Interrelationships between Cellular Density,Mosaic Patterning,and Dendritic Coverage of VGluT3 Amacrine Cells

Amacrine cells of the retina are conspicuously variable in their morphologies, their population demographics, and their ensuing functions. Vesicular glutamate transporter 3 (VGluT3) amacrine cells are a recently characterized type of amacrine cell exhibiting local dendritic autonomy. The present analysis has examined three features of this VGluT3 population, including their density, local distribution, and dendritic spread, to discern the extent to which these are interrelated, using male and female mice. We first demonstrate that Bax-mediated cell death transforms the mosaic of VGluT3 cells from a random distribution into a regular mosaic. We subsequently examine the relationship between cell density and mosaic regularity across recombinant inbred strains of mice, finding that, although both traits vary across the strains, they exhibit minimal covariation. Other genetic determinants must therefore contribute independently to final cell number and to mosaic order. Using a conditional KO approach, we further demonstrate that Bax acts via the bipolar cell population, rather than cell-intrinsically, to control VGluT3 cell number. Finally, we consider the relationship between the dendritic arbors of single VGluT3 cells and the distribution of their homotypic neighbors. Dendritic field area was found to be independent of Voronoi domain area, while dendritic coverage of single cells was not conserved, simply increasing with the size of the dendritic field. Bax-KO retinas exhibited a threefold increase in dendritic coverage. Each cell, however, contributed less dendrites at each depth within the plexus, intermingling their processes with those of neighboring cells to approximate a constant volumetric density, yielding a uniformity in process coverage across the population.SIGNIFICANCE STATEMENT Different types of retinal neuron spread their processes across the surface of the retina to achieve a degree of dendritic coverage that is characteristic of each type. Many of these types achieve a constant coverage by varying their dendritic field area inversely with the local density of like-type neighbors. Here we report a population of retinal amacrine cells that do not develop dendritic arbors in relation to the spatial positioning of such homotypic neighbors; rather, this cell type modulates the extent of its dendritic branching when faced with a variable number of overlapping dendritic fields to approximate a uniformity in dendritic density across the retina.     

Defining the dimensions of circulating tumor cells in a large series of breast,prostate, colon,and bladder cancer patients

Circulating tumor cells (CTCs) in the blood of cancer patients are of high clinical relevance. Since detection and isolation of CTCs often rely on cell dimensions, knowledge of their size is key. We analyzed the median CTC size in a large cohort of breast (BC), prostate (PC), colorectal (CRC), and bladder (BLC) cancer patients. Images of patient‐derived CTCs acquired on cartridges of the FDA‐cleared CellSearch^® method were retrospectively collected and automatically re‐analyzed using the accept software package. The median CTC diameter (μm) was computed per tumor type. The size differences between the different tumor types and references (tumor cell lines and leukocytes) were nonparametrically tested. A total of 1962 CellSearch^® cartridges containing 71 612 CTCs were included. In BC, the median computed diameter (CD) of patient‐derived CTCs was 12.4 μm vs 18.4 μm for cultured cell line cells. For PC, CDs were 10.3 μm for CTCs vs 20.7 μm for cultured cell line cells. CDs for CTCs of CRC and BLC were 7.5 μm and 8.6 μm, respectively. Finally, leukocytes were 9.4 μm. CTC size differed statistically significantly between the four tumor types and between CTCs and the reference data. CTC size differences between tumor types are striking and CTCs are smaller than cell line tumor cells, whose size is often used as reference when developing CTC analysis methods. Based on our data, we suggest that the size of CTCs matters and should be kept in mind when designing and optimizing size‐based isolation methods.

