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ispartof: FUTURE ONCOLOGY vol:16 issue:3 pages:4419-4423 ispartof: location:England status: published

Two recent policy documents by the European Union, ‘Europe's Beating Cancer Plan’ and its accompanying ‘Conquering Cancer: Mission Possible’ (CCMP), articulate broad policies aimed at reducing cancer mortality across Europe, for example, by promoting prevention and early detection. The focus for cancer treatment in these manifestos is the expansion of personalised cancer medicine (PCM). However, the CCMP document suggests that the uptake of PCM is “hampered by uncertainty about its outcomes”. What are these outcomes and why this uncertainty? We address the limits of PCM in pathology-driven and pathology-agnostic PCM, briefly discussing the results of umbrella and basket trials. We suggest that the complexity, plasticity and genetic heterogeneity of advanced cancers will continue to thwart the impact of PCM, limiting it to specific pathologies, or rare subsets of them. Caution regarding the advancement of PCM is justified, and policymakers should be wary of the hype of lobbyists, who do not acknowledge the limits of PCM.

BackgroundTreatments for pancreatic ductal adenocarcinoma are poorly effective, at least partly due to the tumor’s immune-suppressive stromal compartment. New evidence of positive effects on immune responses in the tumor microenvironment (TME), compelled us to test the combination of gemcitabine (GEM), a standard chemotherapeutic for pancreatic cancer, with nicotinamide (NAM), the amide form of niacin (vitamin B3), in mice with pancreatic cancer.MethodsVarious mouse tumor models of pancreatic cancer, that is, orthotopic Panc-02 and KPC (KrasG12D, p53R172H, Pdx1-Cre) grafts, were treated alternately with NAM and GEM for 2 weeks, and the effects on efficacy, survival, stromal architecture and tumor-infiltrating immune cells was examined by immunohistochemistry (IHC), flow cytometry, Enzyme-linked immunospot (ELISPOT), T cell depletions in vivo, Nanostring analysis and RNAscope.ResultsA significant reduction in tumor weight and number of metastases was found, as well as a significant improved survival of the NAM+GEM group compared with all control groups. IHC and flow cytometry showed a significant decrease in tumor-associated macrophages and myeloid-derived suppressor cells in the tumors of NAM+GEM-treated mice. This correlated with a significant increase in the number of CD4 and CD8 T cells of NAM+GEM-treated tumors, and CD4 and CD8 T cell responses to tumor-associated antigen survivin, most likely through epitope spreading. In vivo depletions of T cells demonstrated the involvement of CD4 T cells in the eradication of the tumor by NAM+GEM treatment. In addition, remodeling of the tumor stroma was observed with decreased collagen I and lower expression of hyaluronic acid binding protein, reorganization of the immune cells into lymph node like structures and CD31 positive vessels. Expression profiling for a panel of immuno-oncology genes revealed significant changes in genes involved in migration and activation of T cells, attraction of dendritic cells and epitope spreading.ConclusionThis study highlights the potential of NAM+GEM as immunotherapy for advanced pancreatic cancer.

BackgroundThe global pandemic caused by SARS-CoV-2 virus has prompted an unprecedented international effort to seek medicines for prevention and treatment of infection. Drug repurposing has played a key part in this response. The rapid increase in trial activity has raised questions about efficiency and lack of coordination. Our objective was to develop a user-friendly, open access database to monitor and rapidly identify trials of medicinal products.MethodsUsing the US clinicaltrials.gov (NCT) registry, the EU Clinical Trials Register (EUCTR) and the WHO International Clinical Trials Registry Platform (WHO ICTRP), we identified all COVID-19 trials of medicinal products. Trials that were out of scope and duplicates were excluded. A manual encoding was performed to ascertain key information (e.g. trial aim, type of intervention etc). The database, Covid19db, is published online at: http://www.redo-project.org/covid19db/.ResultsDescriptive statistics of the database from April 4th 2020 through to August 18th show an increase from 186 to 1618 trials, or an average of 10.5 new trials registered per day. Over this period, the proportion of trials including a repurposing arm decreased slightly (from a maximum of 75% to 64% at the end of the covered period) as did the proportion of trials aiming to prevent infection (from a maximum of 16% to 13%). The most popular trial intervention is hydroxychloroquine (212 trials), followed by azithromycin (64 trials), tocilizumab, favipiravir and chloroquine (145 trials). Total planned enrolment is 1064556 participants as of 18th August 2020.Conclusionswe have developed an open access and regularly updated tool to monitor clinical trials of medicinal products to prevent or treat infection by SARS-CoV-2 globally. Our analysis shows a high number of ‘me-too’ trials, in particular for some repurposed drugs, such as hydroxychloroquine, azithromycin and tocilizumab, substantiating calls for better coordination and better use of trial resources.

Introduction: Despite the relatively high prevalence of rare cancers as a collective group, the low incidence for individual rare and ultrarare cancers continues to act as an impediment to the deve...

The repurposing of existing non-cancer drugs is a potential source of new treatment options for cancer patients with high unmet medical needs. While scientific research is progressing rapidly in the field of drug repurposing, the implementation of drug repurposing still faces important financial and regulatory hurdles that should be addressed to optimise clinical adoption.

Repurposing is a drug development strategy that seeks new medical treatments using existing licensed drugs rather than from new molecular entities. There has been increasing interest in the use of this approach to develop innovative treatments in oncology, particularly with respect to the use of noncancer medications as anticancer therapeutics. The review briefly outlines the rationale for such an approach and discusses the process of selecting potential repurposing candidates and the steps necessary to develop them clinically. The challenges of clinical adoption, including regulatory issues, are also explored. Finally, the future prospects of drug repurposing in oncology are touched upon.

Drug repurposing is a strategy that aims to develop novel cancer treatments through the reuse of existing medicines developed in other disease areas. Such a strategy includes the identification of candidate drugs, clinical development, drug licensing, reimbursement and clinical implementation. This review outlines a literature-based approach to candidate selection with illustrative examples in osteosarcoma, pancreatic cancer and perioperative therapies. Key issues related to the development of clinical trials, drug licensing/approval and clinical adoption are explored to highlight some of the obstacles that must be overcome to successfully repurpose a drug as a new therapeutic option. ispartof: SEMINARS IN CANCER BIOLOGY vol:68 pages:186-191 ispartof: location:England status: published

Background. A number of recent high-profile cases have focused attention on scientific misconduct and other problematic issues with the peer review process. The retraction of journal publications is an important part of the scientific publishing process that serves to remove flawed articles, (including, but not limited to, fraudulent results), from the literature. To date there have been few formal studies of journal retractions in the area of oncology. Methods. This article outlines the results of a bibliometric study of journal retractions from 1983 to 2018. Results. Analysis shows that article lifetime – that is the time period from initial publication to ultimate retraction – has decreased in recent years. It also shows that retraction rate has also increased over the same period. The causes and context of these trends are discussed and reference made to the dangers of scientific misconduct in oncology.

