Maturation Markers Research Articles

Striking a balance: new perspectives on homeostatic dendritic cell maturation.

Dendritic cells (DCs) are crucial gatekeepers of the balance between immunity and tolerance. They exist in two functional states, immature or mature, that refer to an information-sensing versus an information-transmitting state, respectively. Historically, the term DC maturation was used to describe the acquisition of immunostimulatory capacity by DCs following their triggering by pathogens or tissue damage signals. As such, immature DCs were proposed to mediate tolerance, whereas mature DCs were associated with the induction of protective T cell immunity. Later studies have challenged this view and unequivocally demonstrated that two distinct modes of DC maturation exist, homeostatic and immunogenic DC maturation, each with a distinct functional outcome. Therefore, the mere expression of maturation markers cannot be used to predict immunogenicity. How DCs become activated in homeostatic conditions and maintain tolerance remains an area of intense debate. Several recent studies have shed light on the signals driving the homeostatic maturation programme, especially in the conventional type 1 DC(cDC1) compartment. Here, we highlight our growing understanding of homeostatic DC maturation and the relevance of this process for immune tolerance.

Human Skeletal Muscle Myoblast Culture in Aligned Bacterial Nanocellulose and Commercial Matrices.

Bacterial nanocellulose (BNC) is a durable, flexible, and dynamic biomaterial capable of serving a wide variety of fields, sectors, and applications within biotechnology, healthcare, electronics, agriculture, fashion, and others. BNC is produced spontaneously in carbohydrate-rich bacterial culture media, forming a cellulosic pellicle via a nanonetwork of fibrils extruded from certain genera. Herein, we demonstrate engineering BNC-based scaffolds with tunable physical and mechanical properties through postprocessing. Human skeletal muscle myoblasts (HSMMs) were cultured on these scaffolds, and in vitro electrical stimulation was applied to promote cellular function for tissue engineering applications. We compared physiologic maturation markers of human skeletal muscle myoblast development using a 2.5-dimensional culture paradigm in fabricated BNC scaffolds, compared to two-dimensional (2D) controls. We demonstrate that the culture of human skeletal muscle myoblasts on BNC scaffolds developed under electrical stimulation produced highly aligned, physiologic morphology of human skeletal muscle myofibers compared to unstimulated BNC and standard 2D culture. Furthermore, we compared an array of metrics to assess the BNC scaffold in a rigorous head-to-head study with commercially available, clinically approved matrices, Kerecis Omega3 Wound Matrix (Marigen) and Phoenix as well as a gelatin methacryloyl (GelMA) hydrogel. The BNC scaffold outcompeted industry standard matrices as well as a 20% GelMA hydrogel in durability and sustained the support of human skeletal muscle myoblasts in vitro. This work offers a robust demonstration of BNC scaffold cytocompatibility with human skeletal muscle cells and sets the basis for future work in healthcare, bioengineering, and medical implant technological development.

Seminal plasma inhibits Chlamydia trachomatis infection in vitro, and may have consequences on mucosal immunity

Seminal plasma (SP) is the main vector of C. trachomatis (CT) during heterosexual transmission from male to female. It has immunomodulatory properties and impacts the susceptibility to HIV-1 infection, but its role has not been explored during CT infection. In the female reproductive tract (FRT), CT infection induces cytokine production and neutrophil recruitment. The role of neutrophils during CT infection is partially described, they could be at the origin of the pathology observed during CT infection. During this study, we developed an experimental in vitro model to characterize the impact of CT infection and SP on endocervical epithelial cell immune response in the FRT. We also studied the impact of the epithelial cell response on neutrophil phenotype and functions. We showed that the production by epithelial cells of pro-inflammatory cytokines increased during CT infection. Moreover, the pool of SP as well as individuals SP inhibited CT infection in a dose-dependent manner. The pool of SP inhibited cytokine production in a dose-dependent manner. The pool of SP altered gene expression profiles of infected cells. The culture supernatants of cells infected or not with CT, in presence or not of the pool of SP, had an impact on neutrophil phenotype and functions: they affected markers of neutrophil maturation, activation and adhesion capacity, as well as the survival, ROS production and phagocytosis ability. This study proposes a novel approach to study the impact of the environment on the phenotype and functions of neutrophils in the FRT. It highlights the impact of the factors of the FRT environment, in particular SP and CT infection, on the mucosal inflammation and the need to take into account the SP component while studying sexually transmitted infections during heterosexual transmission from male to female.

