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Bullous pemphigoid (BP) is an autoimmune blistering disease resulting from autoantibodies directed against BP180 and/or BP230 proteins. BP is traditionally diagnosed based on clinical features, histologic assessment of cutaneous biopsies, direct (DIF) and indirect immunofluorescence (IIF) studies, and/or enzyme-linked immunoassay (ELISA) analysis. We here report, 6 elderly patients who presented with multiple tense bullae, clinically mimicking BP in the absence of detectable autoantibodies by DIF, IIF, and ELISA for BP-180, BP-230, and type VII collagen autoantibodies. All patients in this case series had histological overlap between BP and eczema, potentially representing a unique form of bullous eczema. Patient demographics are listed in Table I along with clinical images (Fig 1). Patients were evaluated in the Autoimmune Blistering Disease Clinic at Emory University. All patients had at least 2 negative DIFs on 2 separate occasions. Only 1 patient was subject to systemic immune suppression at the time of biopsy, and no history of diabetes, peripheral edema, or arthropod assault was noted. Table I Clinical summary of patients 1 to 6

Autoimmune diseases have been reported to develop months to years following drug rash with eosinophilia and systemic symptoms (DRESS) syndrome. However, the development of autoimmune bullous diseases in a patient with a history of DRESS syndrome is extremely rare. We here report the unusual case of mucous membrane epidermolysis bullosa acquisita (MM-EBA) presenting 2 years after lamotrigine-induced DRESS syndrome in a pediatric patient. The patient also developed nonpathogenic autoantibodies directed against BP 180 and BP 230.

Teratoma is frequently found in mixed germ cell testicular tumors in up to 80% of cases, but as a pure component it is reported in only 2–6% of cases. Metastatic disease may be seen in 29–76% of these cases in the adult population. We present the case of a 32 year old male patient diagnosed with a pure testicular teratoma in clinical stage I and a right renal cyst Bosniak IIF, as part of treatment he received chemotherapy, during which, radiological changes were observed at the level of renal cyst suggesting metastatic tumor.

The immunoassays that are available for the serological diagnosis of the more common subtypes of autoimmune blistering diseases such as pemphigus vulgaris (PV) and pemphigus foliaceus (PF) include enzyme-linked immunosorbent assay (ELISA) testing to specific antigens desmoglein (Dsg)1 and Dsg3, direct immunofluorescence (DIF), indirect immunofluorescence (IIF), and immunoblotting. A review of the literature on the biochip assay was conducted. Six studies investigated the validity of a new biochip, mosaic-based, IIF test in patients with pemphigus and demonstrated its relatively high sensitivity and specificity (Dsg3: 97.62-100%, 99.6-100%; Dsg1: 90%, 100%) in comparison with ELISA (Dsg3: 81-100%, 94-100%; Dsg1: 69-100%, 61.1-100%), and/or IIF (PV: 75-100%, 91.8-100%; PF: 67-100%) using suitable substrates. So far, validation studies of the biochip have been conducted in four countries (Germany, Italy, Turkey, and Poland) but none in the southern hemisphere. Caucasian patients were recruited as normal controls for these studies; thus, the diagnostic value of the biochip remains uncertain in population groups of other ethnicities. A range of disease control patients were recruited including patients with linear immunoglobulin A dermatosis, psoriasis, discoid lupus erythematosus, lichen planus, and noninflammatory skin diseases (e.g., squamous cell carcinoma, basal cell carcinoma, and vascular leg ulcers). Prospective studies with control patients from a diverse range of ethnicities are needed to better validate the biochip.

Solid phase assays (SPAs) and automated microscope systems are increasingly used to screen for antinuclear antibodies (ANAs). The goal of this study was to evaluate the performance of three automated ANA screening assays; NOVA Lite HEp-2 using NOVA View® (NV, Inova Diagnostics), an automated indirect immunofluorescence method, EliA™ CTD Screen (Fluorescence Enzyme Immunoassay, FEIA; Thermo Fisher) and QUANTA Flash® CTD Screen Plus (Chemiluminescence immunoassay, CIA; Inova Diagnostics). The assays were performed on 480 diagnostic samples from patients with an ANA-associated rheumatic disease (AARD; systemic lupus erythematosus, primary Sjögren's syndrome, systemic sclerosis, inflammatory myopathy, mixed connective tissue disease) and on 767 samples from diseased and healthy controls. Using cut-offs proposed by the manufacturers, the sensitivity was 95%, 80.5% and 86% for NV, FEIA and CIA, respectively. The corresponding specificity was 61% (NV), 97.5% (FEIA) and 88% (CIA). The sensitivity associated with a specificity of ~95% was 79%, 82% and 78% for NV, FEIA, and CIA, respectively. Receiver operating characteristics (ROC) curve analysis revealed no differences in area under the curve (AUC) between the 3 assays when all diseases were grouped. For Sjögren's syndrome, the AUC was higher for SPAs than for NV, whereas for systemic sclerosis, the AUC was higher for NV than for CIA. For all assays, the likelihood ratio for AARD increased with increasing antibody levels and for double positivity of NV with SPA. In conclusion, the performance of automated SPA and IIF was assay- and disease-dependent. Taking into account antibody levels and combining IIF with SPA adds value. ispartof: Autoimmunity Reviews vol:17 issue:6 pages:533-540 ispartof: location:Netherlands status: published

