne syndrome (CS) and trichlorothiodystrophy (TTD). XP patients are over 2000 times more susceptible to skin cancer than are normal people. Sufferers from CS and TTD, however, are not particularly cancer prone but their skin is hypersensitive to UV radiation and they have other characteristic physical and developmental defects. The genetics of these syndromes is complex. Mutations in different genes might give rise to essentially the same condition. Conversely, different mutations in the same genes might give rise to different syndromes. Jan Hoeijmakers and colleagues (Center for Biomedical Genetics, Erasmus University, Rotterdam, The Netherlands) have produced transgenic mice with mutations in Csb (recently re-named Ercc) and Xpd (Ref. 2). These mice, and others with mutations in Xpa and Xpc mimic many of the clinical features of the human NER syndromes. Mice defective in the Xpa gene are wholly NER deficient; Csb mice (Fig. 1) are deficient in TCR only; Xpc mice lack the GGR pathway and TTD mice have a partial defect in each pathway. Garssen tested the effect of UV radiation on the immune response in these mouse strains. Mice homozygous for each defective gene, and wild-type littermates, were exposed to five doses of UV-B radiation (wavelength 280–400 nm) and then sensitized with either picryl chloride (PCl) or Listeria monocytogenes. Four days later, they were challenged with PCl or killed Listeria. ‘Using Listeria, which naturally infects both mice and men, makes our experiments with model antigens more *Hoeijmakers, J.H.J. (2000) DNA repair, cancer and ageing. (Abstr. 211), 18th International Congress of Biochemistry and Molecular Biology, Birmingham, UK
MOLECULAR MEDICINE TODAY, NOVEMBER 2000 (VOL. 6)
Figure 1. A mouse deficient in the Csb gene. Transgenic mice deficient in this and other genes in the TFIIH complex mimic the clinical features of human DNA repair syndromes. Image kindly provided by Jan Hoeijmakers.
relevant,’ says Garssen. The immune response, determined from the degree of ear swelling, deteriorated with increasing radiation dose. The results were similar for both antigens. Only in one mouse strain – Xpa – was the minimum UV dose needed to cause immunosuppression significantly lower than in wild type. This is the only strain that is completely defective in both NER pathways. Thus, either pathway will confer protection against UV-induced immunosuppression. To test for acute UV-induced skin effects, transgenic and wild-type mice were exposed to a single radiation dose. The degree of erythema and edema on exposed, shaved skin and the increase in ear skin thickness were measured. The lowest UV dose that could induce a significant ear swelling response was defined as the minimum erythema–edema dose (MED) for each mouse strain. Garssen found that MED values in the Xpa and Csb strains
were significantly lower than in wild type. There was no difference between Xpc and wild type, and results for the TTD mice were ambiguous. As the affected strains are those that are completely deficient in TCR, this must be the dominant NER pathway for protection against acute UV effects. ‘Many people working in cellular immunity use the MED as a measure of the minimum dose needed to induce immunosuppression,’ says Garssen. ‘These results show that this equation is not always valid. Our Csb mice are susceptible to acute UV effects, and so have a very low MED, but their immune response is near normal.’ UV-induced immunosuppression, rather than the acute skin response, is implicated in the development of skin cancers. Thus, different NER defects should result in different cancer susceptibilities. Hoeijmakers explains further: ‘People with CS have defects in the TCR pathway, but their GGR pathway is intact. They are also not particularly cancer prone. Presumably this can be at least partly explained by the protection against immunosuppression that is conferred by GGR.’ Garssen and his group are generating further transgenic mouse models, deficient in specific DNA repair enzymes, to determine the precise roles for immunomodulation in DNA repair mechanisms. 1 Garssen, J. et al. (2000) Transcription-coupled and global genome repair differentially influence UV-B-induced acute skin effects and systemic immunosuppression. J. Immunol. 164, 6199–6205 2 de Boer, J. and Hoeijmakers, J.H.J. (2000) Nucleotide excision repair and human syndromes. Carcinogenesis 21, 453–460
Clare Sansom Freelance science writer
New diagnostic test for lupus A new diagnostic enzyme-linked immunosorbent assay (ELISA) has been created to help diagnose systemic lupus erythematosus (SLE)1. The test, developed by Mark Roth and colleagues at the Fred Hutchinson Cancer Research Center (Seattle, WA, USA), detects a family of highly phosphorylated Ser and Arg-rich (SR) proteins that act as autoantigens. SLE is a chronic idiopathic autoimmune disorder that causes a variety of symptoms, including joint pain, rash, kidney damage and reactions to sunlight, some of which are 412
