In vivo imaging and quantitative analysis of leukocyte directional migration and polarization in inflamed tissue

Directional migration of transmigrated leukocytes to the site of injury is a central event in the inflammatory response. Here, we present an in vivo chemotaxis assay enabling the visualization and quantitative analysis of subtype-specific directional motility and polarization of leukocytes in their natural 3D microenvironment. Our technique comprises the combination of i) semi-automated in situ microinjection of chemoattractants or bacteria as local chemotactic stimulus, ii) in vivo near-infrared reflected-light oblique transillumination (RLOT) microscopy for the visualization of leukocyte motility and morphology, and iii) in vivo fluorescence microscopy for the visualization of different leukocyte subpopulations or fluorescence-labeled bacteria. Leukocyte motility parameters are quantified off-line in digitized video sequences using computer-assisted single cell tracking. Here, we show that perivenular microinjection of chemoattractants [macrophage inflammatory protein-1alpha (MIP-1alpha/Ccl3), platelet-activating factor (PAF)] or E. coli into the murine cremaster muscle induces target-oriented intravascular adhesion and transmigration as well as polarization and directional interstitial migration of leukocytes towards the locally administered stimuli. Moreover, we describe a crucial role of Rho kinase for the regulation of directional motility and polarization of transmigrated leukocytes in vivo. Finally, combining in vivo RLOT and fluorescence microscopy in Cx3CR1(gfp/gfp) mice (mice exhibiting green fluorescent protein-labeled monocytes), we are able to demonstrate differences in the migratory behavior of monocytes and neutrophils.Taken together, we propose a novel approach for investigating the mechanisms and spatiotemporal dynamics of subtype-specific motility and polarization of leukocytes during their directional interstitial migration in vivo

Feasibility study to inform the design of a randomised controlled trial to eradicate Pseudomonas aeruginosa infection in individuals with Cystic Fibrosis

There are controversies about the most effective treatment to eradicate first growth of Pseudomonas aeruginosa (P aeruginosa) from the lower airways of patients with cystic fibrosis (CF). UK guidelines recommend oral treatment, but some advocate intravenous (IV) treatment. The objective of this study was to assess the feasibility of conducting a randomised controlled trial comparing two treatment strategies to eradicate P aeruginosa in CF patients

Dissociable Effects of Reward on Attentional Learning: From Passive Associations to Active Monitoring

Visual selective attention (VSA) is the cognitive function that regulates ongoing processing of retinal input in order for selected representations to gain privileged access to perceptual awareness and guide behavior, facilitating analysis of currently relevant information while suppressing the less relevant input. Recent findings indicate that the deployment of VSA is shaped according to past outcomes. Targets whose selection has led to rewarding outcomes become relatively easier to select in the future, and distracters that have been ignored with higher gains are more easily discarded. Although outcomes (monetary rewards) were completely predetermined in our prior studies, participants were told that higher rewards would follow more efficient responses. In a new experiment we have eliminated the illusory link between performance and outcomes by informing subjects that rewards were randomly assigned. This trivial yet crucial manipulation led to strikingly different results. Items that were associated more frequently with higher gains became more difficult to ignore, regardless of the role (target or distracter) they played when differential rewards were delivered. Therefore, VSA is shaped by two distinct reward-related learning mechanisms: one requiring active monitoring of performance and outcome, and a second one detecting the sheer association between objects in the environment (whether attended or ignored) and the more-or-less rewarding events that accompany them

Promoting Client Participation and Constructing Decisions in Mental Health Rehabilitation Meetings

