Demelza J. Ireland

Induction of necrosis and cell cycle arrest in murine cancer cell lines by Melaleuca alternifolia (tea tree) oil and terpinen-4-ol

PurposeTo examine the in vitro anticancer activity of Melaleuca alternifolia (tea tree) oil (TTO), and its major active terpene component, terpinen-4-ol, against two aggressive murine tumour cell lines, AE17 mesothelioma and B16 melanoma.MethodsEffects of TTO and terpinen-4-ol on the cellular viability of two tumour cell lines and fibroblast cells were assessed by MTT assay. Induction of apoptotic and necrotic cell death was visualised by fluorescent microscopy and quantified by flow cytometry. Tumour cell ultrastructural changes were examined by transmission electron microscopy and changes in cell cycle distribution were assessed by flow cytometry, with changes in cellular morphology monitored by video time lapse microscopy.ResultsTTO and terpinen-4-ol significantly inhibited the growth of two murine tumour cell lines in a dose- and time-dependent manner. Interestingly, cytotoxic doses of TTO and terpinen-4-ol were significantly less efficacious against non-tumour fibroblast cells. TTO and terpinen-4-ol induced necrotic cell death coupled with low level apoptotic cell death in both tumour cell lines. This primary necrosis was clarified by video time lapse microscopy and also by transmission electron microscopy which revealed ultrastructural features including cell and organelle swelling following treatment with TTO. In addition, both TTO and terpinen-4-ol induced their inhibitory effect by eliciting G1 cell cycle arrest.ConclusionTTO and terpinen-4-ol had significant anti-proliferative activity against two tumour cell lines. Moreover, the identification of primary necrotic cell death and cell cycle arrest of the aggressive tumour cells highlights the potential anticancer activity of TTO and terpinen-4-ol.

Drugs to block cytokine signaling for the prevention and treatment of inflammation induced preterm birth

Preterm birth (PTB) at less than 37 weeks of gestation is the leading cause of neonatal morbidity and mortality. Intrauterine infection (IUI) due to microbial invasion of the amniotic cavity is the leading cause of early PTB (<32 weeks). Commensal genital tract Ureaplasma and Mycoplasma species, as well as Gram-positive and Gram-negative bacteria, have been associated with IUI-induced PTB. Bacterial activation of Toll-like receptors and other pattern recognition receptors initiates a cascade of inflammatory signaling via the NF-κB and p38 mitogen-activated protein kinase (MAPK) signaling pathways, prematurely activating parturition. Antenatal antibiotic treatment has had limited success in preventing PTB or fetal inflammation. Administration of anti-inflammatory drugs with antibiotics could be a viable therapeutic option to prevent PTB and fetal complications in women at risk of IUI and inflammation. In this mini-review, we will discuss the potential for anti-inflammatory drugs in obstetric care, focusing on the class of drugs termed “cytokine suppressive anti-inflammatory drugs” or CSAIDs. These inhibitors work by specifically targeting the NF-κB and p38 MAPK inflammatory signaling pathways. Several CSAIDs are discussed, together with clinical and toxicological considerations associated with the administration of anti-inflammatory agents in pregnancy.

Topically applied Melaleuca alternifolia (tea tree) oil causes direct anti-cancer cytotoxicity in subcutaneous tumour bearing mice

