Supporting microglial niches for therapeutic benefit in psychiatric disorders.

Inflammation is an essential tissue response to injury, stress, or infection resulting in debris and/or pathogen clearance intended to promote healing and recovery. Due to the status as an immune 'privileged' tissue, microglia serve as endogenous regulators of inflammation in the central nervous system, but maintain communication with peripheral immune system to enable recruitment of peripheral immune cells in case of injury or infection. While microglia retain the functional capacity for a full range of inflammatory functions - microglia express a range of pattern-recognition receptors and function as innate immune cells, carry out phagocytosis of pathogens, and act as antigen presenting cells - in the healthy central nervous system (CNS) these functions are rarely engaged. Subsequently microglia are being recognized to occupy an increasing number of homeostatic niches, and in many cases have adopted immune or inflammatory mechanisms to carry out these niche functions absent immune activation. These sterile inflammatory functions are challenging long-held views of the role of inflammation in the central nervous system while simultaneously expanding the potential for the development of truly novel therapeutic interventions for a range of neuroinflammatory, neurodegenerative, and neuropsychiatric disorders. In the present review we discuss recent preclinical evidence for conserved niche functions for microglia whose disruption may causally contribute to various psychiatric disorders, and prospective targets for restoring disrupted niches.

An update on molecular cat allergens: Fel d 1 and what else? Chapter 1: Fel d 1, the major cat allergen.

BACKGROUND: Cats are the major source of indoor inhalant allergens after house dust mites. The global incidence of cat allergies is rising sharply, posing a major public health problem. Ten cat allergens have been identified. The major allergen responsible for symptoms is Fel d 1, a secretoglobin and not a lipocalin, making the cat a special case among mammals. MAIN BODY: Given its clinical predominance, it is essential to have a good knowledge of this allergenic fraction, including its basic structure, to understand the new exciting diagnostic and therapeutic applications currently in development. The recent arrival of the component-resolved diagnosis, which uses molecular allergens, represents a unique opportunity to improve our understanding of the disease. Recombinant Fel d 1 is now available for in vitro diagnosis by the anti-Fel d 1 specific IgE assay. The first part of the review will seek to describe the recent advances related to Fel d 1 in terms of positive diagnosis and assessment of disease severity. In daily practice, anti-Fel d 1 IgE tend to replace those directed against the overall extract but is this attitude justified? We will look at the most recent arguments to try to answer this question. In parallel, a second revolution is taking place thanks to molecular engineering, which has allowed the development of various forms of recombinant Fel d 1 and which seeks to modify the immunomodulatory properties of the molecule and thus the clinical history of the disease via various modalities of anti-Fel d 1-specific immunotherapy. We will endeavor to give a clear and practical overview of all these trends.

Peanut gastrointestinal delivery oral immunotherapy in adolescents: Results of the build-up phase of a randomized, double-blind, placebo-controlled trial (PITA study).

BACKGROUND: Oral immunotherapy to peanut is effective in desensitizing patients but has significant side effects including anaphylaxis and gastrointestinal symptoms. In most protocols, peanut is administered in a vehicle food. OBJECTIVE: In an exclusively adolescent population, we tested a new approach using sealed capsules of peanut (gastrointestinal delivery oral immunotherapy or GIDOIT) to bypass the upper gastrointestinal tract. The primary aim was to assess the efficacy of the oral build-up phase of GIDOIT and the secondary aim to analyse its safety. METHODS: Adolescents with a history of a clinical allergic reaction after peanut ingestion were included in a 2-armed, parallel-design, individually randomized, double-blind, placebo-controlled, multicentre trial after a positive double-blind placebo-controlled oral food challenge (DBPCFC1). A central randomization centre used computer-generated tables to allocate treatments. Peanut (or placebo) capsules were ingested daily over a period of 24 weeks with increments every 2 weeks from 2 to 400 mg of peanut protein (pp). Primary outcome was tolerance of 400 mg of pp at DBPCFC2. RESULTS: Thirty patients were included between September 2013 and May 2014. At DBPCFC2, unresponsiveness to 400 mg of pp was achieved in 17/21 peanut group patients (2 withdrawn patients) and 1/9 in the placebo group (Intention-to-treat analysis, P < .001, absolute difference = 0.7, 95%IC 0.43 0.96). Oropharyngeal symptoms were equally frequent in both groups. No dysphagia or other signs of eosinophilic oesophagitis occurred. Digestive adverse events (AE) were more frequent in the treated group (P = .02), but mild and without compliance issues. Only one severe advent event led to withdrawal in a patient who ingested twice the investigated treatment. Peanut-specific humoral immune responses were modulated. CONCLUSION: The GIDOIT protocol demonstrated clinical and immunological efficacy and had an acceptable level of safety with weak oropharyngeal symptoms, no dysphagia, mild digestive events and few severe systemic AE.

