BACKGROUND

Craniosynostosis is a relatively rare disease. Recently, several studies have investigated the etiology of craniosynostosis using animal models; however, the etiology remains unknown. In this study, we examined transforming growth factor (TGF) βs immunostaining from coronal sutures in patients with plagiocephaly.

METHODS

The examined materials were obtained from 3 patients who had undergone surgery for plagiocephaly. The sections were obtained from the normal patent side and the abnormal fused side of the coronal suture. The subjects included 2 girls and 1 boy with ages ranging from 1 to 4 years. Osteoblasts and connective tissue were observed with hematoxylin and eosin stain. Immunohistochemistry of the TGF-β isoforms was performed to investigate the difference between the patent and fused sutures.

RESULTS

No connective tissue was observed in the fused suture. The osteoblasts in the patent suture were activated, whereas the osteoblasts in the fused suture were inactivated. The osteoblasts were positive for TGF-β1, -β2, and -β3. The periosteum tended to be positive for TGF-β2 and negative for TGF-β1 and -β3. There was no distinct difference between the patent and fused sutures in this study.

CONCLUSIONS

In this study, all sutures had fused completely, and therefore, we may have missed the period when there are differences in protein manifestation. The modulation of the growth factor profile at the suture site may have a potential therapeutic value.

Osteochondral repair remains a major challenge in current clinical practice despite significant advances in tissue engineering. In particular, the lateral integration of neocartilage into surrounding native cartilage is a difficult and inadequately addressed problem that determines the success of tissue repair. Here, a novel design of an integral bilayer scaffold combined with a photocurable silk sealant for osteochondral repair is reported. First, we fabricated a bilayer silk scaffold with a cartilage layer resembling native cartilage in surface morphology and mechanical strength and a BMP-2-loaded porous subchondral bone layer that facilitated the osteogenic differentiation of BMSCs. Second, a TGF-β3-loaded methacrylated silk fibroin sealant (Sil-MA) exhibiting biocompatibility and good adhesive properties was developed and confirmed to promote chondrocyte migration and differentiation. Importantly, this TGF-β3-loaded Sil-MA hydrogel provided a bridge between the cartilage layer of the scaffold and the surrounding cartilage and then guided new cartilage to grow towards and replace the degraded cartilage layer from the surrounding native cartilage in the early stage of knee repair. Thus, osteochondral regeneration and superior lateral integration were achieved in vivo by using this composite. These results demonstrate that the new approach of marginal sealing around the cartilage layer of bilayer scaffolds with Sil-MA hydrogel has tremendous potential for clinical use in osteochondral regeneration.

Keywords: Integral bilayer scaffold; Lateral integration; Marginal sealing; Osteochondral regeneration; Photocurable Sil-MA hydrogel.

BACKGROUND

Dark skin has different properties in comparison to fair skin. Melanocytes have been shown to partly contribute to these differences, however, the involvement of keratinocytes from dark or fair skin is not well demonstrated.

OBJECTIVE

This study investigated the proliferation and barrier function of dark keratinocytes (DK) and fair keratinocytes (FK), and the role of protease activated receptor (PAR)1 and PAR2.

METHODS

DK and FK were isolated from human neonatal foreskins. Cells were treated with PAR1/PAR2 agonists or antagonists, proliferation was measured by BrdU assay; permeability by the flux of FITC-dextran; protein expression by immunostaining or western blot.

RESULTS

When compared to FK, DK proliferated significantly slower; had higher cell permeability; expressed less phosphorylated (P)-ERK/ERK, caspase-14, E-cadherin, tissue growth factor (TGF)-β3 and PAR1; and expressed more PAR2, and matrix metalloproteinase (MMP)-9. Activation of PAR1 or inhibition of PAR2 stimulated cell proliferation and ERK activation, and in concordance inhibition of PAR1 or activation of PAR2 suppressed cell proliferation and ERK activation in both DK and FK. Inhibition of PAR2 decreased and inhibition of PAR1 increased cell permeability. In foreskin sections, the epidermis of dark foreskin expressed less caspase-14 and the same level but different distribution of E-cadherin, when compared to fair foreskin.

CONCLUSIONS

These data highlight functional differences in proliferation and barrier integrity between HK and FK that are partly associated with their differential expression of PAR1 and PAR2.

