Cancer Spheroids Embedded in Tissue-Engineered Skin Substitutes: A New Method to Study Tumorigenicity In Vivo.

Tumorigenic assays are used during a clinical translation to detect the transformation potential of cell-based therapies. One of these in vivo assays is based on the separate injection of each cell type to be used in the clinical trial. However, the injection method requires many animals and several months to obtain useful results. In previous studies, we showed the potential of tissue-engineered skin substitutes (TESs) as a model for normal skin in which cancer cells can be included in vitro. Herein, we showed a new method to study tumorigenicity, using cancer spheroids that were embedded in TESs (cTES) and grafted onto athymic mice, and compared it with the commonly used cell injection assay. Tumors developed in both models, cancer cell injection and cTES grafting, but metastases were not detected at the time of sacrifice. Interestingly, the rate of tumor development was faster in cTESs than with the injection method. In conclusion, grafting TESs is a sensitive method to detect tumor cell growth with and could be developed as an alternative test for tumorigenicity.

Production of Tissue-Engineered Skin Substitutes for Clinical Applications: Elimination of Serum.

Tissue-engineered skin substitutes (TESs) are used as a treatment for severe burn injuries. Their production requires culturing both keratinocytes and fibroblasts. The methods to grow these cells have evolved over the years, but bovine serum is still commonly used in the culture medium. Because of the drawbacks associated with the use of serum, it would be advantageous to use serum-free media for the production of TESs. In a previous study, we developed a serum-free medium (Surge SFM) for the culture of keratinocytes. Herein, we tested the use of this medium, together with a commercially available serum-free medium for fibroblasts (Prime XV), to produce serum-free TESs. Our results show that serum-free TESs are macroscopically and histologically similar to skin substitutes produced with conventional serum-containing media. TESs produced with either culture media expressed keratin 14, Ki-67, transglutaminase 1, filaggrin, type I and IV collagen, and fibronectin comparably. Mechanical properties, such as contraction and tensile strength, were comparable between TESs cultured with and without serum. Serum-free TESs were also successfully grafted onto athymic mice for a six-month period. In conclusion, Surge SFM and Prime XV serum-free media could be used to produce high quality clinical-grade skin substitutes.

Cumulative childhood trauma, sex motives and sexual satisfaction among emerging adults.

BACKGROUND: To better understand sexual satisfaction in emerging adults, previous research has emphasized the importance of considering the role of developmental experiences, such as cumulative childhood trauma (CCT; the number of different forms of abuse and neglect sustained during childhood). However, the mechanisms by which CCT and sexual satisfaction are related remain unknown. Sex motives are suggested as an explanatory mechanism given the previously found associations between sex motives and both sexual satisfaction and CCT. OBJECTIVE: This study examined the direct associations between CCT and sexual satisfaction, as well as indirect associations through sex motives, in emerging adults. PARTICIPANTS AND SETTING: A sample of 437 French Canadian emerging adults (76 % women, mean age of 2.3) were recruited. METHODS: Participants completed online self-reported validated questionnaires assessing CCT, sex motives and sexual satisfaction. RESULTS: Results from a path analysis revealed that CCT was associated with a higher endorsement of the self-affirmation sex motive (ß = 0.25, p < .001) which was linked to lower sexual satisfaction (ß = -0.13, p < .001). CCT was also associated with a higher endorsement of the coping (ß = 0.25, p < .001) and partner approval (ß = 0.09, p < .05) sex motives. A higher endorsement of the intimacy (ß = 0.28, p < .001) and pleasure (ß = 0.24, p < .001) sex motives, and a lower endorsement of the partner approval sex motive (ß =  -0.13, p < .001) were linked to greater sexual satisfaction. CONCLUSIONS: Results suggest education and intervention targets to improve emerging adults' sexuality.

A Newly Developed Chemically Defined Serum-Free Medium Suitable for Human Primary Keratinocyte Culture and Tissue Engineering Applications.

