Lipoxygenase metabolism : roles in tumor progression and survival

The metabolism of arachidonic acid through lipoxygenase pathways leads to the generation of various biologically active eicosanoids. The expression of these enzymes vary throughout the progression of various cancers, and thereby they have been shown to regulate aspects of tumor development. Substantial evidence supports a functional role for lipoxygenase-catalyzed arachidonic and linoleic acid metabolism in cancer development. Pharmacologic and natural inhibitors of lipoxygenases have been shown to suppress carcinogenesis and tumor growth in a number of experimental models. Signaling of hydro[peroxy]fatty acids following arachidonic or linoleic acid metabolism potentially effect diverse biological phenomenon regulating processes such as cell growth, cell survival, angiogenesis, cell invasion, metastatic potential and immunomodulation. However, the effects of distinct LOX isoforms differ considerably with respect to their effects on both the individual mechanisms described and the tumor being examined. 5-LOX and platelet type 12-LOX are generally considered pro-carcinogenic, with the role of 15-LOX-1 remaining controversial, while 15-LOX-2 suppresses carcinogenesis. In this review, we focus on the molecular mechanisms regulated by LOX metabolism in some of the major cancers. We discuss the effects of LOXs on tumor cell proliferation, their roles in cell cycle control and cell death induction, effects on angiogenesis, migration and the immune response, as well as the signal transduction pathways involved in these processes. Understanding the molecular mechanisms underlying the anti-tumor effect of specific, or general, LOX inhibitors may lead to the design of biologically and pharmacologically targeted therapeutic strategies inhibiting LOX isoforms and/or their biologically active metabolites, that may ultimately prove useful in the treatment of cancer, either alone or in combination with conventional therapies. © 2007 Springer Science+Business Media, LLC.

Epidermal growth factor receptors and cyclooxygenase-2 in the pathogenesis of non-small cell lung cancer : potential targets for chemoprevention and systemic therapy

The epidermal growth factor receptor (EGFR) is part of a family of plasma membrane receptor tyrosine kinases that control many important cellular functions, from growth and proliferation to cell death. Cyclooxygenase (COX)-2 is an enzyme which catalyses the conversion of arachidonic acid to prostagladins and thromboxane. It is induced by various inflammatory stimuli, including the pro-inflammatory cytokines, Interleukin (IL)-1β, Tumour Necrosis Factor (TNF)-α and IL-2. Both EGFR and COX-2 are over-expressed in non-small cell lung cancer (NSCLC) and have been implicated in the early stages of tumourigenesis. This paper considers their roles in the development and progression of lung cancer, their potential interactions, and reviews the recent progress in cancer therapies that are directed toward these targets. An increasing body of evidence suggests that selective inhibitors of both EGFR and COX-2 are potential therapeutic agents for the treatment of NSCLC, in the adjuvant, metastatic and chemopreventative settings. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Small RNA viruses of insects : expression in plants and RNA silencing

Interest in insect small RNA viruses (SRVs) has grown slowly but steadily. A number of new viruses have been analyzed at the sequence level, adding to our knowledge of their diversity at the level of both individual virus species and families. In particular, a number of possible new virus families have emerged. This research has largely been driven by interest in their potential for pest control, as well as in their importance as the causal agents of disease in beneficial arthropods. At the same time, research into known viruses has made valuable contributions to our understanding of an emerging new field of central importance to molecular biology-the existence of RNA-based gene silencing, developmental control, and adaptive immune systems in eukaryotes. Subject to RNA-based adaptive immune responses in their hosts, viruses have evolved a variety of genes encoding proteins capable of suppressing the immune response. Such genes were first identified in plant viruses, but the first examples known from animal viruses were identified in insect RNA viruses. This chapter will address the diversity of insect SRVs, and attempts to harness their simplicity in the engineering of transgenic plants expressing viruses for resistance to insect pests. We also describe RNA interference and antiviral pathways identified in plants and animals, how they have led viruses to evolve genes capable of suppressing such adaptive immunity, and the problems presented by these pathways for the strategy of expressing viruses in transgenic plants. Approaches for countering these problems are also discussed. © 2006 Elsevier Inc. All rights reserved.