The chapter analyzes practices by which support workers promote client participation in mental health rehabilitation meetings at the Clubhouse. While promoting client participation, the support workers also need to ascertain that at least some decisions get constructed during the meetings. This combination of goals—promoting participation and constructing decisions—leads to a series of dilemmatic practices, the dynamics of which the chapter focuses on analyzing. The support workers may treat clients’ turns retrospectively as proposals, even if the status of these turns as such is ambiguous. In the face of a lack of recipient uptake, the support workers may remind the clients about their epistemic access to the content of the proposal or pursue their agreement or commitment to the idea. These practices involve the support workers carrying primary responsibility over the unfolding of interaction, which is argued to compromise the jointness of the decision-making outcome.The chapter analyzes practices by which support workers promote client participation in mental health rehabilitation meetings at the Clubhouse. While promoting client participation, the support workers also need to ascertain that at least some decisions get constructed during the meetings. This combination of goals—promoting participation and constructing decisions—leads to a series of dilemmatic practices, the dynamics of which the chapter focuses on analyzing. The support workers may treat clients’ turns retrospectively as proposals, even if the status of these turns as such is ambiguous. In the face of a lack of recipient uptake, the support workers may remind the clients about their epistemic access to the content of the proposal or pursue their agreement or commitment to the idea. These practices involve the support workers carrying primary responsibility over the unfolding of interaction, which is argued to compromise the jointness of the decision-making outcome.Peer reviewe

Sequencing chemotherapy and radiotherapy in locoregional advanced breast cancer patients after mastectomy – a retrospective analysis

<p>Abstract</p> <p>Background</p> <p>Combined chemo- and radiotherapy are established in breast cancer treatment. Chemotherapy is recommended prior to radiotherapy but decisive data on the optimal sequence are rare. This retrospective analysis aimed to assess the role of sequencing in patients after mastectomy because of advanced locoregional disease.</p> <p>Methods</p> <p>A total of 212 eligible patients had a stage III breast cancer and had adjuvant chemotherapy and radiotherapy after mastectomy and axillary dissection between 1996 and 2004. According to concerted multi-modality treatment strategies 86 patients were treated sequentially (chemotherapy followed by radiotherapy) (SEQgroup), 70 patients had a sandwich treatment (SW-group) and 56 patients had simultaneous chemoradiation (SIM-group) during that time period. Radiotherapy comprised the thoracic wall and/or regional lymph nodes. The total dose was 45–50.4 Gray. As simultaneous chemoradiation CMF was given in 95.4% of patients while in sequential or sandwich application in 86% and 87.1% of patients an anthracycline-based chemotherapy was given.</p> <p>Results</p> <p>Concerning the parameters nodal involvement, lymphovascular invasion, extracapsular spread and extension of the irradiated region the three treatment groups were significantly imbalanced. The other parameters, e.g. age, pathological tumor stage, grading and receptor status were homogeneously distributed. Looking on those two groups with an equally effective chemotherapy (EC, FEC), the SEQ- and SW-group, the sole imbalance was the extension of LVI (57.1 vs. 25.6%, p < 0.0001).</p> <p>5-year overall- and disease free survival were 53.2%/56%, 38.1%/32% and 64.2%/50%, for the sequential, sandwich and simultaneous regime, respectively, which differed significantly in the univariate analysis (p = 0.04 and p = 0.03, log-rank test). Also the 5-year locoregional or distant recurrence free survival showed no significant differences according to the sequence of chemo- and radiotherapy. In the multivariate analyses the sequence had no independent impact on overall survival (p = 0.2) or disease free survival (p = 0.4). The toxicity, whether acute nor late, showed no significant differences in the three groups. The grade III/IV acute side effects were 3.6%, 0% and 3.5% for the SIM-, SW- and SEQ-group. By tendency the SIM regime had more late side effects.</p> <p>Conclusion</p> <p>No clear advantage can be stated for any radio- and chemotherapy sequence in breast cancer therapy so far. This could be confirmed in our retrospective analysis in high-risk patients after mastectomy. The sequential approach is recommended according to current guidelines considering a lower toxicity.</p

Body Mass Index, percent body fat, and regional body fat distribution in relation to leptin concentrations in healthy, non-smoking postmenopausal women in a feeding study

