Corneal nerve structure and function as markers of diabetic neuropathy

Diabetic neuropathy is a significant clinical problem that currently has no effective therapy, and in advanced cases, leads to foot ulceration and lower limb amputation. The accurate detection, characterisation and quantification of this condition are important in order to define at-risk patients, anticipate deterioration, monitor progression and assess new therapies. This thesis evaluates novel corneal methods of assessing diabetic neuropathy. Over the past several years two new non-invasive corneal markers have emerged, and in cross-sectional studies have demonstrated their ability to stratify the severity of this disease. Corneal confocal microscopy (CCM) allows quantification of corneal nerve parameters and non-contact corneal aesthesiometry (NCCA), the presumed functional correlate of corneal structure, assesses the sensitivity of the cornea. Both these techniques are quick to perform, produce little or no discomfort for the patient, and with automatic analysis paradigms developed, are suitable for clinical settings. Each has advantages and disadvantages over established techniques for assessing diabetic neuropathy. New information is presented regarding measurement bias of CCM images, and a unique sampling paradigm and associated accuracy determination method of combinations is described. A novel high-speed corneal nerve mapping procedure has been developed and application of this procedure in individuals with neuropathy has revealed regions of sub-basal nerve plexus that dictate further evaluation, as they appear to show earlier signs of damage than the central region of the cornea that has to date been examined. The discriminative capacity of corneal sensitivity measured by NCCA is revealed to have reasonable potential as a marker of diabetic neuropathy. Application of these new corneal markers for longitudinal evaluation of diabetic neuropathy has the potential to reduce dependence on more invasive, costly, and time-consuming assessments, such as skin biopsy.

Human proximal tubule epithelial cells modulate autologous dendritic cell function

Background We have previously demonstrated that human kidney proximal tubule epithelial cells (PTEC) are able to modulate autologous T and B lymphocyte responses. It is well established that dendritic cells (DC) are responsible for the initiation and direction of adaptive immune responses and that these cells occur in the renal interstitium in close apposition to PTEC under inflammatory disease settings. However, there is no information regarding the interaction of PTEC with DC in an autologous human context. Methods Human monocytes were differentiated into monocyte-derived DC (MoDC) in the absence or presence of primary autologous activated PTEC and matured with polyinosinic:polycytidylic acid [poly(I:C)], while purified, pre-formed myeloid blood DC (CD1c+ BDC) were cultured with autologous activated PTEC in the absence or presence of poly(I:C) stimulation. DC responses were monitored by surface antigen expression, cytokine secretion, antigen uptake capacity and allogeneic T-cell-stimulatory ability. Results The presence of autologous activated PTEC inhibited the differentiation of monocytes to MoDC. Furthermore, MoDC differentiated in the presence of PTEC displayed an immature surface phenotype, efficient phagocytic capacity and, upon poly(I:C) stimulation, secreted low levels of pro-inflammatory cytokine interleukin (IL)-12p70, high levels of anti-inflammatory cytokine IL-10 and induced weak Th1 responses. Similarly, pre-formed CD1c+ BDC matured in the presence of PTEC exhibited an immature tolerogenic surface phenotype, strong endocytic and phagocytic ability and stimulated significantly attenuated T-cell proliferative responses. Conclusions Our data suggest that activated PTEC regulate human autologous immunity via complex interactions with DC. The ability of PTEC to modulate autologous DC function has important implications for the dampening of pro-inflammatory immune responses within the tubulointerstitium in renal injuries. Further dissection of the mechanisms of PTEC modulation of autologous immune responses may offer targets for therapeutic intervention in renal medicine.