Sex hormone regulation of innate immunity in the female reproductive tract : the role of epithelial cells in balancing reproductive potential with protection against sexually transmitted pathogens

The immune system in the female reproductive tract (FRT) does not mount an attack against HIV or other sexually transmitted infections (STI) with a single endogenously produced microbicide or with a single arm of the immune system. Instead, the body deploys dozens of innate antimicrobials to the secretions of the female reproductive tract. Working together, these antimicrobials along with mucosal antibodies attack many different viral, bacterial and fungal targets. Within the FRT, the unique challenges of protection against sexually transmitted pathogens coupled with the need to sustain the development of an allogeneic fetus have evolved in such a way that sex hormones precisely regulate immune function to accomplish both tasks. The studies presented in this review demonstrate that estradiol and progesterone secreted during the menstrual cycle act both directly and indirectly on epithelial cells and other immune cells in the reproductive tract to modify immune function in a way that is unique to specific sites throughout the FRT. As presented in this review, studies from our laboratory and others demonstrate that the innate immune response is under hormonal control, varies with the stage of the menstrual cycle, and as such is suppressed at mid-cycle to optimize conditions for successful fertilization and pregnancy. In doing so, a window of STI vulnerability is created during which potential pathogens including HIV enter the reproductive tract to infect host targets.

Transcutaneous immunization with combined cholera toxin and CpG adjuvant protects against Chlamydia muridarum genital tract infection

Chlamydia trachomatis is a pathogen of the genital tract and ocular epithelium. Infection is established by the binding of the metabolically inert elementary body (EB) to epithelial cells. These are taken up by endocytosis into a membrane-bound vesicle termed an inclusion. The inclusion avoids fusion with host lysosomes, and the EBs differentiate into the metabolically active reticulate body (RB), which replicates by binary fission within the protected environment of the inclusion. During the extracellular EB stage of the C. trachomatis life cycle, antibody present in genital tract or ocular secretions can inhibit infection both in vivo and in tissue culture. The RB, residing within the intracellular inclusion, is not accessible to antibody, and resolution of infection at this stage requires a cell-mediated immune response mediated by gamma interferon-secreting Th1 cells. Thus, an ideal vaccine to protect against C. trachomatis genital tract infection should induce both antibody (immunoglobulin A [IgA] and IgG) responses in mucosal secretions to prevent infection by chlamydial EB and a strong Th1 response to limit ascending infection to the uterus and fallopian tubes. In the present study we show that transcutaneous immunization with major outer membrane protein (MOMP) in combination with both cholera toxin and CpG oligodeoxynucleotides elicits MOMP-specific IgG and IgA in vaginal and uterine lavage fluid, MOMP-specific IgG in serum, and gamma interferon-secreting T cells in reproductive tract-draining caudal and lumbar lymph nodes. This immunization protocol resulted in enhanced clearance of C. muridarum (C. trachomatis, mouse pneumonitis strain) following intravaginal challenge of BALB/c mice.

The role of the arachidonic acid pathway in NSCLC development and progression : novel approaches for therapeutic intervention

Arachidonic acid metabolism through cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P-450 epoxygenase (EPOX) pathways is responsible for the formation of biologically active eicosanoids, including prostanoids, leukotrienes, hydroxyeicosatetraenoic acid, epoxyeicosatrienoic acid and hydroperoxyeicosatetraenoic acids. Altered eicosanoid expression levels are commonly observed during tumour development and progression of a range of malignancies, including non-small cell lung cancer (NSCLC). Arachidonic acid-derived eicosanoids affect a range of biological phenomena to modulate tumour processes such as cell growth, survival, angiogenesis, cell adhesion, invasion and migration and metastatic potential. Numerous studies have demonstrated that eicosanoids modulate NSCLC development and progression, while targeting these pathways has generally been shown to inhibit tumour growth/progression. Modulation of these arachidonic acid-derived pathways for the prevention and/or treatment of NSCLC has been the subject of significant interest over the past number of years, with a number of clinical trials examining the potential of COX and LOX inhibitors in combination with traditional and novel molecular approaches. However, results from these trials have been largely disappointing. Furthermore, enthusiasm for the use of selective COX-2 inhibitors for cancer prevention/treatment waned, due to their association with adverse cardiovascular events in chemoprevention trials. While COX and LOX targeting may both retain promise for NSCLC prevention and/or treatment, there is an urgent need to understand the downstream signalling mechanisms through which these and other arachidonic acid-derived signalling pathways mediate their effects on tumourigenesis. This will allow for development of safer and potentially more effective strategies for NSCLC prevention and/or treatment. Chemoprevention studies with PGI2 analogues have demonstrated considerable promise, while binding to/signalling through PGE2 receptors have also been the subject of interest for NSCLC treatment. In this chapter, the role of the eicosanoid signalling pathways in non-small cell lung cancer will be discussed. In particular, the effect of the eicosanoids on tumour cell proliferation, their roles in induction of cell death, effects on angiogenesis, migration, invasion and their regulation of the immune response will be assessed, with signal transduction pathways involved in these processes also discussed. Finally, novel approaches targeting these arachidonic acid-derived eicosanoids (using pharmacological or natural agents) for chemoprevention and/or treatment of NSCLC will be outlined. Elucidating the molecular mechanisms underlying the effects of specific or general arachidonic acid pathway modulators may lead to the design of biologically and pharmacologically targeted therapeutic strategies for NSCLC prevention/treatment, which may be used alone or in combination with conventional therapies.