Alteration of mature neuronal marker of β-III tubulin expression in differentiated SH-SY5Y cells by refinement of foetal bovine serum concentration

BackgroundThe process of differentiating neuroblastoma cells (SH-SY5Y) is crucial for obtaining mature neuronal markers. However, there is variability in the concentrations of foetal bovine serum (FBS) used in the differentiation media, ranging from 1 to 10%. This inconsistency in FBS concentrations may contribute to the inconsistent differentiation of cells. To improve the utility of the SH-SY5Y cell line as a model for neuronal cell culture, we investigated the impact of FBS concentrations in the differentiation media using Dulbecco's modified eagle medium and 10 μM all-trans-retinoic acid (ATRA). The aim of this study was to optimise the concentrations of FBS in the differentiation media of SH-SY5Y cells. Our study focused on assessing the length of dendrites in neuronal cells and the expression of β-III tubulin, a marker indicative of mature neurons. SH-SY5Y cells were differentiated with 10 µM of ATRA for 7 days. Four treatment groups with different FBS concentrations (1%, 3%, 5%, and 10%) were examined to assess the alteration of cellular morphology and dendritic length. The expression of the mature neuron marker β-III tubulin was evaluated using immunocytochemistry technique.ResultsSH-SY5Y cells' dendrite length was significantly longer (p < 0.05) when there was a higher concentration of FBS in the differentiation medium. The result was confirmed with the significant increase of β-III tubulin expression (p < 0.001) of the differentiated SH-SY5Y cells that have been incubated with higher concentrations of FBS in the differentiation medium.ConclusionsWe concluded that optimised concentrations of FBS in the differentiation media display longer length of the dendrites and express higher production of β-III tubulin in the differentiated SH-SY5Y cells. The consistency of serum concentration used in the differentiation media is important to produce a sustainable in vitro neuronal model of SH-SY5Y cells for neurodegenerative studies.

Accelerated maturation of ARPE-19 cells for the translational assessment of gene therapy.

The human retinal pigment epithelium (RPE) cell line ARPE-19 is widely used as an alternative to primary RPE despite losing many features of primary RPE. We aimed to determine whether a combination of RPE-specific laminin (LN) and nicotinamide (NAM) could improve ARPE-19 redifferentiation to resemble mature RPE and improve the assessment of RPE-specific gene therapy strategies. ARPE-19 cells were propagated on tissue culture plastic supplemented with NAM and human recombinant LN521-coating. RPE maturation was performed by immunocytochemistry and gene expression by qPCR. Viral transduction experiments with adeno-associated virus (AAV)1 or AAV2, carrying a VMD2-driven GFP, were assessed at 2- and 4-weeks post-plating in the different culturing conditions with a low multiplicity of infection. The combination of LN521 coating with NAM supplementation promoted cytoskeletal and tight junction protein reorganization. The expression of maturation markers bestrophin-1 and RPE 65 was promoted concomitantly with a reduction of several epithelial-mesenchymal transition markers, such as TNF-α, TGF-β, CDH2, and vimentin. Redifferentiated ARPE-19 transduced at low multiplicity of infection of both AAV1- and AAV2-VMD2-GFP. Expression of GFP was detected at 2 weeks and increased at 4 weeks post-plating. AAV1 exhibited a greater expression efficacy compared to AAV2 in maturated ARPE-19 cells already after 2 weeks with increased efficiency after 4 weeks. Our study demonstrates an improved maturation protocol for ARPE-19 cells invitro, mimicking an invivo phenotype with the expression of signature genes and improved morphology. Viral-mediated RPE-specific gene expression demonstrates that the combination cultures mimic invivo AAV tropism essential to test new gene therapies for RPE-centered diseases.

NingXueShengBan Decoction Regulates Immune Homeostasis by PI3 K/Akt/mTOR and TLR4/NF-κB Signaling Pathways in Immune Thrombocytopenia Patients