In our experience, keratinocytes cultured in feeder-free conditions and in commercially available defined and serum-free media cannot be as efficiently massively expanded as their counterparts grown in conventional bovine serum-containing medium, nor can they properly form a stratified epidermis in a skin substitute model. We thus tested a new chemically defined serum-free medium, which we developed for massive human primary keratinocyte expansion and skin substitute production. Our medium, named Surge Serum-Free Medium (Surge SFM), was developed to be used alongside a feeder layer. It supports the growth of keratinocytes freshly isolated from a skin biopsy and cryopreserved primary keratinocytes in cultured monolayers over multiple passages. We also show that keratin-19-positive epithelial stem cells are retained through serial passaging in Surge SFM cultures. Transcriptomic analyses suggest that gene expression is similar between keratinocytes cultured with either Surge SFM or the conventional serum-containing medium. Additionally, Surge SFM can be used to produce bilayered self-assembled skin substitutes histologically similar to those produced using serum-containing medium. Furthermore, these substitutes were grafted onto athymic mice and persisted for up to six months. In conclusion, our new chemically defined serum-free keratinocyte culture medium shows great promise for basic research and clinical applications.

Blockade of Serotonin 5-HT6 Receptor Constitutive Activity Alleviates Cognitive Deficits in a Preclinical Model of Neurofibromatosis Type 1.

Neurofibromatosis type 1 (NF1) is a common inherited disorder caused by mutations of the NF1 gene that encodes the Ras-GTPase activating protein neurofibromin, leading to overactivation of Ras-dependent signaling pathways such as the mTOR pathway. It is often characterized by a broad range of cognitive symptoms that are currently untreated. The serotonin 5-HT6 receptor is a potentially relevant target in view of its ability to associate with neurofibromin and to engage the mTOR pathway to compromise cognition in several cognitive impairment paradigms. Here, we show that constitutively active 5-HT6 receptors contribute to increased mTOR activity in the brain of Nf1+/- mice, a preclinical model recapitulating some behavioral alterations of NF1. Correspondingly, peripheral administration of SB258585, a 5-HT6 receptor inverse agonist, or rapamycin, abolished deficits in long-term social and associative memories in Nf1+/- mice, whereas administration of CPPQ, a neutral antagonist, did not produce cognitive improvement. These results show a key influence of mTOR activation by constitutively active 5-HT6 receptors in NF1 cognitive symptoms. They provide a proof of concept that 5-HT6 receptor inverse agonists already in clinical development as symptomatic treatments to reduce cognitive decline in dementia and psychoses, might be repurposed as therapies alleviating cognitive deficits in NF1 patients.

Early 5-HT6 receptor blockade prevents symptom onset in a model of adolescent cannabis abuse.

Cannabis abuse during adolescence confers an increased risk for developing later in life cognitive deficits reminiscent of those observed in schizophrenia, suggesting common pathological mechanisms that remain poorly characterized. In line with previous findings that revealed a role of 5-HT6 receptor-operated mTOR activation in cognitive deficits of rodent developmental models of schizophrenia, we show that chronic administration of ∆9-tetrahydrocannabinol (THC) to mice during adolescence induces a long-lasting activation of mTOR in prefrontal cortex (PFC), alterations of excitatory/inhibitory balance, intrinsic properties of layer V pyramidal neurons, and long-term depression, as well as cognitive deficits in adulthood. All are prevented by administrating a 5-HT6 receptor antagonist or rapamycin, during adolescence. In contrast, they are still present 2 weeks after the same treatments delivered at the adult stage. Collectively, these findings suggest a role of 5-HT6 receptor-operated mTOR signaling in abnormalities of cortical network wiring elicited by THC at a critical period of PFC maturation and highlight the potential of 5-HT6 receptor antagonists as early therapy to prevent cognitive symptom onset in adolescent cannabis abusers.

Systematic Analysis of the Stress-Induced Genomic Instability of Type Three Secretion System in Aeromonas salmonicida subsp. salmonicida.

The type three secretion system (TTSS) locus of Aeromonas salmonicida subsp. salmonicida, located on the plasmid pAsa5, is known to be lost when the bacterium is grown at temperatures of 25 °C. The loss of the locus is due to the recombination of the insertion sequences flanking the TTSS region. However, the mechanism involved in this recombination is still elusive. Here, we analyzed 22 A. salmonicida subsp. salmonicida strains that had already lost their TTSS locus, and we systematically explored another 47 strains for their susceptibility to lose the same locus when grown at 25 °C. It appeared that strains from Europe were more prone to lose their TTSS locus compared to Canadian strains. More specifically, it was not possible to induce TTSS loss in Canadian strains that have AsaGEI2a, a genomic island, and prophage 3, or in Canadian strains without a genomic island. A comparative genomic approach revealed an almost perfect correlation between the presence of a cluster of genes, not yet characterized, and the susceptibility of various groups of strains to lose their locus. This cluster of genes encodes putative proteins with DNA binding capacity and phage proteins. This discovery creates new opportunities in the study of pAsa5 thermosensitivity.

