Gene expression signature as a surrogate marker of microvascular invasion on routine hepatocellular carcinoma biopsies.

BACKGROUND & AIMS: Microvascular invasion (MVI), a major risk factor for tumor recurrence after surgery in hepatocellular carcinoma (HCC), is only detectable by microscopic examination of the surgical specimen. We aimed to define a transcriptomic signature associated with MVI in HCC than can be applied to formalin-fixed paraffin-embedded (FFPE) biopsies for use in clinical practice. METHODS: To identify a gene expression signature related to MVI by using NanoString technology, we selected a set of 200 genes according to the literature and RNA-sequencing data obtained from a cohort of 150 frozen HCC samples previously published. We used 178 FFPE-archived HCC samples, including 109 surgical samples for the training set and 69 paired pre-operative biopsies for the validation set. In 14 cases of the training set, a paired biopsy was available and was also analyzed. RESULTS: We identified a 6-gene signature (ROS1, UGT2B7, FAS, ANGPTL7, GMNN, MKI67) strongly associated with MVI in the training set of FFPE surgical HCC samples, with 82% accuracy (sensitivity 82%, specificity 81%, AUC 0.82). The NanoString gene expression was highly correlated in 14 paired surgical/biopsy HCC samples (mean R: 0.97). In the validation set of 69 FFPE HCC biopsies, the 6-gene NanoString signature predicted MVI with 74% accuracy (sensitivity 73%, specificity 76%, AUC 0.74). Moreover, on multivariate analysis, the MVI signature was associated with overall survival in both sets (hazard ratio 2.29; 95% CI 1.03-5.07; p = 0.041). CONCLUSION: We defined a 6-gene signature that can accurately predict MVI in FFPE HCC biopsy samples, which is also associated with overall survival, although its survival impact must be confirmed by extensive study with further clinical data. LAY SUMMARY: Microvascular invasion, a major risk factor for tumor recurrence after surgery in hepatocellular carcinoma, is only detectable by microscopic examination of a surgical specimen. In this study, we defined a relevant surrogate signature of microvascular invasion in hepatocellular carcinoma that may be applied in clinical practice with routine tumor biopsy and integrated into the therapeutic strategy.

Results of Complex Immunopharmacological Analysis of the Mechanisms of Action of Bioregulation Agents.

Elucidation of the pharmacodynamic mechanisms of drugs capable of potentiating the effects of non-steroidal anti-inflammatory drugs is an important task. In this in vitro study, the ability of Traumeel S to influence the innate and acquired immunity was evaluated. Traumeel S was found to reduce activities of NADPH oxidase and neutrophil extracellular traps, as well as to evoke anti-inflammatory activity of lymphocyte subpopulations.

Association between the FAS/FASL Variants and Risk of Male Infertility in Asian Populations; A Systematic Review and Meta-Analysis.

Background and Objectives: Studies suggest that FAS/FASL polymorphisms are associated with male infertility; however, their results are still inconclusive. Therefore, this systematic review and meta-analysis aimed to summarize and clarify the overall association of FAS/FASL polymorphisms and risk of male infertility. Materials and Methods: Our search was conducted on the databases of Science Direct, PubMed and Google Scholar. For performing the meta-analysis, pooled odds ratio (OR) values with 95% confidence interval (CI) was applied in order to analyze the strength of association between the FAS/FASL polymorphisms and risk of male infertility. A total of seven relevant studies published up to September 2018 were considered. Results: FASL-844C/T genotype results of 559 patients and 623 healthy individuals were included in our study. For FAS-670A/G genotype effect, 751 patients and 821 healthy individuals were explored. Results showed that all analysis models including dominant, recessive and allelic models of FASL-844C/T and FAS-670A/G polymorphism had no significant effect on infertility in men (p > 0.05 and p > 0.05, respectively). According to sensitivity analysis, our results were stable. Conclusion: We demonstrated that FAS/FASL polymorphisms might not be an effective factor on male reproductive health. For precise determination of FAS/FASL polymorphisms effects on male infertility, large-scale case-control studies should be performed.

Alteration of CCR6+CD95+CD4+ naïve T cells in HIV-1 infected patients: Implication for clinical practice.

