Western blot analysis of immune response to Legionella bozemanii antigens.

Abstract BACKGROUND: Large Granular Lymphocyte (LGL) leukemia is an indolent clonal disorder arising from either CD3+ or CD3− immunophenotypes characterized as T-cell and Natural-Killer (NK)-cell malignancies, respectively. Due to a rare incidence, all therapeutic investigations in LGL leukemia are generated from single institution experiences, which consist primarily of retrospective cohort analyses. Pathogenesis may relate to an underlying autoimmune mechanism with low-dose methotrexate (10mg/m2 weekly), cyclophosphamide (50–100mg daily), and cyclosporine A (5–10mg/kg daily) used as first-line agents for the treatment of associated cytopenias. The mechanism(s) controlling survival of leukemic LGL are not fully characterized. We hypothesize that agents that target survival signaling will serve as effective therapeutics. Using pharmacologic inhibitors of the Mitogen Activated Protein Kinase (MAPK/ERK) signaling cascase, we found that ERK/Ras drives NK leukemia survival (Epling-Burnette, et al. Oncogene, 23:9220, 2004). The goal of this study was to investigate the in vitro actions of the farnysltransferase-inhibitor R115777 (tipifarnib, Zarnestra®, Johnson and Johnson) on survival and immune response leukemic T cells. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from patients with T LGL leukemia. All patients had increased numbers of CD3+ αβ T lymphocytes and evidence of clonality. Western blot analysis was used to examine the protein levels of phosphorylated (active) ERK1 and ERK2 (MAPK), Bcl-2, and total MAPK. Apoptosis assays were performed by staining with annexin-V-FITC and 7-aminoactinomycin (7-AAD) and flow cytometry analysis. Anti-CD3 plus anti-CD28 was used to stimulate T cells in the presence of brefelden A and IL-2, IFN-γ, TNF-α, IL-4, and IL-10 were determined in distinct T cell subpopulations by intracellular staining. T cell proliferation was assessed by Brdu incorporation in CD4+ and CD8+ T cells. RESULTS: PBMCs from patients with T LGL displayed constitutively-active MAPK/ERK expression as determined by Western blot analysis (n=5). Using MEK pharmacological inhibitors, we found that blockade of the active MEK/ERK signaling pathway induced apoptosis in leukemic T LGL. Bcl-2 was expressed in 80% of patients and when MEK/ERK was inhibited there was a corresponding reduction in expression. Targeted disruption of farnesyl-transferase with two different inhibitors (FTI2153 and R115777, n=9) led to a dose-dependent increase in apoptosis with an apoptotic index that was significantly greater than normal T cells. In addition to apoptosis, we found that antigen-induced proliferation of CD4+ and CD8+ T cells was impaired by R115777 (11% ± 7 vs. 3% ±1.7 and 11% ± 9 vs. 3% ± 1.6, respectively). Furthermore, R115777 biased antigen-dependent cytokine response to a Th2 type with increased expressionof IL-4 and IL-10 after drug treatment (average increase 100% and 43%, respectively). CONCLUSIONS: These findings suggest that FTIs regulate immune response and lead to apoptosis of leukemic T LGL cells. A pilot trial of R115777 (tipifarnib, Zarnestra®) is ongoing for the treatment of LGL leukemia.

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Effects of Neuromedin S on the Proliferation of Splenic Lymphocytes and the Cytokine Secretion by Pulmonary Alveolar Macrophages in Pigs in vitro

Polish Journal of Veterinary Sciences ◽

10.1515/pjvs-2016-0061 ◽

2016 ◽

Vol 19 (3) ◽

pp. 485-494 ◽

Cited By ~ 1

Author(s):

R. Lin ◽

Q. Wang ◽

B. Qi ◽

Y. Huang ◽

G. Yang

Keyword(s):

Immune Response ◽

Western Blot ◽

Alveolar Macrophages ◽

Blot Analysis ◽

Western Blot Analysis ◽

Splenic Lymphocytes ◽

Rt Pcr ◽

Tnf Α ◽

Pulmonary Alveolar Macrophages

Abstract Neuromedin S (NMS), a 36-amino acid neuropeptide, has been found to be involved in the regulation of the endocrine activity. It has been also detected in immune tissues in mammals, what suggests that NMS may play an important role in the regulation of immune response. The aim of this study was to demonstrate the presence of NMS receptor 1 (NMU1R) and effect of NMS in pig splenic lymphocytes (SPLs) and pulmonary alveolar macrophages (PAMs). The presence of NMU1R in pig SPLs and PAMs was respectively confirmed by reverse transcription-polymerase chain reaction (RT-PCR), western blot analysis and immunocytochemical methods. Furthermore, SPL proliferation was analyzed using the 3-(4,5)-dimethyl-thiahiazo-(-2-yl)-3,5-di-phenytetrazoliumromide (MTT) method. Additionally, the secretion of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) in PAMs was all measured by enzyme-linked immunosorbent assay (ELISA) kits. In the present study, the results of RT-PCR and western blot analysis revealed that NMU1R mRNA and protein were both expressed in pig SPLs and PAMs, and the immunocytochemical investigations further revealed that the positive signal of NMU1R immunoreactivity was observed in plasma membranes of both SPLs and PAMs. In the in vitro study, we found that at concentrations of 0.001-1000 nM NMS alone or combined with lipopolysaccharide or phytohemagglutinin significantly increased SPL proliferation. Application of ELISA method showed that NMS could induce the secretion of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in PAMs. These results suggest that NMS can act as a potently positive pro-inflammatory factor and immunomodulatory agent that affects the immune response of immune cells by combining with its receptor NMU1R.

