Screening of biomarkers for prediction of multisite artery disease in patients with recent myocardial infarction.

A few studies have examined biomarkers in patients with myocardial infarction (MI) and peripheral artery disease (PAD), i.e. multisite artery disease (MSAD). The aim of the study was firstly, to associate biomarkers with the occurrence of PAD/MSAD and secondly, if those can, in addition to clinical characteristics, identify MI patients with MSAD.In two prospectively observational studies including unselected patients with recent MI, PAD was defined as an abnormal ankle-brachial index (ABI) score (<0.9 or >1.4). The proximity extension assay (PEA) technique was used, simultaneously analyzing 92 biomarkers with association to cardiovascular disease. Biomarkers were tested for univariate associations with PAD. Random forest was used to identify biomarkers with a higher association to PAD. The additional discriminatory accuracy of adding biomarkers to clinical characteristics was analyzed by the c-statistics. Nine biomarkers were identified as significantly associated with MSAD/PAD in the primary patient cohort, analyzed early after the MI. In the prediction analysis, six biomarkers were identified associated with PAD. Three of these; Tumor necrosis factor receptor (TNFR-1), Tumor necrosis factor receptor 2 (TNFR-2) and Growth Differentiation Factor 15 (GDF-15) improved c-statistics when added to clinical characteristics from 0.683 (95% CI 0.610-0.756) to 0.715 (95% CI 0.645-0.784) in the primary patient cohort with a similar result, 0.729 (95% CI 0.687-0.770) to 0.752 (95% CI 0.771-0.792) in the secondary patient cohort. Biomarkers associated with inflammatory pathways are associated with MSAD in MI patients. Three biomarkers of 92; TNFR-1, TNFR-2 and GDF-15, in this exploratory added information in the prediction of MSAD and emphasis the importance of further studies.

IL-6, sTNF-R2, and CRP in the context of sleep, circadian preference, and health in adolescents with eveningness chronotype: Cross-sectional and longitudinal treatment effects.

BACKGROUND: Inflammation-related processes have emerged as a biological pathway related to adolescent development. This study examined cross-sectional and longitudinal associations of baseline inflammatory markers with sleep, circadian preference, and health at baseline and following treatment. METHODS: Participants included 165 adolescents (58.2% female, mean age 14.7 years, 42.4% taking medication) "at-risk" in at least one domain (emotional, cognitive, behavioral, social, and physical health) who received a sleep-based intervention. Self-reported eveningness as well as total sleep time (TST) and bedtime from sleep diary were assessed at baseline and following treatment. Baseline soluble tumor necrosis factor receptor-2 (sTNF-R2) and interleukin (IL)-6 were assayed from oral mucosal transudate. Baseline C-reactive protein (CRP) was assayed from saliva. RESULTS: At baseline, shorter TST was associated with more emotional risk among adolescents with higher CRP (b = -0.014, p = 0.007). Greater eveningness was related to more behavioral risk in the context of lower IL-6 (b = -0.142, p = 0.005). Following treatment, lower baseline IL-6 was associated with reduced behavioral risk (Χ2 = 8.06, p = 0.045) and lower baseline CRP was related to reduced physical health risk (Χ2 = 9.34, p = 0.025). Baseline inflammatory markers were not significantly associated with sleep, circadian, or other health domain change following treatment. CONCLUSIONS: There was cross-sectional evidence that sleep and circadian dysfunction differentially relate to emotional and behavioral health risk for high and low levels of inflammatory markers. Longitudinal analyses indicated that lower levels of baseline inflammatory markers may be related to better treatment response to a sleep-based intervention.

Effects of TNFα receptor TNF-Rp55- or TNF-Rp75- deficiency on corneal neovascularization and lymphangiogenesis in the mouse.