Preliminary characterisation of tumor necrosis factor alpha and interleukin-10 responses to chlamydia pecorum infection in the koala (Phascolarctos cinereus)

Debilitating infectious diseases caused by Chlamydia are major contributors to the decline of Australia's iconic native marsupial species, the koala (Phascolarctos cinereus). An understanding of koala chlamydial disease pathogenesis and the development of effective strategies to control infections continue to be hindered by an almost complete lack of species-specific immunological reagents. The cell-mediated immune response has been shown to play an influential role in the response to chlamydial infection in other hosts. The objective of this study, hence, was to provide preliminary data on the role of two key cytokines, pro-inflammatory tumour necrosis factor alpha (TNFα) and anti-inflammatory interleukin 10 (IL10), in the koala Chlamydia pecorum response. Utilising sequence homology between the cytokine sequences obtained from several recently sequenced marsupial genomes, this report describes the first mRNA sequences of any koala cytokine and the development of koala specific TNFα and IL10 real-time PCR assays to measure the expression of these genes from koala samples. In preliminary studies comparing wild koalas with overt chlamydial disease, previous evidence of C. pecorum infection or no signs of C. pecorum infection, we revealed strong but variable expression of TNFα and IL10 in wild koalas with current signs of chlamydiosis. The description of these assays and the preliminary data on the cell-mediated immune response of koalas to chlamydial infection paves the way for future studies characterising the koala immune response to a range of its pathogens while providing reagents to assist with measuring the efficacy of ongoing attempts to develop a koala chlamydial vaccine.

Immunization with a MOMP-based vaccine protects mice against a Pulmonary Chlamydia challenge and identifies a disconnection between infection and pathology

Chlamydia pneumoniae is responsible for up to 20% of community acquired pneumonia and can exacerbate chronic inflammatory diseases. As the majority of infections are either mild or asymptomatic, a vaccine is recognized to have the greatest potential to reduce infection and disease prevalence. Using the C. muridarum mouse model of infection, we immunized animals via the intranasal (IN), sublingual (SL) or transcutaneous (TC) routes, with recombinant chlamydial major outer membrane protein (MOMP) combined with adjuvants CTA1-DD or a combination of cholera toxin/CpG-oligodeoxynucleotide (CT/CpG). Vaccinated animals were challenged IN with C. muridarum and protection against infection and pathology was assessed. SL and TC immunization with MOMP and CT/CpG was the most protective, significantly reducing chlamydial burden in the lungs and preventing weight loss, which was similar to the protection induced by a previous live infection. Unlike a previous infection however, these vaccinations also provided almost complete protection against fibrotic scarring in the lungs. Protection against infection was associated with antigen-specific production of IFNγ, TNFα and IL-17 by splenocytes, however, protection against both infection and pathology required the induction of a similar pro-inflammatory response in the respiratory tract draining lymph nodes. Interestingly, we also identified two contrasting vaccinations capable of preventing infection or pathology individually. Animals IN immunized with MOMP and either adjuvant were protected from infection, but not the pathology. Conversely, animals TC immunized with MOMP and CTA1-DD were protected from pathology, even though the chlamydial burden in this group was equivalent to the unimmunized controls. This suggests that the development of pathology following an IN infection of vaccinated animals was independent of bacterial load and may have been driven instead by the adaptive immune response generated following immunization. This identifies a disconnection between the control of infection and the development of pathology, which may influence the design of future vaccines.

Elucidating the host–pathogen interaction between human colorectal cells and invading Enterovirus 71 using transcriptomics profiling

Enterovirus 71 (EV71) is one of the main etiological agents for Hand, Foot and Mouth Disease (HFMD) and has been shown to be associated with severe clinical manifestation. Currently, there is no antiviral therapeutic for the treatment of HFMD patients owing to a lack of understanding of EV71 pathogenesis. This study seeks to elucidate the transcriptomic changes that result from EV71 infection. Human whole genome microarray was employed to monitor changes in genomic profiles between infected and uninfected cells. The results reveal altered expression of human genes involved in critical pathways including the immune response and the stress response. Together, data from this study provide valuable insights into the host–pathogen interaction between human colorectal cells and EV71.

