Neutrophil-Mediated Experimental Metastasis Is Enhanced by VEGFR Inhibition in a Zebrafish Xenograft Model

Inhibition of VEGF signalling effectively suppresses localized tumour growth but accelerates tumour invasiveness and micrometastasis by unknown mechanisms. To study the dynamic and reciprocal interactions between tumour cells and their microenvironment during these processes, we established a xenograft model by injecting tumour cells into the blood circulation of transparent zebrafish embryos. This reproducibly results in rapid simultaneous formation of a localized tumour and experimental micrometastasis, allowing time-resolved imaging of both processes at single-cell resolution within 1 week. The tumour vasculature was initiated de novo by remodelling of primitive endothelial cells into a functional network. Roles of myeloid cells in critical tumourigenesis steps such as vascularization and invasion were revealed by genetic and pharmaceutical approaches. We discovered that the physiological migration of neutrophils controlled tumour invasion by conditioning the collagen matrix and forming the metastatic niche, as detected by two-photon confocal microscopy and second harmonic generation. Administration of VEGFR inhibitors blocked tumour vascularization and a localized tumour growth but enhanced migration of neutrophils, which in turn promoted tumour invasion and formation of micrometastasis. This demonstrates the in vivo cooperation between VEGF signalling and myeloid cells in metastasis and provides a new mechanism underlying the recent findings that VEGFR targeting can promote tumour invasiveness

HIV-1 Nef Protein Structures Associated with Brain Infection and Dementia Pathogenesis

The difference between regional rates of HIV-associated dementia (HAD) in patients infected with different subtypes of HIV suggests that genetic determinants exist within HIV that influence the ability of the virus to replicate in the central nervous system (in Uganda, Africa, subtype D HAD rate is 89%, while subtype A HAD rate is 24%). HIV-1 nef is a multifunctional protein with known toxic effects in the brain compartment. The goal of the current study was to identify if specific three-dimensional nef structures may be linked to patients who developed HAD. HIV-1 nef structures were computationally derived for consensus brain and non-brain sequences from a panel of patients infected with subtype B who died due to varied disease pathologies and consensus subtype A and subtype D sequences from Uganda. Site directed mutation analysis identified signatures in brain structures that appear to change binding potentials and could affect folding conformations of brain-associated structures. Despite the large sequence variation between HIV subtypes, structural alignments confirmed that viral structures derived from patients with HAD were more similar to subtype D structures than to structures derived from patient sequences without HAD. Furthermore, structures derived from brain sequences of patients with HAD were more similar to subtype D structures than they were to their own non-brain structures. The potential finding of a brain-specific nef structure indicates that HAD may result from genetic alterations that alter the folding or binding potential of the protein

Treatment of uterine prolapse stage 2 or higher: a randomized multicenter trial comparing sacrospinous fixation with vaginal hysterectomy (SAVE U trial)

Contains fulltext : 97471.pdf (publisher's version ) (Open Access)BACKGROUND: Pelvic organ prolapse is a common health problem, affecting up to 40% of parous women over 50 years old, with significant negative influence on quality of life. Vaginal hysterectomy is currently the leading treatment method for patients with symptomatic uterine prolapse. Several studies have shown that sacrospinous fixation in case of uterine prolapse is a safe and effective alternative to vaginal hysterectomy. However, no large randomized trials with long-term follow-up have been performed to compare efficacy and quality of life between both techniques.The SAVE U trial is designed to compare sacrospinous fixation with vaginal hysterectomy in the treatment of uterine prolapse stage 2 or higher in terms of prolapse recurrence, quality of life, complications, hospital stay, post-operative recovery and sexual functioning. METHODS/DESIGN: The SAVE U trial is a randomized controlled multi-center non-inferiority trial. The study compares sacrospinous fixation with vaginal hysterectomy in women with uterine prolapse stage 2 or higher. The primary outcome measure is recurrence of uterine prolapse defined as: uterine descent stage 2 or more assessed by pelvic organ prolapse quantification examination and prolapse complaints and/or redo surgery at 12 months follow-up. Secondary outcomes are subjective improvement in quality of life measured by generic (Short Form 36 and Euroqol 5D) and disease-specific (Urogenital Distress Inventory, Defecatory Distress Inventory and Incontinence Impact Questionnaire) quality of life instruments, complications following surgery, hospital stay, post-operative recovery and sexual functioning (Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire). Analysis will be performed according to the intention to treat principle. Based on comparable recurrence rates of 3% and considering an upper-limit of 7% to be non-inferior (beta 0.2 and one sided alpha 0.025), 104 patients are needed per group. DISCUSSION: The SAVE U trial is a randomized multicenter trial that will provide evidence whether the efficacy of sacrospinous fixation is similar to vaginal hysterectomy in women with uterine prolapse stage 2 or higher. TRIAL REGISTRATION: Netherlands Trial Register (NTR): NTR1866