Postoperative reossification is a common clinical correlate following surgery. It has been suggested that an underexpression of transforming growth factor-β3 (TGF-β3) may be related to craniosynostosis and postoperative reossification. Adding TGF-β3 may delay reossification and improve postoperative growth. The present study was designed to test this hypothesis. Thirty 10-day-old New Zealand white rabbits with hereditary coronal suture synostosis were divided into three groups: (1) suturectomy controls (n = 14), (2) suturectomy treated with bovine serum albumin (n = 8), and (3) suturectomy treated with TGF-β3 protein (n = 8). At 10 days of age, a 3-mm × 15-mm coronal suturectomy was performed, and serial three-dimensional (3D) computed tomography (CT) scans and cephalographs were taken at 10, 25, 42, and 84 days of age. Calvaria were harvested at 84 days of age for histomorphometric analysis. Mean differences were analyzed using a group by age analysis of variance. Analysis of the 3D CT scan data revealed that sites treated with TGF-β3 had significantly (P < .05) greater defect areas and significantly (P < .05) greater intracranial volumes through 84 days of age compared with controls. Histomorphometry showed that sites treated with TGF-β3 had patent suturectomy sites and significantly (P < .001) less new bone in the suturectomy site compared with controls. Serial radiograph data revealed significant (P < .05) differences in craniofacial growth from 25 to 84 days in TGF-β3-treated rabbits compared with controls. Data show that TGF-β3 administration delayed reossification and improved craniofacial growth in this rabbit model. These findings also suggest that this molecular-based therapy may have potential clinical use.

OBJECTIVE

To observe the expression and distribution of transforming growth factor β3 (TGF-β3) in the mouse tooth germ after advanced bell stage, and to discuss the role of TGF-β3 during the development of tooth germs.

METHODS

BALB/C's mouse tooth germs at 4, 11, and 18 days postnatal (4dpn,11dpn,and 18dpn) were collected and processed for routine fixation, decalcification, embedding, and slicing. The expression of TGF-β3 was detected by immunohistochemisty.

RESULTS

As to 4dpn tooth germ: Positive expression of TGF-β3 was found in enameloblasts, odontoblasts, ambitus of dental pupilla, with weak positive expression in the intermedial of dental papilla. As to 11dpn tooth germ: Positive expression was seen in enameloblasts, with negative expression in odontoblasts and dental papilla. As to 18dpn tooth, positive expression of TGF-β3 was showed in the vessel wall and its surrounding, with negative expression in other areas.

CONCLUSIONS

The distribution of TGF-β3 expression showed a time-space characteristic during the mouse tooth germ development after advanced bell stage, which may exert a regulatory effect on tooth development and this effect is gradually getting weak with the development of tooth germs.

In stem cell-based chondrogenesis for articular cartilage regeneration, TGF-β3 is dosed to the stem cells to drive differentiation into chondrocytic cells. Meanwhile, type I collagen, which is endogenously expressed in some stem cells (e.g., synovium-derived mesenchymal stem cells) and upregulated by TGF-β3, poses a threat to chondrogenesis, as type I collagen may alter the components and stiffness of articular cartilage. Therefore, a wiser strategy would be to feed the cells with TGF-β3 while at the same time silencing the expression of type I collagen. In this chapter, methods for construction of adenoviral vectors and lentiviral vectors having both of the above functions are given. Their transduction into synovium-derived mesenchymal stem cells for articular cartilage engineering and following characterizations are also described.

Objective: To explore the effect of mmu-circRNA_016901 on the regulation of radiation injury of bone marrow stem cells and its mechanism.

Methods: Bone marrow stem cells were exposed to different dose of X-ray, then the expression level of mmu-circRNA_016901 in bone marrow cells treated with different doses of X-ray was detected. The luciferase reporter gene assay was used to confirm that miRNA1249-5p is the target of mmu-circRNA_016901, and RNA Binding Protein Immunoprecipitation was used to confirm that TGF-β3 is the targeted on miRNA1249-5p，the expression of TGF-β3 and cell proliferation were detected after the expression of mmu-circRNA_01690 was regulated.

