Brain tumour differentiation: rapid stratified serum diagnostics via attenuated total reflection Fourier-transform infrared spectroscopy.

The ability to diagnose cancer rapidly with high sensitivity and specificity is essential to exploit advances in new treatments to lead significant reductions in mortality and morbidity. Current cancer diagnostic tests observing tissue architecture and specific protein expression for specific cancers suffer from inter-observer variability, poor detection rates and occur when the patient is symptomatic. A new method for the detection of cancer using 1 µl of human serum, attenuated total reflection-Fourier transform infrared spectroscopy and pattern recognition algorithms is reported using a 433 patient dataset (3897 spectra). To the best of our knowledge, we present the largest study on serum mid-infrared spectroscopy for cancer research. We achieve optimum sensitivities and specificities using a Radial Basis Function Support Vector Machine of between 80.0 and 100 % for all strata and identify the major spectral features, hence biochemical components, responsible for the discrimination within each stratum. We assess feature fed-SVM analysis for our cancer versus non-cancer model and achieve 91.5 and 83.0 % sensitivity and specificity respectively. We demonstrate the use of infrared light to provide a spectral signature from human serum to detect, for the first time, cancer versus non-cancer, metastatic cancer versus organ confined, brain cancer severity and the organ of origin of metastatic disease from the same sample enabling stratified diagnostics depending upon the clinical question asked.

Growth hormone and cancer: GH production and action in glioma?

The hypersecretion of pituitary growth hormone (GH) is associated with an increased risk of cancer, while reducing pituitary GH signaling reduces this risk. Roles for pituitary GH in cancer are therefore well established. The expression of the GH gene is, however, not confined to the pituitary gland and it is now known to occur in many extrapituitary tissues, in which it has local autocrine or paracrine actions, rather than endocrine function. It is, for instance, expressed in cancers of the prostate, lung, skin, endometrium and colon. The oncogenicity of autocrine GH may also be greater than that induced by endocrine or exogenous GH, as higher concentrations of GHR antagonists are required to inhibit its actions. This may reflect the fact that autocrine GH is thought to act at intracellular receptors directly after synthesis, in compartments not readily accessible to endocrine (or exogenous) GH. The roles and actions of extrapituitary GH in cancer may therefore differ from those of pituitary GH. The possibility that GH may be expressed and act in glioma tumors was therefore examined by immunohistochemistry. These results demonstrate, for the first time, the presence of abundant GH- and GH receptor (GHR-) immunoreactivity in glioma, in which they were co-localized in cytoplasmic but not nuclear compartments. These results demonstrate that glioma differs from most cancers in lacking nuclear GHRs, but GH is nevertheless likely to have autocrine or paracrine actions in the induction and progression of glioma.

Effect of substrate choice and tissue type on tissue preparation for spectral histopathology by Raman microspectroscopy.

Raman spectroscopy is a non-destructive, non-invasive, rapid and economical technique which has the potential to be an excellent method for the diagnosis of cancer and understanding disease progression through retrospective studies of archived tissue samples. Historically, biobanks are generally comprised of formalin fixed paraffin preserved tissue and as a result these specimens are often used in spectroscopic research. Tissue in this state has to be dewaxed prior to Raman analysis to reduce paraffin contributions in the spectra. However, although the procedures are derived from histopathological clinical practice, the efficacy of the dewaxing procedures that are currently employed is questionable. Ineffective removal of paraffin results in corruption of the spectra and previous experiments have shown that the efficacy can depend on the dewaxing medium and processing time. The aim of this study was to investigate the influence of commonly used spectroscopic substrates (CaF2, Spectrosil quartz and low-E slides) and the influence of different histological tissue types (normal, cancerous and metastatic) on tissue preparation and to assess their use for spectral histopathology. Results show that CaF2 followed by Spectrosil contribute the least to the spectral background. However, both substrates retain paraffin after dewaxing. Low-E substrates, which exhibit the most intense spectral background, do not retain wax and resulting spectra are not affected by paraffin peaks. We also show a disparity in paraffin retention depending upon the histological identity of the tissue with abnormal tissue retaining more paraffin than normal.

Cn-AMP2 from green coconut water is an anionic anticancer peptide.

