A novel cell source for therapy of knee osteoarthritis using atelocollagen microsphere-adhered adipose-derived stem cells: Impact of synovial fluid exposure on cell activity.

Introduction: Administration of adipose-derived stem cells (ADSCs) into the joint cavity has been shown to alleviate the symptoms of knee osteoarthritis (OA) by releasing exosomes and anti-inflammatory cytokines. However, the therapeutic effect of these cells is limited by their rapid disappearance after administration. Thus, it is necessary to prolong cell survival in the joint cavity. This study aimed to investigate the potential application of ADSCs adhered to atelocollagen microspheres (AMSs) for cell therapy of knee OA. Methods: ADSCs were cultured for 2, 4, and 7 days in AMS suspension or adherent culture dishes. The supernatants were analyzed for IL-10 and exosome secretion via enzyme-linked immunosorbent assay and Nanosight. The effect of AMS was compared with that of adherent-cultured ADSCs (2D-cultured ADSCs) using transcriptome analysis. Moreover, the solubility of AMS and viability of ADSCs were evaluated using synovial fluid (SF) from patients with knee OA. Results: Compared with 2D-cultured ADSCs, AMS-cultured ADSCs exhibited a significant increase in secretion of exosomes and IL-10, and the expression of several genes involved in extracellular matrix and immune regulation were altered. Furthermore, when AMS-cultured ADSCs were cultured in SF from knee OA patients to mimic the intra-articular environment, the SF dissolved the AMSs and released viable ADSCs. In addition, AMS-cultured ADSCs showed significantly higher long-term cell viability than 2D-cultured ADSCs. Conclusion: Increased survival of AMS-adhered ADSCs was observed in the intra-articular environment, and AMSs were found to gradually dissipate. These results suggest that AMS-adhered ADSCs are promising source for cell therapy of knee OA.

l-Type amino acid transporter 1 in hypothalamic neurons in mice maintains energy and bone homeostasis.

Hypothalamic neurons regulate body homeostasis by sensing and integrating changes in the levels of key hormones and primary nutrients (amino acids, glucose, and lipids). However, the molecular mechanisms that enable hypothalamic neurons to detect primary nutrients remain elusive. Here, we identified l-type amino acid transporter 1 (LAT1) in hypothalamic leptin receptor-expressing (LepR-expressing) neurons as being important for systemic energy and bone homeostasis. We observed LAT1-dependent amino acid uptake in the hypothalamus, which was compromised in a mouse model of obesity and diabetes. Mice lacking LAT1 (encoded by solute carrier transporter 7a5, Slc7a5) in LepR-expressing neurons exhibited obesity-related phenotypes and higher bone mass. Slc7a5 deficiency caused sympathetic dysfunction and leptin insensitivity in LepR-expressing neurons before obesity onset. Importantly, restoring Slc7a5 expression selectively in LepR-expressing ventromedial hypothalamus neurons rescued energy and bone homeostasis in mice deficient for Slc7a5 in LepR-expressing cells. Mechanistic target of rapamycin complex-1 (mTORC1) was found to be a crucial mediator of LAT1-dependent regulation of energy and bone homeostasis. These results suggest that the LAT1/mTORC1 axis in LepR-expressing neurons controls energy and bone homeostasis by fine-tuning sympathetic outflow, thus providing in vivo evidence of the implications of amino acid sensing by hypothalamic neurons in body homeostasis.

Olfactory Regeneration with Nasally Administered Murine Adipose-Derived Stem Cells in Olfactory Epithelium Damaged Mice.

In this study, we aimed to determine whether nasally administered murine adipose-derived stem cells (ADSCs) could support olfactory regeneration in vivo. Olfactory epithelium damage was induced in 8-week-old C57BL/6J male mice by intraperitoneal injection of methimazole. Seven days later, OriCell adipose-derived mesenchymal stem cells obtained from green fluorescent protein (GFP) transgenic C57BL/6 mice were nasally administered to the left nostril of these mice, and their innate odor aversion behavior to butyric acid was assessed. Mice showed significant recovery of odor aversion behavior, along with improved olfactory marker protein (OMP) expression on both sides of the upper-middle part of the nasal septal epithelium assessed by immunohistochemical staining 14 d after the treatment with ADSCs compared with vehicle control animals. Nerve growth factor (NGF) was detected in the ADSC culture supernatant, NGF was increased in the nasal epithelium of mice, and GFP-positive cells were observed on the surface of the left side nasal epithelium 24 h after left side nasal administration of ADSCs. The results of this study suggest that the regeneration of olfactory epithelium can be stimulated by nasally administered ADSCs secreting neurotrophic factors, thereby promoting the recovery of odor aversion behavior in vivo.