The profound deficiency of Th17 cells contributes to HIV disease progression. The mechanisms of their perturbation remain unclear. Recently, CCR6+CD95+CD4+ naïve T cells (CCR6+CD95+CD4+ TNA), identified as pre-committed Th17 precursors, were recognized as a subpopulation of CD4+ T cells with stem cell properties. Following phenotypical identification, we evaluated their level in patients during chronic HIV infection and following antiretroviral therapy (ART) using flow cytometry. The levels of CCR6+CD95+CD4+ TNA were decreased during chronic HIV infection and correlated with CD4+ T cell counts. Immunological responders harbored higher frequency of CCR6+CD95+CD4+ TNA, which was associated with CD4/CD8 T cell ratio. Immunological non-responders with lower frequency of CCR6+CD95+CD4+ TNA failed to exhibit a correlation between CCR6+CD95+CD4+ TNA and CCR6+CD95+CD4+ TCM, and displayed elevated ratio of CCR6+CD95+CD4+ TCM/TNA. The number of CCR6+CD95+CD4+ TNA was increased following early ART. These findings shed light on the importance of targeting pre-committed Th17 precursors that enhance immune reconstitution.

Expression of Fas, FasL, caspase-8 and other factors of the extrinsic apoptotic pathway during the onset of interdigital tissue elimination.

Elimination of the interdigital web is considered to be the classical model for assessing apoptosis. So far, most of the molecules described in the process have been connected to the intrinsic (mitochondrial) pathway. The extrinsic (receptor mediated) apoptotic pathway has been rather neglected, although it is important in development, immunomodulation and cancer therapy. This work aimed to investigate factors of the extrinsic apoptotic machinery during interdigital regression with a focus on three crucial initiators: Fas, Fas ligand and caspase-8. Immunofluorescent analysis of mouse forelimb histological sections revealed abundant expression of these molecules prior to digit separation. Subsequent PCR Array analyses indicated the expression of several markers engaged in the extrinsic pathway. Between embryonic days 11 and 13, statistically significant increases in the expression of Fas and caspase-8 were observed, along with other molecules involved in the extrinsic apoptotic pathway such as Dapk1, Traf3, Tnsf12, Tnfrsf1A and Ripk1. These results demonstrate for the first time the presence of extrinsic apoptotic components in mouse limb development and indicate novel candidates in the molecular network accompanying the regression of interdigital tissue during digitalisation.

Circulating levels of soluble Fas (sCD95) are associated with risk for development of a nonresolving acute kidney injury subphenotype.

BACKGROUND: Critically ill patients with acute kidney injury (AKI) can be divided into two subphenotypes, resolving or nonresolving, on the basis of the trajectory of serum creatinine. It is unknown if the biology underlying these two AKI recovery patterns is different. METHODS: We measured eight circulating biomarkers in plasma obtained from a cohort of patients admitted to an intensive care unit (ICU) (n = 1241) with systemic inflammatory response syndrome. The biomarkers were representative of several biologic processes: apoptosis (soluble Fas), inflammation (soluble tumor necrosis factor receptor 1, interleukin 6, interleukin 8) and endothelial dysfunction, (angiopoietin 1, angiopoietin 2, and soluble vascular cell adhesion molecule 1). We tested for associations between biomarker levels and AKI subphenotypes using relative risk regression accounting for multiple hypotheses with the Bonferroni correction. RESULTS: During the first 3 days of ICU admission, 868 (70%) subjects developed AKI; 502 (40%) had a resolving subphenotype, and 366 (29%) had a nonresolving subphenotype. Hospital mortality was 12% in the resolving subphenotype and 21% in the nonresolving subphenotype. Soluble Fas was the only biomarker associated with a nonresolving subphenotype after adjustment for age, body mass index, diabetes, and Acute Physiology and Chronic Health Evaluation III score (p = 0.005). CONCLUSIONS: Identifying modifiable targets in the Fas-mediated pathway may lead to strategies for prevention and treatment of a clinically important form of AKI.

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.

Changes of sFas and sFasL, oxidative stress markers in serum and follicular fluid of patients undergoing IVF.