Tumor necrosis factor (TNF)α is an inflammatory cytokine likely to be involved in the process of corneal inflammation and neovascularization. In the present study we evaluate the role of the two receptors, TNF-receptor (TNF-R)p55 and TNF-Rp75, in the mouse model of suture-induced corneal neovascularization and lymphangiogenesis. Corneal neovascularization and lymphangiogenesis were induced by three 11-0 intrastromal corneal sutures in wild-type (WT) C57BL/6J mice and TNF-Rp55-deficient (TNF-Rp55d) and TNF-Rp75-deficient (TNF-Rp75d) mice. The mRNA expression of VEGF-A, VEGF-C, Lyve-1 and TNFα and its receptors was quantified by qPCR. The area covered with blood- or lymphatic vessels, respectively, was analyzed by immunohistochemistry of corneal flatmounts. Expression and localization of TNFα and its receptors was assessed by immunohistochemistry of sagittal sections and Western Blot. Both receptors are expressed in the murine cornea and are not differentially regulated by the genetic alteration. Both TNF-Rp55d and TNF-Rp75d mice showed a decrease in vascularized area compared to wild-type mice 14 days after suture treatment. After 21 days there were no differences detectable between the groups. The number of VEGF-A-expressing macrophages did not differ when comparing WT to TNF-Rp55d and TNF-Rp75d. The mRNA expression of lymphangiogenic markers VEGF-C or LYVE-1 does not increase after suture in all 3 groups and lymphangiogenesis showed a delayed effect only for TNF-Rp75d. TNFα mRNA and protein expression increased after suture treatment but showed no difference between the three groups. In the suture-induced mouse model, TNFα and its ligands TNF-Rp55 and TNF-Rp75 do not play a significant role in the pathogenesis of neovascularisation and lymphangiogenesis.

Meta-Analysis of Expression Profiling Data Indicates Need for Combinatorial Biomarkers in Pediatric Ulcerative Colitis.

BACKGROUND: Unbiased studies using different genome-wide methods have identified a great number of candidate biomarkers for diagnosis and treatment response in pediatric ulcerative colitis (UC). However, clinical translation has been proven difficult. Here, we hypothesized that one reason could be differences between inflammatory responses in an inflamed gut and in peripheral blood cells. METHODS: We performed meta-analysis of gene expression microarray data from intestinal biopsies and whole blood cells (WBC) from pediatric patients with UC and healthy controls in order to identify overlapping pathways, predicted upstream regulators, and potential biomarkers. RESULTS: Analyses of profiling datasets from colonic biopsies showed good agreement between different studies regarding pathways and predicted upstream regulators. The most activated predicted upstream regulators included TNF, which is known to have a key pathogenic and therapeutic role in pediatric UC. Despite this, the expression levels of TNF were increased in neither colonic biopsies nor WBC. A potential explanation was increased expression of TNFR2, one of the membrane-bound receptors of TNF in the inflamed colon. Further analyses showed a similar pattern of complex relations between the expression levels of the regulators and their receptors. We also found limited overlap between pathways and predicted upstream regulators in colonic biopsies and WBC. An extended search including all differentially expressed genes that overlapped between colonic biopsies and WBC only resulted in identification of three potential biomarkers involved in the regulation of intestinal inflammation. However, two had been previously proposed in adult inflammatory bowel diseases (IBD), namely, MMP9 and PROK2. CONCLUSIONS: Our findings indicate that biomarker identification in pediatric UC is complicated by the involvement of multiple pathways, each of which includes many different types of genes in the blood or inflamed intestine. Therefore, further studies for identification of combinatorial biomarkers are warranted. Our study may provide candidate biomarkers for such studies.

Effects of One Year of Vitamin D and Marine Omega-3 Fatty Acid Supplementation on Biomarkers of Systemic Inflammation in Older US Adults.

BACKGROUND: Observational studies suggest vitamin D and marine ω-3 fatty acid (n-3 FA) supplements are associated with lower systemic inflammation. However, past trials have been inconsistent. METHODS: The randomized, double-blind, placebo-controlled VITamin D and OmegA-3 TriaL (VITAL) tested vitamin D (2000 IU/day) and/or n-3 FA (1 g/day) supplementation in a 2 × 2 factorial design among women ≥55 and men ≥50 years of age. We assessed changes in interleukin (IL)-6, tumor necrosis factor receptor 2 (TNFR2), and high-sensitivity C-reactive protein (hsCRP) concentrations from baseline to 1 year among participants randomized to vitamin D + n-3 FA (392), vitamin D (392), n-3 FA (392), or placebo only (385). Geometric means and percent changes were compared, adjusting for baseline factors. RESULTS: Baseline characteristics were well balanced. In the active arms, 25-OH vitamin D rose 39% and n-3 FA rose 55% vs minimal change in placebo arms. Neither supplement reduced biomarkers at 1 year. Vitamin D resulted in 8.2% higher IL-6 (95% CI, 1.5%-15.3%; adjusted P = 0.02), but TNFR2 and hsCRP did not. Among 784 receiving vitamin D, hsCRP increased 35.7% (7.8%-70.9%) in those with low (<20 ng/mL) but not with higher baseline serum 25(OH) vitamin D [0.45% (-8.9% to 10.8%); P interaction = 0.02]. Among 777 randomized to n-3 FA, hsCRP declined [-10.5% (-20.4% to 0.8%)] in those with baseline low (<1.5 servings/week), but not with higher fish intake [6.4% (95% CI, -7.11% to 21.8%); P interaction = 0.06]. CONCLUSIONS: In this large sample from a population-based randomized controlled trial, neither vitamin D nor n-3 FA supplementation over 1 year decreased these biomarkers of inflammation. CLINICALTRIALSGOV IDENTIFIER: NCT01169259; NCT01351805.

