scholarly journals Stimulation of Toll-like receptor 3 and 4 induces interleukin-1β maturation by caspase-8

2008 ◽  
Vol 205 (9) ◽  
pp. 1967-1973 ◽  
Author(s):  
Jonathan Maelfait ◽  
Elisabeth Vercammen ◽  
Sophie Janssens ◽  
Peter Schotte ◽  
Mira Haegman ◽  
...  
Keyword(s):  
Active Form ◽  
Biologically Active ◽  
Poly I:C ◽  
Caspase 8 ◽  
Caspase 1 ◽  
Polyinosinic:Polycytidylic Acid ◽  
Membrane Bound ◽  
Acute And Chronic Inflammation ◽  
Stimulation Of

The cytokine interleukin (IL)-1β is a key mediator of the inflammatory response and has been implicated in the pathophysiology of acute and chronic inflammation. IL-1β is synthesized in response to many stimuli as an inactive pro–IL-1β precursor protein that is further processed by caspase-1 into mature IL-1β, which is the secreted biologically active form of the cytokine. Although stimulation of membrane-bound Toll-like receptors (TLRs) up-regulates pro–IL-1β expression, activation of caspase-1 is believed to be mainly initiated by cytosolic Nod-like receptors. In this study, we show that polyinosinic:polycytidylic acid (poly[I:C]) and lipopolysaccharide stimulation of macrophages induces pro–IL-1β processing via a Toll/IL-1R domain–containing adaptor-inducing interferon-β–dependent signaling pathway that is initiated by TLR3 and TLR4, respectively. Ribonucleic acid interference (RNAi)–mediated knockdown of the intracellular receptors NALP3 or MDA5 did not affect poly(I:C)-induced pro–IL-1β processing. Surprisingly, poly(I:C)- and LPS-induced pro–IL-1β processing still occurred in caspase-1–deficient cells. In contrast, pro–IL-1β processing was inhibited by caspase-8 peptide inhibitors, CrmA or vFLIP expression, and caspase-8 knockdown via RNAi, indicating an essential role for caspase-8. Moreover, recombinant caspase-8 was able to cleave pro–IL-1β in vitro at exactly the same site as caspase-1. These results implicate a novel role for caspase-8 in the production of biologically active IL-1β in response to TLR3 and TLR4 stimulation.

scholarly journals Activation of GSDME compensates for GSDMD deficiency in a mouse model of NLRP3 inflammasomopathy

2021 ◽  
Author(s):  
Chun Wang ◽  
Tong Yang ◽  
Jianqiu Xiao ◽  
Canxin Xu ◽  
Yael Alippe ◽  
...  
Keyword(s):  
Inflammatory Disease ◽  
Early Death ◽  
Organ Damage ◽  
Caspase 8 ◽  
Inflammasome Activation ◽  
Inflammatory Pathway ◽  
Disease Symptoms ◽  
Caspase 1 ◽  
Inflammatory Stimuli

AbstractThe D301N NLRP3 mutation in mice (D303N in humans) causes severe multi-organ damage and early death driven by the constitutively activated NLRP3 (NLRP3ca) inflammasome. Triggered inflammasomes activate caspase-1 to process IL-1 family cytokines and gasdermin D (GSDMD), generating N-terminal fragments, which oligomerize within the plasma membrane to form pores, which cause inflammatory cell death (pyroptosis) and through which IL-1β and IL-18 are secreted. GSDMD activation is central to disease symptoms since spontaneous inflammation in Nlrp3ca;Gsdmd-/- mice is negligible. Unexpectedly, when Nlrp3ca;Gsdmd-/- mice were challenged with LPS or TNF-α, they secreted high amounts of IL-1β and IL-18, suggesting an alternative GSDMD-independent inflammatory pathway. Here we show that GSDMD deficient macrophages subjected to inflammatory stimuli activate caspase-8, -3 and GSDME-dependent cytokine release and pyroptosis. Caspase-8, -3 and GSDME also activated pyroptosis when NLRP3 was stimulated in caspase-1 deficient macrophages. Thus, a salvage caspase-8, -3-GSDME inflammatory pathway is activated following NLRP3 activation when the canonical NLRP3-caspase-1-GSDMD is blocked. Surprisingly, the active metabolite of the GSDMD-inhibitor disulfiram, inhibited not only GSDMD but also GSDME-mediated inflammation in vitro and suppressed severe inflammatory disease symptoms in Nlrp3ca mice, a model for severe neonatal multisystem inflammatory disease. Although disulfiram did not directly inhibit GSDME, it suppressed inflammasome activation in GSDMD-deficient cells. Thus, the combination of inflammatory signals and NLRP3ca overwhelmed the protection provided by GSDMD deficiency, rewiring signaling cascades through caspase-8, -3 and GSDME to propagate inflammation. This functional redundancy suggests that concomitant inhibition of GSDMD and GSDME may be necessary to suppress disease in inflammasomopathy patients.

