Examination of the stimulatory signaling potential of a channel catfish leukocyte immune-type receptor and associated adaptor

Expressed by various subsets of myeloid and lymphoid immune cells, channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are predicted to play a key role in the initiation and termination of teleost cellular effector responses. These type I transmembrane proteins belong to the immunoglobulin superfamily and display features of immunoregulatory receptors with inhibitory and/or stimulatory signaling potential. Expanding on our previous work, which demonstrated that putative stimulatory IpLITR-types associated with the catfish adaptor proteins IpFcRγ and FcRγ-L, this study focuses on the functional significance of this immune receptor-adaptor signaling complex. Specifically, we generated an epitope-tagged chimeric receptor construct by fusing the extracellular domain of IpLITR 2.6b with the transmembrane region and cytoplasmic tail of IpFcRγ-L. This chimera was stably expressed in a rat basophilic leukemia (RBL) cell line, RBL-2H3, and following cross-linking of the surface receptor with an anti-hemagglutinin monoclonal antibody or opsonized microspheres, the chimeric teleost receptor induced cellular degranulation and phagocytic responses, respectively. Site-directed mutagenesis of the immunoreceptor tyrosine-based activation motif encoded within the cytoplasmic tail of the chimera confirmed that these functional responses were dependent on the phosphorylated tyrosines within this motif. Using a combination of phospho-specific antibodies and pharmacological inhibitors, we also demonstrate that the IpLITR/IpFcRγ-L-induced degranulation response requires the activity of Src homology 2 domain containing protein tyrosine phosphatases, phosphatidylinositol 3-kinase, protein kinase C, and mitogen-activated protein kinases but appears independent of the c-Jun N-terminal kinase and p38 MAP kinase pathways. In addition to this first look at stimulatory IpLITR-mediated signaling and its influence on cellular effector responses, the advantage of generating RBL-2H3 cells stably expressing a functional IpLITR-adaptor chimera will be discussed.     

Role of oxygen in the regulation of Leydig tumor derived MA-10 cell steroid production: the effect of cobalt chloride

Abstract

We have earlier shown that cobalt chloride (CoCl₂)-induced hypoxia and second messenger 8-bromoadenosine 3′, 5′-cyclic adenosine monophosphate (8-Br-cAMP) stimulates vascular endothelial growth factor (VEGF) production in Leydig tumor cell derived MA-10 cells. Both stimuli follow common signal transduction pathways including protein kinase A (PK-A), extracellular regulated kinase 1/2 (ERK1/2), and phosphatidyl inositol-3 kinase/akt (PI3-K/Akt) pathways in the stimulation of VEGF by MA-10 cells. In the present study we investigated the role of CoCl₂ and 8-Br-cAMP on steroid production in MA-10 cells. The MA-10 cells were cultured in Waymouth MB 752/1 medium, supplemented with 15% heat inactivated horse serum. Progesterone was estimated by radioimmunoassay (RIA).We report that 8-Br-cAMP stimulated progesterone production by the MA-10 cells whereas CoCl₂ inhibited the same. Also, 8-Br-cAMP stimulated steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage enzyme (P450scc) mRNAs expression. However, CoCl₂ had no effect on StAR mRNA. Cobalt chloride directly inhibited the expression of P450scc mRNA. The decrease in progesterone production could be attributed to three different mechanisms, (1) an increase in production of reactive oxygen species (ROS), (2) an increase in HIF-1α activity, and (3) ultimately a decrease in the level of cytochrome P450 side chain cleavage (CYT P450scc). Hypoxia has an action and mechanism of action similar to that of gonadotropins on VEGF production, whereas they have a contrasting effect on steroidogenesis. This study suggests that hypoxia could be as important as gonadotropins in regulating Leydig cell steroidogenesis.     

