New trends in food science: the use of nutraceuticals as an antiinflammatory therapeutic tool in exercise

Prolonged high-intensity training seems to result in increased systemic inflammation, which might explain muscle injury, delayed onset muscle soreness, and overtraining syndrome in athletes. Furthermore, an impaired immune function caused by strenuous exercise leads to the development of upper respiratory tract infections in athletes. Nutraceuticals might help counteract these performance-lowering effects. The use of nanotechnology is an interesting alternative to supply athletes with nutraceuticals, as many of these substances are insoluble in water and are poorly absorbed in the digestive tract. The present chapter starts with a brief review of the effects of exercise on immunity, followed by an analysis on how nutraceuticals such as omega-3 fatty acids, glutamine, BCAAs, or phytochemicals can counteract negative effects of strenuous exercise in athletes. Finally, how nanostructured delivery systems can constitute a new trend in enhancing bioavailability and optimizing the action of nutraceuticals will be discussed, using the example of food beverages.

The epigenome as a therapeutic target for Parkinson's disease

Parkinson’s disease (PD) is a common, progressive neurodegenerative disease characterised by degeneration of nigrostriatal dopaminergic neurons, aggregation of α-synuclein and motor symptoms. Current dopamine-replacement strategies provide symptomatic relief, however their effectiveness wear off over time and their prolonged use leads to disabling side-effects in PD patients. There is therefore a critical need to develop new drugs and drug targets to protect dopaminergic neurons and their axons from degeneration in PD. Over recent years, there has been robust evidence generated showing that epigenetic dysregulation occurs in PD patients, and that epigenetic modulation is a promising therapeutic approach for PD. This article first discusses the present evidence implicating global, and dopaminergic neuron-specific, alterations in the methylome in PD, and the therapeutic potential of pharmacologically targeting the methylome. It then focuses on another mechanism of epigenetic regulation, histone acetylation, and describes how the histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes that mediate this process are attractive therapeutic targets for PD. It discusses the use of activators and/or inhibitors of HDACs and HATs in models of PD, and how these approaches for the selective modulation of histone acetylation elicit neuroprotective effects. Finally, it outlines the potential of employing small molecule epigenetic modulators as neuroprotective therapies for PD, and the future research that will be required to determine and realise this therapeutic potential.

Predictors of in-hospital mortality among cardiogenic shock patients. Prognostic and therapeutic implications

Cardiogenic shock (CS) has a poor prognosis. The heterogeneity in the mortality through different subgroups suggests that some factors can be useful to perform risk stratification and guide management. We aimed to find predictors of in-hospital mortality in these patients. We analyzed all cases of cardiogenic shock due to medical conditions admitted in our intensive acute cardiovascular care unity from November 2010 till November 2015. Clinical, biochemical and hemodynamic variables were registered, as was the Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) profile at 24 h of CS diagnosis. From a total of 281 patients, 28 died within the first 24 h and were not included in the analysis. A total of 253 patients survived the first 24 h, mean age was 68.8 ± 14.4 years, and 174 (68.8%) were men. Etiologies: acute coronary syndrome 146 (57.7%), acute heart failure 60 (23.7%), arrhythmias 35 (13.8%), and others 12 (4.8%). A total of 91 patients (36.0%) died during hospitalization. We found the following independent predictors of in-hospital mortality: age (odds ratio [OR] 1.032, 95% confidence interval [CI] 1.003–1.062), blood glucose (OR 1.004, 95% CI 1.001–1.008), heart rate (OR 1.014, 95% CI 1.001–1.028), and INTERMACS profile (OR 0.168, 95% CI 0.107–0.266). In patients with CS the INTERMACS profile at 24 h of diagnosis was associated with higher in-hospital mortality. This and other prognostic variables (age, blood glucose, and heart rate) may be useful for risk stratification and to select appropriate medical or invasive interventions.

Microglia as therapeutic targets in retinal degeneration: role of translocator protein (18 κDa) (TSPO) and minocycline.