scholarly journals Erythropoiesis in steel mutant mice: effects of erythropoietin in vitro

Blood ◽  
1975 ◽  
Vol 45 (3) ◽  
pp. 427-433 ◽  
Author(s):  
DH Chui ◽  
BV Loyer
Keyword(s):  
Culture System ◽  
Marrow Cell ◽  
Biologically Active ◽  
Mutant Mice ◽  
Erythroid Cell ◽  
Biochemical Modification ◽  
Normochromic Anemia ◽  
Stimulation Of

Abstract Adult SI/SI-d mutant mice have severe macrocytic, normochromic anemia. Moreover these animals are unresponsive to the stimulation of erythropoietin in vivo. By means of a bone marrow cell suspension culture system, the present investigation shows that in adult SI/SI-d marrow, there are cells capable of responding in vitro to erythropoietin in a normal fashion. Moreover, the erythropoietin present in SI/SI-d serum is biologically active in vitro without any prior biochemical modification. These observations support the suggestion that there is a defect in differentiation in the erythroid cell lines of SI/SI-d mice in vivo due to an abnormal hemopoietic microenvironment.

A non-covalent peptide-based strategy for siRNA delivery

2007 ◽  
Vol 35 (1) ◽  
pp. 44-46 ◽  
Author(s):  
L. Crombez ◽  
A. Charnet ◽  
M.C. Morris ◽  
G. Aldrian-Herrada ◽  
F. Heitz ◽  
...  
Keyword(s):  
Sirna Delivery ◽  
Active Form ◽  
Short Review ◽  
Clinical Development ◽  
Biologically Active ◽  
Amphipathic Peptide ◽  
New Strategy ◽  
Endosomal Pathway

The major obstacle to clinical development of siRNAs (short interfering RNAs), like for most of the nucleic-acid-based strategies, is their poor cellular uptake and bioavailability. Although several viral and non-viral strategies have been proposed to improve siRNA delivery, their applications in vivo remain a major challenge. We have developed a new strategy, based on a short amphipathic peptide, MPG, that is able to form stable nanoparticles with siRNA. MPG-based particles enter the cell independently of the endosomal pathway and can efficiently deliver siRNA in a fully biologically active form into a variety of cell lines and in vivo. This short review will discuss the mechanism and the potency of the MPG strategy for siRNA delivery both in vitro and in vivo.

scholarly journals Vitamin D Enhances Radiosensitivity of Colorectal Cancer by Reversing Epithelial-Mesenchymal Transition

2021 ◽  
Vol 9 ◽  
Author(s):  
Xinyue Yu ◽  
Qian Wang ◽  
Baocai Liu ◽  
Ning Zhang ◽  
Guanghui Cheng
Keyword(s):  
Colorectal Cancer ◽  
Vitamin D ◽  
Epithelial Mesenchymal Transition ◽  
Active Form ◽  
Plasminogen Activator Inhibitor ◽  
Biologically Active ◽  
Plasminogen Activator Inhibitor 1 ◽  
Mesenchymal Transition