Correlation between biomarkers of pain in saliva and PAINAD scale in elderly people with cognitive impairment and inability to communicate: descriptive study protocol.

INTRODUCTION: Pain is an under-diagnosed problem in elderly people, especially in those with cognitive impairment who are unable to verbalise their pain. Although the Pain assessment in advanced dementia scale (PAINAD) scale is a tool recognised for its clinical interest in this type of patients, its correlation with the saliva biomarkers reinforced its utility. The aim of this research will be to correlate the scores of this scale with the levels of biomarkers of pain found in saliva samples of patients with cognitive impairment and inability to communicate. METHODS AND ANALYSIS: This is an observational study. The level of pain will be evaluated using the PAINAD scale. Moreover, pain biomarkers, in particular secretory IgA and soluble tumour necrosis factor receptor type II, will be determined in saliva. Both assessments will be conducted in 75 patients aged over 65 years with advanced cognitive impairment and inability to communicate. The PAINAD scores will be correlated with the levels of these biomarkers of pain. A control group consisting of 75 healthy subjects aged over 65 years will be included in the study. Moreover, sociodemographic variables and variables related to pain, dementia and other clinical conditions will be recorded. The analysis will be performed with the statistical package SPSS V.22 and the software R. ETHICS AND DISSEMINATION: The study has been reviewed and approved by the Andalusian Human Research Ethics Committee. In addition, this study has been financed by the Junta de Andalucía through a regional health research fund (Research code: PI-0357-2017). The results will be actively disseminated trough a high-impact journal in our study area, conference presentations and social media.

Development of a biomarker panel to predict cardiac resynchronization therapy response: Results from the SMART-AV trial.

BACKGROUND: Predicting a favorable cardiac resynchronization therapy (CRT) response holds great clinical importance. OBJECTIVE: The purpose of this study was to examine proteins from broad biological pathways and develop a prediction tool for response to CRT. METHODS: Plasma was collected from patients before CRT (SMART-AV [SmartDelay Determined AV Optimization: A Comparison to Other AV Delay Methods Used in Cardiac Resynchronization Therapy] trial). A CRT response was prespecified as a ≥15-mL reduction in left ventricular end-systolic volume at 6 months, which resulted in a binary CRT response (responders 52%, nonresponders 48%; n = 758). RESULTS: Candidate proteins (n = 74) were evaluated from the inflammatory, signaling, and structural domains, which yielded 12 candidate biomarkers, but only a subset of these demonstrated predictive value for CRT response: soluble suppressor of tumorgenicity-2, soluble tumor necrosis factor receptor-II, matrix metalloproteinase-2, and C-reactive protein. These biomarkers were used in a composite categorical scoring algorithm (Biomarker CRT Score), which identified patients with a high/low probability of a response to CRT (P <.001) when adjusted for a number of clinical covariates. For example, a Biomarker CRT Score of 0 yielded 5 times higher odds of a response to CRT compared to a Biomarker CRT Score of 4 (P <.001). The Biomarker CRT Score demonstrated additive predictive value when considered against a composite of clinical variables. CONCLUSION: These unique findings demonstrate that developing a biomarker panel for predicting individual response to CRT is feasible and holds potential for point-of-care testing and integration into evaluation algorithms for patients presenting for CRT.

Antibody-Array-Based Proteomic Screening of Serum Markers in Systemic Lupus Erythematosus: A Discovery Study.