PURPOSE: The Fas-Fas Ligand interaction is one of the essential events for the induction of apoptosis whereas the exact role of their soluble forms in the reproductive system is still not fully understood. Also oxidative stress in the pathogenesis of infertility causing diseases in women and has been suggested as one of the important factors that negatively affect IVF outcome. In this study, our aim was to evaluate serum and follicular fluid levels of soluble Fas soluble Fas Ligand, malondialdehyde, superoxide dismutase and total antioxidant capacity in patients undergoing IVF and compared with controls. METHODS: This study included 109 patients. Patients were classified as unexplained infertility (N = 31), PCOS (N = 19), tubal factor (N = 9) and endometriosis (N = 10) and compared with male factor infertility (N = 40) that was the control group. sFas and sFasL levels were measured by immunoassay method. MDA, SOD and TAC levels were measured by colorimetric method. RESULTS: Patients with unexplained infertility, PCOS and tubal factor had significantly lower sFas levels compared with their controls (respectively, p < 0.01, p < 0.05, p < 0.05). However, SOD activity in unexplained infertility, PCOS and endometriosisgroupswere significantly higher than control group (p < 0.01).Decreased follicular fluid TAC levels were found in all patient groups compared with controls (respectively, p < 0.01, p < 0.05, p < 0.01, p < 0.01).Patients with tubal factor had significantly higher serum sFasL (p < 0.05), but lower follicular fluid sFasL levels (p < 0.05) compared with unexplained infertility. Tubal factor and endometriosis groups had lowerfollicular fluid TAC levels compared to unexplained infertility and PCOSgroups (p < 0.01). CONCLUSION(S): In this study, serum and follicular fluid sFas levels were decreased and antioxidant activity was impaired in infertility, possibly implying increased apoptosis. Especially in unexplained infertility group changes in this parametres more remarkable.

Autoimmune thyroid disease: new models of cell death in autoimmunity.

Autoimmunity to thyroid antigens leads to two distinct pathogenic processes with opposing clinical outcomes: hypothyroidism in Hashimoto's thyroiditis and hyperthyroidism in Graves' disease. The high frequency of these diseases and easy accessibility of the thyroid gland has allowed the identification of key pathogenic mechanisms in organ-specific autoimmune diseases. In early investigations, antibody- and T-cell-mediated death mechanisms were proposed as being responsible for autoimmune thyrocyte depletion. Later, studies on apoptosis have provided new insights into autoimmune target destruction, indicating the involvement of death receptors and cytokine-regulated apoptotic pathways in the pathogenesis of thyroid autoimmunity.

Fatal combined immunodeficiency associated with heterozygous mutation in STAT1.

BACKGROUND: Mutations in the gene for the signal transducer and activator of transcription 1, STAT1, have been shown to be associated with death at an early age due to overwhelming viral infection (complete STAT1 deficiency) or, more commonly, selective deficiencies to mycobacterial or fungal infection (typically heterozygous STAT1 mutations). OBJECTIVES: To define the molecular basis of progressive combined immunodeficiency in a group of patients with fatal infections. METHODS: We studied a group of unrelated patients who displayed an unusual progressive form of combined immunodeficiency. Whole exome sequencing assisted in confirming a common genetic defect in this group, which consisted of a heterozygous mutation of the STAT1 gene. STAT1 protein level as well as function was assessed, and a detailed evaluation of the immune system, including analysis of thymus tissue, was performed. RESULTS: Patients were found to carry de novo heterozygous mutations in STAT1 encoding T385A, I294T, or C284R amino acid substitutions. STAT1 expression appeared significantly decreased as a result of these changes but not completely absent, with diminished signaling responses. This group display progressive loss in lymphocyte number and function accompanied by increasing autoimmune features as well as severe, fatal infections. CONCLUSIONS: These findings show that some heterozygous aberrations of STAT1 can be associated with progressive combined immunodeficiency, quite distinct from the limited susceptibilities to infection previously reported for heterozygous STAT1 mutations. These mutations were not inherited, rather, arose de novo in each case. Accompanied by significant patient mortality, this finding suggests that this class of STAT1 mutation is ultimately fatal due to overwhelming infection.

Expression of apoptosis regulating proteins identifies stage II and III colon cancer patients with high risk of recurrence.