Vaccination of koalas with a recombinant Chlamydia pecorum major outer membrane protein induces antibodies of different specificity compared to those following a natural live infection

Chlamydial infection in koalas is common across the east coast of Australia and causes significant morbidity, infertility and mortality. An effective vaccine to prevent the adverse consequences of chlamydial infections in koalas (particularly blindness and infertility in females) would provide an important management tool to prevent further population decline of this species. An important step towards developing a vaccine in koalas is to understand the host immune response to chlamydial infection. In this study, we used the Pepscan methodology to identify B cell epitopes across the Major Outer Membrane Protein (MOMP) of four C. pecorum strains/genotypes that are recognized, either following (a) natural live infection or (b) administration of a recombinant MOMP vaccine. Plasma antibodies from the koalas naturally infected with a C. pecorum G genotype strain recognised the epitopes located in the variable domain (VD) four of MOMP G and also VD4 of MOMP H. By comparison, plasma antibodies from an animal infected with a C. pecorum F genotype strain recognised epitopes in VD1, 2 and 4 of MOMP F, but not from other genotype MOMPs. When Chlamydia-free koalas were immunised with recombinant MOMP protein they produced antibodies not only against epitopes in the VDs but also in conserved domains of MOMP. Naturally infected koalas immunised with recombinant MOMP protein also produced antibodies against epitopes in the conserved domains. This work paves the way for further refinement of a MOMP-based Chlamydia vaccine that will offer wide cross-protection against the variety of chlamydial infections circulating in wild koala populations.

Chlamydia pneumoniae infection of Dentritic cells and its role in asthma exacerbations

Chlamydia infections are associated with exacerbations of asthma however the mechanisms are poorly understood. In this thesis we infected dendritic cells from healthy controls and asthmatic patients to determine if the immune response to chlamydial infection by these key immune cells could explain this association of chlamydial infection with asthma attacks. Infected dendritic cells from asthmatic patients showed increased expression of multiple inflammatory cytokine genes and genes for several tissue remodelling proteins, suggesting that infected dendritic cells play a central role in driving the airways damage associated with asthma. The findings provide a greater understanding of the role of infections in asthma and may provide a basis for new therapies to treat this important disease.

Osteoimmunomodulatory properties of magnesium scaffolds coated with β-tricalcium phosphate

The osteoimmunomodulatory property of bone biomaterials is a vital property determining the in vivo fate of the implants. Endowing bone biomaterials with favorable osteoimmunomodulatory properties is of great importance in triggering desired immune response and thus supports the bone healing process. Magnesium (Mg) has been recognized as a revolutionary metal for applications in orthopedics due to it being biodegradable, biocompatible, and having osteoconductive properties. However, Mg's high rate of degradation leads to an excessive inflammatory response and this has restricted its application in bone tissue engineering. In this study, β-tricalcium phosphate (β-TCP) was used to coat Mg scaffolds in an effort to modulate the detrimental osteoimmunomodulatory properties of Mg scaffolds, due to the reported favorable osteoimmunomodulatory properties of β-TCP. It was noted that macrophages switched to the M2 extreme phenotype in response to the Mg-β-TCP scaffolds, which could be due to the inhibition of the toll like receptor (TLR) signaling pathway. VEGF and BMP2 were significantly upregulated in the macrophages exposed to Mg-β-TCP scaffolds, indicating pro-osteogenic properties of macrophages in β-TCP modified Mg scaffolds. This was further demonstrated by the macrophage-mediated osteogenic differentiation of bone marrow stromal cells (BMSCs). When BMSCs were stimulated by conditioned medium from macrophages cultured on Mg-β-TCP scaffolds, osteogenic differentiation of BMSCs was significantly enhanced; whereas osteoclastogenesis was inhibited, as indicated by the downregualtion of MCSF, TRAP and inhibition of the RANKL/RANK system. These findings suggest that β-TCP coating of Mg scaffolds can modulate the scaffold's osteoimmunomodulatory properties, shift the immune microenvironment towards one that favors osteogenesis over osteoclastogenesis. Endowing bone biomaterials with favorable osteoimmunomodulatory properties can be a highly valuable strategy for the development or modification of advanced bone biomaterials.