Delta opioid-receptor function in the dorsal striatum plays a role in high levels of ethanol consumption in rats

Binge-like patterns of excessive drinking during young adulthood increase the propensity for alcohol use disorders (AUDs) later in adult life; however, the mechanisms that drive this are not completely understood. Previous studies showed that the δ-opioid peptide receptor (DOP-R) is dynamically regulated by exposure to ethanol and that the DOP-R plays a role in ethanol-mediated behaviors. The aim of this study was to determine the role of the DOP-R in high ethanol consumption from young adulthood through to late adulthood by measuring DOP-R-mediated [(35)S]GTPγS binding in brain membranes and DOP-R-mediated analgesia using a rat model of high ethanol consumption in Long Evans rats. We show that DOP-R activity in the dorsal striatum and DOP-R-mediated analgesia changes during development, being highest during early adulthood and reduced in late adulthood. Intermittent access to ethanol but not continuous ethanol or water from young adulthood leads to an increase in DOP-R activity in the dorsal striatum and DOP-R-mediated analgesia into late adulthood. Multiple microinfusions of naltrindole into the dorsal striatum or multiple systemic administration of naltrindole reduces ethanol consumption, and following termination of treatment, DOP-R activity in the dorsal striatum is attenuated. These findings suggest that DOP-R activity in the dorsal striatum plays a role in high levels of ethanol consumption and suggest that targeting the DOP-R is an alternative strategy for the treatment of AUDs.

Exercise for health : a randomized, controlled trial evaluating impact of a pragmatic, translational exercise intervention on quality of life, function and treatment-related side effects following breast cancer

Purpose Exercise for Health was a randomized, controlled trial designed to evaluate two modes of delivering (face-to-face [FtF] and over-the-telephone [Tel]) an 8-month translational exercise intervention, commencing 6-weeks post-breast cancer surgery (PS). Methods Outcomes included quality of life (QoL), function (fitness and upper-body) and treatment-related side effects (fatigue, lymphoedema, body mass index, menopausal symptoms, anxiety, depression and pain). Generalised estimating equation modelling determined time (baseline [5-weeks PS], mid-intervention [6-months PS], post-intervention [12-months PS]), group (FtF, Tel, Usual Care [UC]) and time-by-group effects. 194 women representative of the breast cancer population were randomised to the FtF (n=67), Tel (n=67) and UC (n=60) groups. Results: There were significant (p<0.05) interaction effects on QoL, fitness and fatigue, with differences being observed between the treatment groups and the UC group. Trends observed for the treatment groups were similar. The treatment groups reported improved QoL, fitness and fatigue over time and changes observed between baseline and post-intervention were clinically relevant. In contrast, the UC group experienced no change, or worsening QoL, fitness and fatigue, mid-intervention. Although improvements in the UC group occurred by 12-months post-surgery, the change did not meet the clinically relevant threshold. There were no differences in other treatment-related side-effects between groups. Conclusion This translational intervention trial, delivered either face-to-face or over-the-telephone, supports exercise as a form of adjuvant breast cancer therapy that can prevent declines in fitness and function during treatment and optimise recovery post-treatment.

Evidence for a dual function of EphB4 as tumor promoter and suppressor regulated by the absence or presence of the ephrin-B2 ligand

Overexpression of the receptor tyrosine kinase EphB4 is common in epithelial cancers and linked to tumor progression by promoting angiogenesis, increasing survival and facilitating invasion and migration. However, other studies have reported loss of EphB4 suggesting a tumor suppressor function in some cancers. These opposing roles may be regulated by (i) the presence of the primary ligand ephrin-B2 that regulates pathways involved in tumor suppression or (ii) the absence of ephrin-B2 that allows EphB4 signaling via ligand-independent pathways that contribute to tumor promotion. To explore this theory, EphB4 was overexpressed in the prostate cancer cell line 22Rv1 and the mammary epithelial cell line MCF-10A. Overexpressed EphB4 localized to lipid-rich regions of the plasma membrane and confirmed to be ligand-responsive as demonstrated by increased phosphorylation of ERK1/2 and internalization. EphB4 overexpressing cells demonstrated enhanced anchorage-independent growth, migration and invasion, all characteristics associated with an aggressive phenotype, and therefore supporting the hypothesis that overexpressed EphB4 facilitates tumor promotion. Importantly, these effects were reversed in the presence of ephrin-B2 which led to a reduction in EphB4 protein levels, demonstrating that ligand-dependent signaling is tumor suppressive. Furthermore, extended ligand stimulation caused a significant decrease in proliferation that correlated with a rise in caspase-3/7 and -8 activities. Together, these results demonstrate that overexpression of EphB4 confers a transformed phenotype in the case of MCF-10A cells and an increased metastatic phenotype in the case of 22Rv1 cancer cells and that both phenotypes can be restrained by stimulation with ephrin-B2, in part by reducing EphB4 levels.

