Objective To investigate the neutrophil extracellular traps (NETs) formation and their molecular mechanisms induced by serum amyloid A (SAA) in rheumatoid arthritis (RA).Methods Neutrophils were isolated from peripheral blood of RA and healthy volunteers.① Neutrophils were cultured in vitro,the formation of NETs was observed and their percentage was calculated.② Neutrophils were cultured in vitro,divided into six groups:control,SAA,[SAA+anti-Toll like receptro4 (TLR4)-Ab],LPS,(LPS+anti-TLR4-Ab) and anti-TLR4-Ab.Appropriate stimulation was conducted for each group.NETs formation and their percentages were investigated.The concentration of DNA in supernatant was detected by fluorescent staining.F test and t test were used for statistical analysis.Results ① The purification of isolated neutrophils was higher than 95％.The network which was collocated with the spreading neutrophils nucleus and neutrophil elastase under the microscope,was NETs.In the RA group,the formation of NETs induced by SAA was significantly more than control [(19.1±0.8) vs (7.4±0.5),t=12.30,P＜0.05].② However,after pretreated with anti-TLR4 antibody,NETs formation was significantly less than the SAA group [(5.7±0.4) vs (14.7±1.1),t=7.825,P＜0.05].Moreover,the fluorescence intensity of DNA in supernatant was significantly higher in SAA group than that of anti-TLR4-Ab pretreatment group [(18.7 ±0.7) vs (12.9±0.8),t=5.552,P＜0.05].The concentration of DNA in supernatant of SAA group was higher than that of anti-TLR4-Ab pretreatment group [(36.9±1.3) μg/ml and (16.3±0.6) μ,g/ml,t=14.41,P＜0.05].Conclusion SAA can induce the formation of NETs from neutrophils by binding to TLR4 in RA.

Objective To investigate the effect of neutrophil extracellular traps (NETs) on inflammation of rheumatoid arthritis (RA), especially angiogenesis. Methods The presence of NETs in synovial tissues of RA and osteoarthritis (OA) was observed by immunofluorescence assay. Neutrophils were isolated from peripheral blood of health volunteers. Neutrophils were cultured in vitro, the formation of NETs was observed. NETs were extracted as a stimulating agent. The effects of NETs on the proliferation of human umbilical vein endothelial cells (HUVECs) and synovial fibroblasts (RAFLS) were evaluated by MTT, and which were classified into two groups: HUVECs group and RAFLS group, with the following treatment: control and NETs (0.28 mg/L). Wound repair assay was employed to evaluate the effect on the cell migration stimulated with NETs. The experiment was divided into three groups:control, VEGF (40μg/L VEGF) and NETs (0.28 mg/L NETs). Results (1) Compared with OA, NETs were found more in the synovial tissue of RA. (2) NETs formation was induced by stimulator in vitro. The concentration of extracted NETs-DNA was 27.8 mg/L. (3) MTT assay showed that compared with the control groups, low concentration of NETs (0.28 mg/L) promoted the proliferation of HUVECs (0.499 ± 0.011 vs. 0.393 ± 0.009, P<0.05) and RAFLS (0.266 ± 0.007 vs. 0.192 ± 0.007, P<0.05). (4) It was showed that a significant wound closure induced by low concentration of NETs (0.28 mg/L) was found compared with control. Conclusion Our present study suggests that NETs are found more in the synovial tissue of RA, and low concentration of NETs can promote angiogenesis in RA.

This paper presents an automatic calculation method for angiography image, which enables programs to intellectively acquire several parameters of blood vessels, such as contours, segments, widths, etc. and then intellectively identify the angiostenosis parts. This method is a kind of automatic optic inspection (AOI) technology. Blood vessels usually distribute as curves and have a fastening direction. According to this feature, the approach performs inspection automatically using improved Steger algorithm, which firstly computes the convolution between image and Gaussian function kernel, and then computes second order Taylor expansion at eac pixel. And further the eigenvalues and eigenvectors of Hessian matrix are calculated on each pixel to obtain the direction of lines and local maximum of second derivative at that point. Hysteresis threshold and directional connection operators are then used to generate blood vessel skeleton. Finally we can compute the blood vessel widths for every sub-pixel object points on blood vessel curve. For given digital X-ray images of hearts with blood vessel local straitness, experiments showed that this method had the ability of getting all the data we need and could find the local confined parts in blood vessels. This approach is proved to have a good effort for angiography images, and it has some advantages such as fast speed, high accuracy, good robustness and no need for human interventions. It could also be a promising computer aided diagnosis method.
Algorithms ; Angiography ; instrumentation ; methods ; Artifacts ; Blood Vessels ; pathology ; Constriction, Pathologic ; diagnostic imaging ; Contrast Media ; Humans ; Pattern Recognition, Automated ; methods ; Radiographic Image Interpretation, Computer-Assisted ; methods

