Astrocyte–microglial association and matrix composition are common events in the natural history of primary familial brain calcification

Primary familial brain calcification (PFBC) is an age‐dependent and rare neurodegenerative disorder characterized by microvascular calcium phosphate deposits in the deep brain regions. Known genetic causes of PFBC include loss‐of‐function mutations in genes involved in either of three processes—platelet‐derived growth factor (PDGF) signaling, phosphate homeostasis or protein glycosylation—with unclear molecular links. To provide insight into the pathogenesis of PFBC, we analyzed murine models of PFBC for the first two of these processes in Pdgfb^ret/ret and Slc20a2^?/? mice with regard to the structure, molecular composition, development and distribution of perivascular calcified nodules. Analyses by transmission electron microscopy and immunofluorescence revealed that calcified nodules in both of these models have a multilayered ultrastructure and occur in direct contact with reactive astrocytes and microglia. However, whereas nodules in Pdgfb^ret/ret mice were large, solitary and smooth surfaced, the nodules in Slc20a2^?/? mice were multi‐lobulated and occurred in clusters. The regional distribution of nodules also differed between the two models. Proteomic analysis and immunofluorescence stainings revealed a common molecular composition of the nodules in the two models, involving proteins implicated in bone homeostasis, but also proteins not previously linked to tissue mineralization. While the brain vasculature of Pdgfb^ret/ret mice has been reported to display reduced pericyte coverage and abnormal permeability, we found that Slc20a2^?/? mice have a normal pericyte coverage and no overtly increased permeability. Thus, lack of pericytes and increase in permeability of the blood–brain barrier are likely not the causal triggers for PFBC pathogenesis. Instead, gene expression and spatial correlations suggest that astrocytes are intimately linked to the calcification process in PFBC.     

NTRK testing: First results of the QuiP‐EQA scheme and a comprehensive map of NTRK fusion variants and their diagnostic coverage by targeted RNA‐based NGS assays

Gene fusions involving the three neurotrophic tyrosine receptor kinase genes NTRK1, NTRK2, or NTRK3 were identified as oncogenic drivers in many cancer types. Two small molecule inhibitors have been tested in clinical trials recently and require the detection of a NTRK fusion gene prior to therapeutic application. Fluorescence in situ hybridization (FISH) and targeted next‐generation sequencing (tNGS) assays are commonly used for diagnostic profiling of gene fusions. In the presented study we applied an external quality assessment (EQA) scheme in order to investigate the suitability of FISH and RNA‐/DNA‐based tNGS for detection of NTRK fusions in a multinational and multicentric ring trial. In total 27 participants registered for this study. Nine institutions took part in the FISH‐based and 18 in the NGS‐based round robin test, the latter additionally subdivided into low‐input and high‐input NGS methods (regarding nucleic acid input). Regardless of the testing method applied, all participants received tumor sections of 10 formalin‐fixed and paraffin‐embedded (FFPE) tissue blocks for in situ hybridization or RNA/DNA extraction, and the results were submitted via an online questionnaire. For FISH testing, eight of nine (88.8%) participants, and for NGS‐based testing 15 of 18 (83.3%) participants accomplished the round robin test successfully. The overall high success rate demonstrates that FISH‐ and tNGS‐based NTRK testing can be well established in a routine diagnostic setting. Complementing this dataset, we provide an updated in silico analysis on the coverage of more than 150 NTRK fusion variants by several commercially available RNA‐based tNGS panels.     

Clinical Factors Associated with Biochemical Adrenal-cortisol Insufficiency in Hospitalized Patients

Background

Diagnosis of adrenal-cortisol insufficiency is often misleading in hospitalized patients, as clinical and biochemical features overlap with comorbidities. We analyzed clinical determinants associated with a biochemical diagnosis of adrenal-cortisol insufficiency in non-intensive care unit (ICU) hospitalized patients.

Methods

In a retrospective cohort study we reviewed 4668 inpatients with random morning cortisol levels ≤15 μg/dL hospitalized in our center between 2003 and 2010. Using serum cortisol threshold level of 18 μg/dL 30 or 60 minutes after Cortrosyn (250 μg; Amphastar Pharmaceuticals, Inc., Rancho Cucamonga, Calif) injection to define biochemical adrenal-cortisol status, we characterized and compared insufficient (n = 108, serum cortisol ≤18 μg/dL) and sufficient (n = 394; serum cortisol >18 μg/dL) non-ICU hospitalized patients.