Objective Immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disease. The immune imbalance of T cells is an essential pathological mechanism of ITP, more specific, the balance of Effector T cells (Teff)/Regulatory T cell (Treg) maintains T cell immune homeostasis. Dendritic cells (DC), the largest antigen-presenting cell, interfere with T cell differentiation pathways and exert immune regulatory functions. Our team created and applied the “ NingXueShengBan” (NXSB) decoction. According to our prior studies, we found that NXSB decoction could restore Th17/Treg balance in ITP mice by regulating protein expression in Notch pathway. Therefore, we further research the mechanism of NXSB decoction that regulates ITP immune homeostasis through affecting the interaction between DC and T cells. Methods In this study, we collected peripheral blood from health donors and ITP patients and extracted DC and CD4 + T cells then grouped in control group, model group and NXSB group. Moreover, we established a co-culture model simulating the immune environment in ITP patient to further investigate whether NXSB had therapeutic effects by the interaction between DC and CD4 + T cells. Results In the current study, we found that NXSB decoction potently regulated interaction between DC and T cells by inhibiting DC development and function, specifically, the differentiation of CD4+ T cells was inhibited after NXSB decoction intervened. Our data showed that NXSB decoction inhibited Toll-like receptor 4 (TLR-4), reduced the expression of Nuclear factor kappa-B (NF-κB) and led to the reduction of downstream inflammatory factors, thereby inhibiting the inflammatory response. More importantly, NXSB decoction reduced the DC surface expression of activation and maturation markers. Moreover, we detected that the decreased expression of the phosphorylated Phosphatidylinositol 3-kinase (PI3 K), Protein kinase B (Akt), Mammalian target of rapamycin (mTOR), and their downstream effectors indicated, we found that NXSB decoction could inhibit PI3 K/Akt/mTOR signaling pathways significantly. Conclusions Collectively, our data suggested that NXSB decoction ameliorates ITP-induced immune imbalance via its anti-inflammation and immunosuppressive by regulating TLR-4/ NF-κB and PI3 K/Akt/mTOR signaling pathways.

Identification of key regulatory genes involved in myelination after spinal cord injury by GSEA analysis

Multilayer dense myelin tissue provides insulating space and nutritional support for axons in healthy spinal cord tissue. Oligodendrocyte precursor cells (OPCs) are the main glial cells that complement myelin loss in the central nervous system and play an important role in the repair of spinal cord injury (SCI). However, the regulation of axonal remyelination after SCI is still insufficient. In this study, we focused on the changes in genes related to myelin repair after rat hemisection SCI by gene set enrichment analysis (GSEA). Key genes proteolipid protein 1 (Plp1), hexosaminidase subunit alpha (Hexa), and hexosaminidase subunit beta (Hexb) during remyelination after SCI were found. Through quantitative real-time polymerase chain reaction (qPCR) experiments, we confirmed that within 28 days after rat hemisection SCI, the mRNA expression of gene Plp1 gradually decreased, while the expressions of gene Hexa and Hexb gradually increased, which was consistent with RNA sequencing results. In vitro, we performed EdU proliferation assays on OPC cell line OLN-93 and primary rat OPCs. We found that interference of Plp1 promoted OPC proliferation, while interference of Hexa and Hexb inhibited OPC proliferation. In addition, we performed in vitro differentiation experiments on primary rat OPCs. By measuring myelin sheath branch outgrowth and the fluorescence intensity of the mature myelin sheath marker myelin basic protein (MBP), we found that interference of Hexa or Hexb promoted OPC differentiation and maturation, but interference of Plp1 inhibited this process. Finally, we injected Hexb siRNA in vivo and found that interfering Hexb could improve motor movements and myelin regeneration after SCI in rats. Our results provide new target genes that can selectively regulate the proliferation and differentiation of endogenous OPCs, providing new ideas for promoting remyelination and functional recovery after SCI.

Integrative analysis of long isoform sequencing and functional data identifies distinct cortical layer neuronal subtypes derived from human iPSCs.

Generation of human induced pluripotent stem cells (iPSCs) through reprogramming was a transformational change in the field of regenerative medicine that led to new possibilities for drug discovery and cell replacement therapy. Several protocols have been established to differentiate hiPSCs into neuronal lineages. However, low differentiation efficiency is one of the major drawbacks of these approaches. Here, we compared the efficiency of two methods of neuronal differentiation from iPSCs cultured in two different culture media, StemFlex Medium (SFM) and Essential 8 Medium (E8M). The results indicated that iPSCs cultured in E8M efficiently generated different types of neurons in a shorter time and without the growth of undifferentiated nonneuronal cells in the culture as compared with those generated from iPSCs in SFM. Furthermore, these neurons were validated as functional units immunocytochemically by confirming the expression of mature neuronal markers (i.e., NeuN, β tubulin, and Synapsin I) and whole cell patch-clamp recordings. Long-read single-cell RNA sequencing confirms the presence of upper and deep layer cortical layer excitatory and inhibitory neuronal subtypes in addition to small populations of GABAergic neurons in day 30 neuronal cultures. Pathway analysis indicated that our protocol triggers the signaling transcriptional networks important for the process of neuronal differentiation in vivo.NEW & NOTEWORTHY Low differentiation efficiency is one of the major drawbacks of the existing protocols to differentiate iPSCs into neuronal lineages. Here, we present time-efficient and robust approach of neuronal differentiation leading to the generation of functional brain units, cortical layer neurons. We found iPSCs cultured in Essential 8 media (E8M) resulted in neuronal differentiation without the signs of growth of spontaneously differentiated cells in culture at any point in 35 days compared with Stemflex media (SFM).