Results: When the irradiation dose＜6 Gy, there were significant difference in the expression of mmn-circRNA-016901 after the bone marrow mesenchymal stem cells were treated by different doses of irradiation, which showed a statistically significant (P＜0.05). The luciferase reporter gene detection and co-immunoprecipitation experiments confirmed that Mmu-circRNA_016901 could binds to miRNA1249-5p specifically, and overexpression of mmu-circRNA_016901 could regulate miRNA1249-5p negatively, which resulted in a significant increase in TGF-β3 expression and promoting of cell proliferation.

Conclusion: mmu-circRNA_016901 affects the expression of TGF-β3 through miRNA1249-5p, and thus participates in the regulation of the radiation damage mechanism of bone marrow mesenchymal stem cells.

题目: Mmu-circRNA_016901对骨髓间充质干细胞放射敏感性的影响.

目的: 探索mmu-circRNA_016901对调控骨髓间充质干细胞放射损伤的作用及其机制.

方法: 通过用不同剂量X射线辐照骨髓间充质干细胞，检测不同剂量处理后骨髓间充质干细胞内mmu-circRNA_016901表达水平，利用荧光素酶报告基因检测验证miRNA1249-5p为mmu-circRNA_016901的靶点，免疫共沉淀实验验证miRNA1249-5p 为TGF-β3的靶点，调节mmu-circRNA_01690表达水平后，检测转化生长因子-β3（transforming growth factor-β, TGF-β3）的表达和细胞增殖情况.

结果: 当辐照剂量＜6 Gy时，不同辐照剂量处理的骨髓间充质干细胞，mmu-circRNA_016901表达差异显著，具有统计学意义（P＜0.05)，荧光素酶报告基因检测和免疫共沉淀实验证实mmu-circRNA_016901可特异性结合miRNA1249-5p，过表达mmu-circRNA_016901可以负性调节miRNA1249-5p， 可导致TGF-β3表达明显升高，且促进细胞增殖.

结论: mmu-circRNA_016901通过miRNA1249-5p影响TGF-β3的表达，从而参与调节骨髓间充质干细胞放射损伤机制.

Transforming growth factor-β (TGF-β)-related genes have been postulated as possible mesoderm inducers in lower vertebrates. Activin, a member of the TGF-β family has been recently suggested to be directly involved in the induction of axial mesoderm in frogs and birds. Nodal, a novel TGF-β-like gene, seems to be essential for mesoderm formation in mice. In this work we have tried to characterize which growth factors of the TGF-β family are involved in the initial stages of differentiation of the chick embryo. Using degenerate primers to specific regions of the TGF-β super family in polymerase chain reactions (PCR), we have identified both TGF-β2 and TGF-β3 transcripts in early stages of chick development. In addition, we have identified a new gene which is transcribed during early chick development. We have called this gene Vgc1 since it is probably the chick homologue of the mouse Vg-related gene Vgrl. RNA coding for the Vgcl gene was detected by RNAse protection in early stage X blastoderms, gastrula and 3-day-old embryos. In situ hybridization experiments further indicate that Vgcl RNA is distributed uniformly throughout the various regions of the chick gastrula. The role of Vgcl in early chick development remains to be established.

Seminal plasma (SP) regulates immune responses in the female reproductive tract through specific cytokines. It is not known whether SP from high fertility bulls (H) differs from SP from low fertility bulls (L). In this study, the cytokine response of bovine endometrial epithelial cells (bEEC) in culture was investigated after challenge with SP from two bulls of below average (L) or three bulls of above average fertility (H). The bEECs were challenged with 1% or 4% SP from l- or H-fertility bulls (L1, L4, H1, H4, respectively) or 1% or 4% PBS as control (C1, C4) for 72 h. The culture media were analysed for concentrations (pg/million cells) of transforming growth factor beta (TGF-β1, TGF-β2 and TGF-β3) by Luminex, and Interleukin 6 and 8 (IL-6, IL-8) by ELISA. Challenge significantly affected production of TGF-ß1, TGF-ß2 and IL-8 compared to controls and was affected by bull fertility (p < 0.0001), SP concentration (p < 0.0001) and their interaction (p < 0.0001). A higher production of TGF-β1, TGF-β2 and IL-8 (p < 0.0001), and also IL-6 (p < 0.01), resulted from challenge with high doses of SP, being higher for L than H (p < 0.05). For TGF-β3, fertility of bull (p < 0.05). For TGF-B3, fertility of bull (p < 0.05) and the interaction between fertility and concentration of SP were significant (p < 0.01). In conclusion, 4% SP from L bulls stimulated more TGF-β1, TGF-β2, TGF-β3, IL-6 and IL-8 production than SP from H bulls, indicating that stimulation of the endometrium is relevant for fertility. Seminal plasma from high fertility bulls seems to affect cytokine production in utero positively in inseminated cows.

