International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies.

Anti-nuclear antibodies (ANA) are fundamental for the diagnosis of autoimmune diseases, and have been determined by indirect immunofluorescence assay (IIFA) for decades. As the demand for ANA testing increased, alternative techniques were developed challenging the classic IIFA. These alternative platforms differ in their antigen profiles, sensitivity and specificity, raising uncertainties regarding standardisation and interpretation of incongruent results. Therefore, an international group of experts has created recommendations for ANA testing by different methods. Two groups of experts participated in this initiative. The European autoimmunity standardization initiative representing 15 European countries and the International Union of Immunologic Societies/World Health Organization/Arthritis Foundation/Centers for Disease Control and Prevention autoantibody standardising committee. A three-step process followed by a Delphi exercise with closed voting was applied. Twenty-five recommendations for determining ANA (1-13), anti-double stranded DNA antibodies (14-18), specific antibodies (19-23) and validation of methods (24-25) were created. Significant differences between experts were observed regarding recommendations 24-25 (p<0.03). Here, we formulated recommendations for the assessment and interpretation of ANA and associated antibodies. Notably, the roles of IIFA as a reference method, and the importance of defining nuclear and cytoplasmic staining, were emphasised, while the need to incorporate alternative automated methods was acknowledged. Various approaches to overcome discrepancies between methods were suggested of which an improved bench-to-bedside communication is of the utmost importance. These recommendations are based on current knowledge and can enable harmonisation of local algorithms for testing and evaluation of ANA and related autoantibodies. Last but not least, new more appropriate terminologies have been suggested.

Human antibodies induce arthritis in mice deficient in the low-affinity inhibitory IgG receptor Fc gamma RIIB.

Rheumatoid arthritis (RA) is a complex autoimmune disease with a poorly understood pathogenesis. The disease is associated with polyclonal B cell activation and the production of autoantibodies (autoAbs), but there is a longstanding controversy as to whether such Abs contribute to, or are secondary to, the pathogenesis of RA. To address the potential pathogenicity of human RA-associated Abs, we developed a passive transfer model involving mice deficient in the low-affinity inhibitory Fc receptor, FcgammaRIIB. We report that plasma or serum from patients with active RA can induce inflammation and histological lesions in FcgammaRIIB-/- mice consistent with arthritis, and that this pathogenic activity is caused by the immunoglobulin G-rich fraction. Our results suggest that humoral autoimmunity can contribute directly to autoimmune arthritis, and that FcgammaRIIB-/- mice are a promising model to evaluate the arthritogenic potential of human autoAbs.

Epstein-Barr virus reactivation induced myeloperoxidase-specific antineutrophil cytoplasmic antibody (MPO-ANCA)-associated vasculitis.

We present a patient with systemic symptoms including 4 months of dyspnoea worsened with exertion, fatigue, rhinorrhoea, intermittent facial swelling, generalised lymphadenopathy and weight loss. Laboratory studies demonstrated proteinuria and eosinophilia. His serology was consistent with Epstein-Barr Virus (EBV) reactivation. A lymph node biopsy was consistent with EBV-associated reactive lymphoid hyperplasia. He was told to continue symptomatic treatment for EBV infection. After several admissions, vasculitis workup and myeloperoxidase-antineutrophil cytoplasmic autoantibody (ANCA) studies were positive. Evolution of clinical symptoms, laboratory parameters and our literature review suggested the diagnosis of EBV-associated ANCA vasculitis. Steroids were started after the patient continued to deteriorate; the viral load started increasing, so we added valganciclovir with favourable clinical response and no relapse during the follow-up for 6 months. This suggests that with evidence of viraemia (primary or reactivation), antiviral treatment likely has clinical benefit while immunosuppression is being considered.

Tumoral calcinosis without hyperparathyroidism in a patient on continuous ambulatory peritoneal dialysis.