Repurposing is a drug development strategy that seeks to use existing medications for new indications. In oncology, there is an increased level of activity looking at the use of non-cancer drugs as possible cancer treatments. The Repurposing Drugs in Oncology (ReDO) project has used a literature-based approach to identify licensed non-cancer drugs with published evidence of anticancer activity. Data from 268 drugs have been included in a database (ReDO_DB) developed by the ReDO project. Summary results are outlined and an assessment of clinical trial activity also described. The database has been made available as an online open-access resource (http://www.redo-project.org/db/). ispartof: ECANCERMEDICALSCIENCE vol:12 ispartof: location:England status: published

GENimmune NV is a newly created biopharmaceutical company, based in Belgium, that focuses on developing immune therapeutics against chronic infectious diseases characterized by inadequate immune responses. The company has already developed an early-stage pipeline of therapeutic vaccine candidates against the hepatitis B and C viruses and human papillomavirus. GENimmune’s goal is to optimize the human immune response through rational vaccine design. Its vaccine-development strategy is based on the use of polyepitopes that take immunological plasticity and viral diversity into account. This represents an original approach to medical treatment. The company’s R&D activities are supported by a biomanufacturing unit that also has an established track record of providing customized third-party contract services.

Selective phosphodiesterase 5 inhibitors, including sildenafil, tadalafil and vardenafil, are widely-used in the treatment of erectile dysfunction and pulmonary arterial hypertension. They are also well-known as examples of successful drug repurposing in that they were initially developed for angina and only later developed for erectile dysfunction. However, these drugs may also be effective cancer treatments. A range of evidentiary sources are assessed in this paper and the case made that there is pre-clinical and clinical evidence that these drugs may offer clinical benefit in a range of cancers. In particular, evidence is presented that these drugs have potent immunomodulatory activity that warrants clinical study in combination with check-point inhibition.

Selective phosphodiesterase 5 inhibitors, including sildenafil, tadalafil and vardenafil, are widely-used in the treatment of erectile dysfunction and pulmonary arterial hypertension. They are also well-known as examples of successful drug repurposing in that they were initially developed for angina and only later developed for erectile dysfunction. However, these drugs may also be effective cancer treatments. A range of evidentiary sources are assessed in this paper and the case made that there is pre-clinical and clinical evidence that these drugs may offer clinical benefit in a range of cancers. In particular, evidence is presented that these drugs have potent immunomodulatory activity that warrants clinical study in combination with check-point inhibition.

BackgroundDrug repurposing can speed up access to new therapeutic options for cancer patients. With more than 2000 drugs approved worldwide and 6 relevant targets per drug on average, the potential is quantitatively important. In this paper, we have attempted to quantify the number of non-cancer drugs supported by either preclinical or clinical cancer data.MethodsA PubMed search was performed to identify non-cancer drugs which could be repurposed in one or more cancer types. Drugs needed at least one peer-reviewed article showing an anticancer effect in vitro, in vivo or in humans.ResultsA total of 235 eligible non-cancer drugs were identified (Table 1). Main charateirstics of the drugs are summarized in Table 2. 67 (29%) are on the WHO list of essential medicines and 176 (75%) are off-patent. 133 (57%) had human data in cancer patient(s). Four were listed in clinical guidelines, namely thalidomide, all-trans retinoic acid, zoledronic acid and non-steroidal anti-inflammatory drugs (NSAID). Several drugs have shown a survival benefit in randomized trials such as cimetidine (colorectal cancer), progesterone (breast cancer) or itraconazole (lung cancer). Several other drugs induced responses in rare tumours, like clarithromycin, timolol or propranolol.ConclusionWe have found that the number of off-patent repurposing opportunities is large and increasing. Joint non-commercial clinical development (academics, governments, charities) may bring new therapeutic options to patients at low cost, especially in indications for which the industry has no incentive to invest in.

Surgical resection remains the major modality for modern curative treatment for solid tumours. However, post-surgical recurrence, even following clear-margin resection and adjuvant treatment, remains common in many types of cancer. Reducing recurrence rates, therefore, offers the potential to increase cure rates and increase overall survival. Perioperative therapies, simple interventions during the perioperative period, are designed to address some of the factors which influence post-surgical recurrence. A range of perioperative therapies are introduced and the rationale for further clinical investigation outlined.

Propranolol (PRO) is a well-known and widely used non-selective beta-adrenergic receptor antagonist (beta-blocker), with a range of actions which are of interest in an oncological context. PRO displays effects on cellular proliferation and invasion, on the immune system, on the angiogenic cascade, and on tumour cell sensitivity to existing treatments. Both pre-clinical and clinical evidence of these effects, in multiple cancer types, is assessed and summarised and relevant mechanisms of action outlined. In particular there is evidence that PRO is effective at multiple points in the metastatic cascade, particularly in the context of the post-surgical wound response. Based on this evidence the case is made for further clinical investigation of the anticancer effects of PRO, particularly in combination with other agents. A number of trials are on-going, in different treatment settings for various cancers.