caused by deposition of autoantibody–antigen complexes in the tissues. SR proteins are essential pre-mRNA splicing factors. As mRNA splicing is blocked in patients with SLE by antibodies specific to other splicing factors, such as Sm epitope-containing small nuclear ribonucleoproteins (RNP), it was logical to investigate whether the SR proteins could also be autoimmune targets. The new ELISA uses SR proteins purified from HeLa cells and the specific monoclonal antibody, 1H4. In a blinded study, it positively identified about 50% of
patients with SLE. There were few falsepositive results among healthy people or those with other autoimmune disorders, such as antiphospholipid syndrome and rheumatoid arthritis, confirming that patients with SLE are more likely to develop autoantibodies to SR proteins than are people with other autoimmune diseases. SLE symptoms often develop slowly and the disease can be difficult to spot. If the illness is suspected, a battery of tests is performed, including: blood counts and tests for antinuclear antibodies (ANA), anti-native
1357-4310/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
N
MOLECULAR MEDICINE TODAY, NOVEMBER 2000 (VOL. 6)
DNA autoantibodies, and anti-Sm autoantibodies. However, none of these tests is 100% effective and a proportion of patients are missed. ‘There are many tests that, in aggregate, are used to diagnose SLE so it is difficult to quantitate accuracy. Another way of putting it is that we don’t know how many patients are currently being missed,’ says Roth. This was confirmed when the SR-protein ELISA was compared with the ANA test. In all, 81% of patients identified as positive with the new test also tested positive for ANA and 67% of ANA-positive patients also had positive SR-protein ELISAs.
Now that the team has identified the SR proteins as specific autoantigens, they want to identify the precise structural features of these molecules that act as autoepitopes and to identify what triggers autoimmunity to these proteins. One possibility involves retrovirally encoded group specific antigen (GAG) protein, which contains a domain with stretches of alternating phosphorylated Ser and Arg similar to those found in U170K protein and the SR proteins. The U170K protein is an mRNA-splicing factor already known to be a target for the immune response in patients with SLE (see Ref. 1). The team suggests that
e
w
s
retroviral infection and immunity to GAG could also lead to autoimmunity to the alternating Arg-phosphorylated Ser epitopes in the SR-proteins. In the meantime, Roth has applied for a patent for the test kit, although it will not be available commercially for at least a year. 1 Neugebauer, K.M. et al. (2000) SR proteins are autoantigens in patients with systemic lupus erythematosus. Arthritis Rheum. 43, 1768–1778
Sharon Dorrell Freelance science writer
Molecular Medicine Today: Editorial Policy Molecular Medicine Today (MMT) is a magazine-style journal from Elsevier Trends, which publishes Trends in Biochemical Sciences and Immunology Today, among others. MMT provides a forum for the explanation and discussion of ideas and issues that relate to the multidisciplinary area of molecular medicine. It is designed for students and professionals who have information needs that transcend the traditional ‘clinical’ or ‘scientific’ categorization. Its broad appeal is to clinicians, medical/clinical and basic science researchers who are concerned with: elucidating and understanding the aetiology of disease at a molecular level; developing new diagnostics, therapies and preventive medicine based on these; and applying these technologies in healthcare. Short Reviews form the foundation of each issue. All reviews are commissioned, peer reviewed and edited to ensure that they are timely, accurate, interesting and relevant to the audience of MMT. Every effort is made to explain specialist terms and illustrate concepts and hypotheses. The review sections are balanced by topical features, which may be commissioned from freelance writers or may be written by researchers in the field. These include News, Literature, Letters, Meetings, Books, Disease models and Opinion articles. The Literature alerts readers to recent primary papers that are important to biomedical researchers, particularly innovative, likely to have significant clinical impact, or controversial. It is written by a panel of active researchers. If you would like to join this panel, please contact the Editor (see below for details).