BACKGROUND Melaleuca alternifolia (tea tree) oil (TTO) applied topically in a dilute (10%) dimethyl sulphoxide (DMSO) formulation exerts a rapid anti-cancer effect after a short treatment protocol. Tumour clearance is associated with skin irritation mediated by neutrophils which quickly and completely resolves upon treatment cessation. OBJECTIVE To examine the mechanism of action underlying the anti-cancer activity of TTO. METHODS Immune cell changes in subcutaneous tumour bearing mice in response to topically applied TTO treatments were assessed by flow cytometry and immunohistochemistry. Direct cytotoxicity of TTO on tumour cells in vivo was assessed by transmission electron microscopy. RESULTS Neutrophils accumulate in the skin following topical 10% TTO/DMSO treatment but are not required for tumour clearance as neutrophil depletion did not abrogate the anti-cancer effect. Topically applied 10% TTO/DMSO, but not neat TTO, induces an accumulation and activation of dendritic cells and an accumulation of T cells. Although topical application of 10% TTO/DMSO appears to activate an immune response, anti-tumour efficacy is mediated by a direct effect on tumour cells in vivo. The direct cytotoxicity of TTO in vivo appears to be associated with TTO penetration. CONCLUSION Future studies should focus on enhancing the direct cytotoxicity of TTO by increasing penetration through skin to achieve a higher in situ terpene concentration. This coupled with boosting a more specific anti-tumour immune response will likely result in long term clearance of tumours.

Intrauterine Candida albicans infection elicits severe inflammation in fetal sheep

Background:Preventing preterm birth and subsequent adverse neonatal sequelae is among the greatest clinical challenges of our time. Recent studies suggest a role for Candida spp. in preterm birth and fetal injury, as a result of their colonization of either the vagina and/or the amniotic cavity. We hypothesized that intraamniotic Candida albicans would cause a vigorous, acute fetal inflammatory response.Methods:Sheep carrying singleton pregnancies received single intraamniotic injections of either saline (control) or 107 colony-forming units C. albicans 1 or 2 d prior to surgical delivery and euthanasia at 124 ± 2 d gestation.Results:Colonization of the amniotic cavity by C. albicans resulted in a modest inflammatory response at 1 d and florid inflammation at 2 d, characterized by fetal thrombocytopenia, lymphopenia, and significant increases of inflammatory cytokines/chemokines in the fetal membranes skin, lung, and the amniotic fluid.Conclusion:Acute colonization of the amniotic cavity by C. albicans causes severe intrauterine inflammation and fetal injury. C. albicans is a potent fetal pathogen that can contribute to adverse pregnancy outcomes.

Effects of cytokine-suppressive anti-inflammatory drugs on inflammatory activation in ex vivo human and ovine fetal membranes

Intrauterine infection and inflammation are responsible for the majority of early (<32 weeks) spontaneous preterm births (PTBs). Anti-inflammatory agents, delivered intra-amniotically together with antibiotics, may be an effective strategy for preventing PTB. In this study, the effects of four cytokine-suppressive anti-inflammatory drugs (CSAIDs: N-acetyl cysteine (NAC), SB239063, TPCA-1 and NEMO binding domain inhibitor (NBDI)) were assessed on human and ovine gestational membrane inflammation. Full-thickness membranes were collected from healthy, term, human placentas delivered by Caesarean section (n=5). Using a Transwell model, they were stimulated ex vivo with γ-irradiation-killed Escherichia coli applied to the amniotic face. Membranes from near-term, ovine placentas were stimulated in utero with lipopolysaccharide, Ureaplasma parvum or saline control and subjected to explant culture. The effects of treatment with CSAIDs or vehicle (1% DMSO) on accumulation of PGE2 and cytokines (human interleukin 6 (IL6), IL10 and TNFα; ovine IL8 (oIL8)) were assessed in conditioned media at various time points (3-20  h). In human membranes, the IKKβ inhibitor TPCA-1 (7  μM) and p38 MAPK inhibitor SB239063 (20  μM) administered to the amniotic compartment were the most effective in inhibiting accumulation of cytokines and PGE2 in the fetal compartment. NAC (10  mM) inhibited accumulation of PGE2 and IL10 only; NBDI (10  μM) had no significant effect. In addition to the fetal compartment, SB239063 also exerted consistent and significant inhibitory effects in the maternal compartment. TPCA-1 and SB239063 suppressed oIL8 production, while all CSAIDs tested suppressed ovine PGE2 production. These results support the further investigation of intra-amniotically delivered CSAIDs for the prevention of inflammation-mediated PTB.

