Risk factors at five-year survival in grade 3 breast cancer: a retrospective observational study of the New Zealand population.

BACKGROUND: Breast cancer is the most common cancer in New Zealand, with approximately 3000 new registrations annually, affecting one in nine women and resulting in more than 600 deaths. This study analyzed data of patients with selected prognostic factors of Nottingham grade 3 tumors over a specified five-year period. The study aimed to identify factors that result in differential survival in the female, New Zealand population. METHOD: This is an observational, retrospective cohort study of prospectively collected data from New Zealand Breast Cancer Register. The selected period of 1st January 2011 to 31st, December 2015 allowed a consistent overlap for a national five-year data of grade 3 breast cancer in New Zealand. Mortality was carried out using univariate Fine-Gray competing risk statistical models. RESULTS: This study showed that women in the older age group (> 70 years) had higher five-year mortality risk (HR: 1.7, 95% CI: 0.9-3.0, p = 0.053). Hormonal receptor analysis showed that ER positive, PR negative, and ER negative, PR negative subjects were at increased risk (HR = 3.5, 95% CI 2.3-5.4, p < 0.001) and (HR = 2.6, 95% CI, 1.8-3.9, p < 0.001) respectively. Molecular subtypes Triple Negative Breast Cancer and Luminal B subjects were at increased risk (HR = 3.0, 95% CI, 1.8-4.7, p < 0.001 and (HR = 3.3, 95% CI, 1.7-6.3, p < 0.001) respectively. HER2 enriched subjects were at a higher, but not significant, risk of five-year mortality compared to luminal A (HR = 1.6, 95% CI, 0.8-3.0, p = 0.10). NZ Europeans were at increased risk (HR = 1.7, 95% CI, 0.8-3.2, p = 0.11), with the highest Cumulative Incidence Function CIF, the largest proportion of HER2 enriched and TNBC across ethnicities.; however, Pacific Islanders experienced the highest HER2 CIF. CONCLUSION: The survival rates for grade 3 breast cancer vary across the selected prognostic factors and ethnicity. The results of this study make an initial contribution to the understanding of grade 3 breast cancer in the New Zealand population.

Transport-Mediated Oxaliplatin Resistance Associated with Endogenous Overexpression of MRP2 in Caco-2 and PANC-1 Cells.

Our recent publications showed that multidrug resistance protein 2 (MRP2, encoded by the ABCC2 gene) conferred oxaliplatin resistance in human liver cancer HepG2 cells. However, the contribution of MRP2 to oxaliplatin resistance remains unclear in colorectal and pancreatic cancer lines. We investigated the effects of silencing MRP2 by siRNA on oxaliplatin accumulation and sensitivity in human colorectal cancer Caco-2 cells and pancreatic cancer PANC-1 cells. We characterized the effects of oxaliplatin on MRP2 ATPase activities using membrane vesicles. Over-expression of MRP2 (endogenously in Caco-2 and PANC-1 cells) was associated with decreased oxaliplatin accumulation and cytotoxicity, but those deficits were reversed by inhibition of MRP2 with myricetin or siRNA knockdown. Silencing MRP2 by siRNA increased oxaliplatin-induced apoptotic rate in Caco-2 and PANC-1 cells. Oxaliplatin stimulated MRP2 ATPase activity with a concentration needed to reach 50% of the maximal stimulation (EC50) value of 8.3 ± 0.7 µM and Hill slope 2.7. In conclusion, oxaliplatin is a substrate of MRP2 with possibly two binding sites, and silencing MRP2 increased oxaliplatin accumulation and cytotoxicity in two widely available gastrointestinal tumour lines (PANC-1 and Caco-2).

Identification of MRP2 as a targetable factor limiting oxaliplatin accumulation and response in gastrointestinal cancer.

