Specificity enhancement of polyclonal antisera by the induction of tolerance to unwanted cross-reacting determinants

Steroids form a structurally closely related group. As a result, antibodies produced for use in immunoassays regularly show unwanted cross-reactivities, These may be reduced by altering hapten-protein coupling procedures, thereby reducing the exposure of the determinants giving rise to the undesirable cross-reaction. However, these procedures carl prove to be complex, expensive and nor totally predictable in outcome. Exploitation of the clonal selection theory is an attractive alternative approach. The host is primed with the interfering cross-reactant coupled to a non-immunogenic amino acid copolymer to inactivate the B-lymphocyte clones specific for this steroid, producing a specific immunotolerance. Then, 3 days Inter, the host is immunized with the steroid against which nn antibody is required. The clones producing antibody to this immunogen are unaffected and the cross-reactivity is significantly reduced or deleted The technique has been applied to the reduction of endogenous sex steroid cross-reactivity from antibodies prepared against synthetic and semi-synthetic androgens (17 alpha-methyltestosterone, 19-nor-beta-testosterone) and the progestogen medroxyprogesterone. Antibodies prepared against the synthetic oestrogen zeranol using this technique have significantly reduced its undesirable cross-reactivity with the fungal metabolite 7 alpha-zearalenol. Highly specific antisera have been generated in all cases, the only adverse effect being a reduction in the titres achieved in comparison with rabbits receiving the conventional immunizing regime.

Evidence for a hypoxic fixation reaction leading to the induction of ssb and dsb in irradiated DNA

Purpose: To measure hypoxic chemical fixation processes of radiation damage in both isolated plasmid DNA and in GSH-depleted E. coli cells.

Anthrax lethal toxin and the induction of CD4 T cell immunity

Bacillus anthracis secretes exotoxins which act through several mechanisms including those that can subvert adaptive immunity with respect both to antigen presenting cell and T cell function. The combination of Protective Antigen (PA) and Lethal Factor (LF) forming Lethal Toxin (LT), acts within host cells to down-regulate the mitogen activated protein kinase (MAPK) signaling cascade. Until recently the MAPK kinases were the only known substrate for LT; over the past few years it has become evident that LT also cleaves Nlrp1, leading to inflammasome activation and macrophage death. The predicted downstream consequences of subverting these important cellular pathways are impaired antigen presentation and adaptive immunity. In contrast to this, recent work has indicated that robust memory T cell responses to B. anthracis antigens can be identified following natural anthrax infection. We discuss how LT affects the adaptive immune response and specifically the identification of B. anthracis epitopes that are both immunogenic and protective with the potential for inclusion in protein sub-unit based vaccines.

Overexpression of the microRNA miR-433 promotes resistance to paclitaxel through the induction of cellular senescence in ovarian cancer cells

Annually, ovarian cancer (OC) affects 240,000 women worldwide and is the most lethal gynecological malignancy. High-grade serous OC (HGSOC) is the most common and aggressive OC subtype, characterized by widespread genome changes and chromosomal instability and is consequently poorly responsive to chemotherapy treatment. The objective of this study was to investigate the role of the microRNA miR-433 in the cellular response of OC cells to paclitaxel treatment. We show that stable miR-433 expression in A2780 OC cells results in the induction of cellular senescence demonstrated by morphological changes, downregulation of phosphorylated retinoblastoma (p-Rb), and an increase in β-galactosidase activity. Furthermore, in silico analysis identified four possible miR-433 target genes associated with cellular senescence: cyclin-dependent kinase 6 (CDK6), MAPK14, E2F3, and CDKN2A. Mechanistically, we demonstrate that downregulation of p-Rb is attributable to a miR-433-dependent downregulation of CDK6, establishing it as a novel miR-433 associated gene. Interestingly, we show that high miR-433 expressing cells release miR-433 into the growth media via exosomes which in turn can induce a senescence bystander effect. Furthermore, in relation to a chemotherapeutic response, quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that only PEO1 and PEO4 OC cells with the highest miR-433 expression survive paclitaxel treatment. Our data highlight how the aberrant expression of miR-433 can adversely affect intracellular signaling to mediate chemoresistance in OC cells by driving cellular senescence.

