Indirect Immunofluorescence Method Research Articles

The protective mechanism of quercetin-3-O-β-D-glucuronopyranoside (QGC) in H2O2-induced injury of feline esophageal epithelial cells.

Quercetin-3-O-β-D-glucuronopyranoside (QGC) is a flavonoid glucoside extracted from Rumex Aquaticus. Recent studies have shown that QGC exhibits anti-inflammatory, anti-oxidateve effect in vivo and cytoprotective effect in vitro. Reactive oxygen species (ROS), at low concentration, play role as a primary signal or second messenger, however, at high concentration, ROS are cytotoxic. In this study, we investigated the protective mechanism of QGC in H2O2-induced injury of Feline Esophageal Epithelial Cells. Primary-cultured feline esophagus cells were identified by an indirect immunofluorescent staining method using a cytokeratin monoclonal antibody. Cell viability was determined by the conventional MTT reduction assay. Western blot analysis was performed with specific antibodies to investigate the activation of MAPKs, NF-κB, and IκB-α, and the expression of COX-2. When the cells were exposed to 600μM H2O2 medium for 24h, cell viability decreased to 54%. However, when cells were pretreated with 50-150μM QGC for 12h, the viability of cells exposed to H2O2 significantly increased in the dose dependent manner. QGC (50μM, 12h) also inhibited the expression of COX-2 induced by 10μM H2O2 for 24h. We found that treatment of H2O2 activated p38 MAPK and JNK, but not ERK. However QGC inhibited the H2O2-induced p38 MAPK and JNK phosphorylation. In addition, NF-κB was activated by H2O2 and translocated into the nucleus, but QGC inhibited the activation of NF-κB by blocking degradation of IκB. These data suggest that QGC reduces H2O2-induced COX-2 production by modulating the p38 MAPK, JNK, NF-κB signal pathway in feline esophageal epithelial cells.

Detection of antineutrophil cytoplasmic antibodies (ANCAs): a multicentre European Vasculitis Study Group (EUVAS) evaluation of the value of indirect immunofluorescence (IIF) versus antigen-specific immunoassays

ObjectiveThis multicentre study was performed to evaluate the diagnostic accuracy of a wide spectrum of novel technologies nowadays available for detection of myeloperoxidase (MPO) and proteinase 3 (PR3)-antineutrophil cytoplasmic antibodies...

Diagnostic accuracy of two tests for determination of anti-m2 in the diagnosis of primary biliary cirrhosis: Is it possible to predict the course of the disease?

To evaluate the analytical agreement between results obtained from the indirect immunofluorescence methods and from the multiplexed line-blot assay and EliA-M2, to analyze the diagnostic accuracy in a cohort of primary biliary cirrhosis (PBC) patients and in control patients of two different types of tests for anti-M2 and assess whether, with the advent of a quantitative test, the possibility exists to correlate disease activity with the value of AMA. Serum analysis of 67 patients with fluorescence patterns detected on Hep-2 cells suggestive of PBC-related antibodies and three groups of patients (15 PBC, 16 PBC suspect and 48 disease controls) was carried out. All samples were tested by both a qualitative test multiplexed line-blot Autoimmune Liver Disease Profile Euroline and by a quantitative test EliA-M2 IgG. In order to evaluate a possible correlation between the quantitative M2 and disease activity, we divided patients mixed in a further three groups based on the value EliA-M2. For each of these groups were calculated the average values of the main indices of cholestasis. A perfect agreement was shown between the EliA-M2 and the multiplexed line-blot method for AMA detection. All sera of patients with PBC were positive with both tests, with a 100% sensitivity. Forty-seven of the 48 sera of the control group were negative for both tests with a 100% next specificity, and only 70% for the AMA-IIF. We had also observed in the other three groups of patients that the average of the values of γ-glutamyl transpeptidase and alkaline phosphatase increases with the increase of the value EliA-M2. The difference between the mean values of the most significant parameter which the alkaline phosphatase of the three groups is significant, with a statistically significant difference between the first and the third group (p value 0.023). Both the qualitative method Profile Euroline and the quantitative EliA-M2 have a high diagnostic accuracy for PBC, with a specificity higher than the immunofluorescence method. These preliminary data might suggest the possibility of using the dosage EliA-M2 not only in the diagnosis phase but also in the monitoring of disease activity.

