STED nanoscopy of actin dynamics in synapses deep inside living brain slices

It is difficult to investigate the mechanisms that mediate long-term changes in synapse function because synapses are small and deeply embedded inside brain tissue. Although recent fluorescence nanoscopy techniques afford improved resolution, they have so far been restricted to dissociated cells or tissue surfaces. However, to study synapses under realistic conditions, one must image several cell layers deep inside more-intact, three-dimensional preparations that exhibit strong light scattering, such as brain slices or brains in vivo. Using aberration-reducing optics, we demonstrate that it is possible to achieve stimulated emission depletion superresolution imaging deep inside scattering biological tissue. To illustrate the power of this novel (to our knowledge) approach, we resolved distinct distributions of actin inside dendrites and spines with a resolution of 60–80 nm in living organotypic brain slices at depths up to 120 μm. In addition, time-lapse stimulated emission depletion imaging revealed changes in actin-based structures inside spines and spine necks, and showed that these dynamics can be modulated by neuronal activity. Our approach greatly facilitates investigations of actin dynamics at the nanoscale within functionally intact brain tissue

Symptomatology in 4-repeat tauopathies is associated with data-driven topology of [18F]-PI-2620 tau-PET signal

In recent years in vivo visualization of tau deposits has become possible with various PET radiotracers. The tau tracer [18F]PI-2620 proved high affinity both to 3-repeat/4-repeat tau in Alzheimer's disease as well as to 4repeat tau in progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS). However, to be clinically relevant, biomarkers should not only correlate with pathological changes but also with disease stage and progression. Therefore, we aimed to investigate the correlation between topology of [18F]PI-2620 uptake and symptomatology in 4-repeat tauopathies. 72 patients with possible or probable 4-repeat tauopathy, i.e. 31 patients with PSP-Richardson's syndrome (PSP-RS), 30 with amyloid-negative CBS and 11 with PSP-non-RS/CBS, underwent [18F]PI-2620-PET. Principal component analysis was performed to identify groups of similar brain regions based on 20-40 min p.i. regional standardized uptake value ratio z-scores. Correlations between component scores and the items of the PSP Rating Scale were explored. Motor signs like gait, arising from chair and postural instability showed a positive correlation with tracer uptake in mesial frontoparietal lobes and the medial superior frontal gyrus and adjacent anterior cingulate cortex. While the signs disorientation and bradyphrenia showed a positive correlation with tracer uptake in the parietooccipital junction, the signs disorientation and arising from chair were negatively correlated with tau-PET signal in the caudate nucleus and thalamus. Total PSP Rating Scale Score showed a trend towards a positive correlation with mesial frontoparietal lobes and a negative correlation with caudate nucleus and thalamus. While in CBS patients, the main finding was a negative correlation of tracer binding in the caudate nucleus and thalamus and a positive correlation of tracer binding in medial frontal cortex with gait and motor signs, in PS

Additive value of [18F]PI-2620 perfusion imaging in progressive supranuclear palsy and corticobasal syndrome

Purpose: Early after [18F]PI-2620 PET tracer administration, perfusion imaging has potential for regional assessment of neuronal injury in neurodegenerative diseases. This is while standard late-phase [18F]PI-2620 tau-PET is able to discriminate the 4-repeat tauopathies progressive supranuclear palsy and corticobasal syndrome (4RTs) from disease controls and healthy controls. Here, we investigated whether early-phase [18F]PI-2620 PET has an additive value for biomarker based evaluation of 4RTs. Methods: Seventy-eight patients with 4RTs (71 ± 7 years, 39 female), 79 patients with other neurodegenerative diseases (67 ± 12 years, 35 female) and twelve age-matched controls (69 ± 8 years, 8 female) underwent dynamic (0-60 min) [18F]PI-2620 PET imaging. Regional perfusion (0.5-2.5 min p.i.) and tau load (20-40 min p.i.) were measured in 246 predefined brain regions [standardized-uptake-value ratios (SUVr), cerebellar reference]. Regional SUVr were compared between 4RTs and controls by an ANOVA including false-discovery-rate (FDR, p < 0.01) correction. Hypoperfusion in resulting 4RT target regions was evaluated at the patient level in all patients (mean value - 2SD threshold). Additionally, perfusion and tau pattern expression levels were explored regarding their potential discriminatory value of 4RTs against other neurodegenerative disorders, including validation in an independent external dataset (n = 37), and correlated with clinical severity in 4RTs (PSP rating scale, MoCA, activities of daily living). Results: Patients with 4RTs had significant hypoperfusion in 21/246 brain regions, most dominant in thalamus, caudate nucleus, and anterior cingulate cortex, fitting to the topology of the 4RT disease spectrum. However, single region hypoperfusion was not specific regarding the discrimination of patients with 4RTs against patients with other neurodegenerative diseases. In contrast, perfusion pattern expression showed promise for discrimination of patients with 4RTs from other neurodegenerative diseases (AUC: 0.850). Discrimination by the combined perfusion-tau pattern expression (AUC: 0.903) exceeded that of the sole tau pattern expression (AUC: 0.864) and the discriminatory power of the combined perfusion-tau pattern expression was replicated in the external dataset (AUC: 0.917). Perfusion but not tau pattern expression was associated with PSP rating scale (R = 0.402; p = 0.0012) and activities of daily living (R = - 0.431; p = 0.0005). Conclusion: [18F]PI-2620 perfusion imaging mirrors known topology of regional hypoperfusion in 4RTs. Single region hypoperfusion is not specific for 4RTs, but perfusion pattern expression may provide an additive value for the discrimination of 4RTs from other neurodegenerative diseases and correlates closer with clinical severity than tau pattern expression

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Examen Examinis Pieriani. Das ist/ Schulfürung und Abfertigung des Examinis, so D. Urbanus Pierius, Calvinischer Prediger zu Bremen/ wider die zu Dreßden/ bey der Begräbnis D. Nicolai Crelli gehalten Leichpredigt/ durch offenen Druck außgesprenget : Zu nothwendiger Verantwortung reiner Lehr der Evangelischen Kirchen in Sachsen/ als auch zu richtiger Entdeckung und gründlicher Widerlegung etlicher Calvinischer Irrthumen

Gestellet durch Philippum Nicolai, der heiligen Schrifft Doctorn und Pastorn der Kirchen zu S. Catharinen in Hambur

Dual Channel RESOLFT Nanoscopy by Using Fluorescent State Kinetics

We show that RESOLFT fluorescence nanoscopy, a low light level scanning superresolution technique employing reversibly switchable fluorescent proteins (rsFPs), is capable of dual-channel live-cell imaging that is virtually free of chromatic errors and temporal offsets. This is accomplished using rsEGFP and Dronpa, two rsFPs having similar spectra but different kinetics of switching and fluorescence emission. Our approach is demonstrated by imaging protein distributions and dynamics in living neurons and neuronal tissues