Reports of tumoral calcinosis (TC) in peritoneal dialysis (PD) patients are rare. Reported PD patients with TC also had hyperparathyroidism. A 67-year-old man on continuous ambulatory PD for almost 3 years developed TC of the right wrist and knee and both shoulders and feet. In the 2 years preceding the diagnosis of TC, this patient's serum parathyroid hormone levels were consistently low (17 +/- 12 pg/ mL). Hypercalcemia had been found in 32% of the serum samples, hyperphosphatemia in 91%, and elevated Ca x P product in 78% of the samples. At presentation with TC, serum C-reactive protein was elevated, and serum levels of vitamin D compounds were below normal. Four months after the diagnosis of TC, the patient died with a combination of gastrointestinal and retroperitoneal bleeding episodes and septic events. Tumoral calcinosis may develop in PD patients without hyperparathyroidism. Sustained hyperphosphatemia and high Ca x P product are important in the pathogenesis of uremic TC. Elevated indices of inflammation may accompany TC. Studies are needed to identify other important factors in the pathogenesis of TC in PD patients and to evaluate treatment methods.

Twenty-first Century ethics of medical research involving human subjects: achievements and challenges.

The field of ethics in medical research has seen important developments in the last three decades, but it also faces great challenges in the new century. The purposes of this report are to examine the current status of ethics of medical research involving human subjects and the nature of the ethical challenges facing this research, to identify the weakness of the current system of safeguards for ethical research, and to stress the importance of the ethical character of the researcher, which is the safeguard that has the greatest potential for protecting the research subjects. Researchers appreciate the risks of human medical research that create ethical dilemmas and the need for an ethical compromise in order to proceed with the research. The main elements of the compromise, formulated primarily from experiences in the Second World War, include: (1) the dominant position of the ethical principle of autonomy; (2) the demand for a signed informed consent; (3) the likelihood of improving health with the research protocol, which must be approved by a duly appointed supervising committee; and (4) an acceptable risk/benefit ratio. The main weakness of this set of safeguards is the difficulty with obtaining a truly informed consent. The new challenges to ethical medical research stem from certain types of research, such as genetic and stem cell research, and from the increasing involvement of the industry in planning and funding the research studies. Developing medical researchers with an ethical character and knowledge about ethics in medicine may be the most effective safeguard in protecting participants of medical research experiments.

Glomerulonephritis causing acute renal failure during the course of bacterial infections. Histological varieties, potential pathogenetic pathways and treatment.

To illustrate diagnostic approaches, potential pathogenetic differences, epidemiological implications and therapeutic dilemmas posed by glomerulonephritis (GN) with acute renal failure (ARF) complicating bacterial infections, we analyzed the course of four male patients, aged 53-71 years, who developed GN and ARF following bacterial infections. The first two patients developed GN with immunoglobulin A (IgA) deposits after infections with hospital-acquired methicillin resistant Staphylococcus aureus (MRSA). Clinical, serologic and histological features, classification of GN and treatment differed between the two patients. In the first patient, serological features (transient hypocomplementemia, normal serum protein electrophoresis) and histological findings were consistent with typical post-infectious GN. Treatment with antibiotics alone resulted in normalization of the renal function despite the severity of ARF, which required temporary hemodialysis. In the second patient, serological features (normal serum complement, polyclonal elevation of gamma globulins) and histological picture of the kidneys were characteristic of IgA nephropathy with fibrocellular crescents, and skin histology was consistent with vasculitis. Cyclophosphamide and corticosteroids were added to the antibiotics, with partial improvement of the renal failure. The third patient developed simultaneous acute rheumatic fever and post-streptococcal GN causing severe ARF requiring hemodialysis. Complete recovery of ARF and migratory polyarthritis followed initiation of corticosteroids. The fourth patient developed ARF and cerebral vasculitis following a prolonged course of Streptococcus mutans endocarditis with delayed diagnosis. He also developed multiple serological abnormalities including elevated titers of antineutrophil cytoplasmic antibodies (ANCA), antinuclear antibodies (ANA), anti-phospholipid antibodies, rheumatoid factor, and modest hypocomplementemia. Kidney biopsy revealed ANCA-mediated focal GN with 10% crescents and acute interstitial nephritis. Treatment with cyclophosphamide plus corticosteroids, but not with antibiotics alone, resulted in resolution of both the ARF and the features of cerebral vasculitis. GN following bacterial infections may have various pathogenetic mechanisms, presents complex diagnostic challenges, may be preventable in the case of hospital-acquired MRSA, and, in addition to antibiotics, may require immunosuppressive therapy in carefully selected and monitored cases.