An Indirect Immunofluorescence Method Facilitates Detection of Thrombospondin Type 1 Domain-Containing 7A-Specific Antibodies in Membranous Nephropathy.

Thrombospondin type 1 domain-containing 7A (THSD7A) is a target antigen identified in adult membranous nephropathy (MN) along with the major antigen phospholipase A2 receptor 1 (PLA2R1). The prevalence of THSD7A-Ab-positive patients is unknown, and it is unclear whether the clinical presentation differs between patients positive for PLA2R1-Ab or THSD7A-Ab. We screened serum samples of 1276 patients with MN from three different cohorts for the presence of THSD7A-Ab by Western blot analysis and a newly developed indirect immunofluorescence test (IFT). Compared with Western blot analysis, the IFT had a 92% sensitivity and a 100% specificity. The prevalence of THSD7A-associated MN in a prospective cohort of 345 patients with MN was 2.6%, and most were women. In this cohort, the percentage of patients with THSD7A-associated MN and malignant disease significantly exceeded that of patients with PLA2R1-associated MN and malignant disease. In all cohorts, we identified 40 patients with THSD7A-associated MN, eight of whom developed a malignancy within a median time of 3 months from diagnosis of MN. In one patient with THSD7A-associated MN and metastases of an endometrial carcinoma, immunohistochemistry showed THSD7A expression on the metastatic cells and within follicular dendritic cells of the metastasis-infiltrated lymph node. We conclude that the IFT allows sensitive and specific measurement of circulating THSD7A-Ab in patients with MN. Patients with THSD7A-associated MN differ in their clinical characteristics from patients with PLA2R1-associated MN, and more intensive screening for the presence of malignancies may be warranted in those with THSD7A-associated MN.

The ANA-reflex test as a model for improving clinical appropriateness in autoimmune diagnostics.

Reflex tests are widely used in clinical laboratories, for example, to diagnose thyroid disorders or in the follow-up of prostate cancer. Reflex tests for antinuclear antibodies (ANA) have recently gained attention as a way to improve appropriateness in the immunological diagnosis of autoimmune rheumatic diseases and avoid waste of resources. However, the ANA-reflex test is not as simple as other consolidated reflex tests (the TSH-reflex tests or the PSA-reflex tests) because of the intrinsic complexity of the ANA test performed by the indirect immunofluorescence method on cellular substrates. The wide heterogeneity of the ANA patterns, which need correct interpretation, and the subsequent choice of the most appropriate confirmatory test (ANA subserology), which depend on the pattern feature and on clinical information, hinder any informatics automation, and require the pathologist’s intervention. In this review, the Study Group on Autoimmune Diseases of the Italian Society of Clinical Pathology and Laboratory Medicine provides some indications on the configuration of the ANA-reflex test, using two different approaches depending on whether clinical information is available or not. We further give some suggestions on how to report results of the ANA-reflex test.

Assessing the itching intensity using visual analogue scales in atopic dermatitis patients against the background of a therapy with calcineurin inhibitors