Microglia are the resident immune cells of the central nervous system (CNS) and play an important role in innate immune defense as well as tissue homeostasis. Chronic microglial reactivity, microgliosis, is a general hallmark of inflammatory and degenerative diseases that affect the CNS, including the retina. There is increasing evidence that chronic microgliosis is more than just a bystander effect, but rather actively contributes to progression of degeneration through processes such as toxic nitric oxide (NO) production and even phagocytosis of stressed but viable photoreceptors. Therefore immunmodulation of microglia presents a possible therapeutic strategy for retinal degenerations. Notably, the expression of the mitochondrial translocator protein 18 (κDa) (TSPO) is highly elevated in reactive microglia as seen in several neuroinflammatory diseases such as Alzheimer’s disease, Parkinson’s disease and multiple sclerosis. Therefore it is used as a gliosis biomarker in the brain. Moreover TSPO ligands show potent effects in resolving neuroinflammatory brain disorders. However, TSPO expression in the eye had not been investigated before. Further, it was unknown whether TSPO ligands’ potent immunomodulatory effects could be used to treat retinal degenerations. To fill this gap, the study aimed to analyze whether TSPO is also a potential biomarker for degenerative processes in the retina. Moreover the thesis attempted to determine whether a specific TSPO ligand, XBD173, might modulate microglial reactivity and is a potent therapeutic, to treat retinal degenerative diseases. The findings revealed that TSPO is strongly upregulated in microglial cells of retinoschisin-deficient (RS1-/y) mice, a model of inherited retinal degeneration and in a murine light damage model. A co-localization of TSPO and microglia was furthermore detectable in human retinal sections, indicating a potential role for TSPO as a biomarker for retinal degenerations. In vitro assays showed that the TSPO ligand XBD173 effectively inhibited features of microglial activation such as morphological transformation into reactive phagocytes and enhanced expression of pro-inflammatory cytokines. XBD173 also reduced microglial migration and proliferation and reduced their neurotoxic potential on photoreceptor cells. In two independent mouse models of light-induced retinal degeneration, the treatment with XBD173 reduced accumulation of amoeboid, reactive microglia in the outer retina and attenuated degenerative processes, indicated by a nearly preserved photoreceptor layer. A further question addressed in this thesis was whether minocycline, an antibiotic with additional anti-inflammatory properties is able to reduce microglial neurotoxicity and to protect the retina from degeneration. Minocycline administration dampened microglial pro-inflammatory gene expression, NO production and neurotoxicity on photoreceptors. Interestingly, in addition to its immunomodulatory effect, minocycline also increased the viability of photoreceptors in a direct manner. In the light damage model, minocycline administration counter-acted microglial activation and blocked retinal degeneration. Taken together these results identified TSPO as a biomarker for microglial reactivity and as therapeutic target in the retina. Targeting TSPO with XBD173 was able to reverse microglial reactivity and could prevent degenerative processes in the retina. In addition, the study showed that the antibiotic minocycline effectively counter-regulates microgliosis and light-induced retinal degeneration. Considering that microgliosis is a major contributing factor for retinal degenerative disorders, this thesis supports the concept of a microglia-directed therapy to treat retinal degeneration.

New prophylactic and therapeutic treatments to combat pathogenic Enterohaemorrhagic Escherichia coli