Oxaliplatin is important for the clinical treatment of colorectal cancer and other gastrointestinal malignancies, but tumour resistance is limiting. Several oxaliplatin transporters were previously identified but their relative contributions to determining oxaliplatin tumour responses and gastrointestinal tumour cell sensitivity to oxaliplatin remains unclear. We studied clinical associations between tumour expression of oxaliplatin transporter candidate genes and patient response to oxaliplatin, then experimentally verified associations found with MRP2 in models of human gastrointestinal cancer. Among 18 oxaliplatin transporter candidate genes, MRP2 was the only one to be differentially expressed in the tumours of colorectal cancer patients who did or did not respond to FOLFOX chemotherapy. Over-expression of MRP2 (endogenously in HepG2 and PANC-1 cells, or induced by stable transfection of HEK293 cells) decreased oxaliplatin accumulation and cytotoxicity but those deficits were reversed by inhibition of MRP2 with myricetin or siRNA knockdown. Mice bearing subcutaneous HepG2 tumour xenografts were sensitised to oxaliplatin antitumour activity by concurrent myricetin treatment with little or no increase in toxicity. In conclusion, MRP2 limits oxaliplatin accumulation and response in human gastrointestinal cancer. Screening tumour MRP2 expression levels, to select patients for treatment with oxaliplatin-based chemotherapy alone or in combination with a MRP2 inhibitor, could improve treatment outcomes.

The Effect of 1,3-Butanediol on Cycling Time-Trial Performance.

This study investigated the effect of the racemic ß-hydroxybutyrate (ßHB) precursor, R,S-1,3-butanediol (BD), on time-trial (TT) performance and tolerability. A repeated-measures, randomized, crossover study was conducted in nine trained male cyclists (age, 26.7 ± 5.2 years; body mass, 69.6 ± 8.4 kg; height, 1.82 ± 0.09 m; body mass index, 21.2 ± 1.5 kg/m2; VO2peak,63.9 ± 2.5 ml·kg-1·min-1; Wmax, 389.3 ± 50.4 W). Participants ingested 0.35 g/kg of BD or placebo 30 min before and 60 min during 85 min of steady-state exercise, which preceded a ∼25- to 35-min TT (i.e., 7 kJ/kg). The ingestion of BD increased blood D-ßHB concentration throughout exercise (0.44-0.79 mmol/L) compared with placebo (0.11-0.16 mmol/L; all p < .001), which peaked 1 hr following the TT (1.38 ± 0.35 vs. 0.34 ± 0.24 mmol/L; p < .001). Serum glucose and blood lactate concentrations were not different between trials (all p > .05). BD ingestion increased oxygen consumption and carbon dioxide production after 20 min of steady-state exercise (p = .002 and p = .032, respectively); however, no further effects on cardiorespiratory parameters were observed. Within the BD trial, moderate to severe gastrointestinal symptoms were reported in five participants, and low levels of dizziness, nausea, and euphoria were reported in two participants. However, this had no effect on TT duration (placebo, 28.5 ± 3.6 min; BD, 28.7 ± 3.2 min; p = .62) and average power output (placebo, 290.1 ± 53.7 W; BD, 286.4 ± 45.9 W; p = .50). These results suggest that BD has no benefit for endurance performance.

Fucoidan Extracted from the New Zealand Undaria pinnatifida-Physicochemical Comparison against Five Other Fucoidans: Unique Low Molecular Weight Fraction Bioactivity in Breast Cancer Cell Lines.

Fucoidan, the complex fucose-containing sulphated polysaccharide varies considerably in structure, composition, and bioactivity, depending on the source, species, seasonality, and extraction method. In this study, we examined five fucoidans extracted from the same seaweed species Undaria pinnatifida but from different geological locations, and compared them to the laboratory-grade fucoidan from Sigma (S). The five products differed in molecular composition. The amount of over 2 kDa low molecular weight fraction (LMWF) of the New Zealand crude fucoidan (S1) was larger than that of S, and this fraction was unique, compared to the other four fucoidans. The difference of molecular compositions between S and S1 explained our previous observation that S1 exhibited different anticancer profile in some cancer cell lines, compared with S. Since we observed this unique LMWF, we compared the cytotoxic effects of a LMWF and a high molecular weight fucoidan (HMWF) in two breast cancer cell lines-MCF-7 and MDA-MB-231. Results indicated that the molecular weight is a critical factor in determining the anti-cancer potential of fucoidan, from the New Zealand U. pinnatifida, as the LMWF exhibited a dose-dependent inhibition on the proliferation of breast cancer cells, significantly better than the HMWF, in both cell lines. A time-dependent inhibition was only observed in the MCF-7. Induction of caspase-dependent apoptosis was observed in the MDA-MB-231 cells, through the intrinsic apoptosis pathway alone, or with the extrinsic pathway. LMWF stimulated a dose-dependent NOS activation in the MDA-MB-231 cells. In conclusion, the fucoidan extracted from the New Zealand U. pinnatifida contains a unique LMWF, which could effectively inhibit the growth of breast cancer cell lines. Therefore, the LMWF from New Zealand U. pinnatifida could be used as a supplement cancer treatment.

