Colorectal cancer liver metastatic growth depends on PAD4-driven citrullination of the extracellular matrix.

Citrullination of proteins, a post-translational conversion of arginine residues to citrulline, is recognized in rheumatoid arthritis, but largely undocumented in cancer. Here we show that citrullination of the extracellular matrix by cancer cell derived peptidylarginine deiminase 4 (PAD4) is essential for the growth of liver metastases from colorectal cancer (CRC). Using proteomics, we demonstrate that liver metastases exhibit higher levels of citrullination and PAD4 than unaffected liver, primary CRC or adjacent colonic mucosa. Functional significance for citrullination in metastatic growth is evident in murine models where inhibition of citrullination substantially reduces liver metastatic burden. Additionally, citrullination of a key matrix component collagen type I promotes greater adhesion and decreased migration of CRC cells along with increased expression of characteristic epithelial markers, suggesting a role for citrullination in promoting mesenchymal-to-epithelial transition and liver metastasis. Overall, our study reveals the potential for PAD4-dependant citrullination to drive the progression of CRC liver metastasis.

Multiple antibody reactivities to citrullinated antigens in sera from patients with rheumatoid arthritis: association with HLA-DRB1 alleles.

BACKGROUND: Autoantibodies to cyclic citrullinated peptides (anti-CCP) are present in most patients with rheumatoid arthritis (RA), and associate with HLA-DRB1 shared epitope (SE) alleles. OBJECTIVE: To investigate reactivities of anti-CCP to various citrullinated proteins/peptides, which represent potential autoantigens in RA, and to examine the relationship between such antibodies, and their association with genetic variants within HLA-DRB1 SE alleles. METHODS: Serum samples from 291 patients with established RA and 100 sex- and age-matched healthy subjects were included in this study. Sera were first analysed for presence of anti-CCP antibodies and further for IgG and IgA antibodies towards candidate autoantigens in both their native and citrullinated form including: fibrinogen, alpha-enolase peptide-1 and the C1-epitope of type II collagen (C1(III)). Antibody specificity was confirmed by cross-reactivity tests. HLA-DR genotyping was performed. RESULTS: 72% of patients with RA were anti-CCP positive. Among the candidate autoantigens examined, IgG antibodies to citrullinated fibrinogen were found in 66% of patients' sera and in 41% for both citrullinated alpha-enolase peptide-1 and citrullinated C1(III). These antibodies were mainly seen in the anti-CCP-positive patient group; they were specific for their respective antigen and displayed limited cross reactivity. IgA responses were also detected, but less frequently than IgG. Anti-CCP and anti-citrullinated protein antibodies were associated with HLA-DRB1*04 rather than with HLA-DRB1*01 alleles. CONCLUSIONS: Antibodies directed against several citrullinated antigens are present in CCP-positive RA, with many patients displaying multireactivity. All specific reactivities were primarily associated with the HLA-DRB1*04 alleles, suggesting common pathways of anti-citrulline immunity.

Management of patients presenting with Sjogren's syndrome.

Sjogren's syndrome is an autoimmune exocrinopathy that predominantly affects salivary and lachrymal glands, leading to dry eyes and mouth. The most common clinical problems faced by the rheumatologist are those of dry eyes and mouth, parotid swelling, fatigue and extraglandular manifestations. The first stage in management is to make an accurate diagnosis based on the American/European consensus criteria. The most frequent differential diagnoses are dry eyes and mouth symptoms, a variant of chronic fatigue syndrome and fibromyalgia, and sialosis, which causes a non-inflammatory enlargement of the parotid glands. The mainstay of treatment for the sicca symptoms is local therapy, and that for the milder systemic symptoms is hydroxychloroquine. Steroids and immunosuppressive drugs are reserved for more severe extraglandular disease. In spite of intensive research in other systemic treatments including biologic therapies, there is limited evidence to support their use in routine clinical practice.

Mixed connective tissue disease.

Mixed connective tissue disease (MCTD) was first described in 1972 as a disease syndrome with overlapping features of systemic sclerosis, systemic lupus erythematosus (SLE) and polymyositis associated with antibodies to RNAse sensitive extractable nuclear antigen. When the antigen was subsequently characterized as polypeptides on the U1 ribonuclear protein component of the splicesosome (U1RNP), MCTD became the first rheumatic disease syndrome to be defined by a serologic test. Clinical features include a high frequency of Raynaud's syndrome, swollen hands, sclerodactyly, arthritis, polymyositis and interstitial lung disease. Over the last 30 years there has been a continuing debate as to whether MCTD constitutes a 'distinct clinical entity'. Here, I will review the pathological, immunogenetic and clinical features of MCTD and conclude that the debate remains unresolved. The early misconception that it has a relatively good prognosis has not stood the test of time with long-term follow-up studies. These have identified a tendency for MCTD to evolve into SLE or systemic sclerosis and highlighted pulmonary hypertension and scleroderma renal crisis as important causes of death. Providing it is realized that our appreciation of the clinical features associated with anti-U1RNP have evolved over time, MCTD remains a useful concept in clinical practice. Whether it can be credited with the term 'disease' awaits the demonstration of common etiopathological events underlying the development of antibodies to U1 RNP and their associated clinical features.