Engineering T cells for cancer therapy

It is generally accepted that the immune system plays an important role in controlling tumour development. However, the interplay between tumour and immune system is complex, as demonstrated by the fact that tumours can successfully establish and develop despite the presence of T cells in tumour. An improved understanding of how tumours evade T-cell surveillance, coupled with technical developments allowing the culture and manipulation of T cells, has driven the exploration of therapeutic strategies based on the adoptive transfer of tumour-specific T cells. The isolation, expansion and re-infusion of large numbers of tumour-specific T cells generated from tumour biopsies has been shown to be feasible. Indeed, impressive clinical responses have been documented in melanoma patients treated with these T cells. These studies and others demonstrate the potential of T cells for the adoptive therapy of cancer. However, the significant technical issues relating to the production of natural tumour-specific T cells suggest that the application of this approach is likely to be limited at the moment. With the advent of retroviral gene transfer technology, it has become possible to efficiently endow T cells with antigen-specific receptors. Using this strategy, it is potentially possible to generate large numbers of tumour reactive T cells rapidly. This review summarises the current gene therapy approaches in relation to the development of adoptive T-cell-based cancer treatments, as these methods now head towards testing in the clinical trial setting

Angiostatin generating capacity and anti-tumour effects of D-penicillamine and plasminogen activators

BACKGROUND: Upregulation of endogenous angiostatin levels may constitute a novel anti-angiogenic, and therefore anti-tumor therapy. In vitro, angiostatin generation is a two-step process, starting with the conversion of plasminogen to plasmin by plasminogen activators (PAs). Next, plasmin excises angiostatin from other plasmin molecules, a process requiring a donor of a free sulfhydryl group. In previous studies, it has been demonstrated that administration of PA in combination with the free sulfhydryl donor (FSD) agents captopril or N-acetyl cysteine, resulted in angiostatin generation, and anti-angiogenic and anti-tumour activity in murine models. METHODS: In this study we have investigated the angiostatin generating capacities of several FSDs. D-penicillamine proved to be most efficient in supporting the conversion of plasminogen to angiostatin in vitro. Next, from the optimal concentrations of tPA and D-penicillamine in vitro, equivalent dosages were administered to healthy Balb/c mice to explore upregulation of circulating angiostatin levels. Finally, anti-tumor effects of treatment with tPA and D-penicillamine were determined in a human melanoma xenograft model. RESULTS: Surprisingly, we found that despite the superior angiostatin generating capacity of D-penicillamine in vitro, both in vivo angiostatin generation and anti-tumour effects of tPA/D-penicillamine treatment were impaired compared to our previous studies with tPA and captopril. CONCLUSION: Our results indicate that selecting the most appropriate free sulfhydryl donor for anti-angiogenic therapy in a (pre)clinical setting should be performed by in vivo rather than by in vitro studies. We conclude that D-penicillamine is not suitable for this type of therapy