Transthyretin Is Commonly Upregulated in the Hippocampus of Two Stress-Induced Depression Mouse Models.

Chronic stress can affect gene expression in the hippocampus, which alters neural and cerebrovascular functions, thereby contributing to the development of mental disorders such as depression. Although several differentially expressed genes in the depressed brain have been reported, gene expression changes in the stressed brain remain underexplored. Therefore, this study examines hippocampal gene expression in two mouse models of depression induced by forced swim stress (FSS) and repeated social defeat stress (R-SDS). Transthyretin (Ttr) was commonly upregulated in the hippocampus of both mouse models, as determined by microarray, RT-qPCR, and Western blot analyses. Evaluation of the effects of overexpressed Ttr in the hippocampus using adeno-associated virus-mediated gene transfer revealed that TTR overexpression induced depression-like behavior and upregulation of Lcn2 and several proinflammatory genes (Icam1 and Vcam1) in the hippocampus. Upregulation of these inflammation-related genes was confirmed in the hippocampus obtained from mice vulnerable to R-SDS. These results suggest that chronic stress upregulates Ttr expression in the hippocampus and that Ttr upregulation may be involved in the induction of depression-like behavior.

Synovial Fluid Derived from Human Knee Osteoarthritis Increases the Viability of Human Adipose-Derived Stem Cells through Upregulation of FOSL1.

Knee osteoarthritis (Knee OA) is an irreversible condition that causes bone deformity and degeneration of the articular cartilage that comprises the joints, resulting in chronic pain and movement disorders. The administration of cultured adipose-derived stem cells (ADSCs) into the knee joint cavity improves the clinical symptoms of Knee OA; however, the effect of synovial fluid (SF) filling the joint cavity on the injected ADSCs remains unclear. In this study, we investigated the effect of adding SF from Knee OA patients to cultured ADSCs prepared for therapeutic use in an environment that mimics the joint cavity. An increase in the viability of ADSCs was observed following the addition of SF. Gene expression profiling of SF-treated ADSCs using DNA microarrays revealed changes in several genes involved in cell survival. Of these genes, we focused on FOSL1, which is involved in the therapeutic effect of ADSCs and the survival and proliferation of cancer stem cells. We confirmed the upregulation of FOSL1 mRNA and protein expression using RT-PCR and western blot analysis, respectively. Next, we knocked down FOSL1 in ADSCs using siRNA and observed a decrease in cell viability, indicating the involvement of FOSL1 in the survival of ADSCs. Interestingly, in the knockdown cells, ADSC viability was also decreased by SF exposure. These results suggest that SF enhances cell viability by upregulating FOSL1 expression in ADSCs. For therapy using cultured ADSCs, the therapeutic effect of ADSCs may be further enhanced if an environment more conducive to the upregulation of FOSL1 expression in ADSCs can be established.

LIF-IGF Axis Contributes to the Proliferation of Neural Progenitor Cells in Developing Rat Cerebrum.

In rodent models, leukemia inhibitory factor (LIF) is involved in cerebral development via the placenta, and maternal immune activation is linked to psychiatric disorders in the child. However, whether LIF acts directly on neural progenitor cells (NPCs) remains unclear. This study performed DNA microarray analysis and quantitative RT-PCR on the fetal cerebrum after maternal intraperitoneal or fetal intracerebral ventricular injection of LIF at day 14.5 (E14.5) and determined that the expression of insulin-like growth factors (IGF)-1 and -2 was induced by LIF. Physiological IGF-1 and IGF-2 levels in fetal cerebrospinal fluid (CSF) increased from E15.5 to E17.5, following the physiological surge of LIF levels in CSF at E15.5. Immunostaining showed that IGF-1 was expressed in the cerebrum at E15.5 to E19.5 and IGF-2 at E15.5 to E17.5 and that IGF-1 receptor and insulin receptor were co-expressed in NPCs. Further, LIF treatment enhanced cultured NPC proliferation, which was reduced by picropodophyllin, an IGF-1 receptor inhibitor, even under LIF supplementation. Our findings suggest that IGF expression and release from the NPCs of the fetal cerebrum in fetal CSF is induced by LIF, thus supporting the involvement of the LIF-IGF axis in cerebral cortical development in an autocrine/paracrine manner.