Chronic hepatitis B virus (HBV) infection, which occurs in 1 to 5% of adult infections and up to 30% of pediatric infections, is characterized by high levels of viral replication averaging a daily production of about 1011 viral particles, hepatic inflammation, necrosis, and ultimately liver failure (36). An estimated 350 million individuals are classified as chronically HBV infected, with the highest concentrations of infection occurring in large parts of Asia and Africa (23). Chronic HBV can be treated with nucleoside analogues that inhibit polymerase activity. Lamivudine was the first licensed polymerase inhibitor, and it results in significant suppression of HBV DNA levels. However, this response, similar to the loss of hepatitis B virus e antigen, is often not sustained upon discontinuation of treatment (11, 28). The emergence of viral resistance in 15 to 20% of treated patients per year clearly pinpoints the limitations of this treatment. Newer drugs such as adefovir dipivoxil, entecavir, and telbivudine can result in less resistance, increased suppression of DNA levels, or in somewhat higher levels of hepatitis B virus e antigen loss. Real long-term treatment data with these drugs are, however, limited, and it is unclear how well these responses would be sustained if therapy were withdrawn. Higher levels of sustained response after the cessation of therapy have been documented for treatment with alpha interferon (IFN-α) and even more so for its pegylated form. This higher efficacy may be related to the fact that interferon has not only an antiviral but also an immunomodulatory function. However, drug cost and toxicities often restrict the use of IFN (11, 28). Thus, the development of supplemental therapies, especially those aiming at an improved immune response, remain a priority, particularly as a combination of lamivudine and IFN failed to improve sustained response rates (19). In acute HBV infection, cellular immune responses are well characterized and temporally associated with the clearance of infection. The HBV-specific cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) responses present during and immediately following resolution of acute infections are readily detected and often broadly specific, targeting epitopes from numerous viral gene products (13, 17). Chronic HBV infection is, however, only rarely resolved by the immune system. When this occurs, viral clearance is associated with increased CTL activity and increased levels of alanine transaminase (ALT), referred to as ALT flares, caused by the destruction of infected hepatocytes by the immune system (40). Viral clearance also can be induced in a significant proportion (10 to 15%) of individuals receiving IFN-α, and, similar to the effect of spontaneous clearance, the effect is correlated with increased CTL and HTL responses. Antiviral effects can be mediated through the action of cytokines, such as IFN-γ, which depress viral replication or inactivate virus particles without killing HBV-infected cells (15, 16). Similarly, in persistent HBV infection, the presence of HBV-specific CTL is associated with lower viral loads, independent from liver damage, further confirming the noncytolytic antiviral activity of CTL (4, 29). Successful vaccine immunotherapy in chronically infected individuals is expected to be characterized by similar CTL and HTL responses. However, in the presence of active HBV replication, the immune system of chronically infected individuals no longer responds vigorously to viral epitopes. The high levels of HBV particles and HBV proteins in the circulation are thought to be responsible for this immune suppression (12, 43). This effect can be so pronounced that it develops into a generalized imbalance of HTL type 1 and 2 responses (Th1/Th2), which manifests as general peripheral tolerance (42). The rare spontaneous resolution of chronic HBV infection and the immune responses observed during IFN-α or lamivudine treatment, in which viral replication is significantly reduced, indicates that deletion of HBV-specific CTL precursors does not occur and that the disease state of immune system tolerance is reversible (7, 8). So far, therapeutic vaccine candidates (6, 20, 25, 38, 44) for HBV have focused on the use of HBsAg and core proteins or parts thereof. However, cellular immune responses to these antigens probably are most prone to suppression, tolerance, or other dysfunctions, since these proteins circulate in very high levels in the infected host (21, 41). This was effectively demonstrated by a single core epitope lipopeptide vaccine candidate, which successfully induced CTL responses in healthy volunteers but failed to do so in chronic HBV patients (24, 25). Even under effective antiviral treatment with polymerase inhibitors or IFN-α, the levels of circulating HBsAg remain very high, possibly pointing to an explanation of why combinations of lamivudine and therapeutic vaccines based on HBsAg so far have failed to show a benefit (44). On the other hand, the polymerase antigen so far has not been as thoroughly investigated as therapeutic vaccine candidate, although the immune system may be more likely to respond to this antigen, which is produced only in minute amounts compared to production of the HBsAg and core antigen. Importantly, polymerase-specific CTL and HTL responses can be readily detected in persons resolving HBV infection (33). The induction of broadly specific CTL and HTL responses against various HBV antigens including the polymerase thus would be a new approach to improve the efficacy of a candidate therapeutic vaccine. Such a vaccine, designed for use in the general population, needs to be based on numerous epitopes restricted by multiple HLA types in order to provide acceptable levels of population coverage. The use of synthetic peptides to deliver large numbers of CTL and HTL epitopes is not feasible, but the technology to design and synthesize genes encoding multiple epitopes for use in DNA plasmid vaccines and in viral vectors is well suited (26, 27). The utility of using genetic constructs, such as DNA vaccines, to deliver multiple epitopes was demonstrated through the development of a vaccine product designed primarily for use with human immunodeficiency virus (HIV)-infected patients (56). This vaccine, which is designated EP HIV-1090, was successfully evaluated for safety and immunogenicity in two phase-1 clinical trials. This first-generation product proved to be safe at the dose levels tested, but immunogenicity was limited. The combined use of DNA vaccines with viral vectors in a heterologous prime-boost sequential immunization format has proved useful for augmenting response levels and rates in some clinical studies. Thus, it was decided that the combined use of DNA and modified vaccinia Ankara (MVA)-vectored vaccines encoding CTL and HTL epitopes warranted investigation. This report describes the design, synthesis, and characterization of a multiepitope-based HBV candidate therapeutic vaccine composed of a plasmid DNA (pDNA) construct (INX102-3697) and a recombinant MVA viral vector (INX102-0557) that each contain a single gene encoding 30 HBV-derived CTL epitopes, 16 HBV-derived HTL epitopes, and the universal pan-DR epitope (PADRE) HTL epitope. These two products are designed for sequential administration using a heterologous prime-boost immunization format to maximize the cellular immune responses induced.

Diclofenac (DCF) is a well-known and widely used non-steroidal anti-inflammatory drug (NSAID), with a range of actions which are of interest in an oncological context. While there has long been an interest in the use of NSAIDs in chemoprevention, there is now emerging evidence that such drugs may have activity in a treatment setting. DCF, which is a potent inhibitor of COX-2 and prostaglandin E2 synthesis, displays a range of effects on the immune system, the angiogenic cascade, chemo- and radio-sensitivity and tumour metabolism. Both pre-clinical and clinical evidence of these effects, in multiple cancer types, is assessed and summarised and relevant mechanisms of action outlined. Based on this evidence the case is made for further clinical investigation of the anticancer effects of DCF, particularly in combination with other agents - with a range of possible multi-drug and multi-modality combinations outlined in the supplementary materials accompanying the main paper.

Nitroglycerin (NTG), a drug that has been in clinical use for more than a century, has a range of actions which make it of particular interest in an oncological setting. It is generally accepted that the main mechanism of action of NTG is via the production of nitric oxide (NO), which improves cardiac oxygenation via multiple mechanisms including improved blood flow (vasodilation), decreased platelet aggregation, increased erythrocyte O2 release and decreased mitochondrial utilization of oxygen. Its vasoactive properties mean that it has the potential to exploit more fully the enhanced permeability and retention effect in delivering anti-cancer drugs to tumour tissues. Moreover NTG can reduce HIF-1α levels in hypoxic tumour tissues and this may have anti-angiogenic, pro-apoptotic and anti-efflux effects. Additionally NTG may enhance anti-tumour immunity. Pre-clinical and clinical data on these anti-cancer properties of NTG are summarised and discussed. While there is evidence of a positive action as a monotherapy in prostate cancer, there are mixed results in NSCLC where initially positive results have yet to be fully replicated. Based on the evidence presented, a case is made that further exploration of the clinical benefits that may accrue to cancer patients is warranted. Additionally, it is proposed that NTG may synergise with a number of other drugs, including other repurposed drugs, and these are discussed in the supplementary material appended to this paper.