Keywords: Artificial insemination; Endometrium; High fertility bulls; Low fertility bulls; Seminal plasma proteins; Uterus.

Defining the best combination of cells and biomaterials is a key challenge for the development of tendon tissue engineering (TE) strategies. Adipose-derived stem cells (ASCs) are ideal candidates for this purpose. In addition, controlled cell-based products adherent to good manufacturing practice (GMP) are required for their clinical scale-up. With this aim, in this study, ASC 3D bioprinting and GMP-compliant tenogenic differentiation were investigated. In detail, primary human ASCs were embedded within a nanofibrillar-cellulose/alginate bioink and 3D-bioprinted into multi-layered square-grid matrices. Bioink viscoelastic properties and scaffold ultrastructural morphology were analyzed by rheology and scanning electron microscopy (SEM). The optimal cell concentration for printing among 3, 6 and 9 × 10⁶ ASC/mL was evaluated in terms of cell viability. ASC morphology was characterized by SEM and F-actin immunostaining. Tenogenic differentiation ability was then evaluated in terms of cell viability, morphology and expression of scleraxis and collagen type III by biochemical induction using BMP-12, TGF-β3, CTGF and ascorbic acid supplementation (TENO). Pro-inflammatory cytokine release was also assessed. Bioprinted ASCs showed high viability and survival and exhibited a tenocyte-like phenotype after biochemical induction, with no inflammatory response to the bioink. In conclusion, we report a first proof of concept for the clinical scale-up of ASC 3D bioprinting for tendon TE.

Keywords: 3D bioprinting; adipose-derived stem cells; alginate; collagen; nanocellulose; scleraxis; tendon tissue engineering; tenogenic differentiation; xenogenic-free.

OBJECTIVE

Transforming growth factor-β3 (TGF-β3) has been proved to perturb the blood-testis barrier (BTB) by accelerating junction protein endocytosis in Sertoli cells (SCs) to accommodate the traversing of preleptotene spermatocytes across the BTB around stage VIII in rat. Yet the molecular network underlying the impairment of TGF-β3 on BTB integrity is not fully elucidated. Our study herein was designed to investigate the participation of microRNA-142-3p (miR-142-3p), which has been reported to affect TGF-β3 signaling via different pathways, during BTB dynamics and the corresponding mechanisms.

METHODS

MiRNA mimic or agomiRNA was co-administered with or without TGF-β3 in the cultured SCs or in the rat testis. The SC permeability barrier function was reflected by measuring the transepithelial resistance (TER) and the permeability of the sodium fluorescein (Na-F). The BTB integrity was detected by the permeation of biotin. A luciferase reporter assay was used to testify the potential target of miR-142-3p, lethal giant larvae 2 (Lgl2). Laser capture microdissection (LCM) was applied to acquire cell components of different stages of seminiferious tubules, followed by detection of the expression levels of miR-142-3p, TGF-β3, and Lgl2 by qPCR. The SC barrier function was also detected as above in the presence of TGF-β3 after Lgl2 knockdown.

RESULTS

We revealed a reversion of TGF-β3-induced BTB impairment after miR-142-3p treatment both in vitro and in vivo. Meanwhile, the activation of Cdc42 and reduction in occludin aroused by TGF-β3 were also reversed by miR-142-3p. The predicted binding of miR-142-3p with 3'-untranslated region (3'-UTR) of Lgl2, was verified by the luciferase assay. Moreover, an increased Lgl2 level in TGF-β3-treated SCs was found and correlated stage-specific expressions of TGF-β3, miR-142-3p, and Lgl2 were revealed. Knockdown of Lgl2 in SCs was shown to partially antagonize the BTB disruption mediated by TGF-β3.