[When and how to report suspected child abuse to child protective services. Construction and evaluation of a specific support tool for primary care physicians]. / Évaluation d'un outil spécifique sur l'information préoccupante pour les médecins généralistes de Loire-Atlantique.

INTRODUCTION: The aim of this study was to create a specific tool and evaluate its impact on the knowledge of primary care physicians (PCPs) in reporting child abuse to child protective services (CPS). MATERIAL AND METHODS: Prospective "before/after" study assessing the knowledge of general practitioners (GPs) registered at the medical board in a French administrative area through anonymous questionnaires. The tool was adapted from the guidelines published in 2014 by the French Health authorities. The main criterion was the median score (/100) calculated for each questionnaire before (Q1) and after (Q2) the dissemination of the tool. These median scores were compared and associations between scores and some PCPs' characteristics were tested through multiple linear regression. RESULTS: A total of 279 GPs answered the first questionnaire (Q1) and 172 answered the second (Q2). PCPs who answered were mainly women (68% and 74%), were between 30 and 50 years old (61% and 66%), practiced in association with other physicians (82% and 84), and had 15-30% children in their patient population. For Q1, the general median was 65 [IQR: 40-81] versus 82 [IQR: 71-91] for Q2 (P<0.001). The PCPs' characteristics leading to significant variations in the scores for Q1 were age older than 50 years, being female, and having been trained in diagnosis and management of child abuse, with the ß coefficient at -16.4 [95% CI: -31.1; -1.69], +8.93 [95% CI: 2.58; 15.27] and +12 [95% CI: 6.33; 17.73], respectively. DISCUSSION: This study confirms the significant impact of this new tool on PCPs' knowledge concerning reporting suspected child abuse to the CPS. CONCLUSION: Wider dissemination of this tool could increase PCPs' awareness and comprehension of when and how to make a report to the CPS.

Characterization of BMS-911543, a functionally selective small-molecule inhibitor of JAK2.

We report the characterization of BMS-911543, a potent and selective small-molecule inhibitor of the Janus kinase (JAK) family member, JAK2. Functionally, BMS-911543 displayed potent anti-proliferative and pharmacodynamic (PD) effects in cell lines dependent upon JAK2 signaling, and had little activity in cell types dependent upon other pathways, such as JAK1 and JAK3. BMS-911543 also displayed anti-proliferative responses in colony growth assays using primary progenitor cells isolated from patients with JAK2(V617F)-positive myeloproliferative neoplasms (MPNs). Similar to these in vitro observations, BMS-911543 was also highly active in in vivo models of JAK2 signaling, with sustained pathway suppression being observed after a single oral dose. At low dose levels active in JAK2-dependent PD models, no effects were observed in an in vivo model of immunosuppression monitoring antigen-induced IgG and IgM production. Expression profiling of JAK2(V617F)-expressing cells treated with diverse JAK2 inhibitors revealed a shared set of transcriptional changes underlying pharmacological effects of JAK2 inhibition, including many STAT1-regulated genes and STAT1 itself. Collectively, our results highlight BMS-911543 as a functionally selective JAK2 inhibitor and support the therapeutic rationale for its further characterization in patients with MPN or in other disorders characterized by constitutively active JAK2 signaling.

Molecular basis for hair loss in mice carrying a novel nonsense mutation (Hrrh-R ) in the hairless gene (Hr).