Globally, death due to cancers is likely to rise to over 20 million by 2030, which has created an urgent need for novel approaches to anticancer therapies such as the development of host defence peptides. Cn-AMP2 (TESYFVFSVGM), an anionic host defence peptide from green coconut water of the plant Cocos nucifera, showed anti-proliferative activity against the 1321N1 and U87MG human glioma cell lines with IC50 values of 1.25 and 1.85 mM, respectively. The membrane interactive form of the peptide was found to be an extended conformation, which primarily included ß-type structures (levels > 45%) and random coil architecture (levels > 45%). On the basis of these and other data, it is suggested that the short anionic N-terminal sequence (TES) of Cn-AMP2 interacts with positively charged moieties in the cancer cell membrane. Concomitantly, the long hydrophobic C-terminal sequence (YFVFSVGM) of the peptide penetrates the membrane core region, thereby driving the translocation of Cn-AMP2 across the cancer cell membrane to attack intracellular targets and induce anti-proliferative mechanisms. This work is the first to demonstrate that anionic host defence peptides have activity against human glioblastoma, which potentially provides an untapped source of lead compounds for development as novel agents in the treatment of these and other cancers.

Investigating the rapid diagnosis of gliomas from serum samples using infrared spectroscopy and cytokine and angiogenesis factors.

The ability to diagnose brain cancer rapidly from serum samples is of great interest; such a diagnosis would allow for rapid testing and time to results providing a responsive diagnostic environment, ability to monitor treatment efficacy, early detection of recurrent tumours and screening techniques. Current methods rely upon subjective, time-consuming tests such as histological grading and are particularly invasive with the diagnostic test requiring hospitalisation of 2-3 days. A rapid diagnostic method based upon serum samples would allow for a relatively non-invasive test and open up the possibility of screening for brain cancer. We report for the first time the use of a Bioplex immunoassay to provide cytokine and angiogenesis factor levels that differ between serum from glioma and non-cancer patients specifically angiopoietin, follistatin, HGF, IL-8, leptin, PDGF-BB and PECAM-1 providing sensitivities and specificities as high as 88 % and 81 %, respectively. We also report, for the first time, the use of serum ATR-FTIR combined with a RBF SVM for the diagnosis of gliomas from non-cancer patients with sensitivities and specificities as high as 87.5 % and 100 %, respectively. We describe the combination of these techniques in an orthogonal diagnostic regime, providing strength to the diagnosis through data combinations, in a rapid diagnostic test within 5 h from serum collection (10 min for ATR-FTIR and 4 h for the Bioplex Immunoassay). This regime has the ability to revolutionise the clinical environment by providing objective measures for diagnosis allowing for increased efficiency with corresponding decreases in mortality, morbidity and economic impact upon the health services.

Effects of orally administered lavender essential oil on responses to anxiety-provoking film clips.

BACKGROUND: Lavender odour is commonly used to alleviate mild anxiety. Double blind studies are difficult to conduct with odours, and there are few reliable investigations of lavender's efficacy. METHOD: Orally administered lavender capsules (placebo, 100, 200 microl) were tested in a randomised between-subjects (n = 97) double-blind study. Film clips were used to elicit anxiety. Measures included anxiety, State Trait Anxiety Inventory (STAI), mood, positive and negative affect scale (PANAS), heart rate (HR), galvanic skin response (GSR), and heart rate variation (HRV). Following baseline measurements capsules were administered. Participants viewed a neutral film clip, then an anxiety-provoking and light-hearted recovery film clip. RESULTS: For the 200 microl lavender dose during the neutral film clip there was a trend towards reduced state anxiety, GSR and HR and increased HRV. In the anxiety-eliciting film, lavender was mildly beneficial in females but only on HRV measures. In males sympathetic arousal increased during the anxiety film (GSR). HRV significantly increased at 200 microl during all three film clips in females, suggesting decreased anxiety. CONCLUSION: These findings suggest that lavender has anxiolytic effects in humans under conditions of low anxiety, but these effects may not extend to conditions of high anxiety.

Increased plasma levels of 8-iso-PGF2alpha and IL-6 in an elderly population with depression.