Itraconazole, a common triazole anti-fungal drug in widespread clinical use, has evidence of clinical activity that is of interest in oncology. There is evidence that at the clinically relevant doses, itraconazole has potent anti-angiogenic activity, and that it can inhibit the Hedgehog signalling pathway and may also induce autophagic growth arrest. The evidence for these anticancer effects, in vitro, in vivo, and clinical are summarised, and the putative mechanisms of their action outlined. Clinical trials have shown that patients with prostate, lung, and basal cell carcinoma have benefited from treatment with itraconazole, and there are additional reports of activity in leukaemia, ovarian, breast, and pancreatic cancers. Given the evidence presented, a case is made that itraconazole warrants further clinical investigation as an anti- cancer agent. Additionally, based on the properties summarised previously, it is proposed that itraconazole may synergise with a range of other drugs to enhance the anti-cancer effect, and some of these possible combinations are presented in the supplementary materials accompanying this paper.

Interleukin-6, a cytokine produced particularly by triple-negative breast cancers, strongly inhibits T cell responses in the tumor microenvironment. Here we tested cryoablation combined with Meriva (a lecithin delivery system of curcumin with improved bioavailability) in mice with metastatic breast cancer (4T1). Cryoablation involves killing of tumor cells through freezing and thawing, resulting in recruitment of tumor-specific T cells, while curcumin stimulates T cells through the reduction of IL-6 in the TME. Cryoablation plus Meriva accumulated and activated CD8+ T cells to multiple tumor-associated antigens such as Mage-b and Survivin (both expressed by 4T1 tumors). This correlated with a nearly complete reduction of 4T1 primary tumors and lung metastases while little effect was observed from saline or Meriva alone (28 d after tumor cell injection). The survival rate in the group of cryoablation plus Meriva was significantly improved compared to all control groups. Using a less aggressive 4T1 model expressing luciferase (4T1.2luc3), we demonstrated that all mice receiving saline or Meriva developed metastases in the lungs and a primary tumor (38 d after tumor cell injection; and died soon after that), but not the mice receiving cryoablation or cryoablation plus Meriva. However, on day 58 the mice receiving cryoablation developed 4T1.2luc3 metastases in the lungs, while mice receiving cryoablation plus Meriva were free of metastases. These results strongly suggest that cryoablation delayed the development of lung metastases on the short-term, but Meriva administered after cryoablation was significantly better in delaying the development of lung metastases and survival on the long-term.

Many forms of cancer lack efficacious treatments, despite continuing advances in our understanding of molecular biology, and the development of precisely targeted agents that exploit relevant drivers and pathways of malignancy. To date the effectiveness of most tumor cell-focused molecularly targeted agents, in terms of event free and overall survival appears to be modest [1]. What is more, these new drugs come with a high price tag as companies seek to recoup development costs and to generate a return on investment (in part a consequence of a high attrition rate in oncological drug development, high regulatory burdens and expensive clinical trial costs) [2]. In the developing economies of the world, where cancer incidence is rising, the problems of unmet patient need is exacerbated by these high costs, putting many cancer treatments out of reach of patients and imposing strains on local health systems [3]. An alternative approach to seeking new drug treatments for cancer is not to start with molecular targets in mind, but to assess those drugs – approved for any indication – in our existing armamentarium which show some evidence of anticancer activity. This is the field of drug repurposing in oncology and there is now an increasing interest in the use of non-cancer drugs as anticancer therapeutics. There are two main advantages of repurposing. Firstly, by starting with well-known and well-characterized drugs we can draw on existing and detailed knowledge of pharmacodynamics, pharmacokinetics, bioavailability, toxicities, established protocols and dosing. This body of knowledge is far in excess of what can be gained in early phase clinical trials of new agents, particularly for first in class drugs. This is not to say that repurposed drugs do not need Phase I trials, since they may be used for cancer in schedules and combinations that differ from their accepted use, but it does represent a considerable short-circuiting of the preclinical phase of the drug d evelopment life cycle [4]. Secondly, many of the candidate drugs for repurposing are available at low cost; indeed many are available as generics. This is in stark contrast to the very high costs associated with the newest agents emerging from current pharmaceutical pipelines.

Objective: To determine the diagnostic distribution in a consecutive anti-SSA and/or anti-SSB positive population. Methods: A total of 15 937 serum samples from 10 550 consecutive patients were analysed for antinuclear antibodies (ANAs) on HEp-2 cells. Serum samples positive for ANAs were analysed by immunodiffusion and line immunoassay with recombinant SSA-Ro52, natural SSA-Ro60, and recombinant SSB. Results: Among ANA positive patients in whom clinical information was available, 181 consecutive patients with anti-SSA and/or anti-SSB antibodies were identified, Disease associations were systemic lupus erythematosus (SLE) (45.3%), primary Sjogren9s syndrome (pSS) (14.4%), scleroderma (8.8%), RA (7.7%), cutaneous lupus (7.7%), and dermatomyositis (2.2%). The ratio of diagnoses differed according to the anti-SSA/anti-SSB serotype. Scleroderma and dermatomyositis were enriched among mono-Ro52 reactive serum samples (34.2% and 10.5% respectively). Single reactivity towards Ro60 or anti-Ro60 with anti-Ro52 predisposed for SLE (80.0% and 52.2% respectively). Triple reactivity towards Ro52, Ro60, and SSB was primarily linked with SLE (55.8%) followed by pSS (20.9%). Anti-SSA on immunodiffusion increased the chance for SLE (62.8%), whereas isolated anti-SSB reactivity on immunodiffusion was less indicative for SLE (14.3%) and predisposed more for cutaneous lupus (23.8%) and pSS (33.3%). Conclusion: The diagnostic range associated with anti-SSA or anti-SSB reactivity differs significantly according to the detailed serotype defined by line immunoassay and immunodiffusion.