BACKGROUND AND OBJECTIVES: Deregulation of apoptosis related genes may be associated with poor outcome in cancer. Aim of the present study was to investigate the prognostic role of expression levels of apoptosis related proteins in stage II and III colon cancer. METHODS: From tumor samples of 386 stage II and III colon cancer patients, DNA was isolated and tissue microarrays were constructed. Expression of Bcl-2, Bcl-X, BAX, XIAP, Fas, FasL and c-FLIP was evaluated and PCR-based microsatellite instability analysis was performed. RESULTS: High FasL expressing tumors were associated with high disease recurrence rates in stage II colon cancer patients overall, as was low Bcl-X expression in microsatellite stable stage II patients. In stage II patients, a multivariable model based on FasL and Bcl-XL expression revealed a significant association with disease free survival (DFS). In stage III colon cancer patients, low Bcl-2, low BAX and low Fas expression levels were associated with worse outcome. In these patients a multivariable model based on angioinvasion and Bcl-2, Fas and FasL expression was significantly associated with DFS. CONCLUSIONS: Stage II patients with low Bcl-X and high FasL protein expression levels and stage III patients with low Fas, high FasL and low Bcl-2 expression could be considered as high risk for disease recurrence.

Cord blood graft composition impacts the clinical outcome of allogeneic stem cell transplantation.

INTRODUCTION: Despite routine use of umbilical cord blood (CB) grafts as stem cell source for allogeneic stem cell transplantations, much remains unknown regarding their cell composition and correlation with clinical outcome. METHODS: We analyzed material from 30 CB units used for allogeneic hematopoietic stem cell transplantation by multicolor flow cytometry. Phenotypic data were correlated with various clinical outcomes such as survival, graft-versus-host disease (GVHD), relapse, rejection, viral reactivation, and bacteremia. RESULTS: We found that above-median frequencies of CD69+ T cells, naïve CD8+ T cells, and CD127+ B cells in the CB graft were each associated with significantly improved patient survival. Moreover, a statistically significant correlation was seen between higher levels of CD94+ T cells and herpes simplex virus and varicella zoster virus reactivation post transplantation. A similar correlation was seen for the frequency of CD95+ cells in total CD3+, as well as CD4+ and CD8+ T-cell subsets, and viral reactivation. Finally, a higher frequency of naïve CD8+ T cells was associated with the incidence of acute GVHD. CONCLUSION: Our study highlights the importance of further exploration of graft composition before CB transplantation as a tool for risk prediction.

Differential expression and localization of ADAM10 and ADAM17 during rat spermatogenesis suggest a role in germ cell differentiation.

BACKGROUND: Extracellular metolloproteases have been implied in different process such as cell death, differentiation and migration. Membrane-bound metalloproteases of the ADAM family shed the extracellular domain of many cytokines and receptor controlling auto and para/juxtacrine cell signaling in different tissues. ADAM17 and ADAM10 are two members of this family surface metalloproteases involved in germ cell apoptosis during the first wave of spermatogenesis in the rat, but they have other signaling functions in somatic tissues. RESULTS: In an attempt to further study these two enzymes, we describe the presence and localization in adult male rats. Results showed that both enzymes are detected in germ and Sertoli cells during all the stages of spermatogenesis. Interestingly their protein levels and cell surface localization in adult rats were stage-specific, suggesting activation of these enzymes at particular events of rat spermatogenesis. CONCLUSIONS: Therefore, these results show that ADAM10 and ADAM17 protein levels and subcellular (cell surface) localization are regulated during rat spermatogenesis.

Associations between indicators of nitrosative stress and levels of soluble HLA-I, CD95 molecules in patients with COPD.