Directed differentiation of human embryonic stem cells into conjunctival epithelial cells

Severe conjunctival damage can lead to extensive ocular cicatrisation, fornix shortening, and even ocular surface failure, resulting in significant vision impairment. Conjunctival reconstruction is the primary therapeutic strategy for these clinical conjunctival diseases. However, there have been limited studies on induced differentiation of conjunctival epithelial cells derived from stem cells. In this study, we established a chemical defined differentiation protocol from human embryonic stem cells (hESCs) into conjunctival epithelial cells. hES cell line H1 was used for differentiation, and RT-qPCR, immunofluorescence staining, Periodic-acid-Schiff staining (PAS), and transcriptome analysis were employed to identify the differentiated cells. Here, to imitate the development of the vertebrate conjunctiva, hESCs were induced using a three-step process involving first chetomin was used to induce ocular surface ectoderm, then nicotinamide was used to induce ocular surface epithelial progenitor cells, and finally epidermal growth factor, keratinocyte growth factor and other factors were used to differentiate mature conjunctival epithelial cells. hESC-derived conjunctival epithelial cells expressed mature conjunctival epithelial lineage markers (including PAX6, P63, K13). The presence of goblet cells was confirmed by positive PAS. Transcriptome analysis revealed that hESC-derived conjunctival epithelial cells possessed a more naïve phenotype, and exhibited greater proliferation capacity compared to mature human conjunctival epithelial cells, suggesting their potential as alternative seed cells for conjunctival reconstruction.

Hippocampal neurogenesis modulated by Quinic acid: A therapeutic strategy for the neurodegenerative disorders.

Neural progenitor cells (NPCs) reside in the brain and participate in the mechanism of neurogenesis that permits the brain to generate the building blocks for enhancement of cognitive abilities and acquisition of new skills. The existence of NPCs in brain has opened a novel dimension of research to explore their potential for treatment of various neurodegenerative disorders. The present study provides novel insights into the intracellular mechanisms in neuronal cells proliferation, maturation and differentiation regulated by Quinic acid (QA). Furthermore, this study might help in discovery and development of lead molecule that can overcome the challenges in the treatment of neurodegenerative diseases. The growth supporting effect of QA was studied using MTT assay. For that purpose, hippocampal cell cultures of neonatal rats were treated with different concentrations of QA and incubated for 24, 48 and 72 h. Gene and protein expressions of the selected molecular markers nestin, neuron-specific class III beta-tubulin (Tuj-1), neuronal nuclear protein (NeuN), neuronal differentiation 1 (NeuroD1), glial fibrillary acidic protein (GFAP), neuroligin (NLGN) and vimentin were analyzed. QA-induced cell proliferation and differentiation of hippocampal progenitor cells was also accompanied by significantly increased expression of progenitor and immature neuronal marker, mature neuronal marker and differentiating factor, that is, nestin, Tuj-1, NeuN and NeuroD1, respectively. On the other hand, vimentin downregulation and constant GFAP expression were observed following QA treatment. Additionally, the effects of QA on the recovery of stressed cells was studied using in vitro model of oxygen glucose deprivation (OGD). It was observed that hippocampal cells were able to recover from OGD following the treatment with QA. These findings suggest that QA treatment promotes hippocampal neurogenesis by proliferating and differentiating of NPCs and recovers neurons from stress caused by OGD. Thus, the neurogenic potential of QA can be explored for the treatment of neurodegenerative disorders.