Animal models carrying mutations in the hairless (Hr) gene provide a rich resource for study of hair follicle biology. A spontaneous mouse mutant with a phenotype strikingly similar to rhino mutants of Hr arose spontaneously in the mouse facility at Oak Ridge National Laboratory. Sequence analysis of Hr in these mutants uncovered a nonsense mutation in exon 12, designated as Hr(rh-R) (rhino, Oak Ridge). The mutation led to significant reduction in Hr mRNA levels, predicted to be due to nonsense-mediated decay. Histological analysis indicated dilated hair follicle infundibula at 14 days of age that rapidly became filled with cornified material. Microarray analyses revealed that expression levels of many genes involved in keratinocyte differentiation, epidermal regeneration, and wound healing were significantly upregulated before morphological detection of the phenotype, suggesting their role in onset of the Hr(rh-R) phenotype. Identification of this new Hr allele and the underlying molecular alterations allows further understanding of the role of Hr in hair follicle biology.

Alternative processing of the human and mouse raly genes(1).

A human homolog(RALY) of the mouse Raly gene was isolated and sequenced, and shown to encode a novel protein isoform containing a 16 amino acid in-frame insert in the variable region of the protein. Analysis of the corresponding region of the mouse Raly gene demonstrated that this novel protein isoform is also present in the mouse. Comparative analysis of RALY cDNA and EST sequences suggests the presence of additional alternatively processed RALY transcripts. As in the mouse, the human RALY gene is widely expressed as a 1.7-kb transcript.

An agouti mutation lacking the basic domain induces yellow pigmentation but not obesity in transgenic mice.

Chronic antagonism of melanocortin receptors by the paracrine-acting agouti gene product induces both yellow fur and a maturity-onset obesity syndrome in mice that ubiquitously express wild-type agouti. Functional analysis of agouti mutations in transgenic mice indicate that the cysteine-rich C terminus, signal peptide, and glycosylation site are required for agouti activity in vivo. In contrast, no biological activity has been ascribed to the conserved basic domain. To examine the functional significance of the agouti basic domain, the entire 29-aa region was deleted from the agouti cDNA, and the resulting mutation (agoutiDeltabasic) was expressed in transgenic mice under the control of the beta-actin promoter (BAPaDeltabasic). Three independent lines of BAPaDeltabasic transgenic mice all developed some degree of yellow pigment in the fur, indicating that the agoutiDeltabasic protein was functional in vivo. However, none of the BAPaDeltabasic transgenic mice developed completely yellow fur, obesity, hyperinsulinemia, or hyperglycemia. High levels of agoutiDeltabasic expression in relevant tissues exceeded the level of agouti expression in obese viable yellow mice, suggesting that suboptimal activity or synthesis of the agoutiDeltabasic protein, rather than insufficient RNA synthesis, accounts for the phenotype of the BAPaDeltabasic transgenic mice. These findings implicate a functional role for the agouti basic domain in vivo, possibly influencing the biogenesis of secreted agouti protein or modulating protein-protein interactions that contribute to effective antagonism of melanocortin receptors.

Calorimetric, Viscosimetric, and Light Scattering Studies of the Aggregation of, and the Solubilization of Water by, Triton X-35 in n-Heptane.

The behavior of a nonionic surfactant TX-35 in solution in n-heptane in the presence and absence of added water has been examined using the microcalorimetric, viscosimetric, and quasielastic light scattering experimental methods. In this paper, we were interested in the aggregation process of the poly(oxyethylene) glycol alkylphenyl ether in n-heptane and in the solubilization of water in the reverse micelle of the surfactant (micellar solubilization). The analysis of the differential molar enthalpies of dilution of TX-35 in dried n-heptane has shown the occurrence of a gradual exothermic aggregation process on a very wide range of concentration which takes place at particular concentration so-called "operational CMC". This operational CMC value has been confirmed by viscosities measurements. The differential molar enthalpies of hydration of TX-35 were also measured and found to be exothermic. The maximal hydration ratio (w0) was found to be equal to 3.2 mol H2O per mole of TX-35 before the point of phase separation. The measurements of the variation of the amount of water contained in TX-35 solutions at different concentrations in n-heptane also show the occurence of a gradual aggregation process and confirm the value of the maximal hydration ratio already determined by microcalorimetry. In the absence of added water, from the quasielastic light scattering experiments, a mean diameter of the aggregates close to 45 Å has been determine, while in the presence of water, a mean diameter of 61 Å was detected and remained unchanged with increasing the hydration ratio indicating that the size of the aggregate is more influenced by the presence of water than by the amount. In the presence of water, it is relevant to discuss aggregates of lamellar or filament shape. Copyright 1999 Academic Press.

Utilization of microhomologous recombination in yeast to generate targeting constructs for mammalian genes.