Abbreviations

ACCEPT

Automated CTC Classification, Enumeration, and PhenoTyping software

BC

breast cancer

BLC

bladder cancer

CD

computed diameter

CEL

cultured tumor cell (cell line)

CK

cytokeratin

CRC

colorectal cancer

CTC‐L

circulating tumor cells derived from cerebrospinal fluid (liquor)

CTCs

circulating tumor cells

DAPI

4′6‐diamidino‐2‐phenylindole

EMT

epithelial–mesenchymal transition

EpCAM

epithelial cell adhesion molecule

IQR

interquartile range

KW test

Kruskal–Wallis test

MWU test

Mann–Whitney U test

NCR

nucleus/cytoplasm ratio

P2A

perimeter to area

PC

prostate cancer

TIF

tagged Image Format files

TXT

text file

μm

micrometer

µm²

square micrometers

     

Traumatic Cerebral Microbleeds in the Subacute Phase Are Practical and Early Predictors of Abnormality of the Normal-Appearing White Matter in the Chronic Phase

BACKGROUND AND PURPOSE:In the chronic phase after traumatic brain injury, DTI findings reflect WM integrity. DTI interpretation in the subacute phase is less straightforward. Microbleed evaluation with SWI is straightforward in both phases. We evaluated whether the microbleed concentration in the subacute phase is associated with the integrity of normal-appearing WM in the chronic phase.MATERIALS AND METHODS:Sixty of 211 consecutive patients 18 years of age or older admitted to our emergency department ≤24 hours after moderate to severe traumatic brain injury matched the selection criteria. Standardized 3T SWI, DTI, and T1WI were obtained 3 and 26 weeks after traumatic brain injury in 31 patients and 24 healthy volunteers. At baseline, microbleed concentrations were calculated. At follow-up, mean diffusivity (MD) was calculated in the normal-appearing WM in reference to the healthy volunteers (MD_z). Through linear regression, we evaluated the relation between microbleed concentration and MD_z in predefined structures.RESULTS:In the cerebral hemispheres, MD_z at follow-up was independently associated with the microbleed concentration at baseline (left: B = 38.4 [95% CI 7.5–69.3], P = .017; right: B = 26.3 [95% CI 5.7–47.0], P = .014). No such relation was demonstrated in the central brain. MD_z in the corpus callosum was independently associated with the microbleed concentration in the structures connected by WM tracts running through the corpus callosum (B = 20.0 [95% CI 24.8–75.2], P < .000). MD_z in the central brain was independently associated with the microbleed concentration in the cerebral hemispheres (B = 25.7 [95% CI 3.9–47.5], P = .023).CONCLUSIONS:SWI-assessed microbleeds in the subacute phase are associated with DTI-based WM integrity in the chronic phase. These associations are found both within regions and between functionally connected regions.