Objective To identify immunodominant epitopes in natural filaggrin that are reactive with antifilaggrin autoantibodies (AFA) in the sera of patients with rheumatoid arthritis (RA) and to explore their use in a diagnostic assay format. Methods Based on the results of epitope mapping of human natural filaggrin as well as molecular modeling and computational chemistry, synthetic peptides together with recombinant citrullinated filaggrin were evaluated by a line immunoassay (LIA) for AFA detection. Diagnostic performance was assessed using 336 RA and 253 disease control sera and was compared with that of reference methods. Results Several immunoreactive epitopes were identified in natural filaggrin, all of which contained at least 1 citrulline residue. Three antigenic substrates, including 2 synthetic peptides and recombinant citrullinated filaggrin showing maximal reactivity on LIA, were finally selected. Using the 3-antigen LIA3, overall sensitivity, specificity, and positive predictive value for RA were 65.2%, 98.0%, and 89.1%, respectively, compared with 61.9%, 98.8%, and 92.8% using the 2-antigen LIA2 (without recombinant protein). Thirty-seven percent of the rheumatoid factor (RF)-negative RA samples (30 of 81) were AFA-positive by LIA2, and 52 of 54 RF-positive control samples had no AFA detected on LIA2. Higher specificity and sensitivity were obtained by LIA2 versus anti-RA33 immunoblot, whereas good agreement was observed with antikeratin antibody testing. LIA performed significantly better than AFA immunoblotting using natural filaggrin, at a specificity level of 99% (P = 0.0047). Conclusion Citrullinated residues are present in immunoreactive epitopes of natural human filaggrin. AFA can be readily detected by citrullinated peptides in an LIA-based test, resulting in high specificity and positive predictive value for RA. The LIA could serve as a user-friendly alternative to existing immunofluorescence tests and AFA immunoblot techniques. Given its complementarity to RF, this test can be a valuable tool in the differential diagnosis of arthritis.

Clarithromycin (CAM) is a well-known macrolide antibiotic available as a generic drug. CAM is traditionally used for many types of bacterial infections, treatment of Lyme disease and eradication of gastric infection with Helicobacter pylori. Extensive preclinical and clinical data demonstrate a potential role for CAM to treat various tumours in combination with conventional treatment. The mechanisms of action underlying the anti-tumour activity of CAM are multiple and include prolonged reduction of pro-inflammatory cytokines, autophagy inhibition, and anti-angiogenesis. Here, we present an overview of the current preclinical (in vitro and in vivo) and clinical evidence supporting the role of CAM in cancer. Overall these findings justify further research with CAM in many tumour types, with multiple myeloma, lymphoma, chronic myeloid leukaemia (CML), and lung cancer having the highest level of evidence. Finally, a series of proposals are being made to further investigate the use of CAM in clinical trials which offer the greatest prospect of clinical benefit to patients.

Cimetidine, the first H2 receptor antagonist in widespread clinical use, has anti-cancer properties that have been elucidated in a broad range of pre-clinical and clinical studies for a number of different cancer types. These data are summarised and discussed in relation to a number of distinct mechanisms of action. Based on the evidence presented, it is proposed that cimetidine would synergise with a range of other drugs, including existing chemotherapeutics, and that further exploration of the potential of cimetidine as an anti-cancer therapeutic is warranted. Furthermore, there is compelling evidence that cimetidine administration during the peri-operative period may provide a survival benefit in some cancers. A number of possible combinations with other drugs are discussed in the supplementary material accompanying this paper.

Objective To investigate the presence of citrullinated proteins in the synovial membrane of patients with rheumatoid arthritis (RA) and controls, and to analyze a possible relationship with antifilaggrin autoantibody (AFA) reactivity. Methods Synovial biopsy samples were obtained from 88 consecutive patients undergoing needle arthroscopy for knee synovitis associated with RA (n = 36), spondylarthropathy (n = 35), osteoarthritis (n = 9), or other diagnoses (n = 8). Tissue sections were stained with 2 different anticitrulline polyclonal antibodies and an antifilaggrin monoclonal antibody (mAb). The phenotype of citrulline-positive cells and the colocalization with affinity-purified AFA were investigated by double immunofluorescence on frozen sections. Results Studies with the first antibody showed that citrulline is expressed intracellularly in the lining and sublining layers of RA synovial tissue. Staining with the second antibody, monospecific for proteins containing modified citrulline, and with anti–inducible nitric oxide synthetase confirmed the presence of citrullinated proteins rather than free citrulline in the synovium. Citrulline-positive cells were detected in 50% of the RA patients (18 of 36) but in none of the controls (0 of 52). The anticitrulline reactivity colocalized with affinity-purified AFA reactivity, although stainings with the antifilaggrin mAb indicated the absence of filaggrin in the synovium. Conclusion Intracellular citrullinated proteins, which are not recognized by an antifilaggrin mAb, are expressed in RA but not in control synovium. The high specificity of this finding and the colocalization with AFA reactivity boost the interest in citrullinated proteins as possible triggers of autoimmune responses in RA. Moreover, this is the first description of a specific histologic marker for RA synovium.

Optimal application of biological mass spectrometry (MS) in combination with two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of human cerebrospinal fluid (CSF) can lead to the identification of new potential biological markers of neurological disorders. To this end, we analyzed a number of 2-D PAGE protein spots in a human CSF pool using spot co-localization, N-terminal sequencing, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and nanoliquid chromatography-electrospray ionization-time of flight-mass spectrometry (nanoLC-ESI-TOF-MS) with tandem MS switching. Our constructed CSF master contained 469 spots after image analysis and processing of 2-D gels. Upon visual inspection of our CSF master with the CSF pattern available on the ExPASy server, it was possible to locate and annotate 15 proteins. N-terminal sequence analysis and MALDI-MS peptide mass fingerprint analysis of both silver- and Coomassie Brilliant Blue (CBB) G-250-stained protein spots after in situ trypsin digest not only confirmed nine of the visually annotated spots but additionally resolved the identity of another 13 spots. Six of these proteins were not annotated on the 2-D ExPASy map: complement C3 alpha-chain (1321-1663), complement factor B, cystatin C, calgranulin A, hemoglobin beta-chain, and beta-2-microglobulin. It was clear that MALDI-MS identification from CBB G-250-stained, rather than from silver-stained, spots was more successful. In cases where no N-terminal sequence and/or no clear MALDI-MS result was available, nanoLC-ESI-TOF-MS and tandem MS automated switching was used to clarify and/or identify these protein spots by generating amino acid sequence tags. In addition, enrichment of the concentration of low-abundant proteins on 2-D PAGE was obtained by removal of albumin and immunoglobulins from the CSF pool using affinity chromatography. Subsequent analysis by 2-D PAGE of the fractionated CSF pool showed various new silver-stainable protein spots, of which four were identified by nanoLC-ESI-TOF-MS and tandem MS switching. No significant homology was found in either protein or DNA databases, indicating than these spots were unknown proteins.