Oxidative damage and immune-inflammatory activation have been suggested to play a role in depression. The purpose of the study was to investigate possible associations and interactions of these pathophysiological mechanisms in geriatric depression by determining the levels of plasma 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) and interleukin-6 (IL-6) in elderly depressed individuals. Subjects over 60 years of age with depression and controls were randomly selected from a population in the community after screening with the Geriatric Depression Scale. Plasma concentrations of 8-iso-PGF2alpha and IL-6 were measured in both groups. Depressed patients had significantly higher mean (+/-S.D.) 8-iso-PGF2alpha levels compared to healthy controls (245.01+/-179.92 pg/ml vs 97.64+/-42.72 pg/ml, respectively). Similarly, the same groups demonstrated significantly elevated IL-6 levels compared with controls (58.73+/-39.90 pg/ml vs 15.41+/-9.27 pg/ml). This study indicates an association between increased levels of plasma 8-iso-PGF2alpha and IL-6 with depressive symptomatology in elderly individuals and indicates the necessity for further investigation, possibly within the framework of an integrated involvement of oxidative damage and inflammation in the pathophysiology of depression in the elderly.

Correlation of folate, vitamin B12 and homocysteine plasma levels with depression in an elderly Greek population.

BACKGROUND: Alterations in folate, vitamin B12 and homocysteine plasma levels have been associated with aging, neuronal development and depressive symptomatology. Nevertheless, the associations are not strong enough to suggest the use of these parameters in every day practice for diagnostic or therapeutic purposes. OBJECTIVES: The aim of the study was to investigate the relationship between plasma folate, vitamin B12 and homocysteine in depressive states in the elderly. METHODS: Community-dwelling, elderly individuals over 60 years of age were screened with the Geriatric Depression Scale. The study population was divided into two groups: (a) 33 subjects with depression and (b) 33 healthy controls. All participants were clinically evaluated and completed a questionnaire for socio-demographic and clinical data. Measurements of folate, vitamin B12 and homocysteine were estimated in all blood samples and results were statistically evaluated at p<0.05 level of significance. RESULTS: No statistical significance emerged for the socio-demographic data between the two groups. Chronic diseases such as stroke, hypercholesterolemia, hypertension and diabetes also did not differ between the depression and control group. Group (a) had significantly lower levels of folate and vitamin B12 than group (b). Homocysteine was significantly higher in depressed individuals than in controls. CONCLUSION: Lower levels of plasma folate and/or vitamin B12, and higher levels of plasma homocysteine are associated with depression in elderly individuals.

Characterization of the lipid profile in dementia and depression in the elderly.

The purpose of the study was to examine the association of plasma lipid concentrations with changes in cognitive function and depressive states in elderly Greek individuals. The study population consisted of 3 groups: A) 37 subjects with dementia, B) 33 subjects with depression, and C) 33 controls. All individuals were screened with the Mini-Mental State Examination (MMSE), the Geriatric Depression Scale (GDS), and an evaluation of their psychiatric state. Lipid profile was assessed in all subjects, and the results were statistically evaluated at P < .05 level of significance. Groups A and B had significantly lower levels of total plasma cholesterol and HDL cholesterol than group C (P < .01). Triglyceride levels did not differ significantly between groups A and C, although they were significantly higher in group B. The results of this study suggest that an association does exist between the plasma concentration of cholesterol and HDL-C and depression and/or cognitive impairment. Further studies are required to explore the significance of these observations and establish if lipid levels could serve as markers for diagnostic and therapeutic purposes.

Indices of low-grade chronic inflammation correlate with early cognitive deterioration in an elderly Greek population.