Bacterial diarrhoeal diseases have significant influence on global human health, and are a leading cause of preventable death in the developing world. Enterohaemorrhagic Escherichia coli (EHEC), pathogenic strains of E. coli that carry potent toxins, have been associated with a high number of large-scale outbreaks caused by contaminated food and water sources. This pathotype produces diarrhoea and haemorrhagic colitis in infected humans, and in some patients leads to the development of haemolytic uremic syndrome (HUS), which can result in mortality and chronic kidney disease. A major obstacle to the treatment of EHEC infections is the increased risk of HUS development that is associated with antibiotic treatment, and rehydration and renal support are often the only options available. New treatments designed to prevent or clear E. coli infections and reduce symptoms of illness would therefore have large public health and economic impacts. The three main aims of this thesis were: to explore mouse models for pre-clinical evaluation in vivo of small compounds that inhibit a major EHEC colonisation factor, to assess the production and role of two proteins considered promising candidates for a broad-spectrum vaccine against pathogenic E. coli, and to investigate a novel compound that has recently been identified as a potential inhibitor of EHEC toxin production. As EHEC cannot be safely tested in humans due to the risk of HUS development, appropriate small animal models are required for in vivo testing of new drugs. A number of different mouse models have been developed to replicate different features of EHEC pathogenesis, several of which we investigated with a focus on colonisation mediated by the Type III Secretion System (T3SS), a needle-like structure that translocates bacterial proteins into host cells, resulting in a tight, intimate attachment between pathogen and host, aiding colonisation of the gastrointestinal tract. As E. coli models were found not to depend significantly on the T3SS for colonisation, the Citrobacter rodentium model, a natural mouse pathogen closely related to E. coli, was deemed the most suitable mouse model currently available for in vivo testing of T3SS-targeting compounds. Two bacterial proteins, EaeH (an outer membrane adhesin) and YghJ (a putative secreted lipoprotein), highly conserved surface-associated proteins recently identified as III protective antigens against E. coli infection of mice, were explored in order to determine their suitability as candidates for a human vaccine against pathogenic E. coli. We focused on the expression and function of these proteins in the EHEC O157:H7 EDL933 strain and the adherent-invasive E. coli (AIEC) LF82 strain. Although expression of EaeH by other E. coli pathotypes has recently been shown to be upregulated upon contact with host intestinal cells, no evidence of this upregulation could be demonstrated in our strains. Additionally, while YghJ was produced by the AIEC strain, it was not secreted by bacteria under conditions that other YghJ-expressing E. coli pathotypes do, despite the AIEC strain carrying all the genes required to encode the secretion system it is associated with. While our findings indicate that a vaccine that raises antibodies against EaeH and YghJ may have limited effect on the EHEC and AIEC strains we used, recent studies into these proteins in different E. coli pathogens have suggested they are still excellent candidates for a broadly effective vaccine against E. coli. Finally, we characterised a small lead compound, identified by high-throughput screening as a possible inhibitor of Shiga toxin expression. Shiga toxin production causes both the symptoms of illness and development of HUS, and thus reduction of toxin production, release, or binding to host receptors could therefore be an effective way to treat infections and decrease the risk of HUS. Inhibition of Shiga toxin production by this compound was confirmed, and was shown to be caused by an inhibitory effect on activation of the bacterial SOS response rather than on the Shiga toxin genes themselves. The bacterial target of this compound was identified as RecA, a major regulator of the SOS response, and we hypothesise that the compound binds covalently to its target, preventing oligomerisation of RecA into an activated filament. Altogether, the results presented here provide an improved understanding of these different approaches to combating EHEC infection, which will aid the development of safe and effective vaccines and anti-virulence treatments against EHEC.

Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

SNP technologies for drug discovery : a current review

Single nucleotide polymorphisms (SNPs) are unique genetic differences between individuals that contribute in significant ways to the determination of human variation including physical characteristics like height and appearance as well as less obvious traits such as personality, behaviour and disease susceptibility. SNPs can also significantly influence responses to pharmacotherapy and whether drugs will produce adverse reactions. The development of new drugs can be made far cheaper and more rapid by selecting participants in drug trials based on their genetically determined response to drugs. Technology that can rapidly and inexpensively genotype thousands of samples for thousands of SNPs at a time is therefore in high demand. With the completion of the human genome project, about 12 million true SNPs have been identified to date. However, most have not yet been associated with disease susceptibility or drug response. Testing for the appropriate drug response SNPs in a patient requiring treatment would enable individualised therapy with the right drug and dose administered correctly the first time. Many pharmaceutical companies are also interested in identifying SNPs associated with polygenic traits so novel therapeutic targets can be discovered. This review focuses on technologies that can be used for genotyping known SNPs as well as for the discovery of novel SNPs associated with drug response.