A procedure for fast and precise molecular weight, purity, and base composition determination of oligonucleotides (in the size range of 20−120 bases) using capillary zone electrophoresis (CZE) coupled by an electrospray interface (ES) on-line to a quadrupole-inlet time-of-flight mass spectrometer (Q-TOFMS) is described. The detection limit for the 5‘-monophosphate nucleotides dAMP and TMP was 30 fmol injected for single MS and 400 fmol for a collision-induced daughter ion spectrum (CID-MS/MS). Injection of 1 pmol was the detection limit for the oligonucleotides. The use of the CZE as an injection interface for oligonucleotides successfully removed metal ion adducts, greatly enhancing the sensitivity and the precision of the mass detection without the need for sample preparation steps. Using electrophoretic separation in an ammonium carbonate buffer system (0.1 M, pH 9.68), these metal adducts were removed and exchanged for volatile ammonium ions. A simple sample stacking technique was developed in order t...

Rheumatoid factor (RF) is found commonly in patients with systemic lupus erythematosus (SLE), and has been associated with a more benign disease course.1,2 Anti-citrullinated peptide antibodies (ACPA) are more specific for rheumatoid arthritis (RA).3–5 Several assays for ACPA detection have been developed: among others, an enzyme linked immunosorbent assay (ELISA) for anti-cyclic citrullinated peptide (anti-CCP) antibodies3 and a line immunoassay (LIA) for antibodies to peptide A (pepA) and peptide B (pepB), two synthetic citrullinated peptides.4 Few reports exist about the presence of ACPA in SLE. Although patients with SLE are often part of the control group when determining the specificity of ACPA for RA, SLE alone is seldom studied. Mediwake et al found that 3/66 …

Despite the widespread use of cyclosporine A (CsA), its mechanism of action and side effects are not yet completely understood. There exists a large body of evidence suggesting that disturbance of calcium homeostasis is a critical step in the cascade of cellular and molecular events induced by the drug. As recently shown in our laboratory by two-dimensional protein gel electrophoresis (2-DE) analysis of kidney homogenates, CsA induced numerous changes in several kidney proteins. One kidney protein in particular was shown to be strongly down-regulated by the drug. In this work we report the identification of the strongly decreased kidney protein as calbindin-D 28kDa, a vitamin D-dependent calcium-binding protein associated with calcium handling by cells. The assignment of the down-regulated protein spot is based on its internal amino acid sequence analysis and its specific reaction with a monoclonal antibody raised against calbindin-D 28kDa. In kidney homogenates of male Wistar rats treated with 50 mg/kg/d CsA for up to 28 days, calbindin levels were measured by ELISA and were shown to be continuously decreased with prolonged CsA treatment. To our knowledge, this is the first report describing the effect of CsA on kidney calbindin-D 28kDa protein levels. Further studies are needed to elucidate whether the CsA-mediated down-regulation of the calcium-binding protein calbindin-D 28kDa may be a critical factor for the renal adverse effects induced by this drug.

Dear Editors, After several patients asked our organization, the Anticancer Fund, www.anticancerfund.org, about GcMAF as a cancer treatment, we looked for the evidence supporting its use in cancer. The literature showed us striking issues and inconsistencies. We would like to comment on the article from Yamamoto et al. published in your journal in 2008 [1]. It is claimed that eight colorectal cancer patients were successfully treated with GcMAF, a protein claimed to be discovered by the authors. “Treatment success” was determined by Nagalase in serum. Nagalase is supposed to deglycosilate naturally occurring GcMAF in cancer patients so that it is incapable of activating macrophages to fight cancer. GcMAF manufactured by Yamamoto might be unaffected by Nagalase. This article was published in parallel to two other articles by the same group in other journals, claiming that their product (GcMAF) successfully treated prostate [2] and breast cancer [3]. In 2009, they published another article reporting that GcMAF successfully treated HIV, again determining success with Nagalase [4]. In the cancer-related articles, the authors claim Nagalase is exclusively produced by cancer cells as opposed to the HIV-related article where they claim Nagalase is a viral component. The authors do not give the most basic information on the disease of these patients: No TNM, no stage, no histology. They determined that these patients had metastatic disease, based exclusively on an elevated level of serum Nagalase. Nagalase is not a criterion to define metastatic disease in the TNM classification of cancer [5]. No key opinion leader has validated its use in oncology. The claim “Although their serum Nagalase activities indicated that they have significant amounts of metastasized tumor cells, CT did not detect metastasized tumor lesions in other organs” means these patients did not have residual disease before starting GcMAF. All evidence cited to justify Nagalase use in this trial is publications by the same group (33 references that are cited 155 times against the 18 times the remaining 15 references are). We have found the following about Yamamoto’s work: The Nagasaki and the Hyogo Immunotherapy Research Groups, that gave IRB approval for these trials, do not exist except in Yamamoto’s clinical papers. Three purported members of these groups, including one chairman, informed us they are not part of these groups and that they have never been involved in Yamamoto’s activities. Other members of these IRBs could not be found. Yamamoto’s co-authors in these papers could not be found. We contacted the sponsors of these trials (US Public Health Service and the Elsa U. Pardee Foundation), and we found that they did not support them. They only supported Yamamoto’s early preclinical work while he was affiliated to other institutions rather than his Socrates Institute for Therapeutic Immunology. This article also contains many mistakes and uses invalid endpoints: Many references are used inappropriately and most do not support the authors’ claims. For example: The assertion “Administration of 100 nanogram (ng) GcMAF to humans results in the maximal activation of macrophages with 30-fold increased ingestion index and 15-fold increased superoxide-generating capacity” has no basis. This statement is supported by reference 33, which is an animal experiment in which these numbers are not mentioned. Furthermore, it has been demonstrated that naturally occurring GcMAF in cancer patients has a concentration of approximately 4 mg/L, making the 100 ng proposed by Yamamoto meaningless, plus it is not deglycosilated [6]. Without adequate randomized controlled clinical trials, the assertion “Since the molecular structure of GcMAF is identical to that of the native human MAF, GcMAF (even 5-fold higher therapeutic dosage) produced no side effects” is wrong and dangerous. It is well established that injection of some human products (i.e., insulin and epinephrine) into patients can be lethal. The conclusions make no sense: “The curative rate measurements of tumors during GcMAF therapy and the estimation of the degree of tumor differentiation have been possible because of the availability of precision measurement of serum Nagalase”. Yamamoto proved that Nagalase failed as a disease measurement method when it was compared to CT scans at the beginning of the study. However, at the end of the study, when the CT scans matched the authors’ speculations, CT scans were again reported. The degree of tumor differentiation can only be determined by histopathology, which was not reported in this or their other articles (prostate and breast cancer articles). These results cannot be scientifically validated as they contradict established tenets in oncology.