CONCLUSIONS

Collectively, our results suggest a resistance of miR-142-3p on the BTB impairment caused by TGF-β3 during the seminiferous epithelial cycle by targeting Lgl2.

Tendon injury occurs frequently and tendon repair is limited by its poor self-healing. The current tissue engineering approach for treating tendon injuries has showed limited success, largely due to the lack of scaffolds with suitable structural and biological properties, and suitable growth factors for differentiation of stem cells into tendon cells. This study investigated if the combination of environmental and biological cues from aligned chitosan-poly-caprolactone (C-PCL) combined with TGF-β3 growth factor can efficiently and rapidly direct the tenogenic differentiation of primary human bone marrow stem cells (BMSCs). C-PCL nanofibers were prepared to have the anisotropic nanostructure, and mechanical and biological properties similar to those of the native tendon extracellular matrix (ECM). The tenogenic commitment of BMSCs was assessed using cell morphology, and gene and protein expressions. BMSCs grown on uniaxially aligned C-PCL nanofibers in a medium containing TGF-β3 displayed an elongated morphology along nanofiber orientation, upregulated expressions of marker genes, and increased collagen production associated with tenogenic differentiation as compared to control substrates. Significantly, this tenogenic microenvironment induced the transcription of tenogenic markers in 5 days and production of a large amount of Collagen I in 10 days, more effective and faster than existing scaffolds combined with growth factors. This research reveals that a combinative effect of aligned C-PCL nanofibers and TGF-β3, as environmental and biological cues, can lead to rapid, effective BMSC differentiation into tenogenic progenitors, offering a potential strategy for managing tendon disorders.

Silk fibroin (SF) and hyaluronic acid (HA) were crosslinked by horseradish peroxidase (HRP)/H₂O₂, and 1,4-Butanediol di-glycidyl ether (BDDE), respectively, to produce HA/SF-IPN (interpenetration network) (HS-IPN) hydrogels. HS-IPN hydrogels consisted of a SF strain with a high content of tyrosine (e.g., strain A) increased viscoelastic modules compared with those with low contents (e.g., strain B and C). Increasing the quantities of SF in HS-IPN hydrogels (e.g., HS7-IPN hydrogels with weight ratio of HA/SF, 5:7) increased viscoelastic modules of the hydrogels. In addition, the mean pores size of scaffolds of the model hydrogels were around 38.96 ± 5.05 μm which was between those of scaffolds H and S hydrogels. Since the viscoelastic modulus of the HS7-IPN hydrogel were similar to those of human nucleus pulposus (NP), it was chosen as the model hydrogel for examining the differentiation of human bone marrow-derived mesenchymal stem cell (hBMSC) to NP. The differentiation of hBMSC induced by transforming growth factor β3 (TGF-β3) in the model hydrogels to NP cells for 7 d significantly enhanced the expressions of glycosaminoglycan (GAG) and collagen type II, and gene expressions of aggrecan and collagen type II while decreased collagen type I compared with those in cultural wells. In summary, the model hydrogels consisted of SF of strain A, and high concentrations of SF showed the highest viscoelastic modulus than those of others produced in this study, and the model hydrogels promoted the differentiation of hBMSC to NP cells.

Keywords: Tyrosine; hBMSC differentiations; hyaluronic acid; nucleus pulposus; silk fibroin; viscoelastic modulus of HS-IPN hydrogels.