Abstract We046: Building Mass or Strength: Stage-specific Effects of Insulin and Wnt on Cell Fate Decisions in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Background: Reactivation of the so-called fetal gene programs and pathways have been reported in failing hearts, however, it remains unclear whether this phenomenon is beneficial or detrimental to the remaining functional myocardium. During cardiac development, the orchestration of these embryonic growth pathways, facilitate temporal and stage-specific cell fate decisions such as required for specification and growth of the various myocardial compartments. Previously, we demonstrated that molecular activation of the Wnt pathway resulted in massive expansion of immature human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Yet, it is unknown what exact cues guide the cell fate decisions of immature hiPSC-CMs to either proliferate or terminally differentiate into more mature cardiomyocytes. Purpose: This work uncovers the embryonic growth pathways that control the cues for proliferation versus terminal differentiation in functional immature hiPSC-CMs. Methods: We utilised spontaneously beating hiPSC-CMs at day 11 of differentiation and performed a combinatory screen for various dosages of CHIR99021/Wnt and Insulin/Akt pathway. Proliferation was determined by quantification of cell number and the proportion of Ki67 positive hiPSC-CMs. Terminal differentiation was evaluated by presence of more mature sarcomere markers and cell cycle exit. Results: Our combinatory screen for Insulin/Akt and CHIR99021/Wnt demonstrates synergistic effects on proliferation of immature hiPSC-CMs, whereas the absence of these pathway activators leads to rapid cell cycle exit and terminal differentiation of hiPSC-CMs. Detailed characterization reveals that CHIR99021/Wnt also importantly regulates sarcomere homeostasis and architecture in hiPSC-CMs. Moreover, we identify a novel temporal interplay between CHIR99021/Wnt via TCF and Insulin/Akt via FOXO as regulators of cell-fate decision in immature hiPSC-CMs. Conclusions: Molecular targeting of the embryonic growth pathways in acute and chronic forms of heart failure requires a detailed understanding of the specific effects on cardiomyocyte function and sarcomere architecture. This work provides a starting point to develop future strategies to target acute and chronic forms of heart failure via modulation of embryonic growth pathways.

Abstract We123: Cardiomyocyte Specific Sustained Atg7 Overexpression Activates Mitochondrial Autophagy in the Heart

Introduction: Accumulating evidence indicates that autophagy-related protein 7 (Atg7) is essential in cellular autophagy. Our earlier studies showed that Atg7 is required to promote macro-autophagy in the heart. However, it is unknown if the activation of Atg7 has a role in the heart to regulate selective mitochondrial autophagy (mitophagy). Objectives: We employed in vitro and in vivo approaches to delineate the molecular function and mechanisms of Atg7-mediated activation of mitophagy in the heart. Methods and Results: Subcellular fractionation and quantum dot-labeled electron microscopy revealed Atg7’s localization on the mitochondrial fraction in the heart. Atg7 co-localization to Mitotracker-labeled mitochondria was confirmed using isolated heart mitochondria. To quantitate the mitophagy, we overexpressed Atg7 in cultured primary cardiomyocytes, treated with cyanide 3-chlorophenylhydrazone (CCCP) to induce mitophagy, and stained with pH-sensitive fluorescent mitophagy dye to enumerate mitophagy dots by microscopy. We found significantly increased mitophagy dots in Atg7 overexpressed cardiomyocytes than in β-gal expressing control. To assess the activation of mitophagy in vivo , we treated the cardiac-specific Atg7 overexpressing transgenic (αMHC-Atg7 Tg) and their littermate non-transgenic (Ntg) mice with CCCP. We found an increased accumulation of mature autophagolysosomal marker protein LC3-II in isolated mitochondria from Atg7 Tg mice hearts post-CCCP treatment. To assess the extent of mitochondrial lysosomal delivery in Atg7 overexpressing hearts, we crossed αMHC-Atg7 Tg mice with cardiac-specific Mito-Keima transgenic (Mt-Keima Tg) mice. Mt-Keima Tg mice bear mitochondria-targeted pH-dependent fluorescent lysosomal protease-resistance Keima protein, enabling the measurement of mitophagy activation in vivo . We observed increased Mt-Keima high red-to-green areas in αMHC-Atg7xMt-Keima Tg mice hearts compared to Mt-Keima mice. Conclusions: Our biochemical and genetic approaches revealed that Atg7 localizes to the mitochondria, leading to the recruitment and initiation of mitophagy machinery. Our data further confirm that genetic activation of Atg7 leads to increased mitophagy activity in the heart.

Experimental study on small molecule combinations inducing reprogramming of rat fibroblasts into functional neurons.