Creatine-induced activation of antioxidative defence in myotube cultures revealed by explorative NMR-based metabonomics and proteomics

<p>Abstract</p> <p>Background</p> <p>Creatine is a key intermediate in energy metabolism and supplementation of creatine has been used for increasing muscle mass, strength and endurance. Creatine supplementation has also been reported to trigger the skeletal muscle expression of insulin like growth factor I, to increase the fat-free mass and improve cognition in elderly, and more explorative approaches like transcriptomics has revealed additional information. The aim of the present study was to reveal additional insight into the biochemical effects of creatine supplementation at the protein and metabolite level by integrating the explorative techniques, proteomics and NMR metabonomics, in a systems biology approach.</p> <p>Methods</p> <p>Differentiated mouse myotube cultures (C2C12) were exposed to 5 mM creatine monohydrate (CMH) for 24 hours. For proteomics studies, lysed myotubes were analyzed in single 2-DGE gels where the first dimension of protein separation was pI 5-8 and second dimension was a 12.5% Criterion gel. Differentially expressed protein spots of significance were excised from the gel, desalted and identified by peptide mass fingerprinting using MALDI-TOF MS. For NMR metabonomic studies, chloroform/methanol extractions of the myotubes were subjected to one-dimensional ¹H NMR spectroscopy and the intracellular oxidative status of myotubes was assessed by intracellular DCFH₂oxidation after 24 h pre-incubation with CMH.</p> <p>Results</p> <p>The identified differentially expressed proteins included vimentin, malate dehydrogenase, peroxiredoxin, thioredoxin dependent peroxide reductase, and 75 kDa and 78 kDa glucose regulated protein precursors. After CMH exposure, up-regulated proteomic spots correlated positively with the NMR signals from creatine, while down-regulated proteomic spots were negatively correlated with these NMR signals. The identified differentially regulated proteins were related to energy metabolism, glucose regulated stress, cellular structure and the antioxidative defence system. The suggested improvement of the antioxidative defence was confirmed by a reduced intracellular DCFH₂oxidation with increasing concentrations of CMH in the 24 h pre-incubation medium.</p> <p>Conclusions</p> <p>The explorative approach of this study combined with the determination of a decreased intracellular DCFH₂oxidation revealed an additional stimulation of cellular antioxidative mechanisms when myotubes were exposed to CMH. This may contribute to an increased exercise performance mediated by increased ability to cope with training-induced increases in oxidative stress.</p

Implementing the chronic care model for frail older adults in the Netherlands: study protocol of ACT (frail older adults: care in transition)

<p>Abstract</p> <p>Background</p> <p>Care for older adults is facing a number of challenges: health problems are not consistently identified at a timely stage, older adults report a lack of autonomy in their care process, and care systems are often confronted with the need for better coordination between health care professionals. We aim to address these challenges by introducing the geriatric care model, based on the chronic care model, and to evaluate its effects on the quality of life of community-dwelling frail older adults.</p> <p>Methods/design</p> <p>In a 2-year stepped-wedge cluster randomised clinical trial with 6-monthly measurements, the chronic care model will be compared with usual care. The trial will be carried out among 35 primary care practices in two regions in the Netherlands. Per region, practices will be randomly allocated to four allocation arms designating the starting point of the intervention. <it>Participants</it>: 1200 community-dwelling older adults aged 65 or over and their primary informal caregivers. Primary care physicians will identify frail individuals based on a composite definition of frailty and a polypharmacy criterion. Final inclusion criterion: scoring 3 or more on a disability case-finding tool. <it>Intervention</it>: Every 6 months patients will receive a geriatric in-home assessment by a practice nurse, followed by a tailored care plan. Expert teams will manage and train practice nurses. Patients with complex care needs will be reviewed in interdisciplinary consultations. <it>Evaluation</it>: We will perform an effect evaluation, an economic evaluation, and a process evaluation. Primary outcome is quality of life as measured with the Short Form-12 questionnaire. Effect analyses will be based on the “intention-to-treat” principle, using multilevel regression analysis. Cost measurements will be administered continually during the study period. A cost-effectiveness analysis and cost-utility analysis will be conducted comparing mean total costs to functional status, care needs and QALYs. We will investigate the level of implementation, barriers and facilitators to successful implementation and the extent to which the intervention manages to achieve the transition necessary to overcome challenges in elderly care.</p> <p>Discussion</p> <p>This is one of the first studies assessing the effectiveness, cost-effectiveness and implementation process of the chronic care model for frail community-dwelling older adults.</p> <p>Trial registration</p> <p>The Netherlands National Trial Register NTR2160.</p

