Effects of High-Temperature-Pressure Polymerized Resin-Infiltrated Ceramic Networks on Oral Stem Cells

International audienceObjectivesThe development of CAD—CAM techniques called for new materials suited to this technique and offering a safe and sustainable clinical implementation. The infiltration of resin in a ceramic network under high pressure and high temperature defines a new class of hybrid materials, namely polymer infiltrated ceramics network (PICN), for this purpose which requires to be evaluated biologically. We used oral stem cells (gingival and pulpal) as an in vitro experimental model.MethodsFour biomaterials were grinded, immersed in a culture medium and deposed on stem cells from dental pulp (DPSC) and gingiva (GSC): Enamic (VITA®), Experimental Hybrid Material (EHM), EHM with initiator (EHMi) and polymerized Z100™ composite material (3M®). After 7 days of incubation; viability, apoptosis, proliferation, cytoskeleton, inflammatory response and morphology were evaluated in vitro.ResultsProliferation was insignificantly delayed by all the tested materials. Significant cytotoxicity was observed in presence of resin based composites (MTT assay), however no detectable apoptosis and some dead cells were detected like in PICN materials. Cell morphology, major cytoskeleton and extracellular matrix components were not altered. An intimate contact appeared between the materials and cells.Clinical SignificanceThe three new tested biomaterials did not exhibit adverse effects on oral stem cells in our experimental conditions and may be an interesting alternative to ceramics or composite based CAD—CAM blocks

Isolation and Characterization of Two Novel Colorectal Cancer Cell Lines, Containing a Subpopulation with Potential Stem-Like Properties: Treatment Options by MYC/NMYC Inhibition

Schulte am Esch J, Windmöller BA, Hanewinkel J, et al. Isolation and Characterization of Two Novel Colorectal Cancer Cell Lines, Containing a Subpopulation with Potential Stem-Like Properties: Treatment Options by MYC/NMYC Inhibition. Cancers. 2020;12(9): 2582.Cancer stem cells (CSC) are crucial mediators of cancer relapse. Here, we isolated two primary human colorectal cancer cell lines derived from a rectal neuroendocrine carcinoma (BKZ-2) and a colorectal adenocarcinoma (BKZ-3), both containing subpopulations with potential stem-like properties. Protein expression of CSC-markers prominin-1 and CD44 antigen was significantly higher for BKZ-2 and BKZ-3 in comparison to well-established colon carcinoma cell lines. High sphere-formation capacity further confirmed the existence of a subpopulation with potential stem-like phenotype. Epithelial–mesenchymal transition markers as well as immune checkpoint ligands were expressed more pronounced in BKZ-2. Both cell populations demonstrated N-myc proto-oncogene (NMYC) copy number gain. Myc proto-oncogene (MYC)/NMYC activity inhibitor all-trans retinoic acid (ATRA) significantly reduced the number of tumor spheres for both and the volume of BKZ-2 spheres. In contrast, the sphere volume of ATRA-treated BKZ-3 was increased, and only BKZ-2 cell proliferation was reduced in monolayer culture. Treatment with KJ-Pyr-9, a specific inhibitor of MYC/NMYC-myc-associated factor X interaction, decreased survival by the induction of apoptosis of both. In summary, here, we present the novel colorectal cancer cell lines BKZ-2 and BKZ-3 as promising cellular in vitro models for colorectal carcinomas and identify the MYC/NMYC molecular pathway involved in CSC-induced carcinogenesis with relevant therapeutic potential

Implantação do programa de adesão ao tratamento de HIV/AIDS - relato de experiência