A discovery study was carried out where serum samples from 22 systemic lupus erythematosus (SLE) patients and matched healthy controls were hybridized to antibody-coated glass slide arrays that interrogated the level of 274 human proteins. On the basis of these screens, 48 proteins were selected for ELISA-based validation in an independent cohort of 28 SLE patients. Whereas AXL, ferritin, and sTNFRII were significantly elevated in patients with active lupus nephritis (LN) relative to SLE patients who were quiescent, other molecules such as OPN, sTNFRI, sTNFRII, IGFBP2, SIGLEC5, FAS, and MMP10 exhibited the capacity to distinguish SLE from healthy controls with ROC AUC exceeding 90%, all with p < 0.001 significance. These serum markers were next tested in a cohort of 45 LN patients, where serum was obtained at the time of renal biopsy. In these patients, sTNFRII exhibited the strongest correlation with eGFR (r = -0.50, p = 0.0014) and serum creatinine (r = 0.57, p = 0.0001), although AXL, FAS, and IGFBP2 also correlated with these clinical measures of renal function. When concurrent renal biopsies from these patients were examined, serum FAS, IGFBP2, and TNFRII showed significant positive correlations with renal pathology activity index, while sTNFRII displayed the highest correlation with concurrently scored renal pathology chronicity index (r = 0.57, p = 0.001). Finally, in a longitudinal cohort of seven SLE patients examined at ∼3 month intervals, AXL, ICAM-1, IGFBP2, SIGLEC5, sTNFRII, and VCAM-1 demonstrated the ability to track with concurrent disease flare, with significant subject to subject variation. In summary, serum proteins have the capacity to identify patients with active nephritis, flares, and renal pathology activity or chronicity changes, although larger longitudinal cohort studies are warranted.

Evaluating mixtures of 14 hygroscopic additives to improve antibody microarray performance.

Microarrays allow the miniaturization and multiplexing of biological assays while only requiring minute amounts of samples. As a consequence of the small volumes used for spotting and the assays, evaporation often deteriorates the quality, reproducibility of spots, and the overall assay performance. Glycerol is commonly added to antibody microarray printing buffers to decrease evaporation; however, it often decreases the binding of antibodies to the surface, thereby negatively affecting assay sensitivity. Here, combinations of 14 hygroscopic chemicals were used as additives to printing buffers for contact-printed antibody microarrays on four different surface chemistries. The ability of the additives to suppress evaporation was quantified by measuring the residual buffer volume in open quill pins over time. The seven best additives were then printed either individually or as a 1:1 mixture of two additives, and the homogeneity, intensity, and reproducibility of both the spotted protein and of a fluorescently labeled analyte in an assay were quantified. Among the 28 combinations on the four slides, many were found to outperform glycerol, and the best additive mixtures were further evaluated by changing the ratio of the two additives. We observed that the optimal additive mixture was dependent on the slide chemistry, and that it was possible to increase the binding of antibodies to the surface threefold compared to 50 % glycerol, while decreasing whole-slide coefficient of variation to 5.9 %. For the two best slides, improvements were made for both the limit of detection (1.6× and 5.9×, respectively) and the quantification range (1.2× and 2.1×, respectively). The additive mixtures identified here thus help improve assay reproducibility and performance, and might be beneficial to all types of microarrays that suffer from evaporation of the printing buffers.

Expression of TNFR2 by regulatory T cells in peripheral blood is correlated with clinical pathology of lung cancer patients.

CD4(+)FoxP3(+) regulatory T cells (Tregs) represent a major cellular mediator of cancer immune evasion. The expression of tumor necrosis factor receptor type II (TNFR2) on Tregs is reported to identify the maximally suppressive Treg population in both mice and human. We therefore investigated the phenotype and function of TNFR2(+) Tregs present in the peripheral blood (PB) of 43 lung cancer patients. Further, the association of TNFR2 expression on Tregs with clinicopathological factors was analyzed. The results showed that in the PB of lung cancer patients, Tregs expressed markedly higher levels of TNFR2 than conventional T cells (Tconvs). Expression of TNFR2 appeared to correlate better than CD25(+) and CD127(-) with FoxP3 expression. PB TNFR2(+) Tregs in lung cancer patients were more proliferative and expressed higher levels of the immunosuppressive molecule CTLA-4, and consequently more potently suppressed IFNγ production by cocultured CD8 CTLs. More importantly, higher TNFR2 expression levels on Tregs were associated with lymphatic invasion, distant metastasis and more advanced clinical stage of lung cancer patients. Therefore, our study suggests that TNFR2(+) Tregs play a role in promoting tumor progressive metastasis and expression of TNFR2 by PB Tregs may prove to be a useful prognostic marker in lung cancer patients.