Tumoral Calcinosis of the Neck in a Patient with Systemic Sclerosis.

Tumoral calcinosis (TC) is rare in patients with systemic sclerosis but is associated with morbidity. Paraspinal TC may cause severe pain and potentially devastating neurological deficits. Surgical decompression by removing the TC masses and applying surgical techniques to support the spine have provided substantial relief of the symptoms in the majority of cases. However, death has occurred in the immediate postoperative period and can even occur after several months. Current indications for surgery include intractable neck pain and, most importantly, the development of neurological deficits. We present a patient with systemic sclerosis and symptomatic paraspinal TC in the neck treated conservatively for two years. This case report illustrates conditions permitting the sustained conservative treatment of paraspinal TC in systemic sclerosis patients.

The universe of ANA testing: a case for point-of-care ANA testing.

Testing for total antinuclear antibodies (ANA) is a critical tool for diagnosis and management of autoimmune diseases at both the primary care and subspecialty settings. Repurposing of ANA from a test for lupus to a test for any autoimmune condition has driven the increase in ANA requests. Changes in ANA referral patterns include early or subclinical autoimmune disease detection in patients with low pre-test probability and use of negative ANA results to rule out underlying autoimmune disease. A positive result can lead to further diagnostic considerations. Currently, ANA tests are performed in centralized laboratories; an alternative would be ANA testing at the clinical point-of-care (POC). By virtue of its near real-time data collection capability, low cost, and ease of use, we believe the POC ANA has the potential to enable a new paradigm shift in autoimmune serology testing.

Biosensor for total antinuclear antibody determination at the point-of-care.

Antinuclear antibodies (ANA) are important in diagnosis and follow-up of patients with autoimmune conditions. The current increase in ANA requests is driven by broadening the use of ANA from a test for lupus to a test for diverse autoimmune diseases, but the standard method is protracted, cumbersome and prone to error. We describe an electrochemical method for quantifying total ANA for use as a point-of-care diagnostic aid. In this technology the target autoantigens are derived from a protein/nucleoprotein mixture prepared from an inexpensive source and adsorbed to a porous membrane with high protein binding capacity. Serum is slowly drawn through the membrane comprising the high density autoantigen mixture to induce rapid binding of patient autoantibodies. After rinsing, peroxidase-conjugated anti-IgG is drawn through the membrane followed by rinsing, insertion of an electrode assembly, and addition of the enzyme substrate. Substrate peroxidation is measured by microamperage-level current accompanying electrochemical reduction of the intermediate product. Values are comparable to a standard ANA test but require a total processing time of ~20min. This method has the promise to greatly expand ANA testing in clinical settings for initial patient assessment of autoimmune disease.

IgG4-Related Hypophysitis: Case Report and Literature Review.

IgG4-related hypophysitis is a rare, inflammatory process of the pituitary that mimics more commonly seen pituitary tumors. We report a case of IgG4-related hypophysitis in a 16-year-old female with diabetes insipidus who was found to have IgG4-related hypophysitis based on tissue diagnosis. This entity has not been previously described in a pediatric patient. Recognition of certain inflammatory processes of the pituitary may lead to alternative means of diagnosis and medical management without a biopsy.

Infections and antineutrophil cytoplasmic antibodies: triggering mechanisms.