Surgical implantation of human adipose derived stem cells attenuates experimentally induced hepatic fibrosis in rats.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stromal cells and could exert hepatoprotective effects against acute liver injury, steatohepatitis, and fibrogenesis. Here, we evaluated the effects of human adipose derived stem cells (hADSCs) to attenuate experimentally induced hepatic fibrosis and early cirrhosis in rats. METHODS: Hepatic fibrosis was induced by intraperitoneal injections of CCl4 (0.1 ml/100 g body weight) twice a week for 8 weeks. hADSCs were isolated and cultured on polyethylene discs coated with hydroxyapatite and 2 cm diameter disc was surgically implanted on the right lateral lobe of the liver. Discs implanted without hADSCs served as control. The animals were injected again with CCl4 once a week for another 8 weeks. All the animals were sacrificed at the end of 16th week. RESULTS: Serial administrations of CCl4 resulted in well developed fibrosis and early cirrhosis at 8th week which maintained until the 16th week. Animals treated with hADSC discs depicted over 50% decrease of collagen with significant increase in serum albumin and total protein levels. Immunohistochemical staining for TGF-ß1, α-smooth muscle actin, and collagen type I and type III demonstrated marked decrease compared to the animals without hADSC treatment. CONCLUSIONS: Treatment with hADSCs improved liver functions, markedly reduced hepatic fibrosis and early cirrhosis. Various pleiotropic and paracrine factors secreted from the hADSCs seem to serve as reparative functions in the attenuation of liver cirrhosis. The data demonstrated that treatment with hADSCs can be successfully used as a potent therapeutic method to prevent progression of hepatic fibrosis and related adverse events.

Different In Vitro-Generated MUTZ-3-Derived Dendritic Cell Types Secrete Dexosomes with Distinct Phenotypes and Antigen Presentation Potencies.

Human dendritic cell (DC) dexosomes were evaluated for their function and preclinical validation for vaccines. Dexosomes are small DC-secreted vesicles that contain absorbing immune signals. Vaccine manufacturing requires a significant number of monocyte-derived DCs (Mo-DCs) from donor blood; thus, Mo-DC dexosomes are expected to serve as novel materials for cancer vaccination. In this study, we characterized a potential dexosome model using immature and mature MUTZ3-derived DCs (M-imIL-4-DC, M-imIFN-DC, M-mIL-4-DC, and M-mIFN-DC) and their dexosomes (M-imIL-4-Dex, M-imIFN-Dex, M-mIL4-Dex, and M-mIFN-Dex). Despite the lack of significant differences in viability, M-mIFN-DC showed a significantly higher level of yield and higher levels of maturation surface markers, such as CD86 and HLA-ABC, than M-mIL-4-DC. In addition, M-mIFN-Dex expressed a higher level of markers, such as HLA-ABC, than M-mIL-4-Dex. Furthermore, M-mIFN-Dex exhibited a higher level of antigen presentation potency, as evaluated using a MART-1 system, than either M-imIFN-Dex or M-mIL-4-Dex. We found that M-mIFN-Dex is one of the four types of MUTZ3-derived DCs that harbor potential immunogenicity, suggesting that DC dexosomes could be useful resources in cancer immunotherapy.

A Case Report on Dysgraphia in a Patient Receiving Blinatumomab: Complex Characters Are Easy to Find in a Handwriting Test.