Objectives: To calculate the probabilities for rheumatoid arthritis in a consecutive cohort of patients during diagnostic investigation. Different logistic regression models evaluating the value of human leucocyte antigen (HLA)-shared epitope determination and testing for rheumatoid factor and anti-citrullinated protein/peptide antibodies (ACPA) were fitted. Methods: 1003 consecutive patients were included in the study, presenting a new diagnostic problem for which rheumatoid arthritis was included in the differential diagnosis. All patients were tested for ACPA, rheumatoid factor and HLA-shared epitope. Results: After 1 year, diagnoses were established: 153 patients had definite rheumatoid arthritis and 629 patients had rheumatoid arthritis excluded. Rheumatoid factor, used as a continuous marker, is useful in evaluating the probability for rheumatoid arthritis. Combined rheumatoid factor and shared epitope testing may provide additional predictive information, but combined ACPA and rheumatoid factor testing is superior. The redundancy of shared epitope testing in a model that includes ACPA testing can be explained by the high association between ACPA and shared epitope both in patients with rheumatoid arthritis and in those with non-rheumatoid arthritis. The value of rheumatoid factor testing increased if patients presented with at least one swollen joint at baseline. Conclusion: Valid probabilities for rheumatoid arthritis during routine diagnostic investigation were calculated, and showed that the potential additional value of shared epitope testing disappears when ACPA testing is available. Combined rheumatoid factor and ACPA testing is useful, especially when rheumatoid factor is considered as a continuous parameter reflecting an increasing probability for rheumatoid arthritis at higher rheumatoid factor titres. The value of (continuous) rheumatoid factor testing increases when the a priori chance is higher.

Background: Autoantibodies such as rheumatoid factor (RF) and anticitrullinated protein antibodies can be detected in rheumatoid arthritis (RA) sera. Objective: To determine the diagnostic values of RF, anticitrullinated protein antibodies, and the shared epitope (SE), and their associations with radiological progression rates and extra-articular manifestations. Methods: Population 1 consisted of sera from 315 patients, consecutively sent for detection of anticitrullinated protein antibodies, of which 264 were used to determine the sensitivity and specificity of RF and of antibodies against three synthetic citrullinated peptides: peptide A (pepA), peptide B (pepB), and CCP2. Population 2 consisted of sera from 180 longstanding RA patients and was used to determine associations of RA associated antibodies and the SE with radiological progression rates and extra-articular manifestations. Antibodies to pepA and pepB were detected by line immunoassay, and antibodies to CCP2 by ELISA. HLA Class II typing was performed by LiPA. Results: In population 1, we defined adapted cut offs corresponding to a specificity of ⩾98.5%. This yielded the following sensitivities: RF 12.8%; anti-pepA antibodies 63.6%; anti-pepB antibodies 54.2%; and anti-CCP2 antibodies 73.7%. In population 2, significant differences in radiological progression rates were found between positive and negative patients for different RA antibodies and the SE. RF, but not anticitrullinated protein antibodies or the SE, were more frequent in patients with extra-articular manifestations. Conclusion: A valid comparison of RA associated antibodies shows superior sensitivity of the anticitrullinated protein antibodies compared with RF. The presence of RA associated antibodies and the SE are indicative for poorer radiological outcome, and presence of extra-articular manifestations is associated with RF but not with anticitrullinated protein antibodies.

Rheumatoid factor (RF) is currently the most accepted laboratory test for rheumatoid arthritis (RA) and is part of the revised American College of Rheumatology (ACR) classification criteria for RA (1). The specificity of RF is, however, often low (2)(3)(4)(5). A newer diagnostic marker for RA is anti-citrullinated peptide antibodies (ACPAs), which can be identified by tests such as a line immunoassay (LIA™) for the detection of anti-pepA and anti-pepB antibodies (6), the anti-cyclic citrullinated peptide ELISA (7), an ELISA using citrullinated recombinant rat filaggrin (8), and an ELISA using deiminated fibrinogen (9). ACPAs have excellent specificity (89–100%) for RA, with good sensitivity (41–80%) (3)(4)(5)(6)(7)(10)(11)(12)(13)(14)(15). Furthermore, the HLA shared epitope (SE) has been described, which is found more often in RA patients than in controls (16)(17)(18). Most studies of these newer tests have used control populations consisting of selected groups of patients with defined diseases and healthy controls. This does not represent real-life clinical practice because the composition of the control group does not reflect the natural prevalences of diseases in cases for which serologic markers for RA are requested. Data about specificity, positive predictive value (PPV), and negative predictive value (NPV) are thus hard to interpret. We designed the present study to reflect everyday rheumatology practice. In this prospective study, we included 1003 consecutive patients in three academic and nonacademic centers: the Department of Rheumatology, Ghent University Hospital (Ghent, Belgium); the Locomotor Center, Elisabeth Hospital (Sijsele, Belgium); and the Department of Rheumatology, St-Augustinus Hospital (Wilrijk, Belgium). Patients were entered in the study when they presented with a new diagnostic problem for which RA was included in the differential diagnosis. This …

Objectives: To study associations between antinuclear antibodies (ANA) and signs/symptoms in patients with systemic lupus erythematosus (SLE). Methods: A consecutive cohort of 289 patients with SLE was included; 235 fulfilled ACR criteria for SLE and were further analysed. ANA profiles were determined by line immunoassay and by indirect immunofluorescence on Crithidia luciliae. An extensive list of signs/symptoms was evaluated. Results: Five clusters of antibodies were defined by cluster analysis: 1 - antibodies to SmB, SmD, RNP-A, RNP-C, and RNP-70k; 2 - antibodies to Ro52, Ro60, and SSB; 3, 4, and 5 - antibodies to ribosomal P, histones and dsDNA, respectively. Significant associations ( pless than or equal to 0.01) were found between anti-RNP-70k, anti-RNP-A, anti-RNP-C and Raynaud's phenomenon, between anti-RNP-A, anti-RNP-70k and leucopenia, and between anti-RNP-A, anti-RNP-C and a lower prevalence of urine cellular casts. Anti-SSA, anti-SSB were associated with xerostomia, and anti-SSB with pericarditis. Antibodies to ribosomal P were associated with haemolytic anaemia, leucopenia, and alopecia. Patients with anti-dsDNA antibodies had a higher risk for cellular casts and a lower risk for photosensitivity. Antihistone antibodies were associated with arthritis. Conclusions: In a large and consecutive cohort of patients with SLE, clusters of antibodies were identified. Previously reported associations of antibodies with symptoms were confirmed and new associations found.