The precise cause of the antineutrophil cytoplasmic antibodies (ANCA) autoimmunity is not known and is likely to be multifactorial. Infections may trigger formation of ANCA and a fraction of the patients with infection-triggered ANCA develop ANCA-associated vasculitis. Here we discuss some of the proposed mechanisms of ANCA formation during the course of infection. They include initiation of autoimmune response by microbial peptides that are complementary to autoantigens; epigenetic silencing and antigen complementarity leading to upregulation of autoantigen genes; molecular mimicry between bacterial and self-antigens; formation of neutrophil extracellular traps that stimulate immune processes including production of ANCA; and interaction of bacterial components with Toll-like receptors, which leads to formation of mediators affecting the immune responses to infections and can trigger ANCA production. Further work is needed to clarify these mechanisms and develop preventive measures and therapeutic interventions.

Electrochemical biosensor for quantitation of anti-DNA autoantibodies in human serum.

Measurement of serum autoantibody is a critical tool in the diagnosis and management of autoimmune diseases. However, rapid and convenient methods at the point-of care have not been achieved in large part because any one antibody species is a heterogeneous and miniscule fraction of the total serum immunoglobulin displaying identical properties other than its antigen-binding specificity. The present system addresses these challenges by vacuum-mediated transport of diluted serum through an antigen-coated porous membrane. To measure anti-DNA autoantibodies, native DNA was immobilized into a poly(vinylidene fluoride) membrane pre-coated with a synthetic phenylalanine/lysine co-polymer. Flow-through of primary and peroxidase-conjugated secondary antibodies over the course of 3 min enhanced productive antibody-antigen interactions by bringing the reactants into close mutual proximity. Signal was quantified electrochemically during the enzymatic conversion of the tetramethylbenzidine substrate to a charge-transfer complex. The electrochemical signals generated by sera from patients with systemic lupus erythematosus using this device showed good quantitative correlation with a standard enzyme-linked immunosorbent assay and displayed similar detection limits. Inter- and intra-assay variability and electrode uniformity were favorable as was a two-month test of the stability of the DNA-coated membrane. While refining the fluidics requirements of this biosensor will be needed, its capacity to quantify over the course of 30 min anti-DNA antibodies in fresh human serum without background reactivity of normal serum makes this a promising technology as a point-of care device of clinical utility.

Nephropathy in dietary hyperoxaluria: A potentially preventable acute or chronic kidney disease.

Hyperoxaluria can cause not only nephrolithiasis and nephrocalcinosis, but also renal parenchymal disease histologically characterized by deposition of calcium oxalate crystals throughout the renal parenchyma, profound tubular damage and interstitial inflammation and fibrosis. Hyperoxaluric nephropathy presents clinically as acute or chronic renal failure that may progress to end-stage renal disease (ESRD). This sequence of events, well recognized in the past in primary and enteric hyperoxalurias, has also been documented in a few cases of dietary hyperoxaluria. Estimates of oxalate intake in patients with chronic dietary hyperoxaluria who developed chronic kidney disease or ESRD were comparable to the reported average oxalate content of the diets of certain populations worldwide, thus raising the question whether dietary hyperoxaluria is a primary cause of ESRD in these regions. Studies addressing this question have the potential of improving population health and should be undertaken, alongside ongoing studies which are yielding fresh insights into the mechanisms of intestinal absorption and renal excretion of oxalate, and into the mechanisms of development of oxalate-induced renal parenchymal disease. Novel preventive and therapeutic strategies for treating all types of hyperoxaluria are expected to develop from these studies.

Detection of autoantibodies in a point-of-care rheumatology setting.

Autoimmune rheumatic diseases are common and confront society with serious medical, social, and financial burdens imposed by their debilitating nature. Many autoimmune diseases are associated with a particular set of autoantibodies, which have emerged as highly useful to define and classify disease, predict flares, or monitor efficacy of therapy. However, current practice for monitoring autoantibodies is protracted, labor-intensive, and expensive. This review provides an overview on the value of point-of-care (POC) biosensor technology in the diagnosis and management of patients with autoimmune rheumatic diseases. Real-time measurement of autoantibodies will clearly benefit the rheumatology practice in emergency and urgent care settings, where definitive diagnosis is essential for initiation of correct critical care therapy. Immediate serological information in clinic will provide considerable value for long-term patient care and an opportunity for an instant, result-deduced therapeutic action, avoiding delays and improving compliance, especially in field-based and remote areas. We describe the particular autoantibodies that are useful disease and activity markers and would, therefore, be attractive to POC applications. Already existing biosensors and platforms that show promise for autoantibody testing are summarized and comparatively evaluated. As POC assessment is gaining momentum in several areas of patient care, we propose that rheumatology is poised to benefit from this innovative and affordable technology.