Goal. To assess the effect of topical treatment of atopic dermatitis patients with the 0.1% tacrolimus ointment on the itching intensity and skin expression level of growth factor proteins affecting the intensity of cutaneous innervation. Materials and methods. Fifteen patients suffering from atopic dermatitis underwent treatment with the 0.1% tacrolimus ointment. The SCORAD index was calculated to assess the severity of clinical manifestations. The itching intensity was assessed using a visual analogue scale. The skin expression of nerve growth factors, amphiregulin, semaphorin 3A and PGP9.5 protein (a nerve fiber marker) was assessed by the indirect immunofluorescence method. Results. An increased expression of the nerve growth factor and reduced semaphorin 3A expression levels were noted in the patients’ epidermis; there was an increase in the quantity, mean length and fluorescence intensity of PGP9.5+ nerve fibers. As a result of the treatment, the disease severity and itching intensity were reduced, the nerve growth factor expression level was reduced while semaphorin 3A expression level increased in the epidermis, and the mean length and fluorescence intensity of PGP9.5+ nerve fibers was also reduced. A positive correlation among the itching intensity and nerve growth factor expression level, quantity and mean length of PGP9.5+ nerve fibers in the epidermis was revealed, and negative correlation between the itching intensity and semaphorin 3A expression level in the epidermis was established. Conclusion. Topical treatment with the 0.1% Tacrolimus ointment reduces the itching intensity in atopic dermatitis patients, which is related to the therapy-mediated reduction in the epidermis innervation level, decreased expression of epidermal nerve growth factor and increased semaphorin 3A expression level.

Rangelia vitalii, Babesia spp. and Ehrlichia spp. in dogs in Passo Fundo, state of Rio Grande do Sul, Brazil.

Pathogens transmitted by ticks are an emerging problem worldwide, this study aimed to diagnose the causal agents of infection in dogs presenting suspected hemoparasitoses. Fifty-eight dogs with clinical signs such as depression, hemorrhagic diathesis and fever were evaluated regarding clinical presentation, hemogram, blood smears and serological tests, using the indirect immunofluorescence method for the agents Babesia vogeli and Ehrlichia canis and conventional PCR for Babesia spp. (gene 18S rRNA), Rangelia vitalii (gene 18S rRNA) and Ehrlichia spp. (gene dsb). Five (8.6%) of the 58 dogs were serologically positive for Babesia spp. and three (5.1%) for E. canis. Four dogs (6.8%) were positive for R. vitalii through the molecular diagnosis. The PCR products were sequenced and the DNA from R. vitalii was found to be 99% genetically identical to samples of R. vitalii that had been isolated in Brazil. No presence of Babesia spp. or E. canis was observed through PCR on the dogs evaluated here. The results indicate the presence of R. vitalii and exposure to Babesia spp. and Ehrlichia spp. among the dogs analyzed.

Using Yeast Transposon-Insertion Libraries for Phenotypic Screening and Protein Localization.

This protocol details how to use a transposon-insertion library for phenotypic screening and protein localization. The insertion library was generated by mutagenesis of a plasmid-based yeast genomic DNA library by using a multipurpose transposon; the transposon produces gene disruptions, and, by Cre-mediated recombination at lox sites incorporated within the transposon, alleles with an in-frame insertion can be truncated to a residual transposon encoding multiple copies of the hemagglutinin epitope. Insertions are generated in yeast by shuttle mutagenesis. Yeast genomic DNA containing a transposon insertion is released from the library, and the mutagenized DNA sequences are introduced into a desired strain of yeast, where the insertion alleles replace native loci by homologous recombination. The insertion mutants can be screened for phenotypes, and the site of transposon insertion can subsequently be identified in selected mutants by inverse polymerase chain reaction (PCR). In-frame insertions within genes of interest can be truncated to an epitope-tagged allele by Cre-lox recombination, and the subcellular localization of the encoded protein product can be identified by standard methods of indirect immunofluorescence. In summary, the transposon-insertion libraries represent an informative resource for large-scale mutagenesis, presenting a straightforward alternative to labor-intensive targeted approaches for the construction of deletion alleles and fluorescent protein fusions.

A New Immunodot Assay for Multiplex Detection of Autoantibodies in a Cohort of Italian Patients With Idiopathic Inflammatory Myopathies.