The aim of the present study was to investigate the role of microRNA (miRNA or miR)-140 in C3H10T1/2 mesenchymal stem cells (MSCs). Cluster analysis was used to evaluate the miRNA expression profile. The expression level of miRNA‑140 was validated by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). TargetScan and microRNA.org databases were used to predict target miRNAs and cartilage‑associated target genes. Binding sites between miR‑140 and the target gene were predicted by bioinformatics software. A dual‑luciferase reporter assay was performed to determine whether miR‑140 could target C‑X‑C motif chemokine ligand 12 (CXCL12). Following the promotion/inhibition of miR‑140, 1, 7 and 14 days following transforming growth factor‑β3 (TGF‑β3)‑induction, western blotting was utilized to evaluate CXCL12 protein levels. MTT assays and alcian blue staining were applied to assess C3H10T1/2 MSC viability and chondrogenic differentiation, respectively. In the TGF‑β3‑induced group, RT‑qPCR verified that the mRNA level of Mus musculus (mmu)‑miR‑140 was significantly elevated when compared with the control group. miR‑140 was predicted to recognize and interact with CXCL12‑3'UTR and the dual luciferase reporter assay further validated that miR‑140 targeted the predicted region of CXCL12. CXCL12 was markedly decreased following miR‑140 overexpression and visibly increased following miR‑140 inhibition. In addition, the level of CXCL12 expression declined as the duration of induction increased. Following the promotion/inhibition of miR‑140, at 1 and 7 days following TGF‑β3‑induction, C3H10T1/2 MSCs inhibited or promoted cell viability, respectively, when compared with the control groups. In addition, in pellets achieved by chondrogenic differentiation following the induction of C3H10T1/2 MSCs for 7 days, alcian blue staining revealed no significant difference in characteristic extracellular matrix glycosaminoglycans between the miR‑140 up and downregulated groups, and their respective control groups. The present study concludes that miRNA‑140 inhibition promoted C3H10T1/2 MSC viability however, not C3H10T1/2 MSC differentiation by targeting and reducing CXCL12 protein levels during the process of TGF‑β3‑induced chondrogenic differentiation. In conclusion, the present study provided a potential target for the treatment of cartilage defection.

Prevalence of neural tube defect (NTD) has reduced after folic acid intake. However; which mechanisms are effective in NTD are not known exactly. In this study; due to the possible effects on hypoxic pathway and embryonic development, particularly on extracellular matrix components, Hif-1α Pro582Ser and TGF-β3 IVS5+104 A/G SfaN1 polymorphisms were studied by PCR-RFLP method both on children with NTDs and mothers. Statistical differences were seen for Hif-1α and TGF-β3 IVS5+104 A/G SfaN1 polymorphisms in children with NTDs but no difference was seen in mothers. Both genes are effective on many pathways and our results suggest that regulation of extracellular matrix components of children during fetal life is important in neural tube defects formation. The results of this study show that Hif-1α Pro582Ser and TGF-β3 IVS5+104 A/G SfaN1 polymorphisms may play a role in NTDs.

<AbstractText>Basic studies have verified that estradiol or androgen is influential on cardioprotection, howeversynergistic effects of estradiol and testosterone on heart failure (HF) are still unknown. This study aimed to evaluate the association among sex hormones and heart failure risk factors.</AbstractText><AbstractText>142 controls and 196 patients with HF were selected for this study. Serum levels of estradiol, testosterone, Brain natriuretic peptide precursor, transforming growth factor-β1, β2 and <em>β3</em>, lipid-lipoprotein profile, glucose, high-sensitivity C-reactive protein, serum creatinine, microglobulin, uric acid, alanine aminotransferase, aspartate aminotransferase were determined. H9c2 cardiac myocytes were used to investigate the effect of estradiol and testosterone on cardiomyocytes apoptotic involved in <em>TGF</em>-β1. Signaling pathway of caspase 3, Bax, Bcl-2, caspase 8 and <em>TGF</em>-β was determined during the IL-6 induced apoptotic.</AbstractText><AbstractText>First, our results showed that compared with the control, the E2/T ratio decreased from 6.32±9.89 to 3.43±3.16 (P <0.001) in female, from 4.00±8.14 to 7.80±11.35 (P<0.001) in male with heart failure, and the level of <em>TGF</em>-β1 increased. What's more, these changes were favorably associated with the cardiac function classification. Univariate and multivariate logistic regression analysis showed that serum E2/T ratio, <em>TGF</em>-β1 and NT-proBNP were independent risk factor in heart failure patients. Second, we found that <em>TGF</em>-β1 was upregulated in rat H9c2 cardiomyocyte induced by IL-6, and <em>TGF</em>-β1 regulates the apoptosis of rat H9c2 cardiomyocyte. Furthermore, we verified the beneficial effects of the defined appropriate E2/T ratio on cardiomyocyte apoptotic mediated by <em>TGF</em>-β1.</AbstractText><AbstractText>The balance of the serum E2/T ratio was broken in patients with heart failure, and an imbalanced E2/T ratio showed a strong association with heart failure risk factors, and E2 combined with T play a synergistic effect on anti-apoptosis involved in <em>TGF</em>-β1.</AbstractText>

OBJECTIVE

To build a murine model for tobacco smoke and electronic cigarette vapor exposure to characterize the inflammatory and immune responses in the larynx.