Elevated serum levels of adhesion molecules (AM) reflect low-grade chronic inflammation and have been associated with several conditions of neuronal damage. The aim of the present study was the investigation of possible correlation between early cognitive decline and inflammatory processes in the elderly as indicated by plasma C-reactive protein (CRP) and AM levels. Thirty-seven subjects with dementia were selected from a community-dwelling, genetically isolated, geriatric population (above 60 years of age) based on the Mini Mental State Examination scale (MMSE) and the Diagnostic and Statistical Manual (DSM-IV) criteria. In parallel, a group of 33 age-matched healthy controls were selected from the same population. The levels of CRP (mg/l), sICAM-1 (ng/ml) and sVCAM-1 (ng/ml) were measured in the serum samples of both groups. Serum concentrations of all three molecules sICAM-1, sVCAM-1 and CRP were significantly higher in the dementia group when compared to controls (656.78 +/- 161.51 versus 467.05 +/- 231.26, p < 0.01; 631.64 +/- 149.76 versus 449.04 +/- 285.27, p < 0.01; 1.53 +/- 0.97 versus 0.7221 +/- 0.61, p < 0.01, respectively). Furthermore, a positive correlation was observed between the three molecules studied and the degree of severity of cognitive impairment. The findings of this study enhance the hypothesis of the presence of an underlying inflammatory process leading to cognitive deterioration and predisposing dementia in the elderly. The present work supports the evaluation of inflammatory molecules as early indicators of cognitive decline in elderly individuals.

Association of cognitive impairment with plasma levels of folate, vitamin B12 and homocysteine in the elderly.

BACKGROUND: Alterations in folate, vitamin B12 and homocysteine plasma levels have been associated with aging, neuronal development and cognitive impairment. The aim of this study was to investigate the relationship between plasma folate, vitamin B12 and homocysteine and cognitive function in the elderly. PATIENTS AND METHODS: Elderly individuals over 60 years of age living in the community, were screened with the Mini-Mental State Examination. The study population was divided into two groups: (a) 37 subjects with dementia; and (b) 33 healthy controls. Blood samples were analyzed with the use of ELISA, and the results were statistically evaluated at p < 0.05 level of significance. RESULTS: Group a had significantly lower levels of folate and vitamin B12 than group b. Homocysteine was significantly higher in demented individuals than in controls (p < 0.01). CONCLUSION: Lower levels of plasma folate and/or vitamin B12 and higher levels of plasma homocysteine are associated with cognitive impairment in elderly individuals.

Circulating microRNA biomarkers for glioma and predicting response to therapy.

The need for glioma biomarkers with improved sensitivity and specificity has sparked research into short non-coding RNA known as microRNA (miRNA). Altered miRNA biogenesis and expression in glioma plays a vital role in important signaling pathways associated with a range of tumor characteristics including gliomagenesis, invasion, and malignancy. This review will discuss current research into the role of miRNA in glioma and altered miRNA expression in biofluids as candidate biomarkers with a particular focus on glioblastoma, the most malignant form of glioma. The isolation and characterization of miRNA using cellular and molecular biology techniques from the circulation of glioma patients could potentially be used for improved diagnosis, prognosis, and treatment decisions. We aim to highlight the links between research into miRNA function, their use as biomarkers, and how these biomarkers can be used to predict response to therapy. Furthermore, increased understanding of miRNA in glioma biology through biomarker research has led to the development of miRNA therapeutics which could restore normal miRNA expression and function and improve the prognosis of glioma patients. A panel of important miRNA biomarkers for glioma in various biofluids discovered to date has been summarized here. There is still a need, however, to standardize techniques for biomarker characterization to bring us closer to clinically relevant miRNA-based diagnostic and therapeutic signatures. A clinically validated biomarker panel has potential to improve time to diagnosis, predicting response to treatment and ultimately the prognosis of glioma patients.

Attenuated total reflection fourier transform infrared (ATR-FTIR) spectral discrimination of brain tumour severity from serum samples.

Gliomas are the most frequent primary brain tumours in adults with over 9,000 people diagnosed each year in the UK. A rapid, reagent-free and cost-effective diagnostic regime using serum spectroscopy would allow for rapid diagnostic results and for swift treatment planning and monitoring within the clinical environment. We report the use of ATR-FTIR spectral data combined with a RBF-SVM for the diagnosis of gliomas (high-grade and low-grade) from non-cancer with sensitivities and specificities on average of 93.75 and 96.53% respectively. The proposed diagnostic regime has the ability to reduce mortality and morbidity rates.

Analysis of interleukin (IL)-8 expression in human astrocytomas: associations with IL-6, cyclooxygenase-2, vascular endothelial growth factor, and microvessel morphometry.