BACKGROUND: The relationship between BMI and leptin has been studied extensively in the past, but previous reports in postmenopausal women have not been conducted under carefully controlled dietary conditions of weight maintenance using precise measures of body fat distribution. The aim of the present study was to examine the association between serum leptin concentration and adiposity as estimated by BMI and dual energy x-ray absorptiometry (DEXA) measures (percent body fat, central and peripheral fat, and lean mass) in postmenopausal women. METHODS: This study was conducted as a cross-sectional analysis within the control segment of a randomized, crossover trial in which postmenopausal women (n = 51) consumed 0 (control), 15 (one drink), and 30 (two drinks) g alcohol (ethanol)/d for 8 weeks as part of a controlled diet. BMIs were determined and DEXA scans were administered to the women during the 0 g alcohol treatment, and a blood sample was collected at baseline and week 8 of each study period for leptin analysis. RESULTS AND DISCUSSION: In multivariate analysis, women who were overweight (BMI > 25 to ≤ 30 kg/m(2)) had a 2-fold increase, and obese women (BMI > 30 kg/m(2)) had more than a 3-fold increase in serum leptin concentrations compared to normal weight (BMI ≤25 kg/m(2)) women. When the models for the different measures of adiposity were assessed by multiple R(2), models which included percent body fat explained the highest proportion (approximately 80%) of the serum leptin variance. CONCLUSION: Under carefully controlled dietary conditions, we confirm that higher levels of adiposity were associated with higher concentrations of serum leptin. It appears that percent body fat in postmenopausal women may be the best adiposity-related predictor of serum leptin

Autoimmune and autoinflammatory mechanisms in uveitis

The eye, as currently viewed, is neither immunologically ignorant nor sequestered from the systemic environment. The eye utilises distinct immunoregulatory mechanisms to preserve tissue and cellular function in the face of immune-mediated insult; clinically, inflammation following such an insult is termed uveitis. The intra-ocular inflammation in uveitis may be clinically obvious as a result of infection (e.g. toxoplasma, herpes), but in the main infection, if any, remains covert. We now recognise that healthy tissues including the retina have regulatory mechanisms imparted by control of myeloid cells through receptors (e.g. CD200R) and soluble inhibitory factors (e.g. alpha-MSH), regulation of the blood retinal barrier, and active immune surveillance. Once homoeostasis has been disrupted and inflammation ensues, the mechanisms to regulate inflammation, including T cell apoptosis, generation of Treg cells, and myeloid cell suppression in situ, are less successful. Why inflammation becomes persistent remains unknown, but extrapolating from animal models, possibilities include differential trafficking of T cells from the retina, residency of CD8(+) T cells, and alterations of myeloid cell phenotype and function. Translating lessons learned from animal models to humans has been helped by system biology approaches and informatics, which suggest that diseased animals and people share similar changes in T cell phenotypes and monocyte function to date. Together the data infer a possible cryptic infectious drive in uveitis that unlocks and drives persistent autoimmune responses, or promotes further innate immune responses. Thus there may be many mechanisms in common with those observed in autoinflammatory disorders

Role of nonhuman primate models in the discovery and clinical development of selective progesterone receptor modulators (SPRMs)

Selective progesterone receptor modulators (SPRMs) represent a new class of progesterone receptor ligands that exert clinically relevant tissue-selective progesterone agonist, antagonist, partial, or mixed agonist/antagonist effects on various progesterone target tissues in an in vivo situation depending on the biological action studied. The SPRM asoprisnil is being studied in women with symptomatic uterine leiomyomata and endometriosis. Asoprisnil shows a high degree of uterine selectivity as compared to effects on ovulation or ovarian hormone secretion in humans. It induces amenorrhea and decreases leiomyoma volume in a dose-dependent manner in the presence of follicular phase estrogen concentrations. It also has endometrial antiproliferative effects. In pregnant animals, the myometrial, i.e. labor-inducing, effects of asoprisnil are blunted or absent. Studies in non-human primates played a key role during the preclinical development of selective progesterone receptor modulators. These studies provided the first evidence of uterus-selective effects of asoprisnil and structurally related compounds, and the rationale for clinical development of asoprisnil