Nutrient element-based bioceramic coatings on titanium alloy stimulating osteogenesis by inducing beneficial osteoimmmunomodulation

A paradigm shift has taken place in which bone implant materials has gone from being relatively inert to having immunomodulatory properties, indicating the importance of immune response when these materials interact with the host tissues. It has therefore become important to endow the implant materials with immunomodulatory properties favouring osteogenesis and osseointegration. Strontium, zinc and silicon are bioactive elements that have important roles in bone metabolism and that also elicit significant immune responses. In this study, Sr-, Zn- and Si-containing bioactive Sr2ZnSi2O7 (SZS) ceramic coatings on Ti–6Al–4V were successfully prepared by a plasma-spray coating method. The SZS coatings exhibited slow release of the bioactive ions with significantly higher bonding strength than hydroxyapatite (HA) coatings. SZS-coated Ti–6Al–4V elicited significant effects on the immune cells, inhibiting the release of pro-inflammatory cytokines and fibrosis-enhancing factors, while upregulating the expression of osteogenic factors of macrophages; moreover, it could also inhibit the osteoclastic activities. The RANKL/RANK pathway, which enhances osteoclastogenesis, was inhibited by the SZS coatings, whereas the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs) was significantly enhanced by the SZS coatings/macrophages conditioned medium, probably via the activation of BMP2 pathway. SZS coatings are, therefore, a promising material for orthopaedic applications, and the strategy of manipulating the immune response by a combination of bioactive elements with controlled release has the potential to endow biomaterials with beneficial immunomodulatory properties.

Human CD141(+) (BDCA-3)(+) dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens

The characterization of human dendritic cell (DC) subsets is essential for the design of new vaccines. We report the first detailed functional analysis of the human CD141(+) DC subset. CD141(+) DCs are found in human lymph nodes, bone marrow, tonsil, and blood, and the latter proved to be the best source of highly purified cells for functional analysis. They are characterized by high expression of toll-like receptor 3, production of IL-12p70 and IFN-beta, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c(+) DC subset. Polyinosine-polycytidylic acid (poly I:C)-activated CD141(+) DCs have a superior capacity to cross-present soluble protein antigen (Ag) to CD8(+) cytotoxic T lymphocytes than poly I:C-activated CD1c(+) DCs. Importantly, CD141(+) DCs, but not CD1c(+) DCs, were endowed with the capacity to cross-present viral Ag after their uptake of necrotic virus-infected cells. These findings establish the CD141(+) DC subset as an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8 alpha(+) DC subset. The data demonstrate a role for CD141(+) DCs in the induction of cytotoxic T lymphocyte responses and suggest that they may be the most relevant targets for vaccination against cancers, viruses, and other pathogens.

Promiscuity, pheromones and pathogenicity : why all Enterococci are not created equal

Enterococcus faecalis is a Gram-positive, coccus shaped, lactic acid bacterium, with demonstrated ubiquity across multiple anatomical sites. Enterococcus faecalis isolates have been isolated from clinical samples as the etiological agent in patients with overt infections, and from body sites previously thought to be sterile but absent of signs and symptoms of infection. E. faecalis is implicated in both human health and disease, recognized as a commensal, a probiotic and an opportunistic multiply resistant pathogen. E. faecalis has emerged as a key pathogen in nosocomial infections. E. faecalis is well equipped to avert recognition by host cell immune mediators. Antigenic cell wall components including lipotechoic acids are concealed from immune detection by capsular polysaccharides produced by some strains. Thereby preventing complement activation, the pro-inflammatory response, opsonisation and phagocytosis. E. faecalis also produces a suite of enzymes including gelatinase and cytolysin, which aid in both virulence and host immune evasion. The ability of enterococci to form biofilms in vivo further increases virulence, whilst simultaneously preventing detection by host cells. E. faecalis exhibits high levels of both intrinsic and acquired antimicrobial resistance. The mobility of the E. faecalis genome is a significant contributor to antimicrobial resistance, with this species also transferring resistance to other Gram-positive bacteria. Whilst E. faecalis is of increasing concern in nosocomial infections, its role as a member of the endogenous microbiota cannot be underestimated. As a commensal and probiotic, E. faecalis plays an integral role in modulating the immune response, and in providing endogenous antimicrobial activity to enhance exclusion or inhibition of opportunistic pathogens in certain anatomical niches. In this chapter we will review possible mediators of enterococcal transition from commensal microbe to opportunistic pathogen, considering isolates obtained from patients diagnosed with pathogenic infections and those obtained from asymptomatic patients.