Opinion articles give the author’s views on a controversial issue; they set these views in context and comment on what questions need to be answered in order to prove or disprove the author’s hypothesis. Disease models summarize the models used to study a particular disease (usually animal models, although in vitro models may also be described when appropriate), and highlight the similarities and differences between each model and the human condition. Letters on any topic of interest may be submitted to the Editor. Appropriate topics include comments, questions and discussion on articles and topics covered by MMT, new Internet resources, ethical and legal issues, regulatory issues, and business and financial issues. Please note that previously unpublished data and criticisms of work published elsewhere cannot be published. Articles for MMT are generally invited by the Editor, but ideas, particularly for Opinion articles, are welcome. Prospective authors should send a brief summary, citing key references, to the Editor, who will supply guidelines on manuscript preparation if the proposal is accepted. The submission of completed articles without prior consultation is strongly discouraged. As much of the journal content is planned in advance, such manuscripts may be rejected primarily because of lack of space. Authors should note that all review articles for MMT are peer reviewed before acceptance and publication cannot be guaranteed.
For any further information, please contact: The Editor, Molecular Medicine Today, Elsevier Science London, 84 Theobald’s Road, London, UK WC1X 8RR. Tel: +44 (0)20 7611 4400. Fax: +44 (0)20 7611 4470. e-mail: [email protected]
413
e
w
s
Defects in the DNA NER pathway cause three separate clinical syndromes: xeroderma pigmentosa (XP), Cockayne syndrome (CS) and trichlorothiodystrophy (TTD). XP patients are over 2000 times more susceptible to skin cancer than are normal people. Sufferers from CS and TTD, however, are not particularly cancer prone but their skin is hypersensitive to UV radiation and they have other characteristic physical and developmental defects. The genetics of these syndromes is complex. Mutations in different genes might give rise to essentially the same condition. Conversely, different mutations in the same genes might give rise to different syndromes. Jan Hoeijmakers and colleagues (Center for Biomedical Genetics, Erasmus University, Rotterdam, The Netherlands) have produced transgenic mice with mutations in Csb (recently re-named Ercc) and Xpd (Ref. 2). These mice, and others with mutations in Xpa and Xpc mimic many of the clinical features of the human NER syndromes. Mice defective in the Xpa gene are wholly NER deficient; Csb mice (Fig. 1) are deficient in TCR only; Xpc mice lack the GGR pathway and TTD mice have a partial defect in each pathway. Garssen tested the effect of UV radiation on the immune response in these mouse strains. Mice homozygous for each defective gene, and wild-type littermates, were exposed to five doses of UV-B radiation (wavelength 280–400 nm) and then sensitized with either picryl chloride (PCl) or Listeria monocytogenes. Four days later, they were challenged with PCl or killed Listeria. ‘Using Listeria, which naturally infects both mice and men, makes our experiments with model antigens more *Hoeijmakers, J.H.J. (2000) DNA repair, cancer and ageing. (Abstr. 211), 18th International Congress of Biochemistry and Molecular Biology, Birmingham, UK
MOLECULAR MEDICINE TODAY, NOVEMBER 2000 (VOL. 6)
Figure 1. A mouse deficient in the Csb gene. Transgenic mice deficient in this and other genes in the TFIIH complex mimic the clinical features of human DNA repair syndromes. Image kindly provided by Jan Hoeijmakers.