The Effects of Synthetically Modified Natural Compounds on ABC Transporters.

Multidrug resistance (MDR) is a major hurdle which must be overcome to effectively treat cancer. ATP-binding cassette transporters (ABC transporters) play pivotal roles in drug absorption and disposition, and overexpression of ABC transporters has been shown to attenuate cellular/tissue drug accumulation and thus increase MDR across a variety of cancers. Overcoming MDR is one desired approach to improving the survival rate of patients. To date, a number of modulators have been identified which block the function and/or decrease the expression of ABC transporters, thereby restoring the efficacy of a range of anticancer drugs. However, clinical MDR reversal agents have thus far proven ineffective and/or toxic. The need for new, effective, well-tolerated and nontoxic compounds has led to the development of natural compounds and their derivatives to ameliorate MDR. This review evaluates whether synthetically modifying natural compounds is a viable strategy to generate potent, nontoxic, ABC transporter inhibitors which may potentially reverse MDR.

Copper-induced immunomodulation in mussel (Perna canaliculus) haemocytes.

Copper is a common contaminant in aquatic environments, which may cause physiological dysfunction in marine organisms. However, the toxicity mechanisms of copper in marine bivalves is not fully understood. In this study, we applied an integrated approach that combines flow cytometry and Gas Chromatography-Mass Spectrometry (GC-MS)-based metabolomics to characterize cellular and molecular mechanisms of copper immunotoxicity in New Zealand Greenshell™ mussel (Perna canaliculus) haemolymph. Flow cytometric results showed significant increases in haemocyte mortality, production of reactive oxygen species and apoptosis (via alteration of caspase 3/7 and mitochondrial membrane potential) of haemocytes exposed to increasing total concentrations of Cu2+ (62.5, 125.0 and 187.5 µM) compared to a low Cu2+ concentration (25.0 µM) and control (0.0 µM). In addition to flow cytometric data, our metabolomics results showed alterations of 25 metabolites within the metabolite profile of Cu2+-exposed haemolymph (125 µM) compared to those of control samples. Changes in levels of these metabolites may be considered important signatures of oxidative stress (e.g., glutathione) and apoptosis processes (e.g., alanine, glutamic acid). This study provides insights into the cellular and molecular mechanisms of oxidative stress and apoptosis in marine bivalves and highlights the applicability and reliability of metabolomic techniques for immunotoxicological studies in marine organisms.

A comparison of the acute physiological responses to BODYPUMP™ versus iso-caloric and iso-time steady state cycling.

OBJECTIVES: Given apparent consumer interest in calorie counting and arguably inadequate understanding of the differential effects of exercise modality despite equivalent caloric expenditure, we sought to quantify and compare the acute physiological responses within and between a BODYPUMP™ (BP) group-fitness class and steady-state cycling (CARDIO), matched for caloric cost (iso-caloric) and time (iso-time). DESIGN: Acute cross-over study design. METHODS: Twelve healthy recreationally active females (30.1±5.8 years [mean±SD]) completed cardiorespiratory fitness and strength tests. Subsequently, BP and CARDIO were performed on separate days in randomized order, during which heart rate was monitored continuously, and rating of perceived exertion solicited. Blood samples were collected immediately pre- and post-trial and at 45min post-trial for determination of human growth hormone (HGH), interleukin 6 (IL-6), and cortisol. Lactate (BL) was determined from capillary blood. All outcome measures were analyzed for within-, and between-trial differences. RESULTS: HGH, IL-6 and BL were significantly elevated immediately post-trial for both BP and cycling; the elevation for HGH and BL was significantly greater for BP than CARDIO. IL6 remained elevated at 45min post-trial for both exercise modes, but there was no significant between trial difference. Mean heart rate for both trials was 68% of individually determined maximum heart rate, and predicted VO2 during BP was 14.9±5.2mlkgmin, or 46.7±19.4%VO2peak. Mean load self-selected by participants during BP ranged from 21±7%1RM to 32±9%1RM across four exercises. CONCLUSIONS: These results indicate that BP provided some more potent acute physiological effects than iso-caloric and iso-time steady-state cycling.