METHODS

In this pilot study, twenty-four wild-type C57BL/6 mice were divided into four groups: smoke, vapor with nicotine, vapor without nicotine, and air only. Following daily exposure for 4 months, larynges were dissected and processed with cytokine detection arrays. Each laryngeal cytokine level between the four different groups was analyzed statistically by using statistical analysis software (SAS) to calculate the analysis of variance (ANOVA).

RESULTS

IL-4 was the only cytokine found to achieve statistically significant different levels in this study, with elevated levels of IL-4 in the tobacco smoke and vapor with nicotine groups compared to the levels found in the vapor without nicotine and air only groups (p = 0.0418). While statistically non-significant, prominent findings revealed up-regulation of TGF-β2 and TGF-β3 in the smoke group, but near-normal levels of TGF-β2 and TGF-β3 and suppression of IL-10 in the vapor groups (p>> 0.05).

CONCLUSIONS

The potential utility of the murine model is established for studying the inflammatory and immune effects of tobacco smoke and vapor on the mammalian larynx. IL-4 levels in mice larynges were significantly elevated in the tobacco smoke and vapor with nicotine groups.

BACKGROUND

The multifunctional transforming growth factor beta (TGF-β) is a glycoprotein that exists in three isoforms. TGF-β3 expression increases in fetal wound healing and reduces fibronectin and collagen I and III deposition, and also improves the architecture of the neodermis which is a combination of blood vessels and connective tissue during wound healing. Fibroblasts are key cells in the wound healing process. TGF-β3 plays a critical role in scar-free wound healing and fibroblast actions in the wound healing process. The aim of this study was to express the TGF-β3 gene (tgf-b3) in human foreskin fibroblasts (HFF's).

METHODS

We obtained HFF's from a newborn and a primary fibroblast culture was prepared. The cells were transfected with TGF-β3-pCMV6-XL5 plasmid DNA by both lipofection and electroporation. Expression of TGF-β3 was measured by enzyme-linked immunosorbent assay (ELISA).

RESULTS

The highest TGF-β3 expression (8.3-fold greater than control) was obtained by lipofection after 72 hours using 3 µl of transfection reagent. Expression was 1.4-fold greater than control by electroporation.

CONCLUSIONS

In this study, we successfully increased TGF-β3 expression in primary fibroblast cells. In the future, grafting these transfected fibroblasts onto wounds can help the healing process without scarring.

Glycoprotein A repetitions predominant (GARP) (encoded by the Lrrc32 gene) plays important roles in cell-surface docking and activation of TGFβ. However, GARP's role in organ development in mammalian systems is unclear. To determine the function of GARP in vivo, we generated a GARP KO mouse model. Unexpectedly, the GARP KO mice died within 24 h after birth and exhibited defective palatogenesis without apparent abnormalities in other major organs. Furthermore, we observed decreased apoptosis and SMAD2 phosphorylation in the medial edge epithelial cells of the palatal shelf of GARP KO embryos at embryonic day 14.5 (E14.5), indicating a defect in the TGFβ signaling pathway in the GARP-null developing palates. Of note, the failure to develop the secondary palate and concurrent reduction of SMAD phosphorylation without other defects in GARP KO mice phenocopied TGFβ3 KO mice, although GARP has not been suggested previously to interact with TGFβ3. We found that GARP and TGFβ3 co-localize in medial edge epithelial cells at E14.5. In vitro studies confirmed that GARP and TGFβ3 directly interact and that GARP is indispensable for the surface expression of membrane-associated latent TGFβ3. Our findings indicate that GARP is essential for normal morphogenesis of the palate and demonstrate that GARP plays a crucial role in regulating TGFβ3 signaling during embryogenesis. In conclusion, we have uncovered a novel function of GARP in positively regulating TGFβ3 activation and function.

Objective: To detect the expression of cartilage oligomeric matrix protein (COMP) in the synovial chondromatosis of the temporomandibular joint (TMJSC), and to discuss the possible interactions between COMP, transforming growth factor (TGF)-β3, TGF-β1 and bone morphogenetic protein-2 (BMP-2) in the development of this neoplastic disease.