Sustained impairment of α2A-adrenergic autoreceptor signaling mediates neurochemical and behavioral sensitization to amphetamine.

BACKGROUND: In rodents, drugs of abuse induce locomotor hyperactivity, and repeating injections enhance this response. This effect, called behavioral sensitization, persists months after the last administration. It has been shown that behavioral sensitization to amphetamine develops parallel to an increased release of norepinephrine (NE) in the prefrontal cortex (PFC). METHODS: Rats and mice were repeatedly treated with amphetamine (1 or 2 mg/kg intraperitoneally, respectively) to obtain sensitized animals. The NE release in the PFC was measured by microdialysis in freely moving mice (n = 55). Activity of locus coeruleus (LC) noradrenergic neurons was determined in anaesthetized rats (n = 15) by in vivo extracellular electrophysiology. The α2A-adrenergic autoreceptor (α2A-AR) expression was assessed by autoradiography on brain slices, and Gαi proteins expression was measured by western blot analysis of LC punches. RESULTS: In sensitized rats LC neurons had a higher spontaneous firing rate, and clonidine-an α2A-adrenergic agonist-inhibited LC neuronal firing less efficiently than in control animals. Clonidine also induced lower levels of NE release in the PFC of sensitized mice. This desensitization was maintained by a lower density of Gαi1 and Gαi2 proteins in the LC of sensitized mice rather than weaker α2A-AR expression. Behavioral sensitization was facilitated by α2A-AR antagonist, efaroxan, during amphetamine injections and abolished by clonidine treatment. CONCLUSIONS: Our data indicate that noradrenergic inhibitory feedback is impaired for at least 1 month in rats and mice repeatedly treated with amphetamine. This work highlights the key role of noradrenergic autoreceptor signaling in the persistent modifications induced by repeated amphetamine administration.

Inhibition of monoamine oxidases desensitizes 5-HT1A autoreceptors and allows nicotine to induce a neurochemical and behavioral sensitization.

Although nicotine is generally considered to be the main compound responsible for addictive properties of tobacco, experimental data indicate that nicotine does not exhibit all the characteristics of other substances of abuse. We recently showed that a pretreatment with mixed irreversible monoamine oxidases inhibitors (MAOIs), such as tranylcypromine, triggers a locomotor response to nicotine in mice and allows maintenance of behavioral sensitization to nicotine in rats. Moreover, we showed by microdialysis in mice that behavioral sensitization induced by compounds belonging to main groups of drugs of abuse, such as amphetamine, cocaine, morphine, or alcohol, was underlain by sensitization of noradrenergic and serotonergic neurons. Here, this neurochemical sensitization was tested after nicotine, tranylcypromine, or a mixture of both compounds. Data indicate that, whereas neither repeated nicotine nor repeated tranylcypromine alone has any effect by itself, a repeated treatment with a mixture of nicotine and tranylcypromine induces both behavioral sensitization and sensitization of noradrenergic and serotonergic neurons. The development of neurochemical and behavioral sensitizations is blocked by prazosin and SR46349B [(1Z,2E)-1-(2-fluoro-phenyl)-3-(4-hydroxyphenyl)-prop-2-en-one-O-(2-dimethylamino-ethyl)-oxime hemifumarate], two antagonists of alpha1b-adrenergic and 5-HT(2A) receptors, respectively, but not by SCH23390 [R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride], a D(1) receptor antagonist. Finally, we found that pretreatments with WAY 100635 [N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclo-hexane carboxamide trihydrochloride], a 5-HT(1A) receptor antagonist, can also induce a behavioral and neurochemical sensitization to repeated nicotine. Complementary experiments with 8-OHDPAT (8-hydroxy-dipropylamino-tetralin), a 5-HT(1A) receptor agonist, and analysis of 5-HT(1A) receptors expression in the dorsal raphe nucleus after a tranylcypromine injection indicate that MAOIs contained in tobacco desensitize 5-HT(1A) autoreceptors to trigger the strong addictive properties of tobacco.