Relationship among CFH and ARMS2 genotypes, macular pigment optical density, and neuroretinal function in persons without age-related macular degeneration

Purpose: To determine whether there is a difference in neuroretinal function and in macular pigment optical density between persons with high- and low-risk gene variants for age-related macular degeneration (AMD) and no ophthalmoscopic signs of AMD, and to compare the results on neuroretinal function to patients with manifest early AMD. Methods and Participants: Neuroretinal function was assessed with the multifocal electroretinogram (mfERG) for 32 participants (22 healthy persons with no AMD and 10 early AMD patients). The 22 healthy participants with no AMD had high- or low-risk genotypes for either CFH (rs380390) and/or ARMS2 (rs10490924). Trough-to-peak response densities and peak-implicit times were analyzed in 5 concentric rings. Macular pigment optical densitometry was assessed by customized heterochromatic flicker photometry. Results: Trough-to-peak response densities for concentric rings 1 to 3 were, on average, significantly greater in participants with high-risk genotypes than in participants with low-risk genotypes and in persons with early AMD after correction for age and smoking (p<0.05). The group peak- implicit times for ring 1 were, on average, delayed in the patients with early AMD compared with the participants with high- or low-risk genotypes, although these differences were not significant. There was no significant correlation between genotypes and macular pigment optical density. Conclusion: Increased neuroretinal activity in persons who carry high-risk AMD genotypes may be due to genetically determined subclinical inflammatory and/or histological changes in the retina. Neuroretinal function in healthy persons genetically susceptible to AMD may be a useful additional early biomarker (in combination with genetics) before there is clinical manifestation.

Insights into the role and function of L2, the minor capsid protein of papillomaviruses

Human papillomaviruses (HPV) are responsible for the most common human sexually transmitted viral infections, and high-risk types are responsible for causing cervical and other cancers. The minor capsid protein L2 of HPV plays important roles in virus entry into cells, localisation of viral components to the nucleus, in DNA binding, capsid formation and stability. It also elicits antibodies that are more cross-reactive between HPV types than does the major capsid protein L1, making it an attractive potential target for new-generation, more broadly protective subunit vaccines against HPV infections. However, its low abundance in natural capsids-12-72 molecules per 360 copies of L1-limits its immunogenicity. This review will explore the biological roles of the protein, and prospects for its use in new vaccines. © 2009 Springer-Verlag.

The cell surface glycoprotein CUB domain-containing protein 1 (CDCP1) contributes to epidermal growth factor receptor-mediated cell migration

Epidermal growth factor (EGF) activation of the EGF receptor (EGFR) is an important mediator of cell migration, and aberrant signaling via this system promotes a number of malignancies including ovarian cancer. We have identified the cell surface glycoprotein CDCP1 as a key regulator of EGF/EGFR-induced cell migration. We show that signaling via EGF/EGFR induces migration of ovarian cancer Caov3 and OVCA420 cells with concomitant up-regulation of CDCP1 mRNA and protein. Consistent with a role in cell migration CDCP1 relocates from cell-cell junctions to punctate structures on filopodia after activation of EGFR. Significantly, disruption of CDCP1 either by silencing or the use of a function blocking antibody efficiently reduces EGF/EGFR-induced cell migration of Caov3 and OVCA420 cells. We also show that up-regulation of CDCP1 is inhibited by pharmacological agents blocking ERK but not Src signaling, indicating that the RAS/RAF/MEK/ERK pathway is required downstream of EGF/EGFR to induce increased expression of CDCP1. Our immunohistochemical analysis of benign, primary, and metastatic serous epithelial ovarian tumors demonstrates that CDCP1 is expressed during progression of this cancer. These data highlight a novel role for CDCP1 in EGF/EGFR-induced cell migration and indicate that targeting of CDCP1 may be a rational approach to inhibit progression of cancers driven by EGFR signaling including those resistant to anti-EGFR drugs because of activating mutations in the RAS/RAF/MEK/ERK pathway.