relevant,’ says Garssen. The immune response, determined from the degree of ear swelling, deteriorated with increasing radiation dose. The results were similar for both antigens. Only in one mouse strain – Xpa – was the minimum UV dose needed to cause immunosuppression significantly lower than in wild type. This is the only strain that is completely defective in both NER pathways. Thus, either pathway will confer protection against UV-induced immunosuppression. To test for acute UV-induced skin effects, transgenic and wild-type mice were exposed to a single radiation dose. The degree of erythema and edema on exposed, shaved skin and the increase in ear skin thickness were measured. The lowest UV dose that could induce a significant ear swelling response was defined as the minimum erythema–edema dose (MED) for each mouse strain. Garssen found that MED values in the Xpa and Csb strains
were significantly lower than in wild type. There was no difference between Xpc and wild type, and results for the TTD mice were ambiguous. As the affected strains are those that are completely deficient in TCR, this must be the dominant NER pathway for protection against acute UV effects. ‘Many people working in cellular immunity use the MED as a measure of the minimum dose needed to induce immunosuppression,’ says Garssen. ‘These results show that this equation is not always valid. Our Csb mice are susceptible to acute UV effects, and so have a very low MED, but their immune response is near normal.’ UV-induced immunosuppression, rather than the acute skin response, is implicated in the development of skin cancers. Thus, different NER defects should result in different cancer susceptibilities. Hoeijmakers explains further: ‘People with CS have defects in the TCR pathway, but their GGR pathway is intact. They are also not particularly cancer prone. Presumably this can be at least partly explained by the protection against immunosuppression that is conferred by GGR.’ Garssen and his group are generating further transgenic mouse models, deficient in specific DNA repair enzymes, to determine the precise roles for immunomodulation in DNA repair mechanisms. 1 Garssen, J. et al. (2000) Transcription-coupled and global genome repair differentially influence UV-B-induced acute skin effects and systemic immunosuppression. J. Immunol. 164, 6199–6205 2 de Boer, J. and Hoeijmakers, J.H.J. (2000) Nucleotide excision repair and human syndromes. Carcinogenesis 21, 453–460
Clare Sansom Freelance science writer
New diagnostic test for lupus A new diagnostic enzyme-linked immunosorbent assay (ELISA) has been created to help diagnose systemic lupus erythematosus (SLE)1. The test, developed by Mark Roth and colleagues at the Fred Hutchinson Cancer Research Center (Seattle, WA, USA), detects a family of highly phosphorylated Ser and Arg-rich (SR) proteins that act as autoantigens. SLE is a chronic idiopathic autoimmune disorder that causes a variety of symptoms, including joint pain, rash, kidney damage and reactions to sunlight, some of which are 412
caused by deposition of autoantibody–antigen complexes in the tissues. SR proteins are essential pre-mRNA splicing factors. As mRNA splicing is blocked in patients with SLE by antibodies specific to other splicing factors, such as Sm epitope-containing small nuclear ribonucleoproteins (RNP), it was logical to investigate whether the SR proteins could also be autoimmune targets. The new ELISA uses SR proteins purified from HeLa cells and the specific monoclonal antibody, 1H4. In a blinded study, it positively identified about 50% of
patients with SLE. There were few falsepositive results among healthy people or those with other autoimmune disorders, such as antiphospholipid syndrome and rheumatoid arthritis, confirming that patients with SLE are more likely to develop autoantibodies to SR proteins than are people with other autoimmune diseases. SLE symptoms often develop slowly and the disease can be difficult to spot. If the illness is suspected, a battery of tests is performed, including: blood counts and tests for antinuclear antibodies (ANA), anti-native
1357-4310/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
N
MOLECULAR MEDICINE TODAY, NOVEMBER 2000 (VOL. 6)
DNA autoantibodies, and anti-Sm autoantibodies. However, none of these tests is 100% effective and a proportion of patients are missed. ‘There are many tests that, in aggregate, are used to diagnose SLE so it is difficult to quantitate accuracy. Another way of putting it is that we don’t know how many patients are currently being missed,’ says Roth. This was confirmed when the SR-protein ELISA was compared with the ANA test. In all, 81% of patients identified as positive with the new test also tested positive for ANA and 67% of ANA-positive patients also had positive SR-protein ELISAs.