Results

Commonly reported clinical and routine biochemical adrenal-cortisol insufficiency features were similar between insufficient and sufficient inpatients. Biochemical adrenal-cortisol insufficiency was associated with increased frequency of liver disease, specifically hepatitis C (P = .01) and prior orthotopic liver transplantation (P <.001), human immunodeficiency virus (HIV; P = .005), and reported pre-existing male hypogonadism (P <.001), as compared with the biochemical adrenal-cortisol sufficiency group. Forty percent of insufficient inpatients were not treated with glucocorticoids after diagnosis. Multivariable logistic analysis demonstrated that inpatients with higher cortisol levels (P = .0001) and higher diastolic blood pressure (P = .05), and females (P = .009) were more likely not to be treated, while those with previous short-term glucocorticoid treatment (P = .002), other coexisting endocrine diseases (P = .005), or who received an in-hospital endocrinology consultation (P <.0001), were more likely to be replaced with glucocorticoids.

Conclusions

Commonly reported adrenal-cortisol insufficiency features do not reliably identify hospitalized patients biochemically confirmed to have this disorder. Comorbidities including hepatitis C, prior orthotopic liver transplantation, HIV, and reported pre-existing male hypogonadism may help identify hospitalized non-ICU patients for more rigorous adrenal insufficiency assessment.     

From the Cover: Inhaled oxytocin increases positive social behaviors in newborn macaques

Early caregiver–infant interactions are critical for infants’ socioemotional and cognitive development. Several hormones and neuromodulators, including oxytocin, affect these interactions. Exogenous oxytocin promotes social behaviors in several species, including human and nonhuman primates. Although exogenous oxytocin increases social function in adults—including expression recognition and affiliation—it is unknown whether oxytocin can increase social interactions in infants. We hypothesized that nebulized oxytocin would increase affiliative social behaviors and such effects would be modulated by infants’ social skills, measured earlier in development. We also hypothesized that oxytocin’s effects on social behaviors may be due to its anxiolytic effects. We tested these hypotheses in a blind study by nebulizing 7- to 14-d-old macaques (n = 28) with oxytocin or saline. Following oxytocin administration, infants’ facial gesturing at a human caregiver increased, and infants’ salivary oxytocin was positively correlated with the time spent in close proximity to a caregiver. Infants’ imitative skill (measured earlier in development: 1–7 d of age) predicted oxytocin-associated increases in affiliative behaviors—lip smacking, visual attention to a caregiver, and time in close proximity to a caregiver—suggesting that infants with higher propensities for positive social interactions are more sensitive to exogenous oxytocin. Oxytocin also decreased salivary cortisol, but not stress-related behaviors (e.g., scratching), suggesting the possibility of some anxiolytic effects. To our knowledge, this study provides the first evidence that oxytocin increases positive social behaviors in newborns. This information is of critical importance for potential interventions aimed at ameliorating inadequate social behaviors in infants with higher likelihood of developing neurodevelopmental disorder.Oxytocin is a neuropeptide that has wide-ranging effects on social behaviors and social perception, including increased emotion recognition and prosocial behavior (1, 2). Animal studies present convergent evidence of oxytocin’s positive effects on social behavior (2–6), including humans (1, 7). In recent years, an increasing literature on human and nonhuman primates suggests an association between oxytocin levels—either endogenous or exogenously administered—and prosocial behaviors (8–10). In both humans and macaques, exogenous oxytocin appears to enhance social attention, prosocial behaviors, sensitivity to gaze, and sensitivity to facial expressions (for reviews, see refs. 1 and 2).Oxytocin, therefore, may be a tool for promoting social behaviors, especially in clinical populations in which social faculties are compromised (8, 11–13). In the last few years, in fact, oxytocin has been tested in autistic individuals, and it appears to increase social attention and improve emotion recognition (e.g., refs. 14–19; although see ref. 20; for a recent review, see ref. 21). Given the importance of early assessments in the diagnosis of autism (22), studies clarifying the role of oxytocin in early development are critically important. For example, human infants actively participate in face-to-face caregiver–infant interactions; failure to engage with caregivers in this way can disrupt the development of healthy emotion regulation and socioemotional skills (23–25). In both caregivers and neonates, complex cortical and limbic brain networks are prepared to sustain such exchanges (26–28), and several hormones and neuromodulators regulate the affective components of face-to-face caregiver–infant interactions (29–32). However, to our knowledge, studies investigating the role of infants’ oxytocin levels in these early intersubjective exchanges have not been carried out. Only one study to date measured endogenous oxytocin levels in newborns and reported that higher levels of oxytocin in newborns’ cerebrospinal fluid (CSF) were associated with higher levels of social engagement, including actively seeking parental social interaction for soothing and a greater interest in social interaction (33). No studies to date, however, have administered oxytocin to infants to determine its effects on social behavior, despite the fact that a more thorough understanding of oxytocin and its behavioral consequences may provide a potential tool for interventions aimed at promoting social affiliation in individuals with social impairments (11–15, 34). The necessity to fill this gap motivated the present study.Our first goal was to determine whether oxytocin influences newborn macaques’ behaviors during an interaction with a human caregiver. We predicted that oxytocin, compared with saline, would increase positive social behavior, including facial gestures [i.e., lip smacking (LPS) and tongue protrusion (TP)], visual attention to a human caregiver, and time spent in close proximity to a human caregiver (35). As adults, macaques display positive behavioral changes in response to exogenous oxytocin (2–6), as in humans (1).