Autoantibody detection has been assessed as tool for the diagnosis and the definition of idiopathic inflammatory myopathies (IIM). The aim of the study was to characterize the autoantibody profiling of a cohort of Italian patients with IIM. Sera of 53 adult patients with definite IIM, according to Bohan-Peter criteria, were tested for anti-nuclear autoantibodies (ANA), using indirect immunofluorescence (IIF) method, and for myositis-specific autoantibodies (MSAs) and myositis-associated autoantibodies (MAAs), using two new commercial immunodot assays. MSAs and/or MAAs were detected in 29 of 53 (54.7%) patients with IIM. Twenty-three patients (43.4%) were positive for at least one MSAs: 13 (24.5%) had anti-histidyl-tRNA synthetase autoantibodies (Jo1), 4 (7.5%) had other anti-aminoacyl-tRNA synthetases autoantibodies (anti-ARS), 1 (1.8%) had anti-transcription intermediary factor 1 gamma autoantibodies (anti-TIF1γ), 2 (3.7%) had anti-nuclear helicase protein Mi-2 autoantibodies (anti-Mi-2), 4 (7.5%) had anti-small ubiquitin like modifier activating enzyme heterodimer autoantibodies (anti-SAE). Moreover, 17 patients (32%) were positive for at least one MAAs. Coexisting MSAs and MAAs were observed in 9 of 53 (16.9%) patients, anti-Jo1/SS-A autoantibodies in most cases. Overall sensitivity of immunodot assays was 54.7%, the specificity was almost absolute. At cut-off value of 1:160, the sensitivity of ANA-IIF was 52.8%, increasing to 66% if cytoplasmatic fluorescence reaction was reported. Notably, two (5.7%) ANA-IIF negative patients had MSAs, detected only by immunodot assays. It was possible to identify MSAs otherwise undetectable because of the use of new assays. Immunodot can reveal MSAs even when IIF results are inconclusive or, in some cases, ANA negative.

Evaluation of automated multi-parametric indirect immunofluorescence assays to detect anti-neutrophil cytoplasmic antibodies (ANCA) in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA).

The aim of this multicenter EUVAS study was to evaluate the diagnostic performance of multi-parametric indirect immunofluorescence (IIF) assays to detect anti-neutrophil cytoplasmic antibodies (ANCA) in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). The study included 912 samples from diseased controls and 249 diagnostic samples from GPA (n=183) and MPA (n=66) patients. The performance of two automated multi-parametric assays [Aklides (Medipan/Generic Assays) and EuroPattern (Euroimmun)] combining IIF on cellular and purified antigen substrates was compared with two manual IIF analyses and with commercially available ELISAs for MPO- and PR3-ANCA (Euroimmun). The area under the curve (AUC) of the receiver operating characteristics (ROC) curve to discriminate AAV from controls was 0.925, 0.848, 0.855 and 0.904 for, respectively, the two manual analyses, Aklides and EuroPattern, and 0.959, 0.921 and 0.886 for, respectively, antigen-specific ELISA, antigen-coated beads, and microdot, respectively. Variation in pattern assignment between IIF methods was observed. The performance of IIF depends on the substrate used and the definition of IIF patterns. The performance of automated IIF is improved by multi-parameter testing (combined IIF and antigen-specific testing). Given the variability between IIF methods, the diagnostic importance of this technique is questioned.

CYTOTOXIC LYMPHOCYTES: THE ROLE IN INFLAMMATION IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE EXACERBATION AND REMISSION