Adherence to antiretroviral treatment is currently one of the points that have the highest impact on reducing complications and improving quality of life of people with HIV/AIDS. With the aim of providing patients and their family with an effective and continuous orientation, an adherence program was created, divided into two strategies. The first is comprised of individual meetings with nurses for education, counseling and an opportunity to share their feelings/doubts with a health professional. To do so, a partnership with a multidisciplinary staff was established, and a nursing schedule, as well as a protocol, was created for these appointments. The second strategy, aimed at a collective approach, took place through a series of free lectures, in which an invited professional discussed issues suggested by the participants (patients, their family, friends and people interested in those issues). Participation was voluntary and anonymous for everyone interested. For a 1-year period, 65 patients were seen in individual appointments; the others have appointments at intervals according to their individual needs. In the same period, 296 people attended the series of lectures in nine meetings. An improvement was noted as to how patients participating in the program face their condition.A adesão ao tratamento anti-retroviral é atualmente um dos pontos de maior impacto na redução de complicações e melhoria na qualidade de vida das pessoas com HIV/AIDS. Com o objetivo de proporcionar aos pacientes e a seus familiares uma forma de orientação efetiva e contínua, criou-se um programa de adesão, que contemplou duas estratégias; a primeira, na forma de consulta de enfermagem, com enfoque individual, visando o aconselhamento, a educação, e o oferecimento de um espaço para escuta. Para tanto, organizou-se uma parceria com uma equipe multidisciplinar, criou-se uma agenda de enfermagem para estes atendimentos, bem como um protocolo. A segunda estratégia, visando uma abordagem coletiva, ocorreu por intermédio do ciclo de palestras gratuitas, em que um profissional convidado abordava assuntos sugeridos pelos participantes (o paciente, sua família, amigos e pessoas interessadas nos temas). A participação foi voluntária e anônima para os interessados. Durante o período de um ano foram atendidos, em consultas individuais, 65 pacientes, os demais seguem em consultas mais espaçadas de acordo com suas necessidades. No ciclo de palestras, no mesmo período, compareceram 296 pessoas em nove encontros. Foi observada uma melhora no enfrentamento à doença por parte dos pacientes assistidos pelo programa

Implantação do programa de adesão ao tratamento de HIV/AIDS - relato de experiência

A adesão ao tratamento anti-retroviral é atualmente um dos pontos de maior impacto na redução de complicações e melhoria na qualidade de vida das pessoas com HIV/AIDS. Com o objetivo de proporcionar aos pacientes e a seus familiares uma forma de orientação efetiva e contínua, criou-se um programa de adesão, que contemplou duas estratégias; a primeira, na forma de consulta de enfermagem, com enfoque individual, visando o aconselhamento, a educação, e o oferecimento de um espaço para escuta. Para tanto, organizou-se uma parceria com uma equipe multidisciplinar, criou-se uma agenda de enfermagem para estes atendimentos, bem como um protocolo. A segunda estratégia, visando uma abordagem coletiva, ocorreu por intermédio do ciclo de palestras gratuitas, em que um profissional convidado abordava assuntos sugeridos pelos participantes (o paciente, sua família, amigos e pessoas interessadas nos temas). A participação foi voluntária e anônima para os interessados. Durante o período de um ano foram atendidos, em consultas individuais, 65 pacientes, os demais seguem em consultas mais espaçadas de acordo com suas necessidades. No ciclo de palestras, no mesmo período, compareceram 296 pessoas em nove encontros. Foi observada uma melhora no enfrentamento à doença por parte dos pacientes assistidos pelo programa.Adherence to antiretroviral treatment is currently one of the points that have the highest impact on reducing complications and improving quality of life of people with HIV/AIDS. With the aim of providing patients and their family with an effective and continuous orientation, an adherence program was created, divided into two strategies. The first is comprised of individual meetings with nurses for education, counseling and an opportunity to share their feelings/doubts with a health professional. To do so, a partnership with a multidisciplinary staff was established, and a nursing schedule, as well as a protocol, was created for these appointments. The second strategy, aimed at a collective approach, took place through a series of free lectures, in which an invited professional discussed issues suggested by the participants (patients, their family, friends and people interested in those issues). Participation was voluntary and anonymous for everyone interested. For a 1-year period, 65 patients were seen in individual appointments; the others have appointments at intervals according to their individual needs. In the same period, 296 people attended the series of lectures in nine meetings. An improvement was noted as to how patients participating in the program face their condition

