The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is required for pro-inflammatory effects of TNFα. Our previous studies demonstrated that PARP-1 mediates TNFα-induced NF-κB activation in glia. Here, we evaluated the mechanisms by which TNFα activates PARP-1 and PARP-1 mediates NF-κB activation.
Primary cultures of mouse cortical astrocytes and microglia were treated with TNFα and suitable signaling pathway modulators (pharmacological and molecular). Outcome measures included calcium imaging, PARP-1 activation status, NF-κB transcriptional activity, DNA damage assesment and cytokine relesease profiling.
TNFα induces PARP-1 activation in the absence of detectable DNA strand breaks, as measured by the PANT assay. TNFα-induced transcriptional activation of NF-κB requires PARP-1 enzymatic activity. Enzymatic activation of PARP-1 by TNFα was blocked in Ca(2+)-free medium, by Ca(2+) chelation with BAPTA-AM, and by D609, an inhibitor of phoshatidyl choline-specific phospholipase C (PC-PLC), but not by thapsigargin or by U73112, an inhibitor of phosphatidyl inisitol-specific PLC (PI -PLC). A TNFR1 blocking antibody reduced Ca(2+) influx and PARP-1 activation. TNFα-induced PARP-1 activation was also blocked by siRNA downregulation of ERK2 and by PD98059, an inhibitor of the MEK / ERK protein kinase cascade. Moreover, TNFα-induced NF-κB (p65) transcriptional activation was absent in cells expressing PARP-1 that lacked ERK2 phosphorylation sites, while basal NF-κB transcriptional activation increased in cells expressing PARP-1 with a phosphomimetic substitution at an ERK2 phophorylation site.
These results suggest that TNFα induces PARP-1 activation through a signaling pathway involving TNFR1, Ca(2+) influx, activation of PC-PLC, and activation of the MEK1 / ERK2 protein kinase cascade. TNFα-induced PARP-1 activation is not associated with DNA damage, but ERK2 mediated phosphorylation of PARP-1.

Schedule your 30 min Free 1stOncology Demo!
Discover why more than 1,500 members use 1stOncology™ to excel in:

Early/Late Stage Pipeline Development - Target Scouting - Clinical Biomarkers - Indication Selection & Expansion - BD&L Contacts - Conference Reports - Combinatorial Drug Settings - Companion Diagnostics - Drug Repositioning - First-in-class Analysis - Competitive Analysis - Deals & Licensing

                  Schedule Your 30 min Free Demo!

Three leucoreduction filters were evaluated – when used alone or combined with centrifuge leucoreduction (C-LR) – to prevent alloimmune platelet refractoriness in a dog platelet transfusion model.
Donor platelet-rich plasma (PRP) or buffy coat (BC) platelets were either filter leucoreduced (F-LR) or F-LR/C-LR, (51) Cr radiolabelled and transfused. Weekly transfusions were given for up to 8 weeks or until platelet refractoriness. Recipients who accepted treated transfusions were then given non-leucoreduced (non-LR) platelets to determine whether donor-specific tolerance had been induced.
Acceptance of F-LR PRP transfusions ranged from 29% to 66%. F-LR/C-LR transfusions prepared from PRP were accepted by 92%, from BC by 63% and from pooled PRP by 75% of recipients (p=NS); overall acceptance rate of F-LR/C-LR transfusions was 83%. Tolerance to subsequent non-LR transfusions occurred in 45% of the F-LR-/C-LR-accepting recipients unrelated to DR-B compatibility between donors and recipients (P = 0·18).
In a dog platelet transfusion model, acceptance of donor platelets required combining F-LR with C-LR as apparently each process removes different immunizing WBCs.
© 2016 International Society of Blood Transfusion.

Schedule your 30 min Free 1stOncology Demo!
Discover why more than 1,500 members use 1stOncology™ to excel in:

Early/Late Stage Pipeline Development - Target Scouting - Clinical Biomarkers - Indication Selection & Expansion - BD&L Contacts - Conference Reports - Combinatorial Drug Settings - Companion Diagnostics - Drug Repositioning - First-in-class Analysis - Competitive Analysis - Deals & Licensing

                  Schedule Your 30 min Free Demo!

A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.

Schedule your 30 min Free 1stOncology Demo!
Discover why more than 1,500 members use 1stOncology™ to excel in:

Early/Late Stage Pipeline Development - Target Scouting - Clinical Biomarkers - Indication Selection & Expansion - BD&L Contacts - Conference Reports - Combinatorial Drug Settings - Companion Diagnostics - Drug Repositioning - First-in-class Analysis - Competitive Analysis - Deals & Licensing

                  Schedule Your 30 min Free Demo!