Now that the team has identified the SR proteins as specific autoantigens, they want to identify the precise structural features of these molecules that act as autoepitopes and to identify what triggers autoimmunity to these proteins. One possibility involves retrovirally encoded group specific antigen (GAG) protein, which contains a domain with stretches of alternating phosphorylated Ser and Arg similar to those found in U170K protein and the SR proteins. The U170K protein is an mRNA-splicing factor already known to be a target for the immune response in patients with SLE (see Ref. 1). The team suggests that
e
w
s
retroviral infection and immunity to GAG could also lead to autoimmunity to the alternating Arg-phosphorylated Ser epitopes in the SR-proteins. In the meantime, Roth has applied for a patent for the test kit, although it will not be available commercially for at least a year. 1 Neugebauer, K.M. et al. (2000) SR proteins are autoantigens in patients with systemic lupus erythematosus. Arthritis Rheum. 43, 1768–1778
Sharon Dorrell Freelance science writer
Molecular Medicine Today: Editorial Policy Molecular Medicine Today (MMT) is a magazine-style journal from Elsevier Trends, which publishes Trends in Biochemical Sciences and Immunology Today, among others. MMT provides a forum for the explanation and discussion of ideas and issues that relate to the multidisciplinary area of molecular medicine. It is designed for students and professionals who have information needs that transcend the traditional ‘clinical’ or ‘scientific’ categorization. Its broad appeal is to clinicians, medical/clinical and basic science researchers who are concerned with: elucidating and understanding the aetiology of disease at a molecular level; developing new diagnostics, therapies and preventive medicine based on these; and applying these technologies in healthcare. Short Reviews form the foundation of each issue. All reviews are commissioned, peer reviewed and edited to ensure that they are timely, accurate, interesting and relevant to the audience of MMT. Every effort is made to explain specialist terms and illustrate concepts and hypotheses. The review sections are balanced by topical features, which may be commissioned from freelance writers or may be written by researchers in the field. These include News, Literature, Letters, Meetings, Books, Disease models and Opinion articles. The Literature alerts readers to recent primary papers that are important to biomedical researchers, particularly innovative, likely to have significant clinical impact, or controversial. It is written by a panel of active researchers. If you would like to join this panel, please contact the Editor (see below for details).
Opinion articles give the author’s views on a controversial issue; they set these views in context and comment on what questions need to be answered in order to prove or disprove the author’s hypothesis. Disease models summarize the models used to study a particular disease (usually animal models, although in vitro models may also be described when appropriate), and highlight the similarities and differences between each model and the human condition. Letters on any topic of interest may be submitted to the Editor. Appropriate topics include comments, questions and discussion on articles and topics covered by MMT, new Internet resources, ethical and legal issues, regulatory issues, and business and financial issues. Please note that previously unpublished data and criticisms of work published elsewhere cannot be published. Articles for MMT are generally invited by the Editor, but ideas, particularly for Opinion articles, are welcome. Prospective authors should send a brief summary, citing key references, to the Editor, who will supply guidelines on manuscript preparation if the proposal is accepted. The submission of completed articles without prior consultation is strongly discouraged. As much of the journal content is planned in advance, such manuscripts may be rejected primarily because of lack of space. Authors should note that all review articles for MMT are peer reviewed before acceptance and publication cannot be guaranteed.
For any further information, please contact: The Editor, Molecular Medicine Today, Elsevier Science London, 84 Theobald’s Road, London, UK WC1X 8RR. Tel: +44 (0)20 7611 4400. Fax: +44 (0)20 7611 4470. e-mail: [email protected]
413