Deficient cytokine control modulates temporomandibular joint pain in rheumatoid arthritis.

The aim was to investigate how endogenous cytokine control of tumor necrosis factor (TNF) influences temporomandibular joint (TMJ) pain in relation to the role of anti-citrullinated peptide antibodies (ACPA) in patients with rheumatoid arthritis (RA). Twenty-six consecutive patients with TMJ RA were included. Temporomandibular joint pain intensity was assessed at rest, on maximum mouth opening, on chewing, and on palpation. Mandibular movement capacity and degree of anterior open bite (a clinical sign of structural destruction of TMJ tissues) were also assessed. Systemic inflammatory activity was assessed using the Disease Activity Score in 28 joints (DAS28) for rheumatoid arthritis. Samples of TMJ synovial fluid and blood were obtained and analyzed for TNF, its soluble receptor, soluble TNF receptor II (TNFsRII), and ACPA. A high concentration of TNF in relation to the concentration of TNFsRII in TMJ synovial fluid was associated with TMJ pain on posterior palpation on maximum mouth opening. The ACPA concentration correlated significantly to the TNF concentration, but not to the TNFsRII concentration, indicating that increased inflammatory activity is mainly caused by an insufficient increase in anti-inflammatory mediators. This study indicates that TMJ pain on palpation in patients with RA is related to a deficiency in local cytokine control that contributes to increased inflammatory activity, including sensitization to mechanical stimuli over the TMJ.

Association between graft function and serum TNF-α, TNFR1 and TNFR2 levels in patients with kidney transplantation.

INTRODUCTION: This prospective observational study aimed to assess the relevance of serial postoperative serum TNF-α, TNFR1 and TNFR2 measurements for predicting graft function and acute rejection episodes (AR) after transplantation. MATERIALS AND METHODS: We studied 50 kidney transplant recipients (31 female, 19 male; mean age: 38.36 ± 12.88). Blood samples were collected immediately before and after surgery at day 7, month 1 and month 3. Serum TNF-α, TNFR1 and TNFR2 levels were measured by ELISA using a commercial kit (Invitrogen ELISA). Serum cystatin-C levels were measured by particle-enhanced immunonephelometric method. Glomerular filtration rate (GFR) was estimated by Chronic Kidney Disease-Epidemiology (CKD-EPI) equation. Patients were assigned to their transplant outcomes in terms of acute rejection [AR(+) and AR(-)] and slow (SGF) or immediate graft function (IGF). RESULTS: Among 50 recipients, six had AR(+) and 44 had AR(-), depending on graft function: 17 had SGF and 33 had IGF. Serum creatinine, cystatin-C, TNF-α, TNFR1 and TNFR2 levels demonstrated consistent significantly decreases after transplantation while GFR values had consistent increases (p = 0.001). Pretransplant levels were not statistically different between AR(+) and AR(-) groups (TNF-α: 30.79 ± 5.96 vs. 27.95 ± 2.43 pg/mL, TNFR1: 55.96 ± 21.6 vs. 40.52 ± 7.41 ng/mL, TNFR2: 58.31 ± 8.06 vs. 50.9 ± 3.34 ng/mL, respectively) (p > 0.05). Serum TNF-α, TNFR1 and TNFR2 levels on day 7 and month 1 were also significantly higher in AR(+) group compared to AR(-) (p = 0.012, p = 0.049 for TNF-α, p = 0.001, p = 0.002 for TNFR1, p = 0.001, p = 0.002 for TNFR2). CONCLUSIONS: Our preliminary findings suggest that serum TNF-α, TNFR1 and TNFR2 levels might be considered useful markers of evaluating graft function after renal transplantation.

Tumor Necrosis Factor (TNF) Receptor Superfamily Member 1b on CD8+ T Cells and TNF Receptor Superfamily Member 1a on Non-CD8+ T Cells Contribute Significantly to Upper Genital Tract Pathology Following Chlamydial Infection.