Associations between immunological function and memory recall in healthy adults.

Studies in clinical and aging populations support associations between immunological function, cognition and mood, although these are not always in line with animal models. Moreover, very little is known about the relationship between immunological measures and cognition in healthy young adults. The present study tested associations between the state of immune system and memory recall in a group of relatively healthy adults. Immediate and delayed memory recall was assessed in 30 participants using the computerised cognitive battery. CD4, CD8 and CD69 subpopulations of lymphocytes, Interleukin-6 (IL-6) and cortisol were assessed with blood assays. Correlation analysis showed significant negative relationships between CD4 and the short and long delay memory measures. IL-6 showed a significant positive correlation with long-delay recall. Generalized linear models found associations between differences in all recall challenges and CD4. A multivariate generalized linear model including CD4 and IL-6 exhibited a stronger association. Results highlight the interactions between CD4 and IL-6 in relation to memory function. Further study is necessary to determine the underlying mechanisms of the associations between the state of immune system and cognitive performance.

The therapeutic potential of targeting ABC transporters to combat multi-drug resistance.

INTRODUCTION: Most disseminated cancers remain fatal despite the availability of a variety of conventional and novel treatments including surgery, chemotherapy, radiotherapy, immunotherapy, and biologically targeted therapy. A major factor responsible for the failure of chemotherapy in the treatment of cancer is the development of multidrug resistance (MDR). The overexpression of various ABC transporters in cancer cells can efficiently remove the anticancer drug from the cell, thus causing the drug to lose its effect. Areas covered: In this review, we summarised the ongoing research related to the mechanism, function, and regulation of ABC transporters. We integrated our current knowledge at different levels from molecular biology to clinical trials. We also discussed potential therapeutic strategies of targeting ABC transporters to reverse MDR in cancer cells. Expert opinion: Involvement of various ABC transporters to cancer MDR lays the foundation for developing tailored therapies that can overcome MDR. An ideal MDR reversal agent should have broad-spectrum ABC-transporter inhibitory activity, be potent, have good pharmacokinetics, have no trans-stimulation effects, and have low or no toxicity. Alternatively, nanotechnology-based drug delivery systems containing both the cytotoxic drug and reversing agent may represent a useful approach to reversing MDR with minimal off-target toxicity.

Low-Active Male Adolescents: A Dose Response to High-Intensity Interval Training.

PURPOSE: High-intensity interval training (HIIT) is a potential alternative to traditionally recommended steady state exercise for providing health benefits in adolescents, yet its dose-response relationship in this cohort remains unclear, as does its translatability to real-world, nonclinical settings. The present study adopts a novel dose-response design to investigate the effects of undertaking 8 wk of HIIT on the cardiometabolic health of low-active male adolescents. METHODS: Twenty-six male adolescents (age 16 ± 1 yr), identified as low active by nonparticipation in structured sport and physical education classes, were randomly assigned to one of five treatment groups. Corresponding with their group numbers (1-5), participants completed a number of HIIT "sets," which consisted of 4 repeated bouts of 20-s near-maximal exertion interspersed with 10-s passive recovery. Participants performed two HIIT sessions and one resistance training session each week for 8 wk. Baseline and follow-up health measures consisted of peak oxygen uptake (V˙O2peak) with an incremental ramp test to volitional exhaustion; body composition (including visceral fat mass, body fat, and lean tissue mass) with dual-energy x-ray absorptiometry; and lipid profile, glucose, insulin, and interleukin-6 from blood analysis. All health outcomes were analyzed as percentage changes, and data were modeled using a quadratic function to explore dose-response relationships. RESULTS: Significant improvements were observed for V˙O2peak (∼6%), body fat percentage (∼4%), visceral fat mass (∼10%), and waist circumference-to-height ratio (∼3%), but there was no clear effect of dose across groups. CONCLUSIONS: Low-active adolescent males performing a single HIIT set twice weekly, in addition to one resistance training session, gained meaningful improvements in fitness and body composition. Performing additional HIIT sets provided no additional improvements to those of the lowest dose in this study.