Factors associated with the wish to hasten death : a study of patients with terminal illness

Background. There is considerable debate regarding the clinical issues surrounding the wish to hasten death (WTHD) in the terminally ill. The clinical factors contributing to the WTHD need further investigation among the terminally ill in order to enhance understanding of the clinical assessment and treatment needs that underlie this problem. A more detailed understanding may assist with the development of appropriate therapeutic interventions. Method. A sample of terminally ill cancer patients (N=256) recruited from an in-patient hospice unit, home palliative care service and a general hospital palliative care consulting service from Brisbane Australia between 1998–2001 completed a questionnaire assessing psychological (depression and anxiety), social (family relationship, social support, level of burden on others) and the impact of physical symptoms. The association between these factors and the WTHD was investigated. Results. A high WTHD was reported by 14% of patients. A discriminant function analysis revealed that the following variables were associated with a high WTHD (P<0·001): higher levels of depressive symptoms, being admitted to an in-patient hospice setting, a greater perception of being a burden on others, lower family cohesion, lower levels of social support, higher levels of anxiety and greater impact of physical symptoms. Conclusions. Psychological and social factors are related to a WTHD among terminally ill cancer patients. Greater attention needs to be paid to the assessment of psychological and social issues in order to provide appropriate therapeutic interventions for terminally ill patients.

How is a fruit tree like you? Using artistic metaphors to explore and develop emotional competence in children

Counselling children often requires the use of supplementary strategies in order to interest and engage the child in the therapeutic process. One such strategy is the Metaphorical Fruit Tree (MFT); an art metaphor suited to exploring and developing self-concept. Quantitative and qualitative data was used to explore the relationships between children’s ability to use metaphor, age, gender, and level of emotional competence (N = 58). Quantitative and qualitative analyses revealed a significant negative relationship between self-reported emotional competence and ability to use the MFT. It is proposed that children rely on different processes to understand self and as children’s ability to cognitively report on their emotional capabilities via the Emotional Competence Questionnaire (ECQ) increases, their ability to report creatively on those capabilities via the MFT is undermined. It is suggested that the MFT may be used, via creative processes and as an alternative to cognitive processes, to increase understanding and awareness of intrapersonal and interpersonal concepts of self in the child during counselling.

Nurse-determined assessment of cardiac output. Comparing a non-invasive cardiac output device and pulmonary artery catheter: a prospective observational study.

Background The accurate measurement of Cardiac output (CO) is vital in guiding the treatment of critically ill patients. Invasive or minimally invasive measurement of CO is not without inherent risks to the patient. Skilled Intensive Care Unit (ICU) nursing staff are in an ideal position to assess changes in CO following therapeutic measures. The USCOM (Ultrasonic Cardiac Output Monitor) device is a non-invasive CO monitor whose clinical utility and ease of use requires testing. Objectives To compare cardiac output measurement using a non-invasive ultrasonic device (USCOM) operated by a non-echocardiograhically trained ICU Registered Nurse (RN), with the conventional pulmonary artery catheter (PAC) using both thermodilution and Fick methods. Design Prospective observational study. Setting and participants Between April 2006 and March 2007, we evaluated 30 spontaneously breathing patients requiring PAC for assessment of heart failure and/or pulmonary hypertension at a tertiary level cardiothoracic hospital. Methods SCOM CO was compared with thermodilution measurements via PAC and CO estimated using a modified Fick equation. This catheter was inserted by a medical officer, and all USCOM measurements by a senior ICU nurse. Mean values, bias and precision, and mean percentage difference between measures were determined to compare methods. The Intra-Class Correlation statistic was also used to assess agreement. The USCOM time to measure was recorded to assess the learning curve for USCOM use performed by an ICU RN and a line of best fit demonstrated to describe the operator learning curve. Results In 24 of 30 (80%) patients studied, CO measures were obtained. In 6 of 30 (20%) patients, an adequate USCOM signal was not achieved. The mean difference (±standard deviation) between USCOM and PAC, USCOM and Fick, and Fick and PAC CO were small, −0.34 ± 0.52 L/min, −0.33 ± 0.90 L/min and −0.25 ± 0.63 L/min respectively across a range of outputs from 2.6 L/min to 7.2 L/min. The percent limits of agreement (LOA) for all measures were −34.6% to 17.8% for USCOM and PAC, −49.8% to 34.1% for USCOM and Fick and −36.4% to 23.7% for PAC and Fick. Signal acquisition time reduced on average by 0.6 min per measure to less than 10 min at the end of the study. Conclusions In 80% of our cohort, USCOM, PAC and Fick measures of CO all showed clinically acceptable agreement and the learning curve for operation of the non-invasive USCOM device by an ICU RN was found to be satisfactorily short. Further work is required in patients receiving positive pressure ventilation.