Post-match changes in neuromuscular function and the relationship to match demands in amateur rugby league matches

Objectives: The current study investigated the change in neuromuscular contractile properties following competitive rugby league matches and the relationship with physical match demands. Design: Eleven trained, male rugby league players participated in 2–3 amateur, competitive matches (n = 30). Methods: Prior to, immediately (within 15-min) and 2 h post-match, players performed repeated counter-movement jumps (CMJ) followed by isometric tests on the right knee extensors for maximal voluntary contraction (MVC), voluntary activation (VA) and evoked twitch contractile properties of peak twitch force (Pt), rate of torque development (RTD), contraction duration (CD) and relaxation rate (RR). During each match, players wore 1 Hz Global Positioning Satellite devices to record distance and speeds of matches. Further, matches were filmed and underwent notational analysis for number of total body collisions. Results: Total, high-intensity, very-high intensity distances covered and mean speed were 5585 ± 1078 m, 661 ± 265, 216 ± 121 m and 75 ± 14 m min−1, respectively. MVC was significantly reduced immediately and 2 h post-match by 8 ± 11 and 12 ± 13% from pre-match (p < 0.05). Moreover, twitch contractile properties indicated a suppression of Pt, RTD and RR immediately post-match (p < 0.05). However, VA was not significantly altered from pre-match (90 ± 9%), immediately-post (89 ± 9%) or 2 h post (89 ± 8%), (p > 0.05). Correlation analyses indicated that total playing time (r = −0.50) and mean speed (r = −0.40) were moderately associated to the change in post-match MVC, while mean speed (r = 0.35) was moderately associated to VA. Conclusions: The present study highlights the physical demands of competitive amateur rugby league result in interruption of peripheral contractile function, and post-match voluntary torque suppression may be associated with match playing time and mean speeds.

Reduced expression of retinoblastoma gene product (pRB) and high expression of p53 are associated with poor prognosis in ovarian cancer

Paraffin sections (n = 168, 27 benign, 16 low malignant potential [LMP] and 125 malignant tumours) from epithelial ovarian tumours were evaluated immunohistochemically for expression of retinoblastoma gene product (pRB) and p53 protein, and the relationship among pRB, p53 and cyclin-dependent kinase inhibitor 2 (CDKN2) gene product p16INK4A (p16) was analysed, following our previous study of p16. Forty-one percent of the benign, 50% of the LMP and most (71%) of the malignant tumours showed high pRB expression. High expression of pRB (>50% pRB-positive cells) significantly correlated with non-mucinous histological subtypes. Reduced pRB expression, substage and residual disease were significant predictors for poor prognosis in stage I patients. All the benign and most of the LMP (81%) tumours were in either the p53-negative or low p53-positive category, but nearly half of the malignant tumours had high p53 expression. High p53 accumulation was found in non-mucinous, high grade and late stage tumours. For well-differentiated carcinomas, high p53 expression was a predictor of poor prognosis. However, even though high p53 expression was not associated with histological subtype, stage or the presence of residual disease, high p53 expression was not an independent predictor when all clinical parameters were combined. For all ovarian cancers, a close correlation was found between high p53 and high p16 expression. The relationship between the expression of pRB and p16 depended on tumour stage. In stage I tumours, high pRB was associated with low p16 reactivity. On the other hand, most advanced tumours showed both high pRB and high p16 reactivity. Int. J. Cancer 74:407–415, 1997. © 1997 Wiley-Liss, Inc.