The yearly incidence of traumatic brain injury (TBI) is around 300 per 100,000 persons.^1,2 Almost three-quarters of patients with moderate to severe TBI have traumatic axonal injury (TAI).³ TAI is a major predictor of functional outcome,^4,5 but it is mostly invisible on CT and conventional MR imaging.^6,7DTI provides direct information on WM integrity and axonal injury.^5,8 However, DTI abnormalities are neither specific for TAI nor stable over time. Possibly because of the release of mass effect and edema and resorption of blood products, the effects of concomitant (non-TAI) injury on DTI are larger in the subacute than in the chronic phase (>3 months).^4,9,10 Therefore, DTI findings are expected to reflect TAI more specifically in the chronic than in the subacute phase (1 week–3 months).⁴ Even in regions without concomitant injury, the effects of TAI on DTI are dynamic, possibly caused by degeneration and neuroplastic changes.^6,11,12 These ongoing pathophysiological processes possibly contribute to the emerging evidence that DTI findings in the chronic phase are most closely associated with the eventual functional outcome.^12,13Although DTI provides valuable information, its acquisition, postprocessing, and interpretation in individual patients are demanding. SWI, with which microbleeds can be assessed with high sensitivity, is easier to interpret and implement in clinical practice. In contrast to DTI, SWI-detected traumatic microbleeds are more stable¹ except in the hyperacute^14,15 and the late chronic phases.¹⁶ Traumatic cerebral microbleeds are commonly interpreted as signs of TAI. However, the relation is not straightforward. On the one hand, nontraumatic microbleeds may be pre-existing. On the other hand, even if traumatic in origin, microbleeds represent traumatic vascular rather than axonal injury.¹⁷ Indeed, TAI is not invariably hemorrhagic.¹⁸ Additionally, microbleeds may secondarily develop after trauma through mechanisms unrelated to axonal injury, such as secondary ischemia.¹⁸DTI is not only affected by pathophysiological changes but also by susceptibility.¹⁹ The important susceptibility-effect generated by microbleeds renders the interpretation of DTI findings at the location of microbleeds complex. In the chronic phase, mean diffusivity (MD) is the most robust marker of WM integrity.^4,6 For these reasons, we evaluated MD in the normal-appearing WM.Much TAI research focuses on the corpus callosum because it is commonly involved in TAI^5,18,20 and it can reliably be evaluated with DTI,^5,21 and TAI in the corpus callosum is related to clinical prognosis.^6,20 The corpus callosum consists of densely packed WM tracts that structurally and functionally connect left- and right-sided brain structures.²² The integrity of the corpus callosum is associated with the integrity of the brain structures it connects.²³ Therefore, microbleeds in brain structures that are connected through the corpus callosum may affect callosal DTI findings. Analogous to this, microbleeds in the cerebral hemispheres, which exert their function through WM tracts traveling through the deep brain structures and brain stem,^24,25 may affect DTI findings in the WM of the latter.Our purpose was to evaluate whether the microbleed concentration in the subacute phase is associated with the integrity of normal-appearing WM in the chronic phase. We investigated this relation within the cerebral hemispheres and the central brain and between regions that are functionally connected by WM tracts.     

Retrospective Analysis Using Pharmacokinetic/Pharmacodynamic Modeling and Simulation Offers Improvements in Efficiency of the Design of Volunteer Infection Studies for Antimalarial Drug Development

Volunteer infection studies using the induced blood stage malaria (IBSM) model have been shown to facilitate antimalarial drug development. Such studies have traditionally been undertaken in single‐dose cohorts, as many as necessary to obtain the dose‐response relationship. To enhance ethical and logistic aspects of such studies, and to reduce the number of cohorts needed to establish the dose‐response relationship, we undertook a retrospective in silico analysis of previously accrued data to improve study design. A pharmacokinetic (PK)/pharmacodynamic (PD) model was developed from initial fictive‐cohort data for OZ439 (mixing the data of the three single‐dose cohorts as: n = 2 on 100 mg, 2 on 200 mg, and 4 on 500 mg). A three‐compartment model described OZ439 PKs. Net growth of parasites was modeled using a Gompertz function and drug‐induced parasite death using a Hill function. Parameter estimates for the PK and PD models were comparable for the multidose single‐cohort vs. the pooled analysis of all cohorts. Simulations based on the multidose single‐cohort design described the complete data from the original IBSM study. The novel design allows for the ascertainment of the PK/PD relationship early in the study, providing a basis for rational dose selection for subsequent cohorts and studies.

Study Highlights

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

☑ Volunteer infection studies are routinely used in antimalarial drug development to generate early pharmacokinetic/pharmacodynamic data for compounds.

• WHAT QUESTION DID THIS STUDY ADDRESS?

☑ Can in silico analyses be used to suggest improvements to volunteer infection study designs?

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

☑ Multiple dose adaptive trial designs can potentially reduce the number of cohorts needed to establish the dose‐response relationship in volunteer infection studies.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

☑ Real time data analyses can be used to recommend doses for adaptive volunteer infection studies.