Acute effects of MDMA (3,4-methylenedioxymethamphetamine) on EEG oscillations: alone and in combination with ethanol or THC (delta-9-tetrahydrocannabinol)

Item does not contain fulltextRATIONALE: Typical users of 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy") are polydrug users, combining MDMA with alcohol or cannabis [most active compound: delta-9-tetrahydrocannabinol (THC)]. OBJECTIVES: The aim of the present study was to investigate whether co-administration of alcohol or THC with MDMA differentially affects ongoing electroencephalogram (EEG) oscillations compared to the administration of each drug alone. METHODS: In two separate experiments, 16 volunteers received four different drug conditions: (1) MDMA (100 mg); (2) alcohol clamp (blood alcohol concentration = 0.6 per thousand) or THC (inhalation of 4, 6 and 6 mg, interval of 1.5 h); (3) MDMA in combination with alcohol or THC; and (4) placebo. Before and after drug administration, electroencephalography was recorded during an eyes closed resting state. RESULTS: Theta and alpha power increased after alcohol intake compared to placebo and reduced after MDMA intake. No interaction between alcohol and MDMA was found. Significant MDMA x THC effects for theta and lower-1-alpha power indicated that the power attenuation after the combined intake of MDMA and THC was less than the sum of each drug alone. For the lower-2-alpha band, the intake of MDMA or THC alone did not significantly affect power, but the intake of combined MDMA and THC significantly decreased lower-2-alpha power. CONCLUSIONS: The present findings indicate that the combined intake of MDMA and THC, but not of MDMA and alcohol, affects ongoing EEG oscillations differently than the sum of either one drug alone. Changes in ongoing EEG oscillations may be related to the impaired task performance that has often been reported after drug intake

Genetic Diversity among Ancient Nordic Populations

Using established criteria for work with fossil DNA we have analysed mitochondrial DNA from 92 individuals from 18 locations in Denmark ranging in time from the Mesolithic to the Medieval Age. Unequivocal assignment of mtDNA haplotypes was possible for 56 of the ancient individuals; however, the success rate varied substantially between sites; the highest rates were obtained with untouched, freshly excavated material, whereas heavy handling, archeological preservation and storage for many years influenced the ability to obtain authentic endogenic DNA. While the nucleotide diversity at two locations was similar to that among extant Danes, the diversity at four sites was considerably higher. This supports previous observations for ancient Britons. The overall occurrence of haplogroups did not deviate from extant Scandinavians, however, haplogroup I was significantly more frequent among the ancient Danes (average 13%) than among extant Danes and Scandinavians (∼2.5%) as well as among other ancient population samples reported. Haplogroup I could therefore have been an ancient Southern Scandinavian type “diluted” by later immigration events. Interestingly, the two Neolithic samples (4,200 YBP, Bell Beaker culture) that were typed were haplogroup U4 and U5a, respectively, and the single Bronze Age sample (3,300–3,500 YBP) was haplogroup U4. These two haplogroups have been associated with the Mesolithic populations of Central and Northern Europe. Therefore, at least for Southern Scandinavia, our findings do not support a possible replacement of a haplogroup U dominated hunter-gatherer population by a more haplogroup diverse Neolithic Culture