At the present stage of study of chronic obstructive pulmonary disease (COPD) one of the problem is the definition of new criteria for the topical and systemic chronic inflammation of this disease. The aim of the research was to study the concentration of nitric oxide metabolites, the level of soluble human leukocyte antigens class I (sHLA-I) and of soluble CD95 molecules (sCD95) in the serum of blood and exhaled breath condensate (EBC) in patients with exacerbation of COPD. We investigated 49 moderate-to-severe COPD patients with exacerbation, and 21 healthy nonsmokers. The concentration of sHLA-I and sCD95 molecules was studied in serum and in EBC using the ELISA method. The nitrosative stress was evaluated by the measurement of NO2(-) levels in the serum and the concentration of ΣNO2(2)/NO3(2) in the EBC. Exacerbation of COPD is associated with increasing concentrations of NO2(2) in the serum and of the levels of ΣNO2(2)/NO3(2) in the EBC, together with the changing concentration of sHLA-I and sCD95 molecules in the both biological liquid. An association was discovered between the exacerbation of COPD and the indicators of nitrosative stress, the parameters of lung function and the concentration of sHLA-I, sCD95 molecules. The findings suggest a pathogenetic role of nitrosative stress and of soluble molecules of HLA-I and CD95 in the progression of COPD. The studied markers can be used as predictors of unfavourable prognoses of COPD and as quantitative criteria in the diagnosis of exacerbation of moderate-to-severe COPD.

[Conjunctival Fas-FasL expression in chronic Stevens-Johnson syndrome].

OBJECTIVE: To investigate the expression level of Fas-FasL in conjunctiva tissue of chronic Stevens-Johnson syndrome (SJS) patients and to analyze the role of Fas-FasL pathway in the mechanism of ocular damage related to SJS. METHODS: Conjunctiva tissue was obtained during symblepharon release operation and cryostat sectioned at 5 µm. The tissue morphology was observed by electron microscopy. The expression of Fas-FasL (CD95/CD178) in conjunctiva was detected by immunohistochemistry (direct immunofluorescence). The conjunctiva tissue of normal people was also observed and tested as contrast group. RESULTS: The pathogenic changes included incrassation of epithelium and cavity of mucous glands in the sub-epithelium. The expression of Fas in SJS patients focusing in the basal layer of epithelium appeared a lower level compared to that in normal human conjunctiva, which distributed equally in all layers of epithelium. The expressions of FasL in SJS patients and normal people were both weak. CONCLUSIONS: The low level expression of Fas-FasL in conjunctiva of SJS patients has certain effect on the chronic ocular damage.

Correlation of High-Grade Osteosarcoma Response to Chemotherapy with Enhanced Tissue Immunological Response: Analysis of CD95R, IFN-γ, Catalase, Hsp70, and VEGF.

High-grade osteosarcoma, a primary malignant bone tumour, is experiencing a global increase in reported incidence with varied prevalence. Despite advances in management, which include surgery and neoadjuvant chemotherapy often an unsatisfactory outcome is found due to poor or heterogeneous response to chemotherapy. Our study delved into chemotherapy responses in osteosarcoma patients and associated molecular expressions, focusing on CD95 receptor (CD95R), interferon (IFN)-γ, catalase, heat-shock protein (Hsp)70, and vascular endothelial growth factor (VEGF). Employing immunohistochemistry and Huvos grading of post-chemo specimens, we analysed formalin-fixed paraffin-embedded (FFPE) osteosarcoma tissue of resected post-chemotherapy specimens from Dr. Soetomo General Academic Hospital in Surabaya, Indonesia (DSGAH), spanning from 2016 to 2020. Results revealed varied responses (poor 40.38%, moderate 48.08%, good 11.54%) and distinct patterns in CD95R, IFN-γ, catalase, Hsp70, and VEGF expression. Significant differences among response groups were observed in CD95R and IFN-γ expression in tumour-infiltrating lymphocytes. The trend of diminishing CD95R expression from poor to good responses, accompanied by an increase in IFN-γ, implied a reduction in the count of viable osteosarcoma cells with the progression of Huvos grading. Catalase expression in osteosarcoma cells was consistently elevated in the poor response group, while Hsp70 expression was highest. VEGF expression in macrophages was significantly higher in the good response group. In conclusion, this study enhances our understanding of immune-chemotherapy interactions in osteosarcoma and identifies potential biomarkers for targeted interventions.

Inflammatory factors gene polymorphism in recurrent oral ulceration.