To establish a methodological system for reprogramming rat embryonic fibroblasts (REF) into chemically induced neurons (ciNCs) via small molecule compounds to provide safe and effective donor cells for treatment of neurodegenerative diseases. Based on the method established by PEI Gang's research group to directly reprogram human fibroblasts into neurons, the induction medium and maturation medium was optimized by replacing the coating solution, mitigating oxidative stress injury, adding neurogenic protective factors, adjusting the concentration of trichothecenes, performing small-molecule removal experiments, and carrying out immunofluorescence and Western blotting on cells at different stages of induction to validate the effect of induction. When the original protocol was used for induction, the cell survival rate was (34.24±2.77)%. After replacing the coating solution gelatin with matrigel, the cell survival rate increased to (45.41±4.27)%; after adding melatonin, the cell survival rate increased to (67.95±5.61)% and (23.43±1.42)% were transformed into neural-like cells; after adding the small molecule P7C3-A20, the cell survival rate was further increased to (76.27±1.41)%, and (39.72±4.75)% of the cells were transformed into neural-like cells. When the concentration of trichothecene was increased to 30 μmol/L, the proportion of neural-like cells reached (55.79±1.90)%; after the removal of SP600125, (86.96±2.15)% of the cells survived, and the rate of neural-like cell production increased to (63.43±1.60)%. With the optimized protocol, REF could be successfully induced into ciNC through the neural precursor cell stage, in which the neural precursor cells were able to highly express the neural precursor cell markers SRY-related HMG-box gene 2 (Sox2) and paired box 6 (Pax6) as well as neuron-specific marker tubulin 1 (Tuj1), while the expression of fiber-associated protein vimentin was reduced. After two weeks of induction of neural precursor cells in a maturation medium, most cells displayed neuronal-like cell morphology. The induced ciNCs were able to highly express the mature neuronal surface markers Tuj1 and microtubule-associated protein 2 (MAP2), while the expression of vimentin was reduced. The small molecule combinations optimized in this study can reprogram REF to ciNCs under normoxic conditions.

Mass cytometric single cell immune profiles of peripheral blood from acute myeloid leukemia patients in complete remission with measurable residual disease.

Measurable residual disease (MRD) is detected in approximately a quarter of AML chemotherapy responders, serving as a predictor for relapse and shorter survival. Immunological control of residual disease is suggested to prevent relapse, but the mechanisms involved are not fully understood. We present a peripheral blood single cell immune profiling by mass cytometry using a 42-antibody panel with particular emphasis on markers of cellular immune response. Six healthy donors were compared with four AML patients with MRD (MRD+) in first complete remission (CR1MRD+). Three of four patients demonstrated a favorable genetic risk profile, while the fourth patient had an unfavorable risk profile (complex karyotype, TP53-mutation) and a high level of MRD. Unsupervised clustering using self-organizing maps and dimensional reduction analysis was performed for visualization and analysis of immune cell subsets. CD57+ natural killer (NK)-cell subsets were found to be less abundant in patients than in healthy donors. Both T and NK cells demonstrated elevated expression of activity and maturation markers (CD44, granzyme B, and phosho-STAT5 Y694) in patients. Although mass cytometry remains an expensive method with limited scalability, our data suggest the utility for employing a 42-plex profiling for cellular immune surveillance in whole blood, and possibly as a biomarker platform in future clinical trials. The findings encourage further investigations of single cell immune profiling in CR1MRD+ AML-patients.

Obesity-induced upregulation of miR-483-5p impairs the function and identity of pancreatic β-cells.

To assess the expression and function of miR-483-5p in diabetic β cells. The expression of miR-483-5p was evaluated in the pancreatic islets of obesity mouse models by quantitative reverse transcription polymerase chain reaction. Dual-luciferase activity, and western blotting assays, were utilized for miR-483-5p target gene verification. Mice with β cell-specific miR-483-5p downregulation were studied under metabolic stress (i.e. a high-fat diet) condition. Lineage tracing was used to determine β-cell fate. miR-483-5p increased in the islets of obese mouse models. Expression levels of miR-483-5p were significantly upregulated with the treatment of high glucose and palmitate, in both MIN6 cells and mouse islets. Overexpression of miR-483-5p in β cells results in impaired insulin secretion and β-cell identity. Cell lineage-specific analyses revealed that miR-483-5p overexpression deactivated β-cell identity genes (insulin, Pdx1 and MafA) and derepressed β-cell dedifferentiation (Ngn3) genes. miR-483-5p downregulation in β cells of high-fat diet-fed mice alleviated diabetes and improved glucose intolerance by enhancing insulin secretory capacity. These detrimental effects of miR-483-5p relied on its seed sequence recognition and repressed expression of its target genes Pdx1 and MafA, two crucial markers of β-cell maturation. These findings indicate that the miR-483-5p-mediated reduction of mRNAs specifies β-cell identity as a contributor to β-cell dysfunction via the loss of cellular differentiation.