Recent advances in chemotherapy have led to the emergence of new types of anticancer agents. With these advances, cases of side effects that have not been witnessed in the past have emerged. The systems of side effect evaluation and their grading have been based on the existing knowledge, such as the CTCAE (Common Terminology Standard for Adverse Events) for evaluating adverse drug reactions in cancer chemotherapy clinical trials. Therefore, new types of side effects may be overlooked or underestimated. Blinatumomab is a bispecific T-cell-engager (BiTE) antibody with specificity for CD19 on B cells and CD3 on T cells. Neurological events, such as neuropathy and encephalopathy, are serious side effects of BiTE antibodies. We encountered a case of a 62-year-old woman who experienced short-term memory impairment and dysgraphia after the first blinatumomab administration for Philadelphia chromosome negative (Ph-) B-cell acute lymphoblastic leukemia (ALL). The CTCAE does not include dysgraphia as a classifier for antibody therapies, such as blinatumomab, and immune effector cell-associated neurotoxicity syndrome, which is defined as a Chimeric antigen receptor T cell therapy-related toxicity; dysgraphia is included in the list of symptoms but is not graded. In this case, the severity of dysgraphia differed depending on the complexity of the letters examined. There is no report that the severity of dysgraphia depends on the letters' complexity, and therefore, it may be overlooked when using simple letters. We have reported the characteristics of dysgraphia in this case and the differences observed when judging different letters.

Multiple Phosphorylations of SR Protein SRSF3 and Its Binding to m6A Reader YTHDC1 in Human Cells.

N6-methyladenosine (m6A) is a well-known RNA modification and has various functions with its binding proteins. Nuclear m6A reader protein YTHDC1 plays a significant role in RNA metabolism including some non-coding RNA such as LINE or circRNA. It is also known to regulate mRNA splicing through recruiting SRSF3 to the targeted mRNAs, which then mediates export of YTHDC1-bound RNA to the cytoplasm. Additionally, it has been indicated that SRSF3 binding to YHTDC1 may be mediated by its dephosphorylated status. However, their binding mechanism, including the positions of dephosphorylated residues of SRSF3, has not been sufficiently investigated. Thus, we explored the mechanism of interaction between SRSF3 and YTHDC1 in human cells. We used co-immunoprecipitation to examine the binding of YTHDC1/SRSF3 through their N- and C-terminal amino-acid residues. Furthermore, dephosphorylation-mimic serine to alanine mutants of SRSF3 indicated the position of phosphorylated residues. Cumulatively, our results demonstrate that YTHDC1 binding to SRSF3 is regulated by not only hypo-phosphorylated residues of arginine/serine-rich (RS) domain of SRSF3 but also other parts of SRSF3 via YTHDC1 N- or C-terminal residues. Our results contribute to the understanding of the complex mechanism of binding between SR protein SRSF3 and the m6A reader YTHDC1 to regulate the expression of mRNA and non-coding RNAs.

A morphological study of adipose-derived stem cell sheets created with temperature-responsive culture dishes using scanning electron microscopy.

Adipose-derived stem cell (ADSC) sheets have potential to be effective in various therapies. In this study, we first demonstrated that a cell sheet composed of human ADSCs could be created using a new temperature-responsive culture dish from the DIC Corporation. The dish can cause detachment of adherent cells due to temperature changes, but a few morphological analyses have evaluated the presence or absence of damage on the detached surface of cell sheet. To characterize our ADSC sheet, we tried to observe the surface of ADSC sheets with scanning electron microscope (SEM) using the ionic liquid, which enables the rapid preparation of samples. No damage was found on the surface of the ADSC sheets on the side that had been in contact with the surface of the culture dishes. In addition, when the transcriptomes of the harvested cell sheets were compared with those of monolayer cultures, no up-regulation of cell death related genes were detected. These results propose that the detachment from temperature-responsive culture dish causes no serious damage on the prepared ADSC sheet. It is also suggested that the SEM with ionic liquids is a useful and rapid method for the analysis of ADSC sheets for therapy.

Bleeding Risk Factors after Endoscopic Submucosal Dissection in Early Gastric Cancer and the Necessity of "Second-Look" Endoscopic Examination on the following Day.