Immune cytolytic activity is associated with reduced intra-tumoral genetic heterogeneity and with better clinical outcomes in triple negative breast cancer

Evaluation of the functional aspects if the tumor immune microenvironment (TIME), such as the recently introduced cytolytic activity score (CYT) index have been under the spotlight in cancer research; however, clinical relevance of immune cell killing activity in breast cancer has never been analyzed in large patient cohorts. We hypothesized that CYT reflects the immune activity of TIME and can predict patient survival. A total of 7533 breast cancer patients were analyzed as both discovery and validation cohorts. We found that high CYT was associated with advanced histological grade and triple-negative breast cancer (TNBC). High CYT in tumors was significantly associated with better survival in TNBC, but unexpectedly, not in other breast cancer subtypes. High CYT TNBC included both favorable immune-related, as well as unfavorable (suppressive) inflammation-related gene sets, and characterized by high infiltration with T cells and B cells. High CYT TNBC was associated with high homologous recombination deficiency and low somatic copy number alteration score and less mutant allele tumor heterogeneity, but not with tumor mutation burden (TMB). Although CYT was not associated with pathological complete response after neoadjuvant chemotherapy, it was significantly associated with high expression of multiple immune checkpoint molecules. In conclusion, CYT of TNBC is associated with enhanced anti-cancer immunity, less intra-tumoral heterogeneity, and with better survival.     

Spleen deflation and beyond: The pros and cons of Janus kinase 2 inhibitor therapy for patients with myeloproliferative neoplasms

The myeloproliferative neoplasms (MPNs) essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (MF) are malignancies that frequently harbor the recurrent somatic point mutation JAK2^V617F. The discovery of this mutation has fueled the development of Janus kinase 2 (JAK2) inhibitors. Available results have indicated that JAK2 inhibitors are particularly effective at reducing spleen size. However, the activity of these agents is multifaceted and also involves a marked improvement of systemic symptoms and, for those agents with dual JAK1 and JAK2 inhibitory activity, a marked reduction in the levels of circulating cytokines involved in the pathogenesis of the disease. Because JAK2 inhibitors are not specific for JAK2^V617F, responses have also been observed in JAK2^V617F‐negative MPNs because of the inhibition of wild‐type JAK2, which is also likely responsible for the induction of cytopenias in patients with MF and for the normalization of peripheral blood counts observed in patients with ET or PV. Given the distinct mortality and morbidity associated with ET, PV, and MF, the use of JAK2 inhibitors appears reasonable for patients with MF as well as for those with ET or PV who have become resistant or intolerant to hydroxyurea. Ongoing randomized, placebo‐controlled, phase 3 trials will further delineate the role of these agents in the management of patients with MPNs. The pros and cons of JAK2 kinase inhibitor therapy are herein discussed. Cancer 2012;. © 2011 American Cancer Society.     

Anthracycline treatment of the human monocytic leukemia cell line THP-1 increases phosphatidylserine exposure and tissue factor activity

Introduction

Cancer associated thrombosis is a well-recognized phenomenon that results in considerable patient morbidity and mortality. Malignancy conveys an increased risk for thrombosis and chemotherapy further elevates this risk. The pathophysiological mechanisms underlying this process remain poorly defined.

Materials and Methods

A human acute monocytic leukemia cell line (THP-1) was treated with commonly used anthracycline chemotherapeutics at concentrations similar to those found in the plasma of cancer patients. Cells were analyzed for tissue factor (TF) mRNA, protein, and activity. Microparticle (MP) TF activity was also measured. Phosphatidylserine (PS) exposure on cells and MPs was analyzed by flow cytometry. PS levels on MPs was also evaluated in an annexin V capture assay.

Results

Anthracycline treatment of THP-1 cells resulted in a concentration-dependent increase in cellular TF activity without a change in TF protein, which was associated with increased PS exposure on the cell surface and apoptosis. The increase in TF activity was abolished by annexin V or lactadherin indicating that PS exposure was required. Anthracycline treatment of THP-1 cells also increased the number of TF-positive MPs.

Conclusion

Treatment of THP-1 cells with anthracyclines induces apoptosis and increases cellular TF activity. The increased activity required an increase in exposure of PS. Additionally, anthracyclines increase the release of TF-positive MPs from THP-1 cells. We propose that the increase in cellular TF activity in circulating leukemic cells, combined with increased numbers of TF-positive MPs, may contribute to thrombosis in cancer patients receiving chemotherapy.     