Background: Despite the number of publications related to the expression of surface antigens of periphery blood lymphocytes in chronic obstructive pulmonary disease (COPD), algorithm for interpreting of the results and implicating pathogenetic treatments still needs to be developed. Aim: To assess the role of cytotoxic lymphocytes in the maintaining of inflammation in COPD. Materials and methods: To examine immune status in 37 patients with COPD exacerbation or remission and 24 healthy donors (control group), blood cytotoxic T-lymphocytes and NK-cells contents were measured using indirect immunofluorescence method. Absolute and relative numbers of lymphocytes expressing CD3, CD4, CD8, CD16, CD20, CD23, CD25, CD54, CD71, CD72, HLA-DR, CD95 antigens, membrane immunoglobulins M (mIgM) and G (mIgG) were estimated. Results: In COPD., significantly increased numbers of blood cytotoxic lymphocytes were demonstrated independently from the disease stage (p < 0.001). During COPD exacerbation, significant elevations of CD4, CD8, CD20, CD72, NK-cells numbers, serum mIgM and mIgG were demonstrated. During remission, CD20 and CD72 content returned to normal, though, increased numbers of other cytotoxic cells persisted promoting inflammation and progressive damage of pulmonary and bronchial tissues. Conclusion: Observed changes may be due to excessive stimulation of T-cell component of immune system in COPD patients both in exacerbation and remission. Relative reduction of total T-lymphocyte numbers indicates non-specific (non-infectious) inflammation type. High cytotoxic potential of immune system results in pulmonary damage and promotes development of pneumosclerosis and emphysema.

Etiologic and clinical epidemiological features of hemorrhagic fever with renal syndrome (HFRS) in the Krasnodar Krai

The purpose of the study is the investigation of etiological, clinical and epidemiological features of HFRS, caused by Sochi virus in the Krasnodar area, as well as a comparative analysis of the data with those of HFRS, caused by Kurkino virus in the Central Russian regions and Puumala virus in the areas of Volga region. Materials and methods For the identification of HFRS patients, sera from more than 800 acute febrile patients residing in Krasnodar area were examined for hantavirus antibody by IFA with Puumala, Hantaan, Seoul, Sochi, Kurkino and Dobrava viruses. For primary screening there was used the indirect immunofluorescence (ELISA) method with polyvalent cultural antigen as for serotyping of positive sera according to affiliation of antibodies to various Hantaviruses species, there were used ELISA method with monovalent cultural antigens and the neutralization reaction with Puumala, Hantaan, Seoul, Sochi, Kurkino and Dobrava viruses with the use of the method of Inhibition of Focus-Forming Units assay. Clinical and epidemiological studies have been performed on the base of history cases and records of the epidemiological examination. Results of the study. During 2000 - 2013 there were identified 64 patients suffered from HFRS caused by the Sochi virus. The patients resided in 36 settlements of 10 administrative districts of the Krasnodar area. A comparative analysis of clinical, laboratory and epidemiological data of patients with HFRS-Sochi, HFRS-Kurkino and HFRS-Puumala viruses allowed to reveal differences between the clinical (frequency of registration and severity of several symptoms, severity of the course and mortality rate) and epidemiological (prevalence in rural and urban residents, occupational pattern, the seasonality of the disease, conditions of contamination) manifestations. Conclusion There was established the etiological and epidemiological importance of Sochi virus. Sochi virus causes sporadic annual incidence of HFRS in the territory of Krasnodar area. Cases of HFRS caused by the Sochi virus are differ in more severe course of the disease and high lethality rate in comparison with the other two forms of HFRS caused by Puumala and Kurkino viruses in the territory of the European part of Russia.

The relationship between the presence of autoantibodies, indicators of local and systemic inflammation, the serum concentration of B-cell activating factor (BAFF) and the intensity of salivary gland infiltration in patients with primary Sjögren's syndrome - a preliminary study.

ObjectivesThe aim of this study was to find markers related to activation of B cells, which show a correlation with the systemic inflammation markers – erythrocyte sedimentation rate and C-reactive protein and with the intensity of in situ inflammation.Material and methodsForty-one primary Sjögren's syndrome (pSS) patients (33 female, 8 male) of the mean age 52.9 ±15 years were included. A group of 20 healthy volunteers was applied as a control. Erythrocyte sedimentation rate (ESR), concentration of gamma-globulins, C-reactive protein (CRP) and rheumatoid factor (RF) were measured by routine laboratory tests. Titres of antinuclear antibodies (ANAs) were determined by the indirect immunofluorescence method, while anti-SS-A/SS-B antibodies were detected by both the dot-blot method and an enzyme immunoassay. The concentrations of BAFF in sera were measured by sandwich ELISA. Biopsies of minor salivary glands were taken and the focus score (FS) was calculated. Correlations between quantitative variables were assessed using the Spearman correlation coefficient (r).ResultsSerum concentrations of BAFF was significantly higher in the pSS patients than in the control group. The study revealed a statistically significant correlation between ANAs titre and the FS (r = 0.421).Anti-SS-A/Ro and anti-SS-B/La antibodies positively correlated with ESR. There was also a positive correlation between the gamma globulin level and the titres of all tested autoantibodies.ConclusionsThe positive correlation between ANAs and FS confirms the importance of these autoantibodies in the local inflammatory process. The positive correlation between anti-SS-A/SS-B antibodies and ESR suggests involvement of these antibodies in generalization of the inflammatory response. In the pSS group serum concentrations of BAFF were statistically significantly higher than healthy volunteers. All presented results confirm the role of activity of B cells in the course of pSS.