BACKGROUND AND AIM: Hemorrhage is often encountered after endoscopic submucosal dissection (ESD). In addition to active bleeding after resection, exposed blood vessels and blood clots without active bleeding on the post-dissection ulcer floor have been recognized within our department. We consider exposed and/or observable vessel findings and clots on the ulcer floor after re-section as important risk factors for hemorrhage. Here, we compared and examined the active bleeding frequency and "post-resection ulcer at risk of bleeding" on the day following ESD, in relation to their risk factors. METHOD: We retrospectively examined 447 patients who underwent second-look endoscopy in our department between August 2008 and March 2018. Logistic regression analyses were performed to determine the hazard ratio and 95% confidence interval. We compared the association of each factor mentioned above with active bleeding on the day after ESD and the presence of ulcers at risk of bleeding after resection. RESULTS: Our retrospective analysis revealed that the risk factors were larger ulcer sizes and the administration of antithrombotic drugs. Additionally, the risk was low for upper body lesions but high for antral lesions. CONCLUSION: Our results may help determine whether second-look endoscopy should be performed to minimize active bleeding after ESD, reduce postoperative complications, and improve medical safety.

Evaluation of the Usefulness of Human Adipose-Derived Stem Cell Spheroids Formed Using SphereRing® and the Lethal Damage Sensitivity to Synovial Fluid In Vitro.

Osteoarthritis (OA) is an irreversible degenerative condition causing bone deformation in the joints and articular cartilage degeneration with chronic pain and impaired movement. Adipose-derived stem cell (ADSC) or crushed adipose tissue injection into the joint cavity reportedly improve knee function and symptoms, including pain. Stem cell spheroids may be promising treatment options due to their anti-inflammatory and enhanced tissue regeneration/repair effects. Herein, to form human ADSC spheroids, we used first SphereRing® (Fukoku Co., Ltd., Ageo, Japan), a newly developed rotating donut-shaped tube and determined their characteristics by DNA microarray of mRNA analysis. The variable gene expression cluster was then identified and validated by RT-PCR. Gene expression fluctuations were observed, such as COL15A1 and ANGPTL2, related to vascular endothelial cells and angiogenesis, and TNC, involved in tissue formation. In addition, multiplex cytokine analysis in the medium revealed significant cytokines and growth factors production increase of IL-6, IL-10, etc. However, ADSC administration into the joint cavity involves their contact with the synovial fluid (SF). Therefore, we examined how SF collected from OA patient joint cavities affect 2D-culture ADSCs and ADSC spheroids and observed SF induced cell death. ADSC spheroids could become promising OA treatment options, although studying the administration methods and consider their interaction with SF is essential.

EPAC2 acts as a negative regulator in Matrigel-driven tubulogenesis of human microvascular endothelial cells.

Angiogenesis is physiologically essential for embryogenesis and development and reinitiated in adult animals during tissue growth and repair. Forming new vessels from the walls of existing vessels occurs as a multistep process coordinated by sprouting, branching, and a new lumenized network formation. However, little is known regarding the molecular mechanisms that form new tubular structures, especially molecules regulating the proper network density of newly formed capillaries. This study conducted microarray analyses in human primary microvascular endothelial cells (HMVECs) plated on Matrigel. The RAPGEF4 gene that encodes exchange proteins directly activated by cAMP 2 (EPAC2) proteins was increased in Matrigel-driven tubulogenesis. Tube formation was suppressed by the overexpression of EPAC2 and enhanced by EPAC2 knockdown in endothelial cells. Endothelial cell morphology was changed to round cell morphology by EPAC2 overexpression, while EPAC2 knockdown showed an elongated cell shape with filopodia-like protrusions. Furthermore, increased EPAC2 inhibited endothelial cell migration, and ablation of EPAC2 inversely enhanced cell mobility. These results suggest that EPAC2 affects the morphology and migration of microvascular endothelial cells and is involved in the termination and proper network formation of vascular tubes.

Rapid generation of conditional knockout mice using the CRISPR-Cas9 system and electroporation for neuroscience research.