Antipsychotic treatment and neuregulin 1-ErbB4 signalling in schizophrenia

Evidence from genetic, transgenic and post-mortem studies has strongly supported the critical role that neuregulin 1 (NRG1) and its ErbB4 receptor plays in the pathophysiology of schizophrenia. This article aims to review current evidence regarding the effects of antipsychotic treatment on NRG1-ErbB4 signalling. NRG1 and ErbB4 knockout mice display abnormal behaviours relevant to certain features of schizophrenia, which could be improved by antipsychotic (clozapine/haloperidol) treatment. In contrast to most NRG1/ErbB4 knockout mice with a decreased NRG1-ErbB4 signalling, the majority post-mortem studies showed an increased NRG1-ErbB4 signalling in schizophrenic patients. These differences could be due to degrees of alteration in risk genes (subtle variations in patients vs pronounced alteration in mutant mice) or the duration of the modification on NRG1 signalling. Various antipsychotics have different effects on NRG1 and ErbB4 expression and signalling that are dependent on treatment duration. Current evidence suggests that a chronic (12 weeks) antipsychotic treatment, at least in animal models, could downregulate NRG1-ErbB4 signalling, although an upregulation is seen for a short-term treatment. These effects may be due to multiple binding profiles with various G-coupled protein receptors (e.g. dopamine, and serotonin receptors) of antipsychotics. Studies are needed to investigate the interactions between NRG1-ErbB4 and the other signalling pathways (such as glutamatergic, GABAergic and dopaminergic). Furthermore, the interactions between NRG1/ErbB4 and other schizophrenia suspensibility genes under antipsychotic treatment also require investigation.     

Phase I trial of CYT997, a novel cytotoxic and vascular-disrupting agent

Background:

CYT997 is a novel microtubule inhibitor and vascular-disrupting agent with marked preclinical anti-tumour activity.

Methods:

This phase I dose-escalation study assessed the safety, tolerability, pharmacokinetics and pharmacodynamics of CYT997 administered by continuous intravenous infusion over 24 h every 3 weeks to patients with advanced solid tumours.

Results:

Thirty-one patients received CYT997 over 12 dose levels (7–358 mg m⁻²). Doses up to 202 mg m⁻² were well tolerated. Dose-limiting toxicities were observed at 269 and 358 mg m⁻², consisting of grade 3 prolonged corrected QT interval in two patients and grade 3 hypoxia and grade 4 dyspnea in one patient. All toxicities were reversible. The pharmacokinetics of CYT997 were linear over the entire dose range. Dynamic contrast-enhanced magnetic resonance imaging scans showed significant changes in tumour K^trans values consistent with vascular disruption in 7 out of 11 evaluable patients treated at CYT997 doses of ⩾65 mg m⁻². Moreover, plasma levels of von Willebrand factor and caspase-cleaved cytokeratin-18 increased post-treatment at higher dose levels. Among 22 patients evaluable for response, 18 achieved stable disease for >2 cycles.

Conclusions:

CYT997 was well tolerated at doses that were associated with pharmacodynamic evidence of vascular disruption in tumours.     

JAK inhibitors in myeloproliferative neoplasms: rationale, current data and perspective

JAK-STAT is an appealing but also problematic drug target in BCR-ABL1-negative myeloproliferative neoplasms (MPN) - it is appealing because the majority of patients with MPN harbor gain-of-function JAK2 or MPL mutations - it is problematic because currently available JAK inhibitors do not distinguish between oncogenic and physiologic JAK-STAT activation. Furthermore, JAK-STAT-relevant mutations in MPN do not always constitute the predominant or ancestral mutant clone. Such complexities undermine the value of JAK-STAT as a robust drug target in MPN and partly explain the hitherto lack of histologic or molecular remissions associated with currently available JAK inhibitors. Most of these drugs were, however, effective in alleviating constitutional symptoms and reducing spleen size; the mechanism of action in this instance includes drug-induced down-regulation of inflammatory cytokine activity. In addition, non-specific myelosuppression contributes to both their salutary and detrimental effects on peripheral blood count. Non-hematologic side effects include gastrointestinal disturbances, asymptomatic elevation of liver and pancreatic enzymes, peripheral neuropathy and hyperacute relapse of symptoms during treatment interruption. It is our impression that many more JAK inhibitors need to be evaluated in order to identify the best-in-class in terms of efficacy, toxicity and suitability for future combination treatment programs.