The automatization of the analysis of Indirect Immunofluorescence (IIF) images is of paramount importance for the diagnosis of autoimmune diseases. This paper proposes a solution to one of the most challenging steps of this process, the segmentation of HEp-2 cells, through an adaptive marker-controlled watershed approach. Our algorithm automatically conforms the marker selection pipeline to the peculiar characteristics of the input image, hence it is able to cope with different fluorescent intensities and staining patterns without any a priori knowledge. Furthermore, it shows a reduced sensitivity to over-segmentation errors and uneven illumination, that are typical issues of IIF imaging.

Over the last two decades, a number of nuclear envelope structures have been recognized as specific targets of antinuclear autoantibodies producing a rim-like staining by indirect immunofluorescence (IIF) and collectively called antinuclear envelope autoantibodies (anti-NE). This peripheral pattern results from autoreactivities directed against proteins within three components of the nuclear envelope: the nuclear lamina (lamins A, B, and C), the nuclear pore complex (glycoprotein (gp)210, p62, and translocated promoter region (Tpr)), and the inner nuclear membrane (lamina-associated polypeptide ( LAP)1, LAP2, and lamin B receptor (LBR)). Although IIF on HEp-2 cells is still the most widely used screening assay for anti-NE, accurate immunochemical tests based on enzyme-linked immunosorbent assay (ELISA) and immunoblotting with purified or recombinant antigens have been recently developed. Anti-NE are generally believed to be not pathogenic, but they can be specific markers of disease, and, therefore, clinically useful as a diagnostic tool. Immunoreactivities against components of the nuclear pore complex have been shown to be associated with primary biliary cirrhosis (PBC), whereas autoantibodies to the lamins are associated with various autoimmune diseases, such as autoimmune hepatitis, systemic lupus erythematosus, and antiphospholipid antibody syndrome. In recent years, data from different groups and countries indicated that particular PBC-specific anti-NE correlate with disease severity and may, therefore, be a marker of poor prognosis. Much less is known, however, about the clinical and prognostic role of anti-NE in other autoimmune conditions.

Antinuclear antibodies were known since 1960s to be common in patients affected by systemic sclerosis (SSc). Anticentromeres (ACA) were first described in 1980 by indirect immunofluorescence (IIF), when dividing human epithelial cultured cells were used as substrate instead of resting rodent liver or kidney cells. The previous use of rat tissue as an antigen source made the identification of ACA very difficult, due to the low numbers of cellular mitosis found on the slide. A large number of cultured cells in metaphase is mandatory for ACA detection, because their IIF pattern is characterized by distinct nuclear speckles in interphase cells, approximately corresponding to the diploid number of chromosomes, with multiple fluorescent speckles in metaphase cells. This IIF pattern marks the centromere of each chromosome. Therefore, if the number of mitotic cells is higher, the possibility of easily discriminating the ACA pattern from other speckled patterns is higher.

Autoantibodies against exocrine pancreas, called pancreatic autoantibodies (PAB), are directed against glycoprotein 2 (GP2), the major zymogen granule membrane protein of pancreatic acinar cells, and presumably other minor components of acinar secretory storage granules of the pancreas such as the CUB and zona pellucida-like domain-containing protein 1 (CUZD1). PAB are routinely detected by indirect immunofluorescence (IIF) using frozen sections of unfixed human pancreas. Their antigen-specific testing is currently based on enzyme-linked immunosorbent assay (ELISA) using eukaryotically expressed recombinant GP2 and IIF based on GP2- and CUZD1-transfected HEK293 cells. PAB are strongly associated with the presence of inflammatory bowel disease (IBD) and in particular Crohn's disease (CD), being present in 29–44% of patients with CD but in less than 10% of ulcerative colitis (UC) patients. Excluding patients with UC and gluten-related disorder (GRD), the specificity of PAB for diagnosing CD is greater than 98%. More studies with assays using PAB-specific targets (GP2 and CUZD1) are necessary to evaluate the clinical and pathophysiological relevance of PAB further, as well as for further stratification of CD patients.