BACKGROUND: We demonstrated previously that tumor necrosis factor α (TNF-α)-producing Chlamydia-specific CD8(+) T cells cause oviduct pathological sequelae. METHODS: In the current study, we used wild-type C57BL/6J (WT) mice with a deficiency in genes encoding TNF receptor superfamily member 1a (TNFR1; TNFR1 knockout [KO] mice), TNF receptor superfamily member 1b (TNFR2; TNFR2 KO mice), and both TNFR1 and TNFR2 (TNFR1/2 double KO [DKO] mice) and mix-match adoptive transfers of CD8(+) T cells to study chlamydial pathogenesis. RESULTS: TNFR1 KO, TNFR2 KO, and TNFR1/2 DKO mice displayed comparable clearance of primary or secondary genital Chlamydia muridarum infection but significantly reduced oviduct pathology, compared with WT animals. The Chlamydia-specific total cellular cytokine response in splenic and draining lymph nodes and the antibody response in serum were comparable between the WT and KO animals. However, CD8(+) T cells from TNFR2 KO mice displayed significantly reduced activation (CD11a expression and cytokine production), compared with TNFR1 KO or WT animals. Repletion of TNFR2 KO mice with WT CD8(+) T cells but not with TNFR2 KO CD8(+) T cells and repletion of TNFR1 KO mice with either WT or TNFR1 KO CD8(+) T cells restored oviduct pathology to WT levels in both KO groups. CONCLUSIONS: Collectively, these results demonstrate that TNFR2-bearing CD8(+) T cells and TNFR1-bearing non-CD8(+) T cells contribute significantly to oviduct pathology following genital chlamydial infection.

Tumor necrosis factor mediates temporomandibular joint bone tissue resorption in rheumatoid arthritis.

OBJECTIVE: To investigate if TNF, IL-1 or their endogenous controls, in relation to ACPA, are associated with radiological signs of ongoing temporomandibular joint (TMJ) bone tissue resorption and disc displacement in RA patients. METHODS: Twenty-two consecutive outpatients with TMJ of RA were included. Systemic inflammatory activity was assessed by DAS28. The number of painful regions in the body and ESR, CRP, RF and ACPA were analyzed. TMJ synovial fluid and blood samples were obtained and analyzed for TNF, TNFsRII, IL-1ra, IL-1sRII and ACPA. The ratios between the mediators and their endogenous control receptors were used in the statistical analysis. Magnetic resonance imaging was performed in closed- and open-mouth positions and evaluated regarding disc position and presence of condylar and temporal erosions of the TMJ. RESULTS: A high TNF level in relation to TNFsRII in TMJ synovial fluid correlated to the degree of TMJ condylar erosion. A high IL-1ra level in relation to TNF in TMJ synovial fluid was also correlated to the degree of TMJ condylar erosion. The total degree of TMJ condylar erosion was correlated with the number of painful regions. CONCLUSION: This study indicates that TNF in TMJ synovial fluid mediates TMJ cartilage and bone tissue resorption in RA. The study also suggests that the degree of endogenous cytokine control is of importance for development of bone tissue destruction.

Determination of soluble tumor necrosis factor receptor 2 produced by alternative splicing.

Soluble cytokine receptors have proven to be very useful biomarkers in a large variety of diseases, including cancer, infections, and chronic inflammatory diseases. These soluble receptors are produced by proteolytic cleavage or alternative splicing. Several cytokine receptors including tumor necrosis factor receptor 2 (TNFR2) can be generated by both mechanisms. However, the conventional ELISA systems do not differentiate between these two types of soluble receptors. We describe a sandwich ELISA to specifically quantify soluble TNFR2 protein generated by alternative splicing. This method requires the use of a capturing monoclonal antibody (mAb) specific of an epitope present in the soluble TNFR2 generated by alternatively splicing but absent in the proteolytically generated isoform. Here we present a detailed protocol for the production and validation of such a mAb. This method has the potential to be applied for measuring other soluble cell surface molecules generated by alternative splicing.