MIR-433 Predicts poor response to chemotherapy in ovarian cancer patients by the induction of cellular senescence

Annually, ovarian cancer (OC) affects 240,000 women worldwide and is the most lethal gynaecological malignancy. Such mortality is predominantly associated with the development of an intrinsic and acquired resistance to chemotherapy, the lack of targeted therapies and the lack of biomarkers predicting response to standard treatment.

Our clinical data demonstrates that increased miR-433 expression in primary high grade serous OC (HGSOCs) is significantly associated with poor PFS (n=46, p=0.024). Interestingly, the IHC analysis of two miR-433 targets: MAD2 [1] and HDAC6 shows that low IHC levels of both proteins is also significantly associated with worse outcome (p=0.002 and 0.002 respectively; n=43). Additionally, the analysis of miR 433 in the publicly available TCGA dataset corroborates that high miR-433 is significantly correlated with worse OS for patients presenting with OC (n=558 and p=0.027). In vito, in a panel of OC cell lines, higher miR-433 and lower MAD2 and HDAC6 levels were associated with resistance to paclitaxel.

To further investigate the role of miR-433 in the cellular response to chemotherapy, we generated an OC cell line stably expressing miR-433 or miR-control. MTT viability assays and Western Blot analyses established that miR-433 cells were more resistant to paclitaxel treatment (50nM) compared to miR-controls. Importantly, we have shown for the first time that miR 433 induced senescence resulting in a chracteristic flattened morphology and down-regulation of phosphorylated Retinoblastoma (p Rb), a molecular marker of senescence. Surprisingly, miR 433 induced senescence was independent from two well recognised senescent drivers: namely p53/p21 and p16. To explore this further we performed an in silico analysis of seven microRNA platforms which indicated that miR 433 potentially targets Cyclin-dependent kinase CDK6, which promotes sustained phosphorylation of Rb and thus cell cycle progression. In vitro, the overexpression of pre-miR-433 resulted in diminished CDK6 expression demonstrating a novel interaction between miR-433 and CDK6.

In conclusion, this study demonstrates that high miR-433 expression predicts poor outcome in OC patients by putatively rendering OC cells resistant to paclitaxel treatment through the induction of cellular senescence identifying this microRNA as a potential marker of chemoresponse.

The Role of Thymidylate Synthase Induction in Modulating p53-regulated Gene Expression in Response to 5-Fluorouracil and Antifolates

Thymidylate synthase (TS) is a critical target for chemotherapeutic agents such as 5-fluorouracil (5-FU) and antifolates such as tomudex (TDX),multitargeted antifolate, and ZD9331. Using the MCF-7 breast cancer line, we have developed p53 wild-type (M7TS90) and null (M7TS90-E6) isogenic lines with inducible TS expression (approximately 6-fold induction compared with control after 48 h). In the M7TS90 line, inducible TS expression resulted in a moderate approximately 3-fold increase in 5-FU IC-50(72 h) dose and a dramatic >20-fold increase in the IC-50(72 h) doses of TDX, multitargeted antifolate, and ZD9331. S-phase cell cycle arrest and apoptosis induced by the antifolates were abrogated by TS induction. In contrast, cell cycle arrest and apoptosis induced by 5-FU was unaffected by TS expression levels. Inactivation of p53 significantly increased resistance to 5-FU and the antifolates with IC-50(72 h) doses for 5-FU and TDX of >100 and >10 microM, respectively, in the M7TS90-E6 cell line. Furthermore, p53 inactivation completely abrogated the cell cycle arrest and apoptosis induced by 5-FU. The antifolates induced S-phase arrest in the p53 null cell line; however, the induction of apoptosis by these agents was significantly reduced compared with p53 wild-type cells. Both inducible TS expression and the addition of exogenous thymidine (10 microM) blocked p53 and p21 induction by the antifolates but not by 5-FU in the M7TS90 cell line. Similarly, inducible TS expression and exogenous thymidine abrogated antifolate but not 5-FU-mediated up-regulation of Fas/CD95 in M7TS90 cells. Our results indicate that in M7TS90 cells, inducible TS expression modulates p53 and p53 target gene expression in response to TS-targeted antifolate therapies but not to 5-FU.