Immunological changes and cytokine gene expression during primary infection with human T-cell leukaemia virus type 1 in squirrel monkeys (Saimiri sciureus).

We have developed an animal model of experimental HTLV-1 infection in Saimiri sciureus monkeys in order to study both the immunological and the virological aspects of the infection. As cytokines expressed by immune cells play an essential role during viral infection, we have studied the correlation between the expression of some Th1/Th2 cytokines, including IL-2, IL-5, IL-6, IL-10, and IFN-gamma, and immunological dynamics during primary and chronic HTLV-1 infection in this model. We first demonstrated that, during primary infection, IFN-gamma, IL-2 and IL-10 are expressed at different times and levels. The expression of these cytokines is concomitant with the increase in the numbers of CD4(+), CD8(+) and CD16(+) cells and with the presence of tax/rex viral mRNA. These data indicate the involvement of various cell types in the antiviral immune response. Subsequently, we showed that peripheral blood mononuclear cells freshly isolated from chronically infected monkeys express IFN-gamma and IL-6 at higher levels than those from uninfected animals. IFN-gamma expression is quantitatively correlated to the proviral load and to the presence of circulating effector T-cells against Tax peptide, as detected by Elispot. Further studies will be needed to determine the effective role of these cytokines and other immune system modulators in the control of viral replication during primary HTLV-1 infection or latency.

Proinflammatory and immunomodulatory cytokine mRNA time course profiles in hamsters infected with a virulent variant of Leptospira interrogans.

In order to quantify in vivo the mRNAs of cytokines which play important roles in leptospirosis, we have developed quantitative real-time PCR assays for interleukin-2 (IL-2), IL-4, IL-10, IL-12p40, tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), transforming growth factor beta, and two housekeeping genes (encoding beta-actin and hypoxanthine phosphoribosyltransferase). We used a lethal hamster model reflecting severe leptospirosis in humans. The LightCycler system was used to quantify the gene expression levels with the SYBR green I detection format using external standard curves for each target. We compared the expression levels of cytokine mRNA in the peripheral blood mononuclear cells of both control (uninfected) hamsters and Leptospira interrogans-inoculated hamsters from 1 to 24 h and then 1 to 4 days postinfection. In this kinetic study, there was pronounced expression of Th1 cytokine mRNA (TNF-alpha, IFN-gamma, and IL-12), with transcripts being detected as early as 1 h postinfection. Expression of anti-inflammatory cytokines, such as IL-4 and IL-10, was prominent in delayed samples from 1 to 4 days postinfection in response to infection with Leptospira interrogans. Our data are the first to establish that pathogenic leptospires can stimulate in vivo the production of type 1 cytokines involved in cellular immunity by using this informative animal model. Measuring and assessing cytokine profiles may provide a useful method for accurate study of the mechanisms of anti-Leptospira immunity, indications of prognosis factors, and prospective evaluation of leptospirosis vaccine efficacy in humans.

Chimeric peptide vaccine composed of B- and T-cell epitopes of human T-cell leukemia virus type 1 induces humoral and cellular immune responses and reduces the proviral load in immunized squirrel monkeys (Saimiri sciureus).

A squirrel monkey model of human T-cell leukemia virus type 1 (HTLV-1) infection was used to evaluate the immunogenicity and protective efficacy of a chimeric peptide vaccine composed of a B-cell epitope from the envelope region (aa 175-218) and three HLA-A*0201-restricted cytotoxic T-lymphocyte epitopes derived from Tax protein (Tri-Tax). These selected Tax peptides induced secretion of gamma interferon (IFN-gamma) in peripheral blood mononuclear cells obtained from monkeys chronically infected with HTLV-1. After immunization, a high titre of antibodies and a high frequency of IFN-gamma-producing cells were detected against the Env and the Tri-Tax immunogens, but not against the individual Tax peptides. This might indicate that epitope(s) distinct from those recognized by humans are recognized by responder monkeys. After challenge, it was shown by competitive PCR that partial protection against HTLV-1 infection could be raised in immunized animals. Further studies should be developed to determine the duration of this protection.