Stem cell technologies : regulation, patents and problems

Human embryonic stem cell research promises to deliver in the future a whole range of therapeutic treatments, but currently governments in different jurisdictions must try to regulate this burgeoning area. Part of the problem has been, and continues to be, polarised community opinion on the use of human embryonic stem cells for research. This article compares the approaches of the Australian, United Kingdom and United States governments in regulating human embryonic stem cell research. To date, these governments have approached the issue through implementing legislation or policy to control research. Similarly, the three jurisdictions have viewed the patentability of human embryonic stem cell technologies in their own ways with different policies being adopted by the three patent offices. This article examines these different approaches and discusses the inevitable concerns that have been raised due to the lack of a universal approach in relation to the regulation of research; the patenting of stem cell technologies; and the effects patents granted are having on further human embryonic stem cell research.

Commercialisation of regenerative human tissue : Regulation and reform in Australia and England, Wales and Northern Ireland

The commercialisation of therapeutic products containing regenerative human tissue is regulated by the common law, statute and ethical guidelines in Australia and England, Wales and Northern Ireland. This article examines the regulatory regimes in these jurisdictions and considers whether reform is required to both support scientific research and ensure conformity with modern social views on medical research and the use of human tissue. The authors consider the crucial role of informed consent in striking the balance between the interests of researchers and the interests of the public.

Self-talk in upper primary school children : its relationship with irrational beliefs, self-esteem and depression

Self-talk, irrational beliefs, self-esteem and depression were measured in a sample of 105 elementary school children in Grades 4 to 7. Sex and grade differences in positive self-talk were found. The pattern of correlation coefficients for positive self-talk supported the substantive position that positive self-talk is positively related to self-esteem and negatively related to irrational beliefs and depression in a non-clinical sample of children. However, the same support was not forthcoming for the reverse relationships for negative self-talk. Therapeutic implications are outlined as are suggestions for future research in the area of children's self-talk.

Translating tissue engineering technology platforms into cancer research

Technology platforms originally developed for tissue engineering applications produce valuable models that mimic three-dimensional (3D) tissue organization and function to enhance the understanding of cell/tissue function under normal and pathological situations. These models show that when replicating physiological and pathological conditions as closely as possible investigators are allowed to probe the basic mechanisms of morphogenesis, differentiation and cancer. Significant efforts investigating angiogenetic processes and factors in tumorigenesis are currently undertaken to establish ways of targeting angiogenesis in tumours. Anti-angiogenic agents have been accepted for clinical application as attractive targeted therapeutics for the treatment of cancer. Combining the areas of tumour angiogenesis, combination therapies and drug delivery systems is therefore closely related to the understanding of the basic principles that are applied in tissue engineering models. Studies with 3D model systems have repeatedly identified complex interacting roles of matrix stiffness and composition, integrins, growth factor receptors and signalling in development and cancer. These insights suggest that plasticity, regulation and suppression of these processes can provide strategies and therapeutic targets for future cancer therapies. The historical perspective of the fields of tissue engineering and controlled release of therapeutics, including inhibitors of angiogenesis in tumours is becoming clearly evident as a major future advance in merging these fields. New delivery systems are expected to greatly enhance the ability to deliver drugs locally and in therapeutic concentrations to relevant sites in living organisms. Investigating the phenomena of angiogenesis and anti-angiogenesis in 3D in vivo models such as the Arterio-Venous (AV) loop mode in a separated and isolated chamber within a living organism adds another significant horizon to this perspective and opens new modalities for translational research in this field.