Extracellular vesicles (including the subclass exosomes) secreted by cells contain specific proteins and RNA that could be of interest in determining new markers. Isolation/characterization of ...PCa‐derived exosomes from bodily fluids enables us to discover new markers for this disease. Unfortunately, isolation with current techniques (ultracentrifugation) is labor intensive and other techniques are still under development. The goal of our study was to develop a highly sensitive time‐resolved fluorescence immunoassay (TR‐FIA) for capture/detection of PCa‐derived exosomes. In our assay, biotinylated capture antibodies against human CD9 or CD63 were incubated on streptavidin‐coated wells. After application of exosomes, Europium‐labeled detection antibodies (CD9 or CD63) were added. Cell medium from 37 cell lines was taken to validate this TR‐FIA. Urine was collected (after digital rectal exam) from patients with PCa (n = 67), men without PCa (n = 76). As a control, urine was collected from men after radical prostatectomy (n = 13), women (n = 16) and patients with prostate cancer without digital rectal exam (n = 16). Signal intensities were corrected for urinary PSA and creatinine. This TR‐FIA can measure purified exosomes with high sensitivity and minimal background signals. Exosomes can be measured in medium from 37 cell lines and in urine. DRE resulted in a pronounced increase in CD63 signals. After DRE and correction for urinary PSA, CD9 and CD63 were significantly higher in men with PCa. This TR‐FIA enabled us to measure exosomes with high sensitivity directly from urine and cell medium. This TR‐FIA forms the basis for testing different antibodies directed against exosome membrane markers to generate disease‐specific detection assays.
What's new?
The vesicles cast off by cancer cells could serve as billboards advertising the cancer's presence – if we knew how to read them. Detecting markers in prostate cancer vesicles is currently labor intensive. These authors set out to change that, by developing an immunoaffinity technique to expose these cellular markers more easily. Using the assay, they detected two cell surface proteins, CD9 and CD63, in the urine of men who had prostate cancer. They found far less of the markers in men without cancer, men without prostates, and women, suggesting CD9 and CD63 could be useful prostate cancer markers.
Abstract Analysis of autoantibodies (AAB) by indirect immunofluorescence (IIF) remains the hallmark of diagnosing autoimmune diseases despite the introduction of multiplex techniques. Non-organ ...specific AAB are screened in routine diagnostics by IIF on HEp-2 cells. However, IIF results vary due to objective (e.g., cell fixation) and subjective factors (e.g., expert knowledge). Therefore, inter- and intralaboratory variance is relatively high. Standardisation of AAB testing by IIF remains a critical issue in and between routine laboratories and may be improved by automated interpretation systems. An overview of existing interpretation techniques will be given taking into account own data of the first fully automated reading system AKLIDES. The novel system provides fully automated reading of IIF images and software algorithms for the mathematical description of IIF AAB patterns. It can be used for screening and preclassification of non-organ specific AAB in routine diagnostics regarding systemic autoimmune and autoimmune liver diseases. Furthermore, this system paves the way for economic data processing of cell-based IIF assays and can contribute to the reduction of interlaboratory variance of AAB testing. More sophisticated pattern recognition algorithms and novel calibration systems will improve standardised quantifications of IIF image interpretation.
Multiplexing, labeling for multiple immunostains in the very same cell or tissue section in situ, has raised considerable interest. The methods proposed include the use of labeled primary antibodies, ...spectral separation of fluorochromes, bleaching of the fluorophores or chromogens, blocking of previous antibody layers, all in various combinations. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, and scarcity of specialized skills or facilities. We have validated a method based on common primary and secondary antibodies and diffusely available fluorescent image scanners. It entails rounds of four-color indirect immunofluorescence, image acquisition, and removal (stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulfide cleavage and a detergent or by a chaotropic salt treatment, this latter followed by antigen refolding. More than 30 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software. Multiplexing on routine tissue sections is a high throughput tool for in situ characterization of neoplastic, reactive, inflammatory, and normal cells.
Canine leishmaniasis is a major veterinary issue and also a public health challenge due to its zoonotic potential. In this context, serological evaluation is essential for Canine leishmaniasis ...management. Several serological alternatives, such as rapid diagnostic tests, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence antibody test (IFAT), are well established. In fact, the capacity of distinct tests and antigens, evaluated by their sensitivity and specificity, to detect disease is normally considered sufficient for diagnosing Canine leishmaniasis. In this context, we evaluated the seropositivity using 8 different serological tests (ELISA with Leishmania recombinant proteins (rK39, LicTXNPx); soluble promastigote Leishmania antigens (SPLA); commercial ELISA test) in 82 clinically suspect animals from Northern Portugal. The obtained serological data originated 50% of inconclusive serological information with a mixture of seropositive and seronegative results for individual animals. Cut-off independent risk groups were then generated from the serological data to evaluate the clustering of the samples. This analysis originated risk groups that correlated with the most seropositive samples, suggesting that this method might be used, in a cut-off independent manner, to improve conventional serological evaluation. Ultimately, given that no test prioritization exists, the use of any single serological test increases the potential for misdiagnosis, along with all associated risks for the dog as well as public health. The use of a cut-off independent analysis has the potential to improve the predictive values of these tests, enabling a more accurate evaluation of the dog's condition.