Pharmacologic Hedgehog inhibition modulates the cytokine profile of osteolytic breast cancer cells

The establishment and progression of bone metastatic breast cancer is supported by immunosuppressive myeloid populations that enable tumor growth by dampening the innate and adaptive immune response. Much work remains to understand how to target these tumor-myeloid interactions to improve treatment outcomes. Noncanonical Hedgehog signaling is an essential component of bone metastatic tumor progression, and prior literature suggests a potential role for Hedgehog signaling and its downstream effector Gli2 in modulating immune responses. In this work, we sought to identify if inhibition of noncanonical Hedgehog signaling alters the cytokine profile of osteolytic breast cancer cells and the subsequent communication between the tumor cells and myeloid cells. Examination of large patient databases revealed significant relationships between Gli2 expression and expression of markers of myeloid maturation and activation as well as cytokine expression. We found that treatment with HPI-1 reduced tumor cell expression of numerous cytokine genes, including CSF1, CSF2, and CSF3, as well as CCL2 and IL6. Secreted CSF-1 (M−CSF) was also reduced by treatment. Changes in tumor-secreted factors resulted in polarization of THP-1 monocytes toward a proinflammatory phenotype, characterized by increased CD14 and CD40 surface marker expression. We therefore propose M−CSF as a novel target of Hedgehog inhibition with potential future applications in altering the immune microenvironment in addition to its known roles in reducing tumor-induced bone disease.

Analysis of beta-cell maturity and mitochondrial morphology in juvenile non-human primates exposed to maternal Western-style diet during development.

Using a non-human primate (NHP) model of maternal Western-style diet (mWSD) feeding during pregnancy and lactation, we previously reported altered offspring beta:alpha cell ratio in vivo and insulin hyper-secretion ex vivo. Mitochondria are known to maintain beta-cell function by producing ATP for insulin secretion. In response to nutrient stress, the mitochondrial network within beta cells undergoes morphological changes to maintain respiration and metabolic adaptability. Given that mitochondrial dynamics have also been associated with cellular fate transitions, we assessed whether mWSD exposure was associated with changes in markers of beta-cell maturity and/or mitochondrial morphology that might explain the offspring islet phenotype. We evaluated the expression of beta-cell identity/maturity markers (NKX6.1, MAFB, UCN3) via florescence microscopy in islets of Japanese macaque pre-adolescent (1 year old) and peri-adolescent (3-year-old) offspring born to dams fed either a control diet or WSD during pregnancy and lactation and weaned onto WSD. Mitochondrial morphology in NHP offspring beta cells was analyzed in 2D by transmission electron microscopy and in 3D using super resolution microscopy to deconvolve the beta-cell mitochondrial network. There was no difference in the percent of beta cells expressing key maturity markers in NHP offspring from WSD-fed dams at 1 or 3 years of age; however, beta cells of WSD-exposed 3 year old offspring showed increased levels of NKX6.1 per beta cell at 3 years of age. Regardless of maternal diet, the beta-cell mitochondrial network was found to be primarily short and fragmented at both ages in NHP; overall mitochondrial volume increased with age. In utero and lactational exposure to maternal WSD consumption may increase mitochondrial fragmentation. Despite mWSD consumption having clear developmental effects on offspring beta:alpha cell ratio and insulin secretory response to glucose, this does not appear to be mediated by changes to beta-cell maturity or the beta-cell mitochondrial network. In general, the more fragmented mitochondrial network in NHP beta cells suggests greater ability for metabolic flexibility.

A Unique Comprehensive Model to Screen Newborns for Severe Combined Immunodeficiency-An Ontario Single-Centre Experience Spanning 2013-2023.