Conservation and loss of the ERV3 open reading frame in primates.

The human endogenous retrovirus ERV3 possesses an open reading frame for a truncated envelope, which is expressed as mRNA and protein. Here we examine the env sequence in primates for evidence of evolutionary conservation. ERV3 sequences were amplified by PCR from genomic DNA of great ape and Old World primates but not from New World primates or gorilla, suggesting an integration event more than 30 million years ago with a subsequent loss in one species. In the chimpanzee, the protein sequence of Env is 98.18% identical to that of human. In other species the identity falls (93.71% in rhesus macaque) in proportion to the separation from the human lineage. Start and stop codons and domains of functional significance in the envelope protein are conserved. The evolutionary conservation of the ERV3 envelope suggests a beneficial function, though the loss from gorilla shows that it is not essential for survival or reproduction.

Glucose-6-phosphate isomerase is not a specific autoantigen in rheumatoid arthritis.

OBJECTIVE: To test the hypothesis that glucose-6-phosphate isomerase (GPI) is a novel autoantigen in RA. METHODS: Eighty-eight serum samples from 23 patients with rheumatoid arthritis (RA), 25 with Sjögren's syndrome, 20 with systemic lupus erythematosus and 20 healthy controls were tested by enzyme-linked immunosorbent assay (ELISA) using a commercially available, partially purified rabbit GPI as antigen. Beside each duplicate well containing antigen (10 micro g/ml), uncoated blocked duplicate wells (phosphate-buffered saline only) were included as controls for non-specific binding for every serum tested. We also examined antibodies binding to various polypeptides in the GPI preparation by immunoblotting in 73 of the sera. RESULTS: By ELISA, binding levels were low and there was no difference between serum from patients with RA, other rheumatic diseases and normal controls. By immunoblotting, antibodies binding to the GPI polypeptide were present in 70-80% of all groups tested. In addition, we showed that another polypeptide identified as phosphoglucomutase was also present in the preparation and reacted with human immunoglobulins. CONCLUSION: Our findings suggest that GPI is not a specific autoantigen in RA.

Autoantibodies to human endogenous retrovirus-K are frequently detected in health and disease and react with multiple epitopes.

A number of studies have found increased levels of antibodies to human endogenous retroviruses (HERVs) in autoimmune rheumatic diseases. It is not clear whether this immune response is driven by the HERV itself or by cross-reactions with an exogenous virus or an autoantigen. To address this question, we examined the antibody response to the Env protein of two closely related members of the HERV-K family, HERV-K10 and IDDMK1,222. By immunoblotting of recombinant proteins, antibodies were found in 32-47% of 84 sera from patients with autoimmune rheumatic disease, and 29% of 35 normal controls. Epitope mapping with overlapping 15mers identified multiple reactive peptides on both antigens, with one (GKTCPKEIPKGSKNT) containing immunodominant epitope(s). By ELISA, the median titre of antibody to this peptide was significantly increased in 39 patients with SLE compared to 39 healthy controls and 86 patients with other rheumatic diseases (P < 0.003). We have shown that there is a high frequency of IgG antibodies to HERV-K env sequences in human sera, both in health and autoimmune rheumatic disease, and that the response is to multiple epitopes. This supports the hypothesis that the autoimmune response to HERV-K is antigen-driven and may be an early stage in the chain of events that leads to tolerance breakdown to other autoantigens.

Dry eyes and mouth syndrome--a subgroup of patients presenting with sicca symptoms.

OBJECTIVE: To evaluate the characteristics of patients presenting with symptoms suggestive of Sjögren's syndrome (SS) but failing to satisfy diagnostic criteria. METHODS: Clinical, serological and histological data were collected on 34 patients presenting with dry eyes and/or mouth who did not satisfy the Vitali criteria for the diagnosis of SS. They were compared with 136 patients with primary SS, 38 patients with secondary SS, and 13 patients without SS. Questionnaires on symptoms from each group were compared with 43 healthy controls. RESULTS: The 34 patients who did not satisfy the diagnostic criteria for SS or any other connective tissue disease were designated dry eyes and mouth syndrome (DEMS). Their demography including age was similar to that of a primary SS group and there was no more atrophy seen on their biopsies compared with SS and non-SS controls. They scored highly on visual analogue scales of symptoms but had few objective signs. All were negative for anti-Ro and anti-La although the prevalence of antinuclear antibodies (19%) was increased compared with a normal population. There was no excess of SS-associated tissue types. CONCLUSION: There was no evidence that age, salivary gland atrophy or subclinical SS accounted for the symptoms in DEMS. Most of the patients fitted into a spectrum of disease which tended more towards fibromyalgia and/or chronic fatigue syndrome.

Delta 32 deletion of CCR5 gene and association with asthma or atopy.