Benchmarking HEp-2 Cells Classification Methods Foggia, Pasquale; Percannella, Gennaro; Soda, Paolo ...
IEEE transactions on medical imaging,
10/2013, Letnik:
32, Številka:
10
Journal Article
In this paper, we report on the first edition of the HEp-2 Cells Classification contest, held at the 2012 edition of the International Conference on Pattern Recognition, and focused on indirect ...immunofluorescence (IIF) image analysis. The IIF methodology is used to detect autoimmune diseases by searching for antibodies in the patient serum but, unfortunately, it is still a subjective method that depends too heavily on the experience and expertise of the physician. This has been the motivation behind the recent initial developments of computer aided diagnosis systems in this field. The contest aimed to bring together researchers interested in the performance evaluation of algorithms for IIF image analysis: 28 different recognition systems able to automatically recognize the staining pattern of cells within IIF images were tested on the same undisclosed dataset. In particular, the dataset takes into account the six staining patterns that occur most frequently in the daily diagnostic practice: centromere, nucleolar, homogeneous, fine speckled, coarse speckled, and cytoplasmic. In the paper, we briefly describe all the submitted methods, analyze the obtained results, and discuss the design choices conditioning the performance of each method.
Immunoglobulin M pemphigoid Boch, Katharina; Hammers, Christoph M.; Goletz, Stephanie ...
Journal of the American Academy of Dermatology,
December 2021, 2021-12-00, 20211201, Letnik:
85, Številka:
6
Journal Article
Recenzirano
Pemphigoid diseases are a heterogeneous group of autoimmune blistering disorders characterized by predominant deposition of immunoglobulin G or immunoglobulin A autoantibodies against structural ...proteins of the dermoepidermal junction (DEJ). Sole linear immunoglobulin M (IgM) deposits at the DEJ in pemphigoid diseases have been observed; however, IgM-specific target antigens have not been identified.
Characterization of patients with IgM pemphigoid.
Skin biopsy specimens and sera from IgM-positive patients were assessed using histopathology, direct and indirect immunofluorescence microscopy, enzyme-linked immunosorbent assays, immunoblotting, cryosection assay, complement fixation test, and internalization assays.
Tissue-bound linear IgM deposits along the DEJ and circulating IgM autoantibodies against type XVII collagen (Col17) were detected. These circulating IgM autoantibodies showed no complement activating or blister inducing capacity, but the ability of Col17 internalization ex vivo.
Limited number of patients.
This study provides further evidence for the role of IgM autoantibodies in pemphigoid disease and highlights Col17 as a target antigen in IgM pemphigoid.
To correlate postmortem histology from a patient with macular telangiectasia (MacTel) type 2 with previously recorded clinical imaging data.
Observational clinicopathologic case report.
The ...distribution of retinal blood vessels was used to map the location of serial wax sections in color fundus and optical coherence tomography (OCT) images. Fluorescent immunohistochemistry was used to visualize markers for Müller's cells (vimentin and retinaldehyde-binding protein 1), photoreceptors (L-M opsin, rhodopsin, and cytochrome oxidase 2), and the outer limiting membrane (OLM) (zonula occludens 1 and occludin).
Distribution of specific markers in immunohistochemistry on retinal sections through the fovea in relation to clinical data.
The clinically recorded region of macular pigment loss in the macula correlated well with Müller's cell depletion. The OCT data showed a loss of the photoreceptor inner segment/outer segment (IS/OS) junction in the central retina, which correlated well with rod loss but not with cone loss. Markers for the OLM were lost where Müller's cells were lost.
We have confirmed our previous finding of Müller's cell loss in MacTel type 2 and have shown that the area of Müller's cell loss matches the area of macular pigment depletion. In this patient, the IS/OS junction seen by OCT was absent in a region where rods were depleted but cones were still present.