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Western blot analysis of the immune response toCandida albicansantigens in 391 long-term intensive care patients

Mycoses ◽

10.1111/j.1439-0507.1997.tb00207.x ◽

1997 ◽

Vol 40 (5-6) ◽

pp. 153-157 ◽

Cited By ~ 9

Author(s):

Christine Weis ◽

R. Kappe ◽

H. G. Sonntag

Keyword(s):

Immune Response ◽

Intensive Care ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Intensive Care Patients

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Bovine Delta Papillomavirus E5 Oncoprotein Interacts With TRIM25 and Hampers Antiviral Innate Immune Response Mediated by RIG-I-Like Receptors

Frontiers in Immunology ◽

10.3389/fimmu.2021.658762 ◽

2021 ◽

Vol 12 ◽

Author(s):

Francesca De Falco ◽

Anna Cutarelli ◽

Ivan Gentile ◽

Pellegrino Cerino ◽

Valeria Uleri ◽

...

Keyword(s):

Immune Response ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Innate Immune ◽

Urothelial Cells ◽

Antiviral Immune Response ◽

Expression Levels ◽

Real Time Quantitative Pcr ◽

E5 Oncoprotein

Persistent infection and tumourigenesis by papillomaviruses (PVs) require viral manipulation of various of cellular processes, including those involved in innate immune responses. Herein, we showed that bovine PV (BPV) E5 oncoprotein interacts with a tripartite motif-containing 25 (TRIM25) but not with Riplet in spontaneous BPV infection of urothelial cells of cattle. Statistically significant reduced protein levels of TRIM25, retinoic acid-inducible gene I (RIG-I), and melanoma differentiation-associated gene 5 (MDA5) were detected by Western blot analysis. Real-time quantitative PCR revealed marked transcriptional downregulation of RIG-I and MDA5 in E5-expressing cells compared with healthy urothelial cells. Mitochondrial antiviral signalling (MAVS) protein expression did not vary significantly between diseased and healthy cells. Co-immunoprecipitation studies showed that MAVS interacted with a protein network composed of Sec13, which is a positive regulator of MAVS-mediated RLR antiviral signalling, phosphorylated TANK binding kinase 1 (TBK1), and phosphorylated interferon regulatory factor 3 (IRF3). Immunoblotting revealed significantly low expression levels of Sec13 in BPV-infected cells. Low levels of Sec13 resulted in a weaker host antiviral immune response, as it attenuates MAVS-mediated IRF3 activation. Furthermore, western blot analysis revealed significantly reduced expression levels of pTBK1, which plays an essential role in the activation and phosphorylation of IRF3, a prerequisite for the latter to enter the nucleus to activate type 1 IFN genes. Our results suggested that the innate immune signalling pathway mediated by RIG-I-like receptors (RLRs) was impaired in cells infected with BPVs. Therefore, an effective immune response is not elicited against these viruses, which facilitates persistent viral infection.

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Quantitative and qualitative analysis of sea bream,Sparus aurata(L.), humoral immune response, vaccinated with commercial and experimental vaccines against vibriosis and photobacteriosis

Journal of the Marine Biological Association of the United Kingdom ◽

10.1017/s0025315417001631 ◽

2017 ◽

Vol 98 (1) ◽

pp. 105-115 ◽

Cited By ~ 2

Author(s):

Vasileios Bakopoulos ◽

Ioanna Kosma ◽

Evita Laspa

Keyword(s):