The Cre/LoxP-based conditional knockout technology is a powerful tool for gene function analysis that allows region- and time-specific gene manipulation. However, inserting a pair of LoxP cassettes to generate conditional knockout can be technically challenging and thus time- and resource-consuming. This study proposes an efficient, low-cost method to generate floxed mice using in vitro fertilization and the CRISPR-Cas9 system over two consecutive generations. This method allowed us to produce floxed mice targeting exons 5 and 6 of CaMK1 in a short period of 125 days, using only 16 mice. In addition, we directly edited the genome of fertilized eggs of mice with our target genetic background, C57BL/6 N, to eliminate additional backcrossing steps. We confirmed that the genome of the generated floxed mice was responsive to the Cre protein. This low-cost, time-saving method for generating conditional knockout will facilitate comprehensive, tissue-specific genome analyses.

Comprehensive analysis of protein-expression changes specific to immunoglobulin G4-related disease.

BACKGROUND AND AIMS: Immunoglobulin 4 (IgG4)-related disease (IgG4-RD) is a lymphoproliferative disorder characterized by elevated serum IgG4 levels and tissue infiltration of IgG4-positive plasma cells. We analyzed the serum proteins, whose levels varied based on the disease state and treatment. MATERIALS AND METHODS: Serum proteins from patients with IgG4-related disease and healthy subjects were resolved using two-dimensional electrophoresis, silver-stained, and scanned. Alternatively, the proteins were labeled with Cy2, Cy3, and Cy5 before electrophoresis. The proteins, whose expression differed significantly between patients and healthy individuals, and between before and after steroid treatment, were identified and validated using enzyme-linked immunosorbent assays. RESULTS: Pre-treatment sera from patients with IgG4-related disease was characterized by increased levels of immunoglobulins such as IgG1, IgG4; inflammatory factors such as α-1 antitrypsin (A1AT); and proteins associated with immune system regulation such as clusterin and leucine-rich α-2-glycoprotein (LRG-1). The serum levels of A1AT, LRG-1 and clusterin, during treatment with prednisolone for up to 12 months revealed that LRG-1 levels were halved after 1 month of treatment, comparable to those in healthy subjects; LRG-1 levels remained normal until the end of treatment. CONCLUSION: LRG-1 could serve as a novel biomarker of IgG4-related diseases.

Protective Effects of Collagen Tripeptides in Human Aortic Endothelial Cells by Restoring ROS-Induced Transcriptional Repression.

Collagen tripeptide (CTP) is defined as a functional food material derived from collagenase digests of type I collagen and contains a high concentration of tripeptides with a Gly-X-Y sequence. CTP has several biological effects, including the acceleration of fracture healing, ameliorating osteoarthritis, and improving dryness and photoaging of the skin. Recently, an antiatherosclerotic effect of CTP has been reported, although its molecular mechanism is yet to be determined. In this study, we examined the effects of CTP on primary cultured human aortic endothelial cells (HAECs) under oxidative stress, because oxidative endothelial dysfunction is a trigger of atherosclerosis. DNA microarray and RT-qPCR analyses showed that CTP treatment recovered the downregulated expression of several genes, including the interleukin-3 receptor subunit alpha (IL3RA), which were suppressed by reactive oxygen species (ROS) treatment in HAECs. Furthermore, IL3RA knockdown significantly decreased the viability of HAECs compared with control cells. RT-qPCR analysis also showed that solute carrier 15 family peptide transporters, which are involved in CTP absorption into cells, were expressed in HAECs at levels more than comparable to those of a CTP-responsive human osteoblastic cell line. These results indicated that CTP exerts a protective effect for HAECs, at least in part, by regulating the recovery of ROS-induced transcriptional repression.

Quality Verification with a Cluster-Controlled Manufacturing System to Generate Monocyte-Derived Dendritic Cells.