Colorectal cancer (CRC) is often resistant to conventional therapies. Previous studies have reported the anticancer effects of vitamin D in several cancers, its role in radiotherapy (RT) remains unknown. We found that 1α, 25-dihydroxyvitamin D3 (VD3), the biologically active form of vitamin D, had antitumor effect on CRC and sensitized CRC cells to ionizing radiation (IR). VD3 demonstrated synergistic effect in combination with IR, which were detected by colony formation and cell proliferation assay. Radiosensitivity restoration induced by VD3 was associated with a series of phenotypes, including apoptosis, autophagy, and epithelial-mesenchymal transition (EMT). Using proteomics, “regulation of cell migration” and “cadherin” were found to be obviously enriched GO terms. Moreover, cystatin D and plasminogen activator inhibitor-1 (PAI-1), the differentially expressed proteins, were associated with EMT. Next, we confirmed the contributions of these two genes in enhancing IR sensitivity of CRC cells upon inhibition of EMT. As determined by proteomics, the mechanism underlying such sensitivity involved partially block of JAK/STAT3 signaling pathway. Furthermore, VD3 also elicited sensitization to RT in xenograft CRC models without additional toxicity. Our study revealed that VD3 was able to act in synergy with IR both in vitro and in vivo and could also confer radiosensitivity by regulating EMT, thereby providing a novel insight for elevating the efficacy of therapeutic regimens.

scholarly journals Vegetal diamine oxidase alleviates histamine-induced contraction of colonic muscles

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Armelle Tchoumi Neree ◽  
Rodolphe Soret ◽  
Lucia Marcocci ◽  
Paola Pietrangeli ◽  
Nicolas Pilon ◽  
...  
Keyword(s):  
Diamine Oxidase ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Active Form ◽  
Distal Colon ◽  
Biologically Active ◽  
In Vitro Assays ◽  
Histaminase Activity ◽  
Irritable Bowel

AbstractExcess of histamine in gut lumen generates a pronounced gastrointestinal discomfort, which may include diarrhea and peristalsis dysfunctions. Deleterious effects of histamine can be alleviated with antihistamine drugs targeting histamine receptors. However, many antihistamine agents come with various undesirable side effects. Vegetal diamine oxidase (vDAO) might be a relevant alternative owing to its histaminase activity. Mammalian intestinal mucosa contains an endogenous DAO, yet possessing lower activity compared to that of vDAO preparation. Moreover, in several pathological conditions such as inflammatory bowel disease and irritable bowel syndrome, this endogenous DAO enzyme can be lost or inactivated. Here, we tested the therapeutic potential of vDAO by focusing on the well-known effect of histamine on gut motility. Using ex vivo and in vitro assays, we found that vDAO is more potent than commercial anti-histamine drugs at inhibiting histamine-induced contraction of murine distal colon muscles. We also identified pyridoxal 5′-phosphate (the biologically active form of vitamin B6) as an effective enhancer of vDAO antispasmodic activity. Furthermore, we discovered that rectally administered vDAO can be retained on gut mucosa and remain active. These observations make administration of vDAO in the gut lumen a valid alternative treatment for histamine-induced intestinal dysfunctions.

Pan-caspase inhibition as a potential host-directed immunotherapy against MRSA and other bacterial skin infections

2021 ◽  
Vol 13 (601) ◽  
pp. eabe9887
Author(s):  
Martin P. Alphonse ◽  
Jessica H. Rubens ◽  
Roger V. Ortines ◽  
Nicholas A. Orlando ◽  
Aman M. Patel ◽  
...  
Keyword(s):  
Caspase 8 ◽  
Skin Infections ◽  
Caspase 9 ◽  
Potential Host ◽  
Caspase 1 ◽  
Caspase Inhibition ◽  
Mrsa Strains ◽  
Bacterial Skin Infections