The efficacy of combined therapy of arsenic trioxide and alpha interferon in human T-cell leukemia virus type-1-infected squirrel monkeys (Saimiri sciureus).

Human T-cell lymphotropic virus type 1 (HTLV-1)-associated adult T-cell leukemia/lymphoma (ATLL) has a poor prognosis owing to its intrinsic resistance to chemotherapy. Although zidovudine (AZT) and alpha interferon (IFN-alpha) give rise to some response and improve the prognosis of ATLL, alternative therapies are needed. Arsenic trioxide (As(2)O(3)) has been shown to synergize with IFN-alpha in arresting cell growth and inducing apoptosis of ATLL cells in vitro. In this study, we evaluated the toxicity and the efficacy of this combined treatment in HTLV-1-infected squirrel monkeys (Saimiri sciureus) and HTLV-1 infected cell lines derived therefrom. We first show that treatment with As(2)O(3) and IFN-alpha can induce growth arrest in HTLV-1-transformed monkey T-cell lines in vitro. We then show that treatment of squirrel monkeys with As(2)O(3) in vivo is highly toxic at 0.9 or 0.3mg/day but not at 0.14mg/day for up to 2 weeks. Although the combination of As(2)O(3) and IFN-alpha did not affect significantly the HTLV-1 proviral load in infected monkeys, it reduced the absolute numbers of CD3(+), CD4(+) and CD8(+) cells during treatment, with a significant reduction in the total number of circulating HTLV-1 flower cells in the infected monkeys with chronic ATLL-like disease.

Reduced cell turnover in lymphocytic monkeys infected by human T-lymphotropic virus type 1.

Understanding cell dynamics in animal models have implications for therapeutic strategies elaborated against leukemia in human. Quantification of the cell turnover in closely related primate systems is particularly important for rare and aggressive forms of human cancers, such as adult T-cell leukemia. For this purpose, we have measured the death and proliferation rates of the CD4+ T lymphocyte population in squirrel monkeys (Saimiri sciureus) infected by human T-lymphotropic virus type 1 (HTLV-1). The kinetics of in vivo bromodeoxyuridine labeling revealed no modulation of the cell turnover in HTLV-1-infected monkeys with normal CD4 cell counts. In contrast, a substantial decrease in the proliferation rate of the CD4+ T population was observed in lymphocytic monkeys (e.g. characterized by excessive proportions of CD4+ T lymphocytes and by the presence of abnormal flower-like cells). Unexpectedly, onset of HTLV-associated leukemia thus occurs in the absence of increased CD4+ T-cell proliferation. This dynamics significantly differs from the generalized activation of the T-cell turnover induced by other primate lymphotropic viruses like HIV and SIV.

Sequencing and analysis of genomic DNA and cDNA encoding TNF-alpha in the squirrel monkey (Saimiri sciureus).

If a number of cytokines and growth factors that have been characterized from human cells were investigated in non-human primates, results from such approaches would allow the development of assays to detect and quantitate cytokines in experimental models. Tumor necrosis factor-alpha (TNF-alpha) is an important pluripotent cytokine which plays a crucial role in host defense. As yet, no complete molecular data have been reported for the squirrel monkey TNF-alpha. Polymerase chain reaction (PCR) primers were used to trace introns, by comparing product sizes obtained using cDNA and genomic DNA as templates. The genomic DNA is composed of four exons and three introns with 1793 nucleotides. The corresponding cDNA is 702 nucleotides and phylogenetic analysis showed that the Saimiri sciureus was most closely related to that of the genus Aotus, a new-world primate, compared to old-world primates (genus Macaca and Papio). The deduced TNF-alpha protein consists of 233 amino acids with 82% identity to human, 95% to new-world monkeys and 79% to old-world monkeys. The cloned TNF-alpha cDNA will be useful to quantitate TNF-alpha at the mRNA level.