Opuntia ficus-indica (L.) is a popular edible plant that possesses considerable nutritional value and exhibits diverse biological actions including anti-inflammatory and antidiabetic activities. In this study, we hypothesized that DWJ504, an extract of O ficus-indica seed, would ameliorate hepatic steatosis and inflammation by regulating hepatic de novo lipogenesis and macrophage polarization against experimental nonalcoholic steatohepatitis. Mice were fed a normal diet or a high-fat diet (HFD) for 10 weeks. DWJ504 (250, 500, and 1000 mg/kg) or vehicle (0.5% carboxymethyl cellulose) were orally administered for the last 4 weeks of the 10-week HFD feeding period. DWJ504 treatment remarkably attenuated HFD-induced increases in hepatic lipid content and hepatocellular damage. DWJ504 attenuated increases in sterol regulatory element-binding protein 1 and carbohydrate-responsive element-binding protein expression and a decrease in carnitine palmitoyltransferase 1A. Although DWJ504 augmented peroxisome proliferator-activated receptor α protein expression, it attenuated peroxisome proliferator-activated receptor γ expression. Moreover, DWJ504 promoted hepatic M2 macrophage polarization as indicated by attenuation of the M1 marker genes and enhancement of M2 marker genes. Finally, DWJ504 attenuated expression of toll-like receptor 4, nuclear factor κB, tumor necrosis factor α, interleukin 6, TIR-domain-containing adapter-inducing interferon β, and interferon β levels. Our results demonstrate that DWJ504 prevented intrahepatic lipid accumulation, induced M2 macrophage polarization, and suppressed the toll-like receptor 4-mediated inflammatory signaling pathway. Thus, DWJ504 has therapeutic potential in the prevention of nonalcoholic fatty liver disease.
Copyright © 2016 Elsevier Inc. All rights reserved.

Schedule your 30 min Free 1stOncology Demo!
Discover why more than 1,500 members use 1stOncology™ to excel in:

Early/Late Stage Pipeline Development - Target Scouting - Clinical Biomarkers - Indication Selection & Expansion - BD&L Contacts - Conference Reports - Combinatorial Drug Settings - Companion Diagnostics - Drug Repositioning - First-in-class Analysis - Competitive Analysis - Deals & Licensing

                  Schedule Your 30 min Free Demo!

Available clinical human papilloma virus (HPV) diagnostics for head and neck cancer have limited sensitivity and/or fail to define the HPV genotype. Common HPV genotyping assays are costly and labor intensive. We sought to develop a next-generation sequencing (NGS)-based HPV genotyping assay that was sensitive enough to work on formalin-fixed paraffin-embedded (FFPE) samples. We developed an ion torrent NGS HPV genotyping assay using barcoded HPV PCR broad-spectrum general primers 5(+)/6(+) (BSGP)5(+)/6(+). To validate genotype specificity and use in archived clinical FFPE tumor samples, we compared NGS HPV genotyping at 2 sequencing centers with typing by Roche Linear Array assay in 42 oropharyngeal and cervical cancer specimens representing 10 HPV genotypes, as well as HPV-negative cases. To demonstrate the detection of a broad range of HPV genotypes, we genotyped a cohort of 266 cervical cancers. A comparison of NGS genotyping of FFPE cancer specimens with genotyping by Linear Array showed concordant results in 34/37 samples (92%) at sequencing site 1 and 39/42 samples (93%) at sequencing site 2. Concordance between sites was 92%. Designed for use with 10 ng genomic DNA, the assay detected HPV using as little as 1.25 ng FFPE-derived genomic DNA. In 266 cervical cancer specimens, the NGS assay identified 20 different HPV genotypes, including all 13 carcinogenic genotypes. This novel NGS assay provides a sensitive and specific high-throughput method to detect and genotype HPV in a range of clinical specimens derived from FFPE with low per-sample cost.

Schedule your 30 min Free 1stOncology Demo!
Discover why more than 1,500 members use 1stOncology™ to excel in:

Early/Late Stage Pipeline Development - Target Scouting - Clinical Biomarkers - Indication Selection & Expansion - BD&L Contacts - Conference Reports - Combinatorial Drug Settings - Companion Diagnostics - Drug Repositioning - First-in-class Analysis - Competitive Analysis - Deals & Licensing

                  Schedule Your 30 min Free Demo!