The clinical and immunological indices and their interaction with thyroid status in patients with Graves’ disease depending on thyrocytes peroxidase autoantibodies level

The process of antibody production against thyroid peroxidase and their relation to the complement-mediated cytotoxicity have the most pronounced cytotoxic effect on thyrocytes in Graves’ disease.Aim.To study the clinical and immunological indices and their interaction with thyroid status in patients with Graves’ disease depending on thyrocytes peroxidase autoantibodies level.Material and methods.The study included 35 women aged 18 to 55 years, mean age of 39.1±7.2, with verified diagnosis of Graves’ disease for the first time, before antithyroid therapy. The clinical and immunological parameters and their relationship with thyroid status studied depending on the level of AT-TPO. The definition of thyroid hormones content was measured using immunoradiometricassay analysis with standard test kits. AT-TPO was assessed by ELISA. Population and subpopulation composition of blood lymphocytes was evaluated using the method of indirect immunofluorescence using monoclonal antibodies to CD3, CD4, CD8, CD16, CD19 and HLA-DR. The humoral subset pattern characterized by the relative synthesis of IgA/CD19+, Ig M/CD19+, IgG/CD19+.Results.The most significant changes in population and subpopulation composition of lymphocytes was found in patients with more than 100 IU/L AT-TPO level and characterized by an increase in absolute lymphocyte, relative and absolute number of CD19+ and HLA-DR+cells, and CD3+ and CD8+cells, as compared to the control range and the values identified in group with AT-TPO less than 100 IU/L. A strong negative relationship AT-TPO with the number of T-lymphocytes (r=–0.75; p&lt;0.001) and moderate with the relative synthesis level of IgM (r=–0,53; p=0.017) have been revealed. In Graves’ disease patients with AT-TPO more than 100 IU/l levels the statistically significant relationships were not found.Conclusion.The immunopathogenesis of Graves’ disease is characterized by positive correlation in AT-TPO and indicators of b-cell immunity, and negative — with the parameters of T-cell immunity, regardless of thyrocytes peroxidase autoantibodies concentration.

СHANGES OF Glut1, mTOR AND AMPK1α GENE EXPRESSION IN PANCREATIC LYMPH NODE LYMPHOCYTES OF RATS WITH EXPERIMENTAL DIABETES MELLITUS