Volunteer infection studies using the induced blood stage malaria (IBSM) model have been recognized as a valuable system for defining the key pharmacokinetic (PK) and pharmacodynamic (PD) relationships for dose selection in antimalarial drug development. ¹ , ² , ³ , ⁴ , ⁵ , ⁶ , ⁷ In such studies, healthy volunteers are inoculated intravenously with a given quantity (with small variability) of Plasmodium‐infected red cells. Parasitemia is then followed by quantitative polymerase chain reaction until a prespecified treatment threshold is reached when the test drug is administered. Parasite and drug concentrations are then measured. These studies are conducted prior to phase II dose‐response (D‐R) trials and can be included in an integrated first‐in‐human study protocol, or after completion of the first‐in‐human PK and safety study. IBSM studies have been typically designed as flexible multiple cohort studies where each volunteer of one cohort receives a single dose of the same amount of drug (“single dose per cohort”). ² , ³ , ⁴ , ⁵ After each cohort, a decision is made to stop or to add a cohort to test a lower or higher dose based on the response observed in the previous cohorts.For the multiple single‐dose‐per‐cohort design, the starting dose is typically selected based on safety and PK information from a phase I single ascending dose (SAD) study and, more recently, on preclinical data from a severe combined immunodeficient mouse model, with the dose selected on the basis of being best able to inform the D‐R relationship, rather than aiming for cure. This approach, where a single dose is tested in all subjects of the initial cohort, risks missing the dose likely to be most informative for defining the PK/PD relationship.An alternative approach is to spread a range of doses across a smaller number of subjects within the initial cohort and use PK/PD models developed based on data from this cohort to support dose selections of subsequent cohorts and studies. Using data from a previous study, ² we undertook an in silico investigation of such an adaptive study design, aiming to reduce the number of subjects exposed to inefficacious doses, and to establish a D‐R relationship. This multiple‐dose‐groups‐per‐cohort design, referred to as the “2‐2‐4” design, is contrasted with the already implemented study design depicted in Figure  1 .Open in a separate windowFigure 1Comparison of standard and adaptive designs of IBSM studies. A/B/C, dose levels to be selected during the progress of the study based on pharmacokinetic/pharmacodynamic results of the initial cohort; CHMI, controlled human malaria infection; D‐R, dose‐response; IBSM, induced blood stage malaria infection; n, number of subjects at each dose.The objectives of this retrospective analysis were to: (i) compare PK/PD parameter estimates from the initial cohort of the 2‐2‐4 study design with the prior results from the data of the full study and (ii) propose a preliminary workflow to establish D‐R early in an IBSM study, and use modeling and simulation (M&S) to support dose selections for subsequent cohorts and later phase clinical trials.     

Questionnaire measures of self-directed ageing stereotype in older adults: a systematic review of measurement properties

The population is ageing, but while average life expectancy continues to increase, healthy life expectancy has not necessarily matched this and negative ageing stereotypes remain prevalent. Self-directed ageing stereotypes are hypothesised to play an important role in older adults’ health and well-being; however, a wide variety of terms and measures are used to explore this construct meaning there is a lack of clarity within the literature. A review was conducted to identify tools used to measure self-directed ageing stereotype in older adults and evaluate their quality. Searches identified 109 papers incorporating 40 different measures. Most common were the Philadelphia Geriatric Centre Morale Scale Attitude Towards Own Ageing (ATOA) subscale, Ageing Perceptions Questionnaire (APQ) and Attitudes to Ageing Questionnaire. Despite being most frequently used, the ATOA was developed to measure morale in older adults rather than self-directed ageing stereotypes. Over 25 terms were used to describe the concept, and it is suggested that for consistency the term “self-directed ageing stereotype” be adopted universally. Across measures, poor reporting of psychometric properties made it difficult to assess scale quality and more research is needed to fully assess measures before conclusions can be drawn as to the best tool; however, the Brief-APQ appears to hold most promise. Future research must address this issue before interventions to reduce negative self-directed ageing stereotypes can be developed and fully evaluated.Electronic supplementary materialThe online version of this article (10.1007/s10433-020-00574-7) contains supplementary material, which is available to authorized users.