The CCR5-delta32 deletion polymorphism (CCR5-delta32) was investigated for linkage and association to asthma and atopy using two panels of nuclear families containing 1284 individuals. No statistically significant linkage to asthma/wheeze or atopy was observed in either of the two panels of families. Multiallelic transmission disequilibrium tests (TDT) of the combined data found no significant association for atopy (52 independent alleles transmitted, 51 non-transmitted) or asthma/wheeze (39 transmitted, 44 non-transmitted). Although functional evidence might suggest that CCR5 is a good candidate gene for atopic asthma, this study provides no genetic evidence from CCR5-delta32 polymorphism to support this hypothesis.

Retroviruses: ancient and modern.

Retroviruses are transmitted in two distinct ways: as infectious virions and as 'endogenous' proviral DNA integrated in the germ line of their hosts. Modern infectious viruses such as HIV recently infected mankind from simian hosts, whereas human endogenous retroviral genomes have been present throughout old world primate evolution. Recently we have characterised novel retroviruses in humans and pigs. Human retrovirus 5 (HRV-5) is detected as an exogenous genome in association with arthritis and systemic lupus erythematosus. Porcine endogenous retroviruses (PERV) are carried in swine DNA but can be activated to produce virions that are infectious for human cells, which has implications for xenotransplantation. A brief account of HRV-5 and PERV is given here.

ERV-3 envelope expression and congenital heart block: what does a physiological knockout teach us.

Congenital heart block is a serious condition with significant mortality due in most cases to the transplacental transfer of autoantibodies from an otherwise asymptomatic mother. Although SSA/Ro and SSB/La autoantibodies have been implicated, attention has focused recently on autoantibodies to envelope proteins of endogenous retrovirus-3 (ERV-3). We have recently identified in 1% of the caucasian population a natural knock out of ERV-3 due to a premature stop mutation generating a severely truncated form of the protein [corrected]. If a pregnant female homozygous for the truncated form of the ERV-3 carries a foetus expressing the entire protein, the mother might be expected to acquire high titre immunity, while the foetus homozygous for the truncated form would not be expected to immunise its mother. In order to test whether this naturally occurring model could shed light on the pathogenesis of CHB, we determined the status of the ERV-3 stop polymorphism in 12 mothers of CHB infants [corrected]. The fact that none was homozygous for the stop mutation tends to rule out a role for the stop polymorphism of the mothers in the generation of the CHB disease, but does not exclude that other polymorphisms might be involved [corrected].

Human retrovirus-5 in rheumatic disease.

It has long been suggested that retroviral infection may play a role in the pathogenesis of autoimmune rheumatic disease. Particles resembling retroviruses have been reported in tissue from patients with Sjögren's syndrome, lupus and rheumatoid arthritis, and molecular mimicry between retroviral antigens and host proteins has been proposed as a mechanism of induction of autoimmunity. Since 1980, four distinct human infectious retroviruses have been discovered, HTLV-I, HTLV-II, HIV-1 and HIV-2. We recently cloned part of a new human retrovirus genome, designated human retrovirus-5 (HRV-5) and demonstrated that this is not endogenous and is therefore a novel infectious retrovirus. Because symptoms resembling arthritis, polymyositis and Sjögren's syndrome occur in individuals infected with HTLV-I and HIV-1, we investigated the possibility that HRV-5 was associated with idiopathic rheumatic disease. Using nested PCR, HRV-5 we demonstrated that proviral DNA was present in approximately 50% of synovial samples of arthritic joints and was also found in over 10% of blood samples of patients with rheumatoid arthritis and systemic lupus erythematosus. HRV-5 proviral DNA was not detectable in affected tissues of autoimmune diseases and was found in only one of over 200 tissues taken at autopsy from non-rheumatoid patients. Sequence analysis of the amplified viral segment showed genetic variation between samples with maintenance of the open reading frame typical of a replicating infectious retrovirus. Thus HRV-5 appears to be a human retrovirus found with a very low genome copy number in most tissues, but which is increased to detectable levels in inflamed joints and blood from patients with rheumatic disease. Whether HRV-5 is aetiologically important in these diseases remains to be determined.

Detection of human retrovirus 5 in patients with arthritis and systemic lupus erythematosus.

OBJECTIVE: To examine whether human retrovirus 5 (HRV-5) infection is associated with autoimmune rheumatic disease. METHODS: DNA from patients with various disorders including inflammatory diseases and from normal subjects was tested by nested polymerase chain reaction (PCR) for HRV-5 proviral DNA. Positive results were confirmed by DNA sequencing. RESULTS: HRV-5 proviral DNA was detected in 53% of synovial samples from arthritic joints, in 12% of blood samples from patients with rheumatoid arthritis (RA), and in 16% of blood samples from patients with systemic lupus erythematosus. In contrast, it was not detectable by PCR of affected tissues from patients with several other autoimmune diseases and was found in only 1 of >200 tissue specimens obtained at autopsy from non-RA patients. Sequence analysis of the amplified viral segment showed genetic variation between samples with maintenance of the open reading frame, typical of a replicating infectious retrovirus. CONCLUSION: This is the first report of the frequent detection of HRV-5 in any disease. We propose that the possible involvement of HRV-5 in autoimmune and rheumatic disease should be investigated further.