Dendritic cell (DC) vaccines for cancer immunotherapy have been actively developed to improve clinical efficacy. In our previous report, monocyte-derived DCs induced by interleukin (IL)-4 with a low-adherence dish (low-adherent IL-4-DCs: la-IL-4-DCs) improved the yield and viability, as well as relatively prolonged survival in vitro, compared to IL-4-DCs developed using an adherent culture protocol. However, la-IL-4-DCs exhibit remarkable cluster formation and display heterogeneous immature phenotypes. Therefore, cluster formation in la-IL-4-DCs needs to be optimized for the clinical development of DC vaccines. In this study, we examined the effects of cluster control in the generation of mature IL-4-DCs, using cell culture vessels and measuring spheroid formation, survival, cytokine secretion, and gene expression of IL-4-DCs. Mature IL-4-DCs in cell culture vessels (cluster-controlled IL-4-DCs: cc-IL-4-DCs) displayed increased levels of CD80, CD86, and CD40 compared with that of la-IL-4-DCs. cc-IL-4-DCs induced antigen-specific cytotoxic T lymphocytes (CTLs) with a human leukocyte antigen (HLA)-restricted melanoma antigen recognized by T cells 1 (MART-1) peptide. Additionally, cc-IL-4-DCs produced higher levels of IFN-γ, possessing the CTL induction. Furthermore, DNA microarrays revealed the upregulation of BCL2A1, a pro-survival gene. According to these findings, the cc-IL-4-DCs are useful for generating homogeneous and functional IL-4-DCs that would be expected to promote long-lasting effects in DC vaccines.

Lens-specific conditional knockout of tropomyosin 1 gene in mice causes abnormal fiber differentiation and lens opacity.

Tropomyosin (Tpm) 1 and 2 are important in the epithelial mesenchymal transition of lens epithelial cells; however, the effect of Tpm1 depletion during aging remains obscure. We analyzed the age-related changes in the crystalline lens of Tpm1- conditional knockout mice (Tpm1-CKO). Floxed alleles of Tpm1 were conditionally deleted in the lens, using Pax6-cre transgenic mice. Lenses of embryonic day (ED) 14, postnatal 1-, 11-, and 48-week-old Tpm1-CKO and wild type mice were dissected to prepare paraffin sections, which subsequently underwent histological and immunohistochemical analysis. Tpm1 and α smooth muscle actin (αSMA) mRNA expression were assessed using RT-PCR. The homozygous Tpm1-CKO (Tpm1-/-) lenses displayed a dramatic reduction in Tpm1 transcript, with no change to αSMA mRNA expression. Tpm1-/- mice had small lenses with disorganized, vesiculated fiber cells, and loss of epithelial cells. The lenses of Tpm1-/- mice had abnormal and disordered lens fiber cells with cortical and peri-nuclear liquefaction. Expression of filamentous-actin was reduced in the equator region of lenses derived from ED14, 1-, 11-, and 48-week-old Tpm1-/- mice. Therefore, Tpm1 plays an integral role in mediating the integrity and fate of lens fiber differentiation and lens homeostasis during aging. Age-related Tpm1 dysregulation or deficiency may induce cataract formation.

Bone formation at Ti-6Al-7Nb scaffolds consisting of 3D honeycomb frame and diamond-like carbon coating implanted into the femur of beagles.

We fabricated Ti-6Al-7Nb bone scaffolds with 5 mm diameter and 20 mm length comprise of a three-dimensional (3D) honeycomb frame structure of truncated octahedra created by selective laser sintering 3D printing. The honeycomb frame was then coated with 0.1 µm thick diamond-like carbon (DLC) to increase biocompatibility. A round rod of Ti-6Al-7Nb alloy (ASTM F1295) was as a control material. They were implanted into the femur bones of beagles to evaluate bone morphometrics and to investigate changes in the transcriptome of the new bone tissue using DNA microarray analysis and real-time polymerase chain reaction (PCR). In the present report, the 3D honeycomb material with and without DLC film consisting of a-C:H is referred to as 3D_a-C:H and 3D_non, respectively. At 3 weeks after implantation, the 3D_non had more contact between the new and artificial bones compared with the control, and the 3D_a-C:H had more contact between the new and artificial bones compared with the control and 3D_non. Furthermore, 3D_a-C:H showed even more new bone compared with the control and 3D_non. At 8 weeks after implantation, more appeared lamellar bone with the 3D_a-C:H implant than those with the control and 3D_non. The real-time PCR results at 1 week of implantation revealed higher expression levels of VEGF, RANKL, and NOTCH2 expression with 3D_a-C:H than with 3D_non and control. As a result of real-time PCR at 2 weeks of implantation, OPN and CTSK expressions were found to be higher with 3D_a-C:H and 3D_non than that with the control.