A New, Potent, and Placenta-Permeable Macrolide Antibiotic, Solithromycin, for the Prevention and Treatment of Bacterial Infections in Pregnancy

Intrauterine infection–inflammation is a major cause of early preterm birth and subsequent neonatal mortality and acute or long-term morbidity. Antibiotics can be administered in pregnancy to prevent preterm birth either prophylactically to women at high risk for preterm delivery, or to women with diagnosed intrauterine infection, prelabor rupture of membranes, or in suspected preterm labor. The therapeutic goals of each of these scenarios are different, with different pharmacological considerations, although effective antimicrobial therapy is an essential requirement. An ideal antibiotic for these clinical indications would be (a) one that is easily administered and orally bioactive, (b) has a favorable adverse effect profile (devoid of reproductive toxicity or teratogenicity), (c) is effective against the wide range of microorganisms known to be commonly associated with intra-amniotic infection, (d) provides effective antimicrobial protection within both the fetal and amniotic compartments after maternal delivery, (e) has anti-inflammatory properties, and (f) is effective against antibiotic-resistant microorganisms. Here, we review the evidence from clinical, animal, and ex vivo/in vitro studies that demonstrate that a new macrolide-derived antibiotic – solithromycin – has all of these properties and, hence, may be an ideal antibiotic for the treatment and prevention of intrauterine infection-related pregnancy complications. While this evidence is extremely encouraging, it is still preliminary. A number of key studies need to be completed before solithromycin’s true potential for use in pregnancy can be ascertained.

Outside-in? Acute fetal systemic inflammation in very preterm chronically catheterized sheep fetuses is not driven by cells in the fetal blood

BACKGROUND The preterm birth syndrome (delivery before 37 weeks gestation) is a major contributor to the global burden of perinatal morbidity and death. The cause of preterm birth is complex, multifactorial, and likely dependent, at least in part, on the gestational age of the fetus. Intrauterine infection is frequent in preterm deliveries that occur at <32 weeks gestation; understanding how the fetus responds to proinflammatory insult will be an important step towards early preterm birth prevention. However, animal studies of infection and inflammation in prematurity commonly use older fetuses that possess comparatively mature immune systems. OBJECTIVE Aiming to characterize acute fetal responses to microbial agonist at a clinically relevant gestation, we used 92-day-old fetuses (62% of term) to develop a chronically catheterized sheep model of very preterm pregnancy. We hypothesized that any acute fetal systemic inflammatory responses would be driven by signaling from the tissues exposed to Escherichia coli lipopolysaccharide that is introduced into the amniotic fluid. STUDY DESIGN Eighteen ewes that were carrying a single fetus at 92 days of gestation had recovery surgery to place fetal tracheal, jugular, and intraamniotic catheters. Animals were recovered for 24 hours before being administered either intraamniotic E coli lipopolysaccharide (n = 9) or sterile saline solution (n = 9). Samples were collected for 48 hours before euthanasia and necroscopy. Fetal inflammatory responses were characterized by microarray analysis, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. RESULTS Intraamniotic lipopolysaccharide reached the distal trachea within 2 hours. Lipopolysaccharide increased tracheal fluid interleukin-8 within 2 hours and generated a robust inflammatory response that was characterized by interleukin-6 signaling pathway activation and up-regulation of cell proliferation but no increases in inflammatory mediator expression in cord blood RNA. CONCLUSIONS In very preterm sheep fetuses, lipopolysaccharide stimulates inflammation in the fetal lung and fetal skin and stimulates a systemic inflammatory response that is not generated by fetal blood cells. These data argue for amniotic fluid-exposed tissues that play a key role in driving acute fetal and intrauterine inflammatory responses.