Objective: To determine whether anti-Ro52 is an independent serum marker in connective tissue disease. Methods: Over a two year period, 1727 consecutive antinuclear antibody (ANA) positive serum samples were analysed in parallel by double immunodiffusion with thymus/spleen nuclear extract and by line immunoassay with recombinant Ro52, recombinant La/SSB, and natural Ro60. Sera that were only reactive towards Ro52 were further analysed by a variety of additional anti-SSA/Ro detection methods and by specific anti-Ro52 and anti-Ro60 assays. Natural purified SSA/Ro was analysed by immunoblot and protein sequencing. Results: Analysis of natural purified SSA/Ro (Immunovision, Springdale, AR) showed only Ro60 and no immunoreactive Ro52. Consequently, assays based on this substrate only identify sera with anti-Ro60 reactivity. Twenty serum samples showed anti-Ro52 without anti-Ro60 and anti-SSB/La on line immunoassay. By additional testing, 2/20 sera were found positive for anti-Ro60 reactivity. The remaining 18 sera were not identified by any of the classical anti-SSA/Ro assays and were considered to be reactive only with Ro52 and not with Ro60. This anti-Ro52 reactivity was confirmed by natural and recombinant Ro52 in 16/18 cases. 12/18 sera corresponded to connective tissue diseases. Conclusion: Anti-Ro52 positive sera without any evidence of anti-Ro60 and anti-La/SSB reactivity can be considered as an independent group that is systematically missed by classical anti-SSA/Ro detection methods owing to a bias towards anti-Ro60 reactivity. The anti-Ro52 sera are precipitin negative, not retrieved by SSA/Ro enzyme linked immunosorbent assays (ELISAs) based on natural SSA/Ro, and show no specific ANA fluorescence staining pattern. These findings together with the clinical data indicate that anti-Ro52 should be considered as an additional and independent serum marker.

Cryoablation of tumors involve the insertion of a probe into a tumor nodule to administer tissue-ablative cold temperatures. Its mechanism of action has been attributed to the mechanical forces of crystallization, the osmotic changes due to crystallization, and the ischemic effects of microvascular injury. This leads to immunogenic tumor cell death resulting in cross presentation of tumor-associated antigens (TAA) of the cryo-killed tumor cells by antigen-presenting cells (APC) to the immune system. This in turn leads to the recruitment of TAA-specific T cells, which are powerless because of the strong immune suppression in the tumor microenvironment. We found that Meriva, a Curcumin derivate with high bioavailability, reduced immune suppression by IL-6, resulting in the activation of T cell responses to TAA Mage-b and Survivin (highly expressed by tumors and metastaess), and a nearly complete elimination of metastatic breast cancer (4T1 model) when combined with Cryoablation. In addition, the overall survival of mice that received Cryoablation and Meriva was significantly increased compared to the saline group. Our results demonstrate that Meriva is a potent agent to reduce immune suppression by IL-6 and to stimulate TAA-specific T cells in a tumor microenvironment. Moreover, Meriva might be useful in other combination therapies that induce immunogenic tumor cell death like adriamycin or radiation, or with vaccines that are hampered by immune suppression in the TME.

Arginine residues in RG-rich proteins are frequently dimethylated posttranslationally by protein arginine methyltransferases (PRMTs). The most common methylation pattern is asymmetrical dimethylation, a modification important for protein shuttling and signal transduction. Symmetrically dimethylated arginines (sDMA) have until now been confined to the myelin basic protein MBP and the Sm proteins D1 and D3. We show here by mass spectrometry and protein sequencing that also the human Sm protein B/B' and, for the first time, one of the Sm-like proteins, LSm4, contain sDMA in vivo. The symmetrical dimethylation of B/B', LSm4, D1, and D3 decisively influences their binding to the Tudor domain of the "survival of motor neurons" protein (SMN): inhibition of dimethylation by S-adenosylhomocysteine (SAH) abolished the binding of D1, D3, B/B', and LSm4 to this domain. A synthetic peptide containing nine sDMA-glycine dipeptides, but not asymmetrically modified or nonmodified peptides, specifically inhibited the interaction of D1, D3, B/B', LSm4, and UsnRNPs with SMN-Tudor. Recombinant D1 and a synthetic peptide could be methylated in vitro by both HeLa cytosolic S100 extract and nuclear extract; however, only the cytosolic extract produced symmetrical dimethylarginines. Thus, the Sm-modifying PRMT is cytoplasmic, and symmetrical dimethylation of B/B', D1, and D3 is a prerequisite for the SMN-dependent cytoplasmic core-UsnRNP assembly. Our demonstration of sDMAs in LSm4 suggests additional functions of sDMAs in tri-UsnRNP biogenesis and mRNA decay. Our findings also have interesting implications for the understanding of the aetiology of spinal muscular atrophy (SMA).

The Sm proteins B/B′, D1, D2, D3, E, F, and G are components of the small nuclear ribonucleoproteins U1, U2, U4/U6, and U5 that are essential for the splicing of pre-mRNAs in eukaryotes. D1 and D3 are among the most common antigens recognized by anti-Sm autoantibodies, an autoantibody population found exclusively in patients afflicted with systemic lupus erythematosus. Here we demonstrate by protein sequencing and mass spectrometry that all arginines in the C-terminal arginine-glycine (RG) dipeptide repeats of the human Sm proteins D1 and D3, isolated from HeLa small nuclear ribonucleoproteins, contain symmetrical dimethylarginines (sDMAs), a posttranslational modification thus far only identified in the myelin basic protein. The further finding that human D1 individually overexpressed in baculovirus-infected insect cells contains asymmetrical dimethylarginines suggests that the symmetrical dimethylation of the RG repeats in D1 and D3 is dependent on the assembly status of D1 and D3. In antibody binding studies, 10 of 11 anti-Sm patient sera tested, as well as the monoclonal antibody Y12, reacted with a chemically synthesized C-terminal peptide of D1 containing sDMA, but not with peptides containing asymmetrically modified or nonmodified arginines. These results thus demonstrate that the sDMA-modified C terminus of D1 forms a major linear epitope for anti-Sm autoantibodies and Y12 and further suggest that posttranslational modifications of Sm proteins play a role in the etiology of systemic lupus erythematosus.