We have developed a new procedure utilizing microhomologous recombination in yeast to generate targeting constructs for producing targeted mutations in mice. This procedure is rapid and efficient, and should be directly applicable to all mammalian genes. Moreover, only minimal information about the locus being targeted is required. The feasibility of this approach was demonstrated by producing another allele of the mouse Tg737 polycystic kidney gene.

Differential rescue of the renal and hepatic disease in an autosomal recessive polycystic kidney disease mouse mutant. A new model to study the liver lesion.

Autosomal recessive polycystic kidney disease (ARPKD) is characterized by biliary and renal lesions that produce significant morbidity and mortality. The biliary ductual ectasia and hepatic portal fibrosis associated with ARPKD have not been well studied even though such lesions markedly affect the clinical course of patients after renal replacement therapy such as dialysis or transplantation. Here we describe the generation of a new mouse model to study the hepatic lesions associated with polycystic kidney disease. This model was generated by differentially rescuing the renal pathology in the orpk mutant mouse that displays a hepatorenal pathology that is similar to that seen in human patients with ARPKD. This was accomplished by expressing, as a transgene in the mutant animals, the cloned wild-type version of the gene associated with the mutant locus in this line of mice. Although renal function in the rescue animals is normal, the liver still exhibits biliary and ductular hyperplasia along with varying degrees of hepatic portal fibrosis that is indistinguishable from that in the mutant animals. Most important, the rescue animals survive significantly longer than mutants and will permit a more detailed analysis of the clinical and cellular pathophysiology of the hepatic defect associated with this disease.

Functional correction of renal defects in a mouse model for ARPKD through expression of the cloned wild-type Tg737 cDNA.

Autosomal recessive polycystic kidney disease (ARPKD) is characterized by the formation of large collecting tubule and ductular cysts that often result in renal insufficiency within the first decade of life. Understanding the process leading to cyst formation will require the identification and characterization of genes involved in the etiology of this disease. In this regard, we previously described the generation of a mouse model (TgN737Rpw) for ARPKD and the cloning of a candidate gene. Here we show direct involvement of the Tg737 gene in collecting duct cyst formation by expressing the wild-type Tg737 cDNA as a transgene in TgN737Rpw mutants. In contrast to TgN737Rpw mutants, the "rescued" animals survive longer, have normal renal function and normal localization of the EGFr to the basolateral surfaces of collecting duct epithelium.

Syntheses and structure-activity relationships of the second-generation antitumor taxoids: exceptional activity against drug-resistant cancer cells.

A series of new 3'-(2-methyl-1-propenyl) and 3'-(2-methylpropyl) taxoids with modifications at C-10 was synthesized by means of the beta-lactam synthon method using 10-modified 7-(triethylsilyl)-10-deacetylbaccatin III derivatives. The new taxoids thus synthesized show excellent cytotoxicity against human ovarian (A121), non-small-cell lung (A549), colon (HT-29), and breast (MCF-7) cancer cell lines. All but one of these new taxoids possess better activity than paclitaxel and docetaxel in the same assay, i.e., the IC50 values of almost all the taxoids are in the subnanomolar level. It is found that a variety of modifications at C-10 is tolerated for the activity against normal cancer cell lines, but the activity against a drug-resistant human breast cancer cell line expressing MDR phenotype (MCF7-R) is highly dependent on the structure of the C-10 modifier. A number of the new taxoids exhibit remarkable activity (IC50 = 2.1-9.1 nM) against MCF7-R. Among these, three new taxoids, SB-T-1213 (4a), SB-T-1214 (4b), and SB-T-1102 (5a), are found to be exceptionally potent, possessing 2 orders of magnitude better activity than paclitaxel and docetaxel. The observed exceptional activity of these taxoids may well be ascribed to an effective inhibition of P-glycoprotein binding by the modified C-10 moieties. The new taxoid SB-T-1213 (4a) shows an excellent activity (T/C = 0% at 12.4 and 7.7 mg/kg/dose, log10 cell kill = 2.3 and 2.0, respectively) against B16 melanoma in B6D2F1 mice via intravenous administration.

Upregulation of adipocyte metabolism by agouti protein: possible paracrine actions in yellow mouse obesity.