Sustained remission of antineutrophil cytoplasmic antibody-mediated glomerulonephritis and nephrotic syndrome in mixed connective tissue disease.

A woman diagnosed with mixed connective tissue disease (MCTD) developed an anti-myeloperoxidase (MPO) antineutrophil cytoplasmic antibody (ANCA) and nephrotic syndrome with normal serum creatinine. Percutaneous kidney biopsy showed pauci-immune glomerulonephritis with superimposed immune complex deposition. After treatment with cyclophophamide and prednisone, proteinuria decreased progressively to a level of 0.4 g/g creatinine, ANCA became undetectable, while serum creatinine remained normal seven years after the beginning of treatment. Sustained remission of nephrotic proteinuria with preserved renal function may follow treatment of ANCA-mediated disease developing in patients with MCTD.

Glomerular disease in patients with infectious processes developing antineutrophil cytoplasmic antibodies.

To identify differences in treatment and outcome of various types of glomerulonephritis developing in the course of infections triggering antineutrophil cytoplasmic antibody (ANCA) formation, we analyzed published reports of 50 patients. Immunosuppressives were added to antibiotics in 22 of 23 patients with pauci-immune glomerulonephritis. Improvement was noted in 85% of 20 patients with information on outcomes. Death rate was 13%. Corticosteroids were added to antibiotics in about 50% of 19 patients with postinfectious glomerulonephritis. Improvement rate was 74%, and death rate was 26%. Two patients with mixed histological features were analyzed under both pauci-immune and post-infectious glomerulonephritis categories. In 9 patients with other renal histology, treatment consisted of antibiotics alone (7 patients), antibiotics plus immunosuppressives (1 patient), or immunosuppressives alone (1 patient). Improvement rate was 67%, permanent renal failure rate was 22%, and death rate was 11%. One patient with antiglomerular basement disease glomerulonephritis required maintenance hemodialysis. Glomerulonephritis developing in patients who became ANCA-positive during the course of an infection is associated with significant mortality. The histological type of the glomerulonephritis guides the choice of treatment. Pauci-immune glomerulonephritis is usually treated with addition of immunosuppressives to antibiotics.

Symptomatic anti-neutrophil cytoplasmic antibody-positive disease complicating subacute bacterial endocarditis: to treat or not to treat?

A 54-year-old man was diagnosed with Streptococcus mutans endocarditis of the mitral valve. Serological tests disclosed the presence of multiple autoantibodies including c-ANCA, anti-PR3 and anti-MPO. While the fever subsided with antibiotics, mental status and renal function deteriorated rapidly. Kidney biopsy revealed pauci-immune glomerulonephritis and acute eosinophilic interstitial nephritis. The abnormal clinical features improved rapidly after addition of corticosteroids and cyclophosphamide to the antibiotics. Immunosuppressive agents may be required in a fraction of the patients with infective endocarditis who develop ANCA and ANCA-mediated renal disease. Histological identification of the type of renal disease is imperative for the choice of the treatment.

Rapid detection of anti-chromatin autoantibodies in human serum using a portable electrochemical biosensor.

The current system employed electrochemical sensor technology for the measurement of autoantibodies in human sera. Anti-chromatin antibodies are an early and sensitive indicator of systemic lupus erythematosus (SLE) and are typically quantified using enzyme-linked immunosorbent assays (ELISA). Electrochemical detection compared favorably with ELISA using a sample of 30 SLE sera (r=0.9), and non-specific binding by normal serum immunoglobulin was undetectable. The electrochemical sensor assay required <20min processing time and utilized a hand-held apparatus with a disposable electrode. These results demonstrate the applicability of this technology to the rapid measurement of a clinically relevant analyte with an apparatus of potential simplicity and low cost.