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

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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Long-term exposure to LPS enhances the rate of stimulated exocytosis and surfactant secretion in alveolar type II cells and upregulates P2Y2receptor expression

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00536.2007 ◽

2008 ◽

Vol 295 (4) ◽

pp. L708-L717 ◽

Cited By ~ 19

Author(s):

Ignacio Garcia-Verdugo ◽

Andrea Ravasio ◽

Elvira Garcia de Paco ◽

Monique Synguelakis ◽

Nina Ivanova ◽

...

Keyword(s):

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Purinergic Receptor ◽

Receptor Subtype ◽

Alveolar Type ◽

Type Ii Cells ◽

Type Ii ◽

Alveolar Type Ii Cells

Bacterial LPS is a potent proinflammatory molecule. In the lungs, LPS induces alterations in surfactant pool sizes and phospholipid (PL) contents, although direct actions of LPS on the alveolar type II cells (AT II) are not yet clear. For this reason, we studied short- and long-term effects of LPS on basal and agonist-stimulated secretory responses of rat AT II by using Ca2+microfluorimetry, a microtiter plate-based exocytosis assay, by quantitating PL and3H-labeled choline released into cell supernatants and by using quantitative PCR and Western blot analysis. Long term, but not short term, exposures to LPS led to prolonged ATP-induced Ca2+signals and an increased rate in vesicle fusions with an augmented release of surfactant PL. Most notably, the stimulatory effect of LPS was ATP-dependent and may be mediated by the upregulation of the purinergic receptor subtype P2Y2. Western blot analysis confirmed higher levels of P2Y2, and suramin, a P2Y receptor antagonist, was more effective in LPS-treated cells. From these observations, we conclude that LPS, probably via Toll-like receptor-4, induces a time-dependent increase in P2Y2receptors, which, by yet unknown mechanisms, leads to prolonged agonist-induced Ca2+responses that trigger a higher activity in vesicle fusion and secretion. We further conclude that chronic exposure to endotoxin sensitizes AT II to increase the extracellular surfactant pool, which aids in the pulmonary host defense mechanisms.

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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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Western blot analysis of immune response to Legionella bozemanii antigens.

Journal of Clinical Pathology ◽

10.1136/jcp.44.11.932 ◽

1991 ◽

Vol 44 (11) ◽

pp. 932-935 ◽

Cited By ~ 2

Author(s):

I Boldur ◽

H Kahana ◽

R Kazak ◽

B Cwikel ◽

I Sarov

Keyword(s):

Immune Response ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis

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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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Characterization of a New Case of Autoimmune Type I Hyperlipidemia: Long-Term Remission under Immunosuppressive Therapy1

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.82.3.3835 ◽

1997 ◽

Vol 82 (3) ◽

pp. 791-796

Author(s):

Valerie Pruneta ◽

Philippe Moulin ◽

Florence Labrousse ◽

Pierre-Jean Bondon ◽

Gabriel Ponsin ◽

...

Keyword(s):

Western Blot ◽

Immunosuppressive Therapy ◽

Blot Analysis ◽

Western Blot Analysis ◽

Dietary Therapy ◽

Type I ◽

Hepatic Triglyceride ◽

Triglyceride Lipase ◽

Hepatic Triglyceride Lipase

Abstract Only a few cases of type I hyperlipidemia occurring in patients with autoimmune disease have been reported. We describe the case of a 35-yr-old woman suffering from severe type I hyperchylomicronemia. A combination of various hypolipidemic treatments, including strict hypolipidemic dietary therapy and administration of fibrates or n-3 fatty acids, was inefficient. Because of a history of familial autoimmunity, we introduced an immunosuppressive therapy that resulted in consistent long term and stable remission. Two attempts to reduce the immunosuppressor dose resulted in major relapses. To explain the defect of chylomicron hydrolysis, we investigated the postheparin plasma lipase activities. Hepatic triglyceride lipase activity was normal, whereas that of lipoprotein lipase (LPL) was reduced to about 30% of normal. Immunosuppressive therapy resulted in a complete and durable normalization of LPL activity. Using Western blot analysis, we found in the plasma of the patient a circulating IgG specifically directed against LPL, which became undetectable during immunosuppressive therapy. Western blot analysis revealed that the whole circulating anti-LPL autoantibody was bound to chylomicrons. Proteins extracted from patient’s chylomicrons were able to induce a dose-related inhibition of LPL activity in vitro, whereas that of hepatic triglyceride lipase remained unchanged. These data constitute the first description of autoimmune hyperchylomicronemia due to an exclusive defect of LPL activity, and they show that a complete remission has been obtained after immunosuppressive therapy. Finally, our finding that the anti-LPL autoantibody is bound to chylomicrons emphasizes their previously unrecognized ability to transport LPL, already described for other lipoprotein fractions.

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Effects of silk solution against laminectomy-induced dural adhesion formation and inflammation in a rat model

Journal of Neurosurgery Spine ◽

10.3171/2018.4.spine171164 ◽

2018 ◽

Vol 29 (5) ◽

pp. 599-607 ◽

Cited By ~ 1

Author(s):

Yong-Jun Cho ◽

Chi Hern Lee ◽

Dae Won Kim ◽

Ki-Yeon Yoo ◽

Won Sik Eum ◽

...

Keyword(s):

Western Blot ◽

Rat Model ◽

Blot Analysis ◽

Western Blot Analysis ◽

Combined Treatment ◽

Adhesion Formation ◽

Long Term Effects ◽

Solution Treated ◽

Grade 3

OBJECTIVEAdhesion formation is one of the most common complications following laminectomy. The efficiency of antiadhesive agents is required to prevent postsurgical adhesion. Therefore, the authors investigated the effects of silk solution against laminectomy-induced dural adhesion formation in a rat model.METHODSThe authors prepared the silk solution from silkworms. Laminectomies were performed between L3 and L5 in all rats. MediShield was used as a control. The effects of silk solution against laminectomy-induced dural adhesion formation and inflammation were confirmed by histological examination and Western blot analysis.RESULTSLaminectomy-induced dural adhesion formation was drastically reduced in silk solution–treated rats (Grade 2) compared to vehicle-treated rats (Grade 3). Dural adhesion formation was significantly reduced in rats that received treatment with silk solution or MediShield, which is a known antiadhesion drug. In addition, combined treatment with silk solution and MediShield showed greater reductions in inflammation compared to the silk solution– or MediShield-treated rats (p > 0.05).CONCLUSIONSIn this study, the authors demonstrated that silk solution effectively reduced postlaminectomy dural adhesion formation in rats. Although further studies on the long-term effects are necessary to validate these results, silk solution may potentially serve as an antiadhesion agent in neurological procedures.

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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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