Background/Aims: Transmembrane 4 L six family member 1 (TM4SF1) is highly expressed and contributes to the progression of various malignancies. However, how it modulates hepatocellular carcinoma (HCC) progression and senescence remains to be elucidated. Methods: TM4SF1 expression in HCC samples was evaluated using immunohistochemistry and flow cytometry. Cellular senescence was assessed through SA-β-gal activity assays and Western blot analysis. TM4SF1-related protein interactions were investigated using immunoprecipitation-mass spectrometry, co-immunoprecipitation, bimolecular fluorescence complementation, and immunofluorescence. Tumor-infiltrating immune cells were analyzed by flow cytometry. The HCC mouse model was established via hydrodynamic tail vein injection. Results: TM4SF1 was highly expressed in human HCC samples and murine models. Knockdown of TM4SF1 suppressed HCC proliferation both in vitro and in vivo, inducing non-secretory senescence through upregulation of p16 and p21. TM4SF1 enhanced the interaction between AKT1 and PDPK1, thereby promoting AKT phosphorylation, which subsequently downregulated p16 and p21. Meanwhile, TM4SF1-mediated AKT phosphorylation enhanced PD-L1 expression while reducing major histocompatibility complex class I level on tumor cells, leading to impaired cytotoxic function of CD8+ T cells and an increased proportion of exhausted CD8+ T cells. In clinical HCC samples, elevated TM4SF1 expression was associated with resistance to anti-PD-1 immunotherapy. Targeting TM4SF1 via adeno-associated virus induced tumor senescence, reduced tumor burden and synergistically enhanced the efficacy of anti-PD-1 therapy. Conclusions Our results revealed that TM4SF1 regulated tumor cell senescence and immune evasion through the AKT pathway, highlighting its potential as a therapeutic target in HCC, particularly in combination with first-line immunotherapy.

Background/Aims: Transmembrane 4 L six family member 1 (TM4SF1) is highly expressed and contributes to the progression of various malignancies. However, how it modulates hepatocellular carcinoma (HCC) progression and senescence remains to be elucidated. Methods: TM4SF1 expression in HCC samples was evaluated using immunohistochemistry and flow cytometry. Cellular senescence was assessed through SA-β-gal activity assays and Western blot analysis. TM4SF1-related protein interactions were investigated using immunoprecipitation-mass spectrometry, co-immunoprecipitation, bimolecular fluorescence complementation, and immunofluorescence. Tumor-infiltrating immune cells were analyzed by flow cytometry. The HCC mouse model was established via hydrodynamic tail vein injection. Results: TM4SF1 was highly expressed in human HCC samples and murine models. Knockdown of TM4SF1 suppressed HCC proliferation both in vitro and in vivo, inducing non-secretory senescence through upregulation of p16 and p21. TM4SF1 enhanced the interaction between AKT1 and PDPK1, thereby promoting AKT phosphorylation, which subsequently downregulated p16 and p21. Meanwhile, TM4SF1-mediated AKT phosphorylation enhanced PD-L1 expression while reducing major histocompatibility complex class I level on tumor cells, leading to impaired cytotoxic function of CD8+ T cells and an increased proportion of exhausted CD8+ T cells. In clinical HCC samples, elevated TM4SF1 expression was associated with resistance to anti-PD-1 immunotherapy. Targeting TM4SF1 via adeno-associated virus induced tumor senescence, reduced tumor burden and synergistically enhanced the efficacy of anti-PD-1 therapy. Conclusions Our results revealed that TM4SF1 regulated tumor cell senescence and immune evasion through the AKT pathway, highlighting its potential as a therapeutic target in HCC, particularly in combination with first-line immunotherapy.

Background/Aims: Transmembrane 4 L six family member 1 (TM4SF1) is highly expressed and contributes to the progression of various malignancies. However, how it modulates hepatocellular carcinoma (HCC) progression and senescence remains to be elucidated. Methods: TM4SF1 expression in HCC samples was evaluated using immunohistochemistry and flow cytometry. Cellular senescence was assessed through SA-β-gal activity assays and Western blot analysis. TM4SF1-related protein interactions were investigated using immunoprecipitation-mass spectrometry, co-immunoprecipitation, bimolecular fluorescence complementation, and immunofluorescence. Tumor-infiltrating immune cells were analyzed by flow cytometry. The HCC mouse model was established via hydrodynamic tail vein injection. Results: TM4SF1 was highly expressed in human HCC samples and murine models. Knockdown of TM4SF1 suppressed HCC proliferation both in vitro and in vivo, inducing non-secretory senescence through upregulation of p16 and p21. TM4SF1 enhanced the interaction between AKT1 and PDPK1, thereby promoting AKT phosphorylation, which subsequently downregulated p16 and p21. Meanwhile, TM4SF1-mediated AKT phosphorylation enhanced PD-L1 expression while reducing major histocompatibility complex class I level on tumor cells, leading to impaired cytotoxic function of CD8+ T cells and an increased proportion of exhausted CD8+ T cells. In clinical HCC samples, elevated TM4SF1 expression was associated with resistance to anti-PD-1 immunotherapy. Targeting TM4SF1 via adeno-associated virus induced tumor senescence, reduced tumor burden and synergistically enhanced the efficacy of anti-PD-1 therapy. Conclusions Our results revealed that TM4SF1 regulated tumor cell senescence and immune evasion through the AKT pathway, highlighting its potential as a therapeutic target in HCC, particularly in combination with first-line immunotherapy.