Avaliacao comparativa entre os metodos objetivo e subjetivo em laminas coradas pela imuno-histoquimica / Comparative assessment between objective and subjective methods in slides stained by immunohistochemistry

Métodos objetivos de avaliação são frequentemente cobrados em estudos científicos. Exames histológicos com coloração imuno-histoquímica podem ser avaliados por meio de fotometria. OBJETIVO: Comparar este método objetivo com a avaliação subjetiva realizada por três observadores independentes, utilizando lâminas de colesteatoma adquirido da orelha média. MÉTODO: Foram selecionadas um total de 54 imagens de colesteatomas imuno-histoquimicamente coradas pelos anticorpos anti-TNF-R2 (32 lâminas) e anti-TGF-α; (22 lâminas). O anticorpo secundário utilizado nos dois grupos foi o Max Polimer Detection System (Kit Novo Link, Novocastra®, UK). As amostras foram processadas por um scanner digital de lâminas (modelo ScanScope - Aperio). As áreas selecionadas foram submetidas à análise por fotometria. RESULTADOS: A avaliação objetiva por fotometria foi comparada com a avaliação subjetiva por três observadores e submetidas à análise estatística. A análise estatística revelou reprodutibilidade moderada (K valores entre 0,41 e 0,60) para os dois grupos. CONCLUSÃO: O presente estudo demonstrou que as características irregulares das lâminas de colesteatoma da orelha média coradas pela imuno-histoquímica impossibilita a sua adequada avaliação objetiva, enquanto a avaliação subjetiva por observadores experientes se mostrou mais confiável. .

Objective methods of assessment are often required in scientific studies. Histological tests with immunohistochemical staining can be assessed by photometry. OBJECTIVE: To compare this objective method with the subjective evaluation performed by three independent examiners, using slides of acquired middle ear cholesteatomas. METHOD: We selected a total of 54 cholesteatoma images, immunohistochemically stained by anti-TNF-R2 (32 slides) and anti-TGF-α, (22 slides). The secondary antibody used in the two groups was the Max Polymer Detection System (Novo Link Kit, Novocastra®, UK). The samples were processed by a digital slide scanner (ScanScope - Aperio). The selected sites were analyzed by photometry. RESULTS: The objective assessment by photometry was compared with the subjective evaluation by three examiners and subjected to statistical analysis. The Statistical analysis revealed moderate reproducibility (K values between 0.41 and 0.60) for both groups. CONCLUSION: Our study showed that the irregular characteristics of middle ear cholesteatoma slides stained by immunohistochemistry prevents its proper objective evaluation, while the subjective assessment by experienced examiners was more reliable. .

Predictive value of conjointly examined IL-1ra, TNF-R I, TNF-R II, and RANTES in patients with primary glomerulonephritis.

Interleukin-1 receptor antagonist (IL-1ra), tumor necrosis factor soluble receptors (sTNF-R) type I and II, and regulated upon activation, normal T-cell expressed and secreted (RANTES) play an important role in the modulation of primary glomerulonephritis (GN) course. The aim of the study was to assess whether pre-treatment measurements of IL-1ra, sTNF-R, and RANTES assessed conjointly may be useful as predicting factors in patients with GN. In 84 patients (45 males and 39 female) serum concentration (pg/mL) and urinary excretion (pg/mgCr) of cytokines were measured. After 12 months of therapy with steroids and cyclophosphamide the patients were divided into two subgroups: Responders (R) and Non-Responders (NR) according to the treatment results. The urinary IL-1ra, TNF-RI and RII were significantly higher in R than NR (1,732 vs 646 with P < 0.001, 13.1 vs 6.3 with P = 0.005, and 33.6 vs 14.4 with P = 0.012). The urinary RANTES excretion was increased in NR (79.6 vs 28.5; P < 0.001). The multivariable analysis showed that if conjointly assessed, only urinary IL-1ra, TNF-R I and R II, RANTES with 85% probability pointed the feature remission (R). In conclusion, the urinary excretion of IL-1ra, TNF-R I and R II, and RANTES examined conjointly are effective in predicting favorable response to immunosuppressive treatment in patients with GN.

Comparative assessment between objective and subjective methods in slides stained by immunohistochemistry.

UNLABELLED: Objective methods of assessment are often required in scientific studies. Histological tests with immunohistochemical staining can be assessed by photometry. OBJECTIVE: To compare this objective method with the subjective evaluation performed by three independent examiners, using slides of acquired middle ear cholesteatomas. METHOD: We selected a total of 54 cholesteatoma images, immunohistochemically stained by anti-TNF-R2 (32 slides) and anti-TGF-α, (22 slides). The secondary antibody used in the two groups was the Max Polymer Detection System (Novo Link Kit, Novocastra®, UK). The samples were processed by a digital slide scanner (ScanScope - Aperio). The selected sites were analyzed by photometry. RESULTS: The objective assessment by photometry was compared with the subjective evaluation by three examiners and subjected to statistical analysis. The Statistical analysis revealed moderate reproducibility (K values between 0.41 and 0.60) for both groups. CONCLUSION: Our study showed that the irregular characteristics of middle ear cholesteatoma slides stained by immunohistochemistry prevents its proper objective evaluation, while the subjective assessment by experienced examiners was more reliable.