Staphylococcus aureus causes most skin infections in humans, and the emergence of methicillin-resistant S. aureus (MRSA) strains is a serious public health threat. There is an urgent clinical need for nonantibiotic immunotherapies to treat MRSA infections and prevent the spread of antibiotic resistance. Here, we investigated the pan-caspase inhibitor quinoline–valine–aspartic acid–difluorophenoxymethyl ketone (Q-VD-OPH) for efficacy against MRSA skin infection in mice. A single systemic dose of Q-VD-OPH decreased skin lesion sizes and reduced bacterial burden compared with vehicle-treated or untreated mice. Although Q-VD-OPH inhibited inflammasome-dependent apoptosis-associated speck-like protein containing caspase activation and recruitment domain (ASC) speck formation and caspase-1–mediated interleukin-1β (IL-1β) production, Q-VD-OPH maintained efficacy in mice deficient in IL-1β, ASC, caspase-1, caspase-11, or gasdermin D. Thus, Q-VD-OPH efficacy was independent of inflammasome-mediated pyroptosis. Rather, Q-VD-OPH reduced apoptosis of monocytes and neutrophils. Moreover, Q-VD-OPH enhanced necroptosis of macrophages with concomitant increases in serum TNF and TNF-producing neutrophils, monocytes/macrophages, and neutrophils in the infected skin. Consistent with this, Q-VD-OPH lacked efficacy in mice deficient in TNF (with associated reduced neutrophil influx and necroptosis), in mice deficient in TNF/IL-1R and anti-TNF antibody-treated WT mice. In vitro studies revealed that combined caspase-3, caspase-8, and caspase-9 inhibition reduced apoptosis, and combined caspase-1, caspase-8, and caspase-11 inhibition increased TNF, suggesting a mechanism for Q-VD-OPH efficacy in vivo. Last, Q-VD-OPH also had a therapeutic effect against Streptococcus pyogenes and Pseudomonas aeruginosa skin infections in mice. Collectively, pan-caspase inhibition represents a potential host-directed immunotherapy against MRSA and other bacterial skin infections.

An Investigational Proteasome Inhibitor MLN9708 (MLN2238) Induces Apoptosis In Human Multiple Myeloma Cells In Vitro

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3066-3066
Author(s):  
Aisha Masood ◽  
Kasyapa Chitta ◽  
Kiersten M Miles ◽  
Nazmul H Khan ◽  
Remi Adelaiye ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Conflicts Of Interest ◽  
Active Form ◽  
Biologically Active ◽  
Mitochondrial Outer Membrane ◽  
Dependent Manner ◽  
In Vivo Models

Abstract Abstract 3066 Targeting the proteasome has proven to be one of the most effective therapeutic strategies in the treatment of multiple myeloma (MM), and the proteasome inhibitor bortezomib is approved for treatment of MM. However its clinical efficacy is compromised by the acquired resistance in patients, necessitating the development of new therapeutics. Several new proteasome inhibitors are under investigation for their therapeutic efficacy in MM. MLN9708 (Millennium Pharmaceuticals, Inc., Cambridge, MA) is a proteasome inhibitor which shows refined pharmacokinetic and pharmacodynamic properties in preclinical studies and is currently in Phase I clinical development. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to MLN2238, the biologically active form. MLN2238 was used for all of the studies reported here, in which we report the efficacy of MLN2238 on three established MM cell lines-KMS11, OPM2 and U266. MLN2238 was found to inhibit the chymotrypsin-like proteasomal activity of all MM cell lines in a dose dependent manner. Investigation of the IC50 of MLN2238 on these cell lines demonstrated that KMS11 is the most sensitive (IC50 of 15.9 nM) while U266 was found to be the least sensitive cell line (IC50 of 511 nM). OPM2 cells also showed intermediate sensitivity with an IC50 of 58.6 nM. MLN2238 induced apoptosis in KMS11 cells as evidenced by annexin V staining and PARP-1 cleavage. Cleavage of caspases 9 and 3 suggested activation of the intrinsic apoptotic pathway by MLN2238. Furthermore, MLN2238 treatment was shown to increase the mitochondrial outer membrane permeability (MOMP) and decrease BCL-2 levels. Evaluation of the expression of PSMB5, the preferred proteasomal subunit target for both bortezomib and MLN2238, revealed that it is expressed at approximately 3 fold more in KMS11 cells as compared to U266, suggesting a possible mechanism for higher sensitivity of KMS11 to the proteasomal inhibitor, MLN2238. This preclinical evaluation confirms the anti-myeloma effects of MLN2238, warranting further in-depth evaluation in both in vitro and in vivo models of MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The induction of secondary cytolytic T lymphocytes by solubilized membrane proteins.