DKK1 inhibits canonical Wnt signaling in human papillomavirus-positive penile cancer cells

Penile squamous cell cancer (PSCC) is the most frequent penile malignant disease. Infections with human papillomaviruses (HPV) are a major etiologic driver of PSCC. However, the molecular details of the underlying carcinogenesis are understudied because of rare clinical specimens and missing cell lines. Here, we investigated if the expression of high-risk HPV16 oncogenes causes an augmentation of the Wnt pathway using unique HPV-positive penile cancer (PeCa) cell lines in monolayer and organotypic 3D raft cultures as well as tissue micro arrays containing clinical tissue specimens. The HPV oncoproteins enhanced the expression of Leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) and the HPV-positive PeCa cells expressed a signature of Wnt target and stemness-associated genes. However, the notable lack of nuclear β-catenin in vitro and in situ raised the question if the enhanced expression of Wnt pathway factors is tantamount to an active Wnt signaling. Subsequent TOP-flash reporter assays revealed Wnt signaling as absent and not inducible by respective Wnt ligands in PeCa cell lines. The HPV-positive PeCa cells and especially HPV-positive PeCa specimens of the tumor core expressed the Wnt antagonist and negative feedback-regulator Dickkopf1 (DKK1). Subsequent neutralization experiments using PeCa cell line-conditioned media demonstrated that DKK1 is capable to impair ligand-induced Wnt signaling. While gene expression analyses suggested an augmented and active canonical Wnt pathway, the respective signaling was inhibited due to the endogenous expression of the antagonist DKK1. Subsequent TMA stainings indicated Dkk1 as linked with HPV-positivity and metastatic disease progression in PeCa suggesting potential as a prognostic marker

Generation of glucocorticoid-producing cells derived from human pluripotent stem cells.

Adrenal insufficiency is a life-threatening condition resulting from the inability to produce adrenal hormones in a dose- and time-dependent manner. Establishing a cell-based therapy would provide a physiologically responsive approach for the treatment of this condition. We report the generation of large numbers of human-induced steroidogenic cells (hiSCs) from human pluripotent stem cells (hPSCs). Directed differentiation of hPSCs into hiSCs recapitulates the initial stages of human adrenal development. Following expression of steroidogenic factor 1, activation of protein kinase A signaling drives a steroidogenic gene expression profile most comparable to human fetal adrenal cells, and leads to dynamic secretion of steroid hormones, in vitro. Moreover, expression of the adrenocorticotrophic hormone (ACTH) receptor/co-receptor (MC2R/MRAP) results in dose-dependent ACTH responsiveness. This protocol recapitulates adrenal insufficiency resulting from loss-of-function mutations in AAAS, which cause the enigmatic triple A syndrome. Our differentiation protocol generates sufficient numbers of hiSCs for cell-based therapy and offers a platform to study disorders causing adrenal insufficiency

Identification of de novo variants in nonsyndromic cleft lip with/without cleft palate patients with low polygenic risk scores

[Background]: Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is a congenital malformation of multifactorial etiology. Research has identified >40 genome-wide significant risk loci, which explain less than 40% of nsCL/P heritability. Studies show that some of the hidden heritability is explained by rare penetrant variants. [Methods]: To identify new candidate genes, we searched for highly penetrant de novo variants (DNVs) in 50 nsCL/P patient/parent-trios with a low polygenic risk for the phenotype (discovery). We prioritized DNV-carrying candidate genes from the discovery for resequencing in independent cohorts of 1010 nsCL/P patients of diverse ethnicities and 1574 population-matched controls (replication). Segregation analyses and rare variant association in the replication cohort, in combination with additional data (genome-wide association data, expression, protein–protein-interactions), were used for final prioritization. [Conclusion]: In the discovery step, 60 DNVs were identified in 60 genes, including a variant in the established nsCL/P risk gene CDH1. Re-sequencing of 32 prioritized genes led to the identification of 373 rare, likely pathogenic variants. Finally, MDN1 and PAXIP1 were prioritized as top candidates. Our findings demonstrate that DNV detection, including polygenic risk score analysis, is a powerful tool for identifying nsCL/P candidate genes, which can also be applied to other multifactorial congenital malformations.The present study was supported by the German Research Foundation (DFG)-Grants BE 3828/8-1, LU 1944/2-1, MA 2546/5-1, and LU1944/3-1