Immune Response ◽

Western Blot ◽

Humoral Immune Response ◽

Blot Analysis ◽

Western Blot Analysis ◽

Sparus Aurata ◽

Sea Bream ◽

Whole Cell ◽

Humoral Immune ◽

Commercial Vaccine

The specific humoral immune response of sea bream,Sparus aurata(L.), againstVibrio anguillarumO1 andPhotobacterium damselaesubsp.piscicida(Phdp) after immunization with commercial and experimental bacterins was analysed quantitatively and qualitatively. Specific anti-V. anguillarumO1 and anti-Phdp levels provoked by the adjuvanted commercial vaccine reached higher levels in comparison to the aqueous commercial and experimental bacterins. Infection of vaccinated fish withV. anguillarumO1 bacterial cells acted as a boost of the humoral immune response, except for the sera of the group vaccinated with the adjuvanted vaccine. Infection with Phdp acted as a boost of the humoral immune response mainly for the group vaccinated with a monovalent Phdp bacterin and to a lesser degree for the group vaccinated with the aqueous commercial vaccine. Western blot analysis of the sera againstV. anguillarumO1 whole cell antigens revealed strong reactions to only a few antigens below 54 kD and above 15 kD and weak reactions to other antigens. Similar reactions were observed from the sera isolated from the controls. Western blot analysis of the sera against Phdp whole cell antigens revealed strong reactions to only a handful of antigens below 20.7 and below 6.4 kD. Sera from the control group, as in the case ofV. anguillarumO1, reacted with Phdp whole cell antigens. No differences regarding antigen reactions between monovalent and bivalent formulations were noted, in contrast to the adjuvanted and aqueous bacterins.

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Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

Journal of Neurosurgery ◽

10.3171/2010.7.gks10972 ◽

2010 ◽

Vol 113 (Special_Supplement) ◽

pp. 228-235 ◽

Cited By ~ 4

Author(s):

Qiang Jia ◽

Yanhe Li ◽

Desheng Xu ◽

Zhenjiang Li ◽

Zhiyuan Zhang ◽

...

Keyword(s):

Western Blot ◽

Gamma Knife ◽

Blot Analysis ◽

Western Blot Analysis ◽

Glioma Cells ◽

C6 Glioma ◽

C6 Glioma Cells ◽

C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

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Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

Current Topics in Medicinal Chemistry ◽

10.2174/1568026620666200731174216 ◽

2020 ◽

Vol 20 (23) ◽

pp. 2070-2079

Author(s):

Srimadhavi Ravi ◽

Sugata Barui ◽

Sivapriya Kirubakaran ◽

Parul Duhan ◽

Kaushik Bhowmik

Keyword(s):

Western Blot ◽

Cancer Cells ◽

Cell Line ◽

Cell Lines ◽

Cancer Cell ◽

Blot Analysis ◽

Western Blot Analysis ◽

Cancer Cell Lines ◽

Atm Kinase ◽

Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

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The Antitumor Effects of Britanin on Hepatocellular Carcinoma Cells and its Real-Time Evaluation by In Vivo Bioluminescence Imaging

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200227092623 ◽

2020 ◽

Vol 20 (9) ◽

pp. 1147-1156

Author(s):

Hanrui Li ◽

GeTao Du ◽

Lu Yang ◽

Liaojun Pang ◽

Yonghua Zhan

Keyword(s):

Hepatocellular Carcinoma ◽

Western Blot ◽

Blot Analysis ◽

Tumor Size ◽

Hepg2 Cells ◽

Western Blot Analysis ◽

Bioluminescence Imaging ◽

Antitumor Effects ◽

In Vivo Bioluminescence Imaging

Background: Hepatocellular carcinoma is cancer with many new cases and the highest mortality rate. Chemotherapy is the most commonly used method for the clinical treatment of hepatocellular carcinoma. Natural products have become clinically important chemotherapeutic drugs due to their great potential for pharmacological development. Many sesquiterpene lactone compounds have been proven to have antitumor effects on hepatocellular carcinoma. Objective: Britanin is a sesquiterpene lactone compound that can be considered for the treatment of hepatocellular carcinoma. The present study aimed to investigate the antitumor effect of britanin. Methods: BEL 7402 and HepG2 cells were used to study the cytotoxicity and antitumor effects of britanin. Preliminary studies on the nuclear factor kappa B pathway were conducted by western blot analysis. A BEL 7402-luc subcutaneous tumor model was established for the in vivo antitumor studies of britanin. In vivo bioluminescence imaging was conducted to monitor changes in tumor size. Results: The results of the cytotoxicity analysis showed that the IC50 values for britanin in BEL 7402 and HepG2 cells were 2.702μM and 6.006μM, respectively. The results of the colony formation demonstrated that the number of cells in a colony was reduced significantly after britanin treatment. And the results of transwell migration assays showed that the migration ability of tumor cells was significantly weakened after treatment with britanin. Tumor size measurements and staining results showed that tumor size was inhibited after britanin treatment. The western blot analysis results showed the inhibition of p65 protein expression and reduced the ratio of Bcl-2/Bax after treatment. Conclusion: A series of in vitro and in vivo experiments demonstrated that britanin had good antitumor effects and provided an option for hepatocellular carcinoma treatment.

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Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway

Nutrients ◽

10.3390/nu11112794 ◽

2019 ◽

Vol 11 (11) ◽

pp. 2794 ◽

Cited By ~ 8

Author(s):

Cao ◽

Chen ◽

Ren ◽

Zhang ◽

Tan ◽

...

Keyword(s):

Western Blot ◽

Signaling Pathway ◽

Inflammatory Cytokines ◽

Blot Analysis ◽

Western Blot Analysis ◽

Inflammatory Responses ◽

Raw264.7 Macrophages ◽

Tnf Α ◽

Autophagy Inhibition ◽

Pro Inflammatory Cytokines

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

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