Anti-myeloperoxidase (MPO) antibodies are found in a substantial number of sera that produce a perinuclear fluorescence pattern (p-ANCA) on ethanol-fixed neutrophils. Many sera that produce a p-ANCA pattern by indirect immunofluorescence (IIF) do, however, not contain autoantibodies to MPO. Therefore, a positive test for ANCA by IIF should be followed by antigen-specific assays—in particular, for anti-MPO as generally performed by enzyme-linked immunosorbent assay (ELISA) using purified, commercially available, MPO. A test for anti-MPO is indicated for every patient suspected of vasculitis or glomerulonephritis of unknown origin. The presence of anti-MPO in the correct clinical context strongly suggests necrotizing vasculitis or idiopathic pauci-immune necrotizing and crescentic glomerulonephritis (NCGN). The specificity of anti-MPO for these disorders amounts to 96.3% as detected by ELISA. A positive ELISA test in combination with a positive p-ANCA test by IIF results in a specificity of 98.6%. Anti-MPO are found in 64% of patients with NCGN, 60% of those with microscopic polyangiitis, 50% of patients with eosinophilic granulomatosis with polyangiitis (Churg-Strauss), and 24% of patients with granulomatosis with polyangiitis (formerly Wegener granulomatosis). Most of the patients with the aforementioned diseases who are negative for anti-MPO, with the exception of patients with eosinophilic granulomatosis with polyangiitis, have antibodies to proteinase 3. With respect to the specificity of anti-MPO for the necrotizing vasculitides, it should be noted that the antibodies do occur in drug-induced lupus erythematosus, in patients using antithyroid drugs, and, occasionally, in certain connective tissue diseases, in particular, systemic lupus erythematosus (SLE). Several in vitro and in vivo data, as well as genetic associations, strongly suggest that anti-MPO are involved in the pathophysiology of the associated vasculitides.

Antineutrophil cytoplasmatic autoantibodies (ANCA) with specificity to Proteinase 3 (PR3-ANCA) are serological marker for Wegener's granulomatosis (WG) and are involved in the pathophysiology of the associated vasculitis. PR3 is a neutral serine protease that is localized in the cytoplasmic granules of neutrophils and the lysosomes of monocytes. Priming and apoptosis of neutrophils results in an increased expression of the membrane bound PR3, making them accessible for ANCA. In vitro studies have shown that simultaneous binding of ANCA to PR3 and Fcγ-RII receptors expressed on the cell surface of previously primed neutrophils causes degranulation and an oxidative burst of the neutrophils. Additionally, recently it was demonstrated that ANCA generated against murine PR3 had significant pro-inflammatory effects in wild-type mice, but did not induce a vasculitic disease reminiscent of human ANCA–associated vasculitis. In the clinical setting, the proof of PR3-ANCA is a strong indication for WG but per se does not allow the diagnosis of WG. The sensitivity of ANCA testing is related to the extent, severity, and activity of disease at the time of sampling. The best diagnostic performance is obtained when IIF is combined with PR3-ANCA ELISA (the diagnostic sensitivity and specificity increased up to 73 and 99%, respectively). Even when there is a good correlation between PR3-ANCA ELISA levels and disease activity during the course of the disease, changes cannot be only the basis for therapeutical decisions.

Publisher Summary This chapter describes anti golgi apparatus antibodies (AGAA). In a murine model, AGAA were induced by an isolate of lactate dehydrogenase-elevating virus. AGAA are found in the patients with Sjogren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, and overlap syndromes. Some reports suggest that AGAA are a hallmark of the relationships between viral infections and autoimmunity. AGAA display a characteristic fluorescent staining located in a limited region of the cytoplasm outside the nuclear membrane by indirect immunofluorescent (IIF) techniques.

Publisher Summary This chapter provides an overview of interferon-inducible protein IFI 16 autoantibodies. Intensive efforts to standardize the assay, define associations with disease or clinical presentation, and characterize antigenic and biological properties of these antibodies are included. Autoantibodies against interferon (IFN) inducible protein IFI 16 (anti-IFI 16) that constitute a new antibody specificity of antinuclear antibodies (ANA), occur in about one-third of systemic lupus erythematosus patients. Anti-IFI 16 antibodies can be detected by immunoblot only in those human sera, which manifest ANA by IIF with an appropriate IFI 16-expressing human substrate. The IFI 16 antigen/antibody system is of special interest in autoimmunity because the expression of IFI 16 in cells involved in inflammatory reactions can be induced or increased by IFNγ–– an important mediator of immune response.