Table 1.

Ethogram for 12 behaviors scored during imitation recognition

Optogenetic control of insulin secretion in intact pancreatic islets with β-cell-specific expression of Channelrhodopsin-2

Insulin is secreted from the pancreatic β-cells in response to elevated glucose. In intact islets the capacity for insulin release is determined by a complex interplay between different cell types. This has made it difficult to specifically assess the role of β-cell defects to the insulin secretory impairment in type 2 diabetes. Here we describe a new approach, based on optogenetics, that enables specific investigation of β-cells in intact islets. We used transgenic mice expressing the light-sensitive cation channel Channelrhodopsin-2 (ChR2) under control of the insulin promoter. Glucose tolerance in vivo was assessed using intraperitoneal glucose tolerance tests, and glucose-induced insulin release was measured from static batch incubations. ChR2 localization was determined by fluorescence confocal microscopy. The effect of ChR2 stimulation with blue LED light was assessed using Ca²⁺ imaging and static islet incubations. Light stimulation of islets from transgenic ChR2 mice triggered prompt increases in intracellular Ca²⁺. Moreover, light stimulation enhanced insulin secretion in batch-incubated islets at low and intermediate but not at high glucose concentrations. Glucagon release was not affected. Beta-cells from mice rendered diabetic on a high-fat diet exhibited a 3.5-fold increase in light-induced Ca²⁺ influx compared with mice on a control diet. Furthermore, light enhanced insulin release also at high glucose in these mice, suggesting that high-fat feeding leads to a compensatory potentiation of the Ca²⁺ response in β-cells. The results demonstrate the usefulness and versatility of optogenetics for studying mechanisms of perturbed hormone secretion in diabetes with high time-resolution and cell-specificity.     

Thalidomide and dexamethasone vs. bortezomib and dexamethasone for melphalan refractory myeloma: a randomized study

Objectives: Thalidomide and bortezomib have been frequently used for second‐line therapy in patients with myeloma relapsing after or refractory to initial melphalan‐based treatment, but no randomized trials have been published comparing these two treatment alternatives. Methods: Thalidomide‐ and bortezomib‐naïve patients with melphalan refractory myeloma were randomly assigned to low‐dose thalidomide + dexamethasone (Thal‐Dex) or bortezomib + dexamethasone (Bort‐Dex). At progression on either therapy, the patients were offered crossover to the alternative drug combination. An estimated 300 patients would be needed for the trial to detect a 50% difference in median PFS between the treatment arms. Results: After inclusion of 131 patients, the trial was prematurely closed because of low accrual. Sixty‐seven patients were randomized to Thal‐Dex and 64 to Bort‐Dex. Progression‐free survival was similar (median, 9.0 months for Thal‐Dex and 7.2 for Bort‐Dex). Response rate was similar (55% for Thal‐Dex and 63% for Bort‐Dex), but time to response was shorter (P < 0.05) and the VGPR rate higher (P < 0.01) for Bort‐Dex. Time‐to‐other treatment after crossover was similar (median, 13.2 months for Thal‐Dex and 11.2 months for Bort‐Dex), as was overall survival (22.8 months for Thal‐Dex and 19.0 for Bort‐Dex). Venous thromboembolism was seen in seven patients and cerebrovascular events in four patients in the Thal‐Dex group. Severe neuropathy, reactivation of herpes virus infections, and mental depression were more frequently observed in the Bort‐Dex group. In the quality‐of‐life analysis, no difference was noted for physical function, pain, and global quality of life. Fatigue and sleep disturbances were significantly more prevalent in the Bort‐Dex group. Conclusions: Thalidomide (50–100 mg daily) in combination with dexamethasone seems to have an efficacy comparable with that of bortezomib and dexamethasone in melphalan refractory myeloma. However, the statistical strength of the results in this study is limited by the low number of included patients.