Malignant astrocytomas are highly vascular neoplasms with potent angiogenic activity. The present study aimed to investigate peripheral and local expression of interleukin (IL)-8 in astrocytomas with possible associations to IL-6, cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF) expression, and microvessel morphometry. IL-6- and IL-8-secreting peripheral blood monocytes (PBMCs) were evaluated in 17 glioblastoma (WHO grade IV), 5 anaplastic astrocytoma (WHO grade III), and 6 diffuse astrocytoma patients (WHO grade II), in parallel with 23 healthy controls using enzyme-linked immunosorbent spot (ELISPOT) assay. The IL-8 expression was assessed immunohistochemically in patients' tumor tissue sections and correlated with the expression of COX-2, VEGF, IL-6, and microvessel morphometry (assessed using CD34 antibody). Eighteen cases were also stained for CD31 and used as an additional vessel marker to validate our results regarding microvessel morphometry. IL-6 and IL-8 were highly secreted in the PBMCs of glioma patients compared with controls (p = 0.0001, p < 0.0001, respectively), with a positive correlation between IL-8 expression and secretion levels (p = 0.001). IL-8 immunoreactivity was detected in malignant cells or macrophages in perivascular areas and in pseudopalisading cells around necrosis and was positively correlated with histological grade (p = 0.0175) and tumor necrosis (p = 0.0793). IL-6 and IL-8 expression levels were positively correlated (p = 0.0036) and associated with COX-2 and VEGF expression (IL-6: p = 0.0133, p = 0.065; IL-8: p = 0.0139, p = 0.0101), but not with microvessel morphometry, by either CD31 or CD34. The coordinate expression and topographical relationship of IL-6, IL-8, COX-2, and VEGF in the same tumor areas (e.g., perinecrotic areas) attest to their intimate liaison in terms of cancer-induced angiogenesis, which is probably secondary to the induction of multiple interdependent molecular pathways. Moreover, our study seems to be the first attempt to link IL-8 expression by tumor cells with histological grade, implicating its potent role in gliomagenesis.

Comparative analysis of peripheral and localised cytokine secretion in glioblastoma patients.

BACKGROUND: Malignant gliomas are the most common primary brain tumours of both children and adults. The unique aspects of their biology and anatomic site render them refractory to conventional therapeutic strategies such as surgery and chemotherapy. Significant attention has been given, recently, to immunotherapy which, although promising in preclinical studies, has not yet enhanced the survival of patients with glioblastomas. METHODS: To further understand the immunobiology of glioblastomas in clinical settings, we examined the secretion of four main cytokines in the peripheral blood and in primary cell cultures of 33 human glioblastoma patients. An ELISPOT methodology was used for the first time to examine Th1, and Th2 cytokine secretion from both peripheral lymphocytes and glioma tumour cells. RESULTS: Th1 cytokines (tumour necrosis factor (TNF-alpha), interferon (IFN-gamma) were markedly reduced compared to control levels (P=0.01 and P<0.001, respectively), whereas in contrast, Th2 (interleukin (IL)-4 and IL-10) were strongly expressed in both peripheral lymphocytes and glioma cell cultures (P=0.05 and P<0.001, respectively). CONCLUSION: This pattern indicates an 'immunosuppressive status' in glioblastomas which is related to their origination and the evasion of glioma cells from immune surveillance and could account for the failure of immunotherapy in such tumours. Furthermore, ELISPOT methodology can be used for monitoring of cytokine secretion from tumour cells, in addition to the well-established peripheral cytokine secretion.

Application of the ELISPOT method for comparative analysis of interleukin (IL)-6 and IL-10 secretion in peripheral blood of patients with astroglial tumors.