1978 ◽  
Vol 147 (3) ◽  
pp. 946-951 ◽  
Author(s):  
M Mescher ◽  
L Sherman ◽  
F Lemonnier ◽  
S Burakoff
Keyword(s):  
Plasma Membranes ◽  
Active Form ◽  
Protein A ◽  
Gel Filtration ◽  
Biologically Active ◽  
Cytolytic T Lymphocytes ◽  
Histocompatibility Complex ◽  
Cell Plasma ◽  
Immunological Specificity ◽  
Membrane Bound

Membrane-bound antigens responsible for induction of a secondary allogeneic murine cytolytic T-cell (CTL) response have been obtained in a soluble, biologically active form by deoxycholate solubilization of tumor cell plasma membranes. The active proteins are soluble by the criteria of both ultracentrifugation and gel filtration. The immunological specificity of the induced CTL and removal of the activity from solution by treatment with B6 anti-P815 (anti-H-2d) antiserum and Protein A-Sepharose demonstrate that the CTL-inducing activity is dependent upon solubilized major histocompatibility complex antigens.

scholarly journals Fyn kinase–tubulin interaction during meiosis of rat eggs

Reproduction ◽  
2004 ◽  
Vol 128 (4) ◽  
pp. 387-393 ◽  
Author(s):  
A Talmor-Cohen ◽  
R Tomashov-Matar ◽  
W B Tsai ◽  
W H Kinsey ◽  
R Shalgi
Keyword(s):  
Potential Role ◽  
Active Form ◽  
Src Family Kinase ◽  
Fyn Kinase ◽  
Phase Arrest ◽  
Vertebrate Eggs ◽  
Rat Eggs ◽  
Stimulation Of

Prior to fertilization, the spindle of vertebrate eggs must remain stable and well organized during the second meiotic meta-phase arrest (MII). In a previous study we have determined that the completion of meiosis is a Src family kinase (SFK)-dependent event. In the current study we have used the SFK inhibitors, SU6656 and PP2, and demonstrated that inhibition of SFKs caused the formation of a disorganized spindle. The observation that proper organization of an MII spindle is an SFK-dependent process, combined with our previous finding that Fyn kinase is localized at the microtubules (MTs), prompted us to examine the potential role of Fyn in MT signaling. Our results show an association between Fyn and tubulin, the ability of Fyn to phosphorylate tubulinin vitroand stimulation of meiosis completion by injection of a constitutively active form of Fyn (CAF).We suggested that SFKs mediate significant functions during the organization of the MII spindle. In view of CAF injection experiments, and of the pronounced concentration of Fyn kinase at the spindle, we propose that Fyn may play an important role in some aspects of the spindle functions, possibly those involving the MTs.

scholarly journals Beta-COP is essential for transport of protein from the endoplasmic reticulum to the Golgi in vitro

1993 ◽  
Vol 122 (6) ◽  
pp. 1155-1167 ◽  
Author(s):  
F Peter ◽  
H Plutner ◽  
H Zhu ◽  
TE Kreis ◽  
WE Balch
Keyword(s):  
Active Form ◽  
Biologically Active ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Vitro Assay ◽  
Vesicle Budding ◽  
Vesicular Carriers ◽  
Golgi Compartment ◽  
Vesicular Stomatitis ◽  
Rat Liver Cytosol

Using a novel in vitro assay which allows us to distinguish vesicle budding from subsequent targeting and fusion steps, we provide the first biological evidence that beta-COP, a component of non-clathrin-coated vesicles believed to mediate intraGolgi transport, is essential for transport of protein from the ER to the cis-Golgi compartment. Incubation in the presence of beta-COP specific antibodies and F(ab) fragments prevents the exit of vesicular stomatitis virus glycoprotein (VSV-G) from the ER. These results demonstrate that beta-COP is required for the assembly of coat complexes mediating vesicle budding. Fractionation of rat liver cytosol revealed that a major biologically active form of beta-COP was found in a high molecular pool (> 1,000 kD) distinct from coatomer and which promoted efficient vesicle budding from the ER. Surprisingly, rab1B could be quantitatively coprecipitated with this beta-COP containing complex and was also essential for function. We suggest that beta-COP functions in an early step during vesicle formation and that rab1B may be recruited as a component of a precoat complex which participates in the export of protein from the ER via vesicular carriers.

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