Activated but not resting regulatory T cells accumulated in tumor microenvironment and correlated with tumor progression in patients with colorectal cancer.

Activated T regulatory (T(reg)) cells are potent suppressors that mediate immune tolerance. We investigated the relationship between activated T(reg) cells and the progression of human colon cancer. We designed a cross-sectional study of CD4(+) Foxp3(+) T cells from peripheral blood, primary tumor and nontumor colon tissue of 42 patients with colon cancer and correlated the percentages of different subgroups of T(reg) cells with colon cancer stage. The phenotypes, cytokine-release patterns and suppression ability of these T(reg) cells were analyzed. We found that T(reg) cells increased significantly in both peripheral blood and cancer tissue. In addition, the T(reg) cells expressed significantly lower levels of CCR7, CD62L and CD45RA in comparison to normal volunteers. Further dividing T(reg) cells into subgroups based on Foxp3 and CD45RA expression revealed that both activated T(reg) cells (Foxp3(hi) CD45RA(-)) and nonsuppressive T(reg) cells (Foxp3(lo) CD45RA(-)), but not resting T(reg) cells (Foxp3(low) CD45RA(+)), increased in the peripheral blood and cancer tissue of patients with colon cancer. Only the activated T(reg) cells expressed significantly higher levels of tumor necrosis factor receptor 2 and cytotoxic T-cell antigen-4. Activated T(reg) cells, however, secreted significantly lower levels of effector cytokines (interleukin-2, tumor necrosis factor-α and interferon-γ) than did resting T(reg) cells and nonsuppressive cells upon ex vivo stimulation. Activated, but not resting, T(reg) cells in cancer tissue correlated with tumor metastases. In summary, we confirmed that activated T(reg) cells are a distinct subgroup with effector memory phenotype and fully functional regulatory activity against human colorectal cancer immunity.

Is there an interaction between polycystic ovary syndrome and gingival inflammation?

BACKGROUND: The aim of this study is to evaluate the gingival crevicular fluid (GCF), saliva, and serum concentrations of tumor necrosis factor-α (TNF-α), TNF-α receptor-1 (TNF-αR1), TNF-αR2, and interleukin-6 (IL-6) in non-obese females with polycystic ovary syndrome (PCOS) and either clinically healthy periodontium or gingivitis. METHODS: Thirty-one females with PCOS and healthy periodontium, 30 females with PCOS and gingivitis, and 12 systemically and periodontally healthy females were included in the study. GCF, saliva, and serum samples were collected, and clinical periodontal measurements, body mass index, and Ferriman-Gallwey score (FGS) were recorded. Sex hormones, cortisol, and insulin levels were measured. TNF-α, TNF-αR1, TNF-αR2, and IL-6 were determined by enzyme-linked immunosorbent assay. Kruskal-Wallis followed by Bonferroni-corrected post hoc Mann-Whitney U tests were used to analyze the data. RESULTS: The PCOS + gingivitis group revealed significantly higher GCF, saliva, and serum IL-6 concentrations than the PCOS + healthy group (P <0.0001). The two PCOS groups exhibited significantly higher saliva TNF-α concentrations than the control group (P = 0.024 and P = 0.013, respectively). The FGS index was significantly higher in the PCOS + gingivitis group than the PCOS + healthy group (P = 0.030). The PCOS + gingivitis group revealed significantly higher insulin concentration than the PCOS + healthy and control groups (P = 0.014 and P <0.0001, respectively). Serum TNF-α, TNF-αRs, and serum, GCF, and salivary IL-6 levels correlated with the clinical periodontal measurements. CONCLUSIONS: PCOS and gingival inflammation appear to act synergistically on the proinflammatory cytokines IL-6 and TNF-α. Thus, PCOS may have an impact on gingival inflammation or vice versa. Additional studies are warranted to clarify the possible relationship between PCOS and periodontal disease.