With the help of molecular genetic method we have investigated the level of mRNA gene expressions Glut1, mTOR and AMPK1α in PLN in pancreatic lymph nodes (PLN) of rats with streptozotocininduced diabetes mellitus (SIDM) and after administration of metformin. The levels of Glut1, mTOR and AMPK1α mRNA were determined by means of RT-PCR using CFX96™ thermocycler (Real-Time PCR Detection Systems, Bio-Rad, USA). Relative gene expression levels were calculated as ratios to GAPDH reference gene using ΔΔCt method. Statistical analysis was performed with available “Bio-Rad СFX Manager 3.1” software (Bio-Rad, USA). The mTOR+ positive lymphocytes were identified by means of monoclonal antibodies, using an indirect immunofluorescence method. Hyperglycemia was accompanied by transcriptional induction of glucose transporter Glut1 gene (9.9 to 28.9-fold, p < 0.05), and mTOR protein kinase gene (5.3 to 3.3-fold, p < 0.05) in PLN. Development of diabetes was also associated with increase by 24-34% in total mTOR+ cell numbers in PLN at the 5th week of developing diabetes (p < 0.05) and increased concentrations of rapamycin target in the immunopositive cells. Metformin administration to diabetic rats was followed by increased AMPK1α mRNA level of by 87% (p < 0,05) at the 3rd week, and 38-fold (p < 0,05), at the 5th week of SIDM development and inhibition of mTOR expression in PLN (3 to 14.7-fold, p < 0.05) accompanied by a 40 per cent decrease (p < 0.05) in total density of mTOR+ cells in PLN lymph cords of the rats following 5 weeks of SIDM.

Investigation of liver autoantibodies in anticentromere antibody positive patients: A preliminary research

Background: Anticentromere antibody (ACA) is regarded to be a serological marker specific to CREST (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactylia, and telangiectasia) syndrome. ACA is also found in the sera of patients with autoimmune liver disease. In the present work, anti-soluble liver antigenliver pancreas antigen (anti-SLALP), anti-liver cytosolic antigen 1 (anti-LC1), anti-liver kidney microsomal antigen 1 (anti-LKM1), and anti-mitochondrial antibody M2 (AMA-M2) were evaluated in the patients who had positive anticentromere antibody. Material and Methods: A total of 39 patients who were positive anticentromere antibody were enrolled in this study undertaken in the Izmir Katip Celebi University, Ataturk Training and Research Hospital, Microbiology laboratory between January and September 2015. Positive anticentromere antibody and liver autoantibodies were analyzed. Anticentromere antibody and liver autoantibodies were studied by indirect immunofluorescence method (IIF) and immunoblotting method (IB), respectively. The patients who had negative anticentromere antibody were used as a control group. Results: According to the study’s results, positivity was detected in 3 of 39 patients (%7.6) in terms of liver autoantibodies, all of which were AMA-M2. There was no statistically significant difference between ACA and autoimmune liver autoantibodies. Conclusion: In this study, we reported our preliminary experience to provide evidence for the detection of various autoantibodies as potential diagnostic or prognostic tests. Further studies that contain a broad range of patients may contribute to the field.

Computer-Assisted Classification Patterns in Autoimmune Diagnostics: The AIDA Project.

Antinuclear antibodies (ANAs) are significant biomarkers in the diagnosis of autoimmune diseases in humans, done by mean of Indirect ImmunoFluorescence (IIF) method, and performed by analyzing patterns and fluorescence intensity. This paper introduces the AIDA Project (autoimmunity: diagnosis assisted by computer) developed in the framework of an Italy-Tunisia cross-border cooperation and its preliminary results. A database of interpreted IIF images is being collected through the exchange of images and double reporting and a Gold Standard database, containing around 1000 double reported images, has been settled. The Gold Standard database is used for optimization of a CAD (Computer Aided Detection) solution and for the assessment of its added value, in order to be applied along with an Immunologist as a second Reader in detection of autoantibodies. This CAD system is able to identify on IIF images the fluorescence intensity and the fluorescence pattern. Preliminary results show that CAD, used as second Reader, appeared to perform better than Junior Immunologists and hence may significantly improve their efficacy; compared with two Junior Immunologists, the CAD system showed higher Intensity Accuracy (85,5% versus 66,0% and 66,0%), higher Patterns Accuracy (79,3% versus 48,0% and 66,2%), and higher Mean Class Accuracy (79,4% versus 56,7% and 64.2%).