Increased expression of cyclin-dependent kinase inhibitor 2 (CDKN2A) gene product P16INK4A in ovarian cancer is associated with progression and unfavourable prognosis

Paraffin sections from 190 epithelial ovarian tumours, including 159 malignant and 31 benign epithelial tumours, were analysed immunohistochemically for expression of cyclin-dependent kinase inhibitor 2 (CDKN2A) gene product p16INK4A (p16). Most benign tumours showed no p16 expression in the tumour cells, whereas only 11% of malignant cancers were p16 negative. A high proportion of p16-positive tumour cells was associated with advanced stage and grade, and with poor prognosis in cancer patients. For FIGO stage 1 tumours, a high proportion of p16-positive tumour cells was associated with poorer survival, suggesting that accumulation of p16 is an early event of ovarian tumorigenesis. In contrast to tumour cells, high expression of p16 in the surrounding stromal cells was not associated with the stage and grade, but was associated with longer survival. When all parameters were combined in multivariate analysis, high p16 expression in stromal cells was not an independent predictor for survival, indicating that low p16 expression in stromal cells is associated with other markers of tumour progression. High expression of p16 survival in the stromal cells of tumours from long-term survivors suggests that tumour growth is limited to some extent by factors associated with p16 expression in the matrix.

Expression of MUC1 and MUC2 mucins in epithelial ovarian tumours

This is the first study to describe the association between expression of MUC1 and MUC2 mucins and prognosis in ovarian cancer. Paraffin sections of epithelial ovarian tumours (n=182: 29 benign, 21 low malignant potential, and 132 invasive tumours) were analysed immunohistochemically for expression of MUC1 and MUC2 mucin core proteins. Most benign, low malignant potential, and invasive tumours showed high MUC1 expression in the cytoplasm. Low cytoplasmic expression of MUC1 was a predictor for good prognosis, particularly within stage III tumours. A minority of benign epithelial tumours, but most low malignant potential and invasive non-mucinous tumours, showed high MUC1 expression on the cell membrane. High apical MUC1 reactivity was associated with non-mucinous tumours. Low expression of MUC1 in the apical membrane was associated with early stage and good outcome for invasive tumours. Most benign and low malignant potential tumours, but only a minority of invasive tumours, showed MUC2 expression. MUC2 was found in non-mucinous as well as in mucinous tumours. The presence of MUC2 was inversely associated with high tumour grade but was not associated with altered survival. These results support experimental evidence that MUC1 influences the metastatic ability of ovarian cancer.

Serine protease inhibition and mitochondrial dysfunction associated with cisplatin resistance in human tumor cell lines : targets for therapy

Indicators of mitochondrial function were studied in two different cell culture models of cis-diamminedichloroplatinum-II (CDDP) resistance: the intrinsically resistant human ovarian cancer cell line CI-80-13S, and resistant clones (HeLa-S1a and HeLa-S1b) generated by stable expression of the serine protease inhibitor—plasminogen activator inhibitor type-2 (PAI-2), in the human cervical cancer cell line HeLa. In both models, CDDP resistance was associated with sensitivity to killing by adriamycin, etoposide, auranofin, bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride {[Au(DPPE)2]Cl}, CdCl2 and the mitochondrial inhibitors rhodamine-123 (Rhl23), dequalinium chloride (DeCH), tetraphenylphosphonium (TPP), and ethidium bromide (EtBr) and with lower constitutive levels of ATP. Unlike the HeLa clones, CI-80-13S cells were additionally sensitive to chloramphenicol, 1-methyl-4-phenylpyridinium ion (MPP+), rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, and showed poor reduction of 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), suggesting a deficiency in NADH dehydrogenase and/or succinate dehydrogenase activities. Total platinum uptake and DNA-bound platinum were slightly lower in CI-80-13S than in sensitive cells. The HeLa-S1a and HeLa-S1b clones, on the other hand, showed poor reduction of triphenyltetrazolium chloride (TTC), indicative of low cytochrome c oxidase activity. Total platinum uptake by HeLa-S1a was similar to HeLa, but DNA-bound platinum was much lower than for the parent cell line. The mitochondria of CI-80-13S and HeLa-S1a showed altered morphology and were fewer in number than those of JAM and HeLa. In both models, CDDP resistance was associated with less platinum accumulation and with mitochondrial and membrane defects, brought about one case with expression of a protease inhibitor which is implicated in tumor progression. Such markers may identify tumors suitable for treatment with gold phosphine complexes or other mitochondrial inhibitors.