Publisher Summary This chapter focuses on antineutrophil cytoplasmic autoantibodies (X-ANCA) that reacts with neutrophil antigens other than proteinase 3 and myeloperoxidase. Detection of ANCA is a useful laboratory diagnostic test for certain small vessel vasculitides and some nonvasculitic clinical syndromes, such as inflammatory bowel disease (IBD). ANCA are identified using indirect immunofluorescence (IIF) techniques. X-ANCA was first applied to the ANCA found in patients with IBD. Most of the autoantigens of the specificities subsumed under the term X-ANCA are found within azurophilic or primary granules of polymorphonuclear leukocytes (PMN). Bactericidal/permeability-increasing protein (BPI) is an important antigen for ANCA in patients with a variety of vasculitides. BPI is also the target for autoantibodies in patients with cystic fibrosis. Anti-BPI antibodies producing characteristic IIF changes are found in some patients with primary bronchiectasis, IBD or certain autoimmune liver diseases.

Publisher Summary This chapter describes the fluorescence methods used for the localization of endogenous calmodulin (CaM) and CaM acceptor or binding sites within fixed tissue sections and cells and in living cells. Indirect immunofluorescence (IIF) consists of incubating fixed tissue sections or cultured cells with a primary antibody directed against the protein of interest—in this case, CaM. Unbound antibody is washed away. A second antibody, directed against the IgG of the animal species in which the primary antibody was developed, is then incubated with the tissue or cells. This second antibody is conjugated with a fluorescent molecule, generally fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (RITC). The fluorochrome conjugated to the second antibody is the entity actually seen in the microscope. After washing to remove unbound antibodies, the preparation is mounted on a microscope slide for viewing. Direct immunofluorescence (DIF) differs from IIF in that the primary antibody is conjugated with fluorochrome rather than a secondary antibody. DIF has the advantage of being a more rapid procedure consisting of only one incubation step. But DIF has several disadvantages. DIF does not give as bright a fluorescence emission as IIF. In addition, DIF necessitates the commitment of primary antibody to fluorescent conjugation; primary antibody cannot be then used for other purposes and its purification is mandatory to maintain acceptably low levels of nonspecific binding.

Summary Mechanisms critical for implantation and survival of normal mouse embryos have been detected by new experimental uses of the homozygous lethal genotype t12/t12. The lethals are known to arrest in the morula stage and are obtained in the same uteri as normal (+/+ and +/t12) blastocysts, from matings between heterozygotes. The zona pellucida was completely lysed on the lethal embryos in utero on day 3 of gestation, whereas their zonas remained entirely intact in vitro. The dead embryos therefore unambiguously demonstrated the existence of a uterine zonalytic factor, possibly an enzyme or enzymic complex. Further experiments in which the dead morulae were employed as an in vivo bioassay, showed a sharp peak of the lysin on day 3; this coincided with a sudden attachment of the accompanying hatched blastocysts to the uterine wall, even though some of the blastocysts had hatched earlier, partly by pulsating and breaking out of their own zonas. The evidence suggests that the uterine lysin acts as an implantation initiating factor (IIF), possibly by changing glycoprotein or other molecules on the surface of blastocyst cells (but not of earlier stages), making the surfaces more adhesive to the uterine wall. The IIF is present in all strains, as shown by zona lysis of unfertilized eggs on day 3. The more rapid lysis of unfertilized than of fertilized zonas reveals a change toward increased resistance of the zona to lysis at fertilization. Deficiency of IIF is the immediate cause of delayed implantation in postpartum and prepuberal pregnancies. IIF production is estrogen dependent and can therefore be influenced or controlled by hormonal manipulation. It is produced in the uterus and is not found in the oviduct. Fractionation of mouse uterine fluid proteins by electrophoresis on polyacrylamide gel columns reveals a number of bands (possibly as many as ten) present in the uterine fluid of various stages but not in serum. Whether any of the bands corresponds to the IIF will be determined from other biochemical studies in progress. The peak IIF time is strain-specific. In some all-normal-genotype strains (e.g., BALB/c), embryos are often still in the morula stage when the IIF is produced. These denuded morulae may become dissociated into two groups of cells, forming viable twins. Alternatively, a pair of whole morulae may adhere, forming one aggregate capable of becoming a single mouse. The latter corresponds to our experimental allophenic mice with genetic mosaicism, produced by aggregating embryos at 37°C after zona removal with pronase. XX ⇆ XY sex chromosomal mosaicism may result; such animals are occasionally intersexes, but usually males or females with sterility infrequent. If still younger (cleavage stage) embryos are denuded by IIF, blastomeres tend to dissociate and die. Thus the IIF constitutes a normal system with an important biological function at implantation. It presents new possibilities for experimental control of fertility, via promotion or prevention of implantation. Some of its effects may, however, cause viable anomalies of development.