Glioblastoma, (grade IV astrocytoma), is characterized by rapid growth and resistance to treatment. Identification of markers of aggressiveness in this tumor could represent new therapeutic targets. Interleukins (IL)-6 and IL-10 may be considered as possible candidates, regulating cell growth, resistance to chemotherapy and angiogenesis. ELISPOT method provides a useful tool for the determination of the exact cell number of peripheral lymphocytes secreting a specific cytokine. IL-6 and IL-10 secretion levels were determined using ELISPOT methodology in peripheral blood mononuclear cells of 18 patients with astrocytic neoplasms (3 grade II and 15 grade IV), in parallel with 18 healthy controls. Additionally, immunohistochemical expression of these two cytokines was performed in paraffin-embedded neoplastic tissue in 12 of these patients. The secretion of IL-6 from peripheral monocytes was significantly higher in glioma patients compared to controls (P = 0.0003). In addition, IL-10 secretion from peripheral mononuclear and tumor cells of glioma patients was also higher as compared to healthy controls (P = 0.0002). Based on immunohistochemical staining, IL-6 expression was localized in tumor cells and macrophages as well as in areas of large ischemic necrosis, while the major source of IL-10 expression in glioblastomas was the microglia/macrophage cells. It is suggested that IL-10 contributes to the progression of astrocytomas by suppressing the patient's immune response, whereas IL-6 provides an additional growth advantage. This study demonstrates for the first time the usefulness of ELISPOT in estimating the secretion of IL-6 and IL-10 from peripheral blood and the correlation of their expression in neoplastic cells.

Elevation of plasma concentration of adhesion molecules in late-life depression.

OBJECTIVES: Late-life depression may be associated with vascular disease. The purpose of the study was to investigate this association by determining the levels of soluble adhesion molecules (sICAM-1 and sVCAM-1) which represent markers of ischemia-induced inflammation in elderly individuals with depression. METHODS: Blood samples were obtained from 33 subjects with depression selected from a community-dwelling population after screening with the Geriatric Depression Scale, and 33 matched controls. Serum concentrations of sICAM-1 (ng/mL) and sVCAM-1 (ng/mL) were measured in both groups. RESULTS: Depressed patients (Group A) possessed significantly higher sICAM-1 levels compared to healthy controls (Group B) (674.94 +/- 166.90 ng/ml vs 467.05 +/- 231.26 ng/ml, respectively, p < 0.01). Similarly the same groups demonstrated elevated sVCAM-1 levels compared to controls (572.14 +/- 182.20 ng/ml vs 449.04 +/- 285.27 ng/ml, p < 0.05); a difference that in both cases remained significant after adjustment for potential confounders (gender, smoking, presence of metabolic syndrome). CONCLUSION: These findings indicate an association between high serum levels of VCAM-1, and ICAM-1 and depression in the elderly and further support the vascular depression hypothesis, which has important implications for the understanding and management of late-life depression.

Effect of melatonin on phagocytic activity and intracellular free calcium concentration in testicular macrophages from normal and streptozotocin-induced diabetic rats.

This study examined the effect of melatonin (MLT) on in vitro phagocytosis of testicular macrophages taken from control and streptozotocin (STZ)-induced diabetic rats and the possible mechanism of its action. The phagocytic activity was measured as a number of latex beads ingested by 100 macrophages (PI, phagocytic index) in consecutive time points of the incubation. Changes in intracellular free calcium level [Ca2+]i in isolated macrophages in vitro were measured with the use of ratio-image fluorescence microscopy (fluorescent dye: Fura2/AM). Phagocytic index in macrophages isolated from healthy rats was 20% higher than in those from diabetic animals. Melatonin in physiological concentration (10(-7) M) significantly (p < 0.05) increased the PI in testicular macrophages from control animals (PI = 68 +/- 5 with MLT compared to PI = 46 +/- 7 without MLT) while no such effect was observed in the cells from diabetic rats (PI = 36 +/- 23 with MLT compared to PI = 31 +/- 11 without MLT). Basal [Ca2+]i was significantly (p < 0.01) higher in macrophages from diabetic rats compared to control. Stimulation of both control and diabetic testicular macrophages with 10(-7) M MLT resulted in a significant (p < 0.05) increase in [Ca2+]i in cells incubated in 2.5 mM calcium solution while no such response was observed in calcium-free Tyrode solution. However, MLT evoked [Ca2+]i response in macrophages isolated from diabetic animals was much lower than in macrophages isolated from age-matched controls and the time needed for maximal response was much longer. Lack of response in calcium-free solution suggests that extracellular calcium may be necessary to trigger MLT response and in its progression.