BACKGROUND: Some inflammatory factors play an important role in recurrent oral ulceration (ROU). The genetics mechanism of expression level of inflammatory factors is not clear in ROU, but from genetics the expression level of inflammatory factors at least partly depend on the gene polymorphisms. Therfore, we decided to investigate inflammatory factors gene polymorphism and its association with the susceptibility of recurrent oral ulceration in Chinese. METHODS: Genomic DNA was obtained from 42 subjects with recurrent oral ulceration, 86 subjects of healthy control individuals.Genotypes and alleles of 10 genes and 17 polymorphisms sites were analyzed by Mass-ARRAY Analyzer method. Then, the differences in distribution of each genotype and allele were compared. RESULTS: The statistical differences in distribution of TNF-α (rs1800629 and rs1800630) genotype and allele were observed among the groups with recurrent oral ulceration and healthy control individuals (P < 0.01), while VEGFA (rs1570360, rs833061, and rs2010963), EGF (rs4444903), TNF (rs361525), IL10 (rs1800896, rs1800872), IL2 (rs2069762), IL4 (rs2243250), Fas (rs1800682, rs2234767), IL12A (rs2243115, rs568408), IL12B (rs3212227), and IFNG (rs2430561) showed no statistical differences of genotype and allele in controls as compared to those in patients. CONCLUSIONS: This study suggests that the TNF-α (rs1800629 and rs1800630) genotype is an indicator for the susceptibility of recurrent oral ulceration.

The study of the Oxytropis kansuensis-induced apoptotic pathway in the cerebrum of SD rats.

BACKGROUND: Locoweeds cause significant livestock poisoning and economic loss all over the world. Animals can develop locoism, a chronic neurological disease, after grazing on locoweeds. Oxytropis kansuensis is a variety of locoweed that contains swainsonine as its main toxic ingredient. The purpose of this study was to investigate the apoptotic pathway induced in the cerebrum by swainsonine. RESULTS: Twenty-four Sprague-Dawley rats were randomly divided into four groups (experimental groups I, II, III and a control group) and 6 SD rats of each group were feed in 3 cages separately. Rats were penned as groups and fed with feeds containing 15% (SW content 0.03‰), 30% (SW content 0.06‰), or 45% (SW content 0.09‰) O. kansuensis for experimental groups I, II, and III, respectively, or complete feed in the case of the control group. One hundred and nineteen days after poisoning, and all rats showed neurological disorders at different degrees, which were considered to be successful established a chronic poisoning model of O. kansuensis. rats were sacrificed and the expression of Fas, FasL, Bcl-2, Bax as well as cleaved caspase-3, -8 and -9 proteins in brain tissues were detected by Western blot. The results showed that SW treatment up-regulated Fas and Fas ligand (FasL) (P < 0.05), and that there was an increase in Bax and a decrease in Bcl-2 protein (P < 0.01). Moreover, SW treatment significantly increases the activation of caspase-3, 8 and -9, the key effectors in apoptosis pathway (P < 0.01). CONCLUSION: Our data suggest that SW induces apoptosis in cells of the brain through death receptor and mitochondria-mediated, caspase-dependent apoptotic pathways in the brain tissue of SD rats.

Apoptotic activity of gingival crevicular fluid from localized aggressive periodontitis.

INTRODUCTION: The aim of this study was to examine a potential link between apoptotic biomarkers in gingival crevicular fluid (GCF) and periodontal destruction in four cases of localized aggressive periodontitis (LAP), diagnostically enhanced by cone beam computed tomography. CASE SERIES: This study examined the GCF in four patients diagnosed with LAP (formerly localized juvenile periodontitis) at a routine periodontal examination. The LAP diseased sites had attachment loss ranging from 5-12 mm. Atotal of 62 samples of GCF were collected from diseased sites and from contralateral, matched healthy sites with minimal or no attachment loss. All samples were assayed for apoptotic markers, including Fas/FasL, DNAfragmentation, and nitric oxide. The GCF samples were analyzed utilizing enzyme-linked immunosorbent assays for DNA fragments and nitric oxide levels, whereas Western blotting was used for Fas/FasL analyses. Our results showed a significant increase in the apoptotic markers Fas/FasL and DNA fragmentation when comparing GCF from diseased versus non-diseased sites in patients with LAP. CONCLUSION: To our knowledge, this is the first report of apoptotic biomarkers associated with patients diagnosed with LAP. Finding significantly increased levels of these markers in localized areas may help us understand the pathophysiology associated with this specific form of periodontitis, and, furthermore, may provide a basis for a quantifiably prognostic test when attempting to treat this disease.