Severe combined immunodeficiency (SCID) is a life-threatening genetic disorder caused by critical defects of the immune system. Almost all cases are lethal if not treated within the first two years of life. Early diagnosis and intervention are thus essential for improving patient outcomes. In 2013, Ontario became the first Canadian province to perform newborn screening (NBS) for SCID by T cell receptor excision circles (TRECs) analysis, a surrogate marker of thymic function and lymphocyte maturation. This retrospective study reports on nearly 10 years of NBS for SCID at a quaternary referral centre. From August 2013 to April 2023, our centre's densely populated catchment area flagged 162 newborns with low TRECs levels, including 10 cases with SCID. Follow-up revealed other causes of low TRECs, including non-SCID T cell lymphopenia (secondary/reversible or idiopathic causes, and syndromic conditions) and prematurity. A small number of cases with normal repeat TRECs levels and/or T cell subsets were also flagged. Province-wide data from around this period revealed at least 24 diagnosed cases of SCID or Leaky SCID. This is the first report of NBS outcomes in a Canadian province describing the causative genetic defects, and the non-SCID causes of a positive NBS for SCID.

Poor Eating Habits and Low Physical Activity Contribute to Weight Excess and Increase Cardiometabolic Risk in Adolescents Practicing Soccer as a Recreational Sport.

Monitoring anthropometry, dietary intake, and physical activity (PA) is essential to prevent/counteract childhood obesity. This study examined dietary intake, PA, and anthropometric characteristics in young boys practicing soccer as a recreational sport. A cross-sectional study included 226 boys aged 8 to 13 years participating in soccer as a recreational activity in football academies located in Tunis, Tunisia. Anthropometric measures allowed the calculation of body mass index, fat mass, and peak height velocity as markers of biological maturity. A three-day food record and a food frequency questionnaire estimated nutrient intake and eating habits. The International Physical Activity Questionnaire (IPAQ) was used to estimate the PA level of the participants. It was found that a high percentage of the children had excess weight (54%) and excess fat mass (47%). The total energy, carbohydrate, and saturated fat intake of the children exceeded the recommended levels by approximately 10%, 15%, and 30%, respectively. However, the intake of unsaturated fat was below the estimated requirements, particularly in obese children. In addition to the unbalanced macronutrient intake, the children also showed an insufficient intake of many essential micronutrients. Around 60% to 70% of the children in all three groups had a low intake of magnesium, n-3 polyunsaturated fatty acids (PUFA), and vitamins B9, B12, and D. Moreover, 20% to 35% of the children in the three groups had an insufficient intake of vitamins A and C. Insufficient vitamin E intake was found in 63% of obese children and 35% of non-obese children. It was observed that the PA level was lower in the overweight/obese group compared to the normal-weight group (p < 0.005). More than three-quarters of overweight/obese children had low PA levels, about 20% were moderately active, and only 1 to 2% were highly active. Conversely, normal-weight children showed moderate to high PA levels. Poor eating behavior, an unbalanced diet, and a low PA level are prevalent in Tunisian boys practicing recreational sports. Such a combination is responsible for a disparity between energy intake and expenditure, contributing to weight excess and increased cardiometabolic risk. The study findings provide meaningful information for practitioners and authorities on applying a balanced diet and adequate PA to prevent and fight against obesity and improve cardiometabolic health in youth.

Proposed complementary osteological indicators: Advancing the estimation of puberty stages in Bioarcheology.

The study of puberty is a well-established area of bioarcheological research, which greatly enhances our understanding of adolescence and growth in the past. Since the publications of Shapland and Lewis' works, which have become "standards" for estimating puberty in skeletal material, no additional osteological indicators of puberty have been proposed. Nevertheless, clinical practice constantly develops skeletal maturation markers that could be useful in bioarcheology. This study aims to assess the applicability and reliability of novel puberty indicators as a complementary tool to estimate puberty in skeletal remains. Four new maturation markers including spheno-occipital synchondrosis, humeral head ossification, calcaneal apophysis ossification, and mandibular premolar mineralization were selected and applied to a sample of 85 adolescents from pre-Roman southern Italy (Pontecagnano, 7th-4th BCE). Despite some limits in adapting the original clinical methods to osteoarcheological material, the use of these novel skeletal indicators had moderate to excellent scoring repeatability and an overall high agreement with the puberty and menarche status previously estimated with standard methods. These results encourage us to apply these markers in bioarcheology. In some cases, minor adaptations of the original scoring systems are suggested to enhance reliability. Including the proposed indicators in routine puberty data collection allows us to refine puberty estimation and improve the ability to identify key growth milestones in poorly preserved skeletons. Further application to osteological collections with diverse chronology and geographical differences is needed to assess how and to what extent the newly proposed maturation markers perform.