Methylation of NR3C1 is related to maternal PTSD, parenting stress and maternal medial prefrontal cortical activity in response to child separation among mothers with histories of violence exposure.

Prior research has shown that mothers with Interpersonal violence-related posttraumatic stress disorder (IPV-PTSD) report greater difficulty in parenting their toddlers. Relative to their frequent early exposure to violence and maltreatment, these mothers display dysregulation of their hypothalamic pituitary adrenal axis (HPA-axis), characterized by hypocortisolism. Considering methylation of the promoter region of the glucocorticoid receptor gene NR3C1 as a marker for HPA-axis functioning, with less methylation likely being associated with less circulating cortisol, the present study tested the hypothesis that the degree of methylation of this gene would be negatively correlated with maternal IPV-PTSD severity and parenting stress, and positively correlated with medial prefrontal cortical (mPFC) activity in response to video-stimuli of stressful versus non-stressful mother-child interactions. Following a mental health assessment, 45 mothers and their children (ages 12-42 months) participated in a behavioral protocol involving free-play and laboratory stressors such as mother-child separation. Maternal DNA was extracted from saliva. Interactive behavior was rated on the CARE-Index. During subsequent fMRI scanning, mothers were shown films of free-play and separation drawn from this protocol. Maternal PTSD severity and parenting stress were negatively correlated with the mean percentage of methylation of NR3C1. Maternal mPFC activity in response to video-stimuli of mother-child separation versus play correlated positively to NR3C1 methylation, and negatively to maternal IPV-PTSD and parenting stress. Among interactive behavior variables, child cooperativeness in play was positively correlated with NR3C1 methylation. Thus, the present study is the first published report to our knowledge, suggesting convergence of behavioral, epigenetic, and neuroimaging data that form a psychobiological signature of parenting-risk in the context of early life stress and PTSD

The association of serotonin receptor 3A methylation with maternal violence exposure, neural activity, and child aggression

Background Methylation of the serotonin 3A receptor gene (HTR3A) has been linked to child maltreatment and adult psychopathology. The present study examined whether HTR3A methylation might be associated with mothers' lifetime exposure to interpersonal violence (IPV), IPV-related psychopathology, child disturbance of attachment, and maternal neural activity. Methods Number of maternal lifetime IPV exposures and measures of maternal psychopathology including posttraumatic stress disorder (PTSD), major depression and aggressive behavior (AgB), and a measure of child attachment disturbance known as “secure base distortion” (SBD) were assessed in a sample of 35 mothers and children aged 12–42 months. Brain fMRI activation was assessed in mothers using 30-s silent film excerpts depicting menacing adult male-female interactions versus prosocial and neutral interactions. Group and continuous analyses were performed to test for associations between clinical and fMRI variables with DNA methylation. Results Maternal IPV exposure-frequency was associated with maternal PTSD; and maternal IPV-PTSD was in turn associated with child SBD. Methylation status of several CpG sites in the HTR3A gene was associated with maternal IPV and IPV-PTSD severity, AgB and child SBD, in particular, self-endangering behavior. Methylation status at a specific CpG site (CpG2_III) was associated with decreased medial prefrontal cortical (mPFC) activity in response to film-stimuli of adult male-female interactions evocative of violence as compared to prosocial and neutral interactions. Conclusions Methylation status of the HTR3A gene in mothers is linked to maternal IPV-related psychopathology, trauma-induced brain activation patterns, and child attachment disturbance in the form of SBD during a sensitive period in the development of self-regulation