Features of cellular and humoral immunity in boxers with traumatic brain injury

We studied the cellular and humoral immunity in boxers suffering from traumatic brain injuries (TBI). To this end we examined 47 highly skilled amateur boxers who had had history of repeated TBI. The control group consisted of 30 people who were not boxing and did not have history of TBI. Immunological investigation of cellular and humoral immunity included quantitative assessment of the various subpopulations of lymphocytes indirect immunofluorescence method using monoclonal antibodies. In boxers with TBI in comparison with the control group we revealed lymphocytosis, an increase in the percentage of CD3+lymphocytes, CD4+lymphocytes, CD8+lymphocytes, CD20+lymphocytes, decrease of CD16+lymphocytes, increased immunoregulatory index. We also marked both an increase and decrease in the number of lymphocytes with activating phenotype in boxers. Boxers with TBI are characterized by disorders of cellular and humoral immunity. The differences in the composition of the main lymphocyte subpopulations showed a diversity of their involvement in the pathogenesis of traumatic disease of the brain.

Benchmarking human epithelial type 2 interphase cells classification methods on a very large dataset.

This paper presents benchmarking results of human epithelial type 2 (HEp-2) interphase cell image classification methods on a very large dataset. The indirect immunofluorescence method applied on HEp-2 cells has been the gold standard to identify connective tissue diseases such as systemic lupus erythematosus and Sjögren's syndrome. However, the method suffers from numerous issues such as being subjective, time consuming and labor intensive. This has been the main motivation for the development of various computer-aided diagnosis systems whose main task is to automatically classify a given cell image into one of the predefined classes. The benchmarking was performed in the form of an international competition held in conjunction with the International Conference of Image Processing in 2013: fourteen teams, composed of practitioners and researchers in this area, took part in the initiative. The system developed by each team was trained and tested on a very large HEp-2 cell dataset comprising over 68,000 images of HEp-2 cell. The dataset contains cells with six different staining patterns and two levels of fluorescence intensity. For each method we provide a brief description highlighting the design choices and an in-depth analysis on the benchmarking results. The staining pattern recognition accuracy attained by the methods varies between 47.91% and slightly above 83.65%. However, the difference between the top performing method and the seventh ranked method is only 5%. In the paper, we also study the performance achieved by fusing the best methods, finding that a recognition rate of 85.60% is reached when the top seven methods are employed. We found that highest performance is obtained when using a strong classifier (typically a kernelised support vector machine) in conjunction with features extracted from local statistics. Furthermore, the misclassification profiles of the different methods highlight that some staining patterns are intrinsically more difficult to recognize. We also noted that performance is strongly affected by the fluorescence intensity level. Thus, low accuracy is to be expected when analyzing low contrasted images.

Dynamics of expression rates of growth factor proteins in psoriatic patients receiving a phototherapy

Goal. To study the dynamics of expression rates of growth factor proteins in psoriatic patients receiving the PUVA therapy. Materials and methods. The authors conducted a study of 30 patients with psoriasis vulgaris treated with the PUVA therapy. The psoriasis severity and extent of itching were assessed prior to and after the treatment by the PASI index and visual analogue scale, respectively. The expression of semaphorin 3A, amphiregulin, nerve growth factor and PGP 9.5 protein (a nerve fiber marker) in the skin was assessed by the indirect immunofluorescence method. The expression of PGP 9.5 protein was used to assess the quantity and mean length as well as average and total fluorescence intensity of nerve fibers. Results. An increased expression of amphiregulin and nerve growth factor as well as increase in the quantity, mean length and average and total fluorescence intensity of nerve fibers were revealed in the epidermis of psoriatic patients. Following a course of the PUVA therapy, a decrease in the PASI index and extent of itching, reduced expression of amphiregulin and nerve growth factor as well as reduced quantity, mean length and average and total fluorescence intensity of nerve fibers in the epidermis were observed. Direct correlation dependence between the extent of itching, amphiregulin and nerve growth factor expression level and quantity and length of nerve fibers in the epidermis was discovered. Direct correlation dependence between the amphiregulin and nerve growth factor expression level, and average length of nerve fibers in the epidermis was discovered. Conclusion. The itching intensity in psoriatic patients receiving the PUVA therapy is reduced due to the decreased skin expression of the nerve growth factor and amphiregulin.