Innate sensing of microbial components is well documented to occur at many cellular sites, including at the cell surface, in the cytosol, and in intracellular vesicles, but there is limited evidence ...of nuclear innate signaling. In this study we have defined the mechanisms of interferon regulatory factor-3 (IRF-3) signaling in primary human foreskin fibroblasts (HFF) infected with herpes simplex virus 1 (HSV-1) in the absence of viral gene expression. We found that the interferon inducible protein 16 (IFI16) DNA sensor, which is required for induction of IRF-3 signaling in these cells, is nuclear, and its localization does not change detectably upon HSV-1 d 109 infection and induction of IRF-3 signaling. Consistent with the IFI16 sensor being nuclear, conditions that block viral DNA release from incoming capsids inhibit IRF-3 signaling. An unknown factor must be exported from the nucleus to activate IRF-3 through cytoplasmic STING, which is required for IRF-3 activation and signaling. However, when the viral ICP0 protein is expressed in the nucleus, it causes the nuclear relocalization and degradation of IFI16, inhibiting IRF-3 signaling. Therefore, HSV-1 infection is sensed in HFF by nuclear IFI16 upon release of encapsidated viral DNA into the nucleus, and the viral nuclear ICP0 protein can inhibit the process by targeting IFI16 for degradation. Together these results define a pathway for nuclear innate sensing of HSV DNA by IFI16 in infected HFF and document a mechanism by which a virus can block this nuclear innate response.
Objective
Growing evidence suggests increasing frequencies of autoimmunity and autoimmune diseases, but findings are limited by the lack of systematic data and evolving approaches and definitions. ...This study was undertaken to investigate whether the prevalence of antinuclear antibodies (ANA), the most common biomarker of autoimmunity, changed over a recent 25‐year span in the US.
Methods
Serum ANA were measured by standard indirect immunofluorescence assays on HEp‐2 cells in 13,519 participants age ≥12 years from the National Health and Nutrition Examination Survey, with approximately one‐third from each of 3 time periods: 1988–1991, 1999–2004, and 2011–2012. We used logistic regression adjusted for sex, age, race/ethnicity, and survey design variables to estimate changes in ANA prevalence across the time periods.
Results
The prevalence of ANA was 11.0% (95% confidence interval 95% CI 9.7–12.6%) in 1988–1991, 11.4% (95% CI 10.2–12.8%) in 1999–2004, and 16.1% (95% CI 14.4–18.0%) in 2011–2012 (P for trend <0.0001), corresponding to ~22.3 million, ~26.6 million, and ~41.5 million affected individuals, respectively. Among adolescents age 12–19 years, ANA prevalence increased substantially, with odds ratios of 2.07 (95% CI 1.18–3.64) and 2.77 (95% CI 1.56–4.91) in the second and third time periods relative to the first (P for trend = 0.0004). ANA prevalence increased in both sexes (especially in men), older adults (age ≥50 years), and non‐Hispanic white individuals. These increases in ANA prevalence were not explained by concurrent trends in weight (obesity/overweight), smoking exposure, or alcohol consumption.
Conclusion
The prevalence of ANA in the US has increased considerably in recent years. Additional studies to determine factors underlying these increases in ANA prevalence could elucidate causes of autoimmunity and enable the development of preventative measures.
To 1) determine, using contemporary recombinant antigen-based assays, the aquaporin-4 (AQP4)-immunoglobulin G (IgG) detection rate in sequential sera of patients assigned a clinical diagnosis of ...neuromyelitis optica (NMO) but initially scored negative by tissue-based indirect immunofluorescence (IIF) assay; and 2) evaluate the impact of serostatus on phenotype and outcome.
From Mayo Clinic records (2005-2011), we identified 163 patients with NMO; 110 (67%) were seropositive by IIF and 53 (33%) were scored seronegative. Available stored sera from 49 "seronegative" patients were tested by ELISA, AQP4-transfected cell-based assay, and in-house fluorescence-activated cell sorting assay. Clinical characteristics were compared based on final serostatus.
Thirty of the 49 IIF-negative patients (61%) were reclassified as seropositive, yielding an overall AQP4-IgG seropositivity rate of 88% (i.e., 12% seronegative). The fluorescence-activated cell sorting assay improved the detection rate to 87%, cell-based assay to 84%, and ELISA to 79%. The sex ratio (female to male) was 1:1 for seronegatives and 9:1 for seropositives (p < 0.0001). Simultaneous optic neuritis and transverse myelitis as onset attack type (i.e., within 30 days of each other) occurred in 32% of seronegatives and in 3.6% of seropositives (p < 0.0001). Relapse rate, disability outcome, and other clinical characteristics did not differ significantly.
Serological tests using recombinant AQP4 antigen are significantly more sensitive than tissue-based IIF for detecting AQP4-IgG. Testing should precede immunotherapy; if negative, later-drawn specimens should be tested. AQP4-IgG-seronegative NMO is less frequent than previously reported and is clinically similar to AQP4-IgG-seropositive NMO.
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