Mutations leading to ectopic expression of the murine agouti gene (a) result in progressive obesity. To further characterize this model, we analyzed adipose and hepatic mRNA levels for fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD), two key enzymes in de novo fatty acid synthesis and desaturation, respectively. FAS and SCD mRNA in both tissues of obese (Avy) mice were dramatically increased relative to lean (ala) controls. Excessive expression of these genes in this model could be due to direct effects of the agouti gene product; to test this possibility we treated 3T3-L1 adipocytes in vitro with recombinant agouti protein. Agouti treatment increased FAS and SCD mRNA levels by 1.5- and 4-fold, respectively. In addition, FAS activity and triglyceride content were 3-fold higher in agoutitreated 3T3-L1 cells relative to controls; these effects were attenuated by simultaneous treatment with a calcium channel blocker (nitrendipine). These data demonstrate that the agouti protein can directly increase lipogenesis in adipocytes and suggest that these effects are mediated through an intracellular calcium-dependent mechanism.

Agouti regulation of intracellular calcium: role in the insulin resistance of viable yellow mice.

Several dominant mutations at the agouti locus in the mouse cause a syndrome of marked obesity, hyperinsulinemia, and insulin resistance. Although it is known that the agouti gene is expressed in an ectopic manner in these mutants, the precise mechanism by which the agouti gene product mediates these effects is unclear. Since intracellular Ca2+ is believed to play a role in mediating insulin action and dysregulation of Ca2+ flux is observed in diabetic animals and humans, we examined the status of intracellular Ca2+ in mice carrying the dominant agouti allele, viable yellow (Avy). We show here that in mice carrying this mutation, the intracellular free calcium concentration ([Ca2+]i) is elevated in skeletal muscle, and the degree of elevation is closely correlated with the degree to which the mutant traits are expressed in individual animals. Moreover, we demonstrate that the agouti gene product is capable of inducing increased [Ca2+]i in cultured and freshly isolated skeletal muscle myocytes from wild-type mice. Based on these findings, we present a model in which we propose that the agouti polypeptide promotes insulin resistance in mutant animals through its ability to increase [Ca2+]i.

Differential expression of a new dominant agouti allele (Aiapy) is correlated with methylation state and is influenced by parental lineage.

The agouti gene normally confers the wild-type coat color of mice. Dominant mutations at the agouti locus result in a pleiotropic syndrome that is characterized by excessive amounts of yellow pigment in the coat, obesity, a non-insulin-dependent diabetic-like condition, and the propensity to form a variety of tumors. Here, we describe a new dominant mutation at the agouti locus in which an intracisternal A-particle (IAP) has integrated in an antisense orientation immediately 5' of the first coding exon of the gene. This mutation, which we have named Aiapy, results in the ectopic expression of the agouti gene through the utilization of a cryptic promoter within the IAP 5' long terminal repeat (LTR). The coat color of Aiapy/-mice ranges from solid yellow to a pigment pattern that is similar to wild type (pseudoagouti), and the expressivity of this mutant phenotype varies with parental inheritance. Those offspring with a yellow coat ectopically express agouti mRNA at high levels and exhibit marked obesity, whereas pseudoagouti mice express agouti mRNA at a very low level and their weights do not differ from wild-type littermates. Data are presented to show that the differential expressivity of the Aiapy allele is correlated with the methylation status of the inserted IAP 5' LTR. These data further support the hypothesis that in dominant yellow mutations at the agouti locus, it is the ubiquitous expression of the wild-type agouti coding sequence that is responsible for the yellow coat color, obesity, diabetes, and tumorigenesis.

A molecular model for the genetic and phenotypic characteristics of the mouse lethal yellow (Ay) mutation.

Lethal yellow (Ay) is a mutation at the mouse agouti locus in chromosome 2 that causes a number of dominant pleiotropic effects, including a completely yellow coat color, obesity, an insulin-resistant type II diabetic condition, and an increased propensity to develop a variety of spontaneous and induced tumors. Additionally, homozygosity for Ay results in preimplantation lethality, which terminates development by the blastocyst stage. The Ay mutation is the result of a 170-kb deletion that removes all but the promoter and noncoding first exon of another gene called Raly, which lies in the same transcriptional orientation as agouti and maps 280 kb proximal to the 3' end of the agouti gene. We present a model for the structure of the Ay allele that can explain the dominant pleiotropic effects associated with this mutation, as well as the recessive lethality, which is unrelated to the agouti gene.