The chemoattractants, IL-8 and formyl-methionyl-leucyl-phenylalanine, regulate granulocyte colony-stimulating factor signaling by inducing suppressor of cytokine signaling-1 expression

Suppressors of cytokine signaling (SOCS) are encoded by immediate early genes known to inhibit cytokine responses in a classical feedback loop. SOCS gene expression has been shown to be induced by many cytokines, growth factors, and innate immune stimuli, such as LPS. In this paper, we report that the chemoattractants, IL-8 and fMLP, up-regulate SOCS1 mRNA in human myeloid cells, primary human neutrophils, PBMCs, and dendritic cells. fMLP rapidly up-regulates SOCS1, whereas the induction of SOCS1 upon IL-8 treatment is delayed. IL-8 and fMLP did not signal via Jak/STATs in primary human macrophages, thus implicating the induction of SOCS by other intracellular pathways. As chemoattractant-induced SOCS1 expression in neutrophils may play an important role in regulating the subsequent response to growth promoting cytokines like G-CSF, we investigated the effect of chemoattractant-induced SOCS1 on cytokine signal transduction. We show that pretreatment of primary human neutrophils with fMLP or IL-8 blocks G-CSF-mediated STAT3 activation. This study provides evidence for cross-talk between chemoattractant and cytokine signal transduction pathways involving SOCS proteins, suggesting that these chemotactic factors may desensitize neutrophils to G-CSF via rapid induction of SOCS1 expression.

BRCA1 Regulates IFN-γ Signaling through a Mechanism Involving the Type I IFNs

BRCA1 encodes a tumor suppressor gene that is mutated in the germ line of women with a genetic predisposition to breast and ovarian cancer. BRCA1 has been implicated in a number of important cellular functions including DNA damage repair, transcriptional regulation, cell cycle control, and ubiquitination. Using an Affymetrix U95A microarray, IRF-7 was identified as a BRCA1 transcriptional target and was also shown to be synergistically up-regulated by BRCA1 specifically in the presence of IFN-gamma, coincident with the synergistic induction of apoptosis. We show that BRCA1, signal transducer and activator of transcription (STAT)-1, and STAT2 are all required for the induction of IRF-7 following stimulation with IFN-gamma. We also show that the induction of IRF-7 by BRCA1 and IFN-gamma is dependent on the type I IFNs, IFN-alpha and IFN-beta. We show that BRCA1 is required for the up-regulation of STAT1, STAT2, and the type I IFNs in response to IFN-gamma. We show that BRCA1 is localized at the promoters of the molecules involved in type I IFN signaling leading to their up-regulation. Blocking this intermediary type I IFN step using specific antisera shows the requirement for IFN-alpha and IFN-beta in the induction of IRF-7 and apoptosis. Finally, we outline a mechanism for the BRCA1/IFN-gamma regulation of target genes involved in the innate immune response, which is dependent on type I IFN signaling.

The 2,5 Oligoadenylate Synthetase/RNaseL pathway is a novel effector of BRCA1 - and interferon-gamma-mediated apoptosis

I discovered that 2,5OAS family of proteins was transcriptionally upregulated by BRCA1 and interferon gamma in a synergistic manner. This correlated with synergistically induced apoptosis and both the induction of 2,5OAS and the accompanying apoptosis could be inhibited by 2,5OAS specific siRNA proving 2,5OAS was the apoptotic effector.