Polymyxin B Agonist Capture Therapy for Intrauterine Inflammation: Proof-of-Principle in a Fetal Ovine Model

Intrauterine infection is a leading cause of preterm birth (PTB), most notably in deliveries occurring before 32 weeks gestation. Preterm infants exposed to intrauterine inflammation are more likely to have a host of neurological, respiratory, gastrointestinal, and visual pathologies. Preventing preterm delivery and protecting the fetus from injury is thus likely to require treatment of both intrauterine infection and inflammation. Polymyxin B (PMXB) is a cationic peptide antibiotic that binds Escherichia coli lipopolysaccharides (LPS) and prevents inflammatory activation. We hypothesized that intraamniotic administration of PMXB would selectively inhibit LPS-driven inflammation, serving as a proof-of-principle for targeted agonist capture therapy as a treatment for PTB and fetal injury. In vitro studies with primary fetal ovine keratinocytes demonstrated a significant and sustained reduction in tumor necrosis factor α and interleukin 8 messenger RNA expression after treatment with PMXB and LPS, relative to cells treated with LPS alone. In vivo studies with fetal sheep demonstrated a significant reduction in proinflammatory cytokines in the amniotic fluid and fetal lung (but not fetal skin or chorioamnion) in LPS + PMXB-treated animals, relative to those treated with LPS alone. These data are consistent with a partial resolution of LPS-driven intrauterine inflammation. They suggest the potential for agonist capture as a conceptual means of resolving the proparturition inflammation caused by infection of the amniotic cavity.

The Role of Regulatory T Cells in Mesothelioma

Malignant mesothelioma (MM) appears to be responsive to immunotherapy. The lack of complete tumour cure as a result of many immunotherapies tested to date suggests that the immune response to MM is complex and multi-parametric. Regulatory T (Treg) cells are prevalent within murine and human mesotheliomas with their removal shown to result in tumour growth inhibition and the release of anti-tumour effector T cells from immunosuppression. The targeting of immune checkpoints as treatments for various solid tumours has recently shown promise in clinical settings. In addition, synergy between chemotherapy and immunotherapy has been demonstrated for many cancers, including mesothelioma. Here we demonstrate Treg cells as critical mediators of the anti-tumour immune response to MM and potential targets for anti-tumour immunotherapy; though the timing and dosage of Treg cell manipulating immunotherapies need to be optimised.

Intrauterine Candida albicans Infection Causes Systemic Fetal Candidiasis with Progressive Cardiac Dysfunction in a Sheep Model of Early Pregnancy

Introduction: Several recent studies have identified a potential role for intrauterine Candida albicans in adverse pregnancy outcomes, including preterm birth. There is, however, a limited understanding of the impact of intrauterine candida infection on fetal well-being in early pregnancy. Using a sheep model of early pregnancy, the aims of this study were to determine (1) the ability of experimentally induced intrauterine C albicans to infect the fetus and (2) whether C albicans exposure in early pregnancy is associated with alterations in fetal cardiac function, as measured by spectral tissue Doppler imaging analysis of fetal cardiac function. Methods: Merino ewes carrying singleton pregnancies at 89 days’ gestation (term is ∼150 days) received C albicans (n = 8) via ultrasound-guided intra-amniotic injection. Saline-exposed fetuses served as controls (n = 6). Spectral tissue Doppler imaging echocardiography and amniotic fluid collection were performed at baseline and 24 and 72 hours after intrauterine C albicans injection. Fetal tissues were collected at postmortem for analysis of infection and inflammation. Results: Relative to saline control, intrauterine C albicans infection resulted in pronounced increases in amniotic fluid tumor necrosis factor α (TNF-α; P < .05) and cytokine/chemokine messenger RNA (interleukin [IL] 1β, IL-6, TNF-α, and monocyte chemoattractant protein 1; P < .05) in the fetal myocardium, lung, skin, and liver at 72 and 96 hours postinfection. Spectral tissue Doppler imaging showed diastolic dysfunction at 24 hours and severe biventricular diastolic dysfunction 72 hours postinfection. Conclusion: Intrauterine C albicans infection in a sheep model of early pregnancy causes systemic fetal candidiasis, which is associated with a robust systemic inflammatory response and progressive cardiac dysfunction detectable by spectral tissue Doppler imaging.