Methods: Patients in Peking University School and Hospital of Stomatology from January 2011 to February 2020 were selected, who had complete medical records, TMJSC was verified histologically after operation. The expressions of COMP, TGF-β3, TGF-β1 and BMP-2 in the TMJSC of the temporomandibular joint were detected by immunohistochemistry and quantitative real-time PCR (RT-PCR) at the protein level and mRNA level respectively, compared with the normal synovial tissue of temporomandibular joint. The histological morphology, protein expression and distribution of TMJSC tissues were observed microscopically, and the positive staining proteins were counted and scored. SPSS 22.0 statistical software was used to analyze the expression differences between the related proteins in TMJSC tissue and the normal synovial tissue of temporomandibular joint and to compare their differences. P < 0.05 indicated that the difference was statistically significant.

Results: Immunohistochemical results showed that the positive expression of COMP in TMJSC tissues was mostly found in synovial tissues and chondrocytes adjacent to synovial tissues, and the difference was statistically significant, compared with the normal temporomandibular joint synovial tissues. The positive expression of COMP was significantly different between recurrent TMJSC and non-recurrent ones. The positive expressions of TGF-β3, TGF-β1 and BMP-2 were higher than the normal synovial tissue, and were also mostly found in the synovial cells and adjacent chondrocytes, which was further confirmed by Western blot. According to the RT-PCR results, the expressions of COMP, TGF-β3, TGF-β1 and BMP-2 in TMJSC were higher than those in the normal synovial tissue.

Conclusion: The expression of COMP in TMJSC of temporomandibular joint increased significantly, compared with the normal synovial tissue. There may be interactions between COMP and cytokines related to the proliferation and differentiation, like TGF-β3, TGF-β1 and BMP-2, which may play a potential role in the pathogenesis of TMJSC.

目的: 检测软骨寡聚基质蛋白(cartilage oligomeric matrix protein，COMP)在颞下颌关节滑膜软骨瘤病(synovial chondromatosis of the temporomandibular joint，TMJSC)中的表达，并初步探讨其在TMJSC发病中可能的作用。

方法: 选取2011年1月至2020年2月于北京大学口腔医院就诊的有完整病历资料、经手术治疗且术后病理诊断为TMJSC的患者，通过免疫组织化学实验、蛋白免疫印记法及实时荧光定量PCR，分别在蛋白、mRNA水平检测TMJSC中COMP、转化生长因子(transforming growth factor，TGF)-β3、TGF-β1、骨形态发生蛋白-2(bone morphogenetic protein-2，BMP-2)等细胞因子的表达情况，并与正常滑膜组织进行比较。显微镜下观察病变的组织形态学特征、蛋白表达及分布情况，对阳性染色蛋白进行半定量分析; 采用SPSS 22.0统计软件对实验结果进行分析，P＜0.05表示差异有统计学意义。

结果: 免疫组织化学实验结果显示，TMJSC组织中COMP阳性表达多见于滑膜组织及与滑膜邻近的软骨细胞内，与正常滑膜组织相比表达明显增多; TMJSC复发病例中COMP阳性表达评分较未复发病例更高; TGF-β3、TGF-β1及BMP-2阳性表达分布与COMP类似，表达水平较正常滑膜组织明显增高; 该结论在蛋白免疫印迹法中得到进一步印证。实时荧光定量PCR检测结果显示，COMP、TGF-β3、TGF-β1及BMP-2的表达较正常滑膜组织均有增高。

结论: COMP在TMJSC中表达显著增高，并与TGF-β3、TGF-β1及BMP-2间存在协同作用，可能在该肿瘤的发病中起一定作用。

Keywords: Bone morphogenetic protein-2; Cartilage oligomeric matrix protein; Synovial chondromatosis; Temporomandibular joint; Transforming growth factor.

Background: Ultraviolet radiation (UVR) is the most well-known cause of skin pigmentation accompanied with photoaging. Transforming growth factor (TGF)-β1 was previously shown to have anti-melanogenic property; however, it can induce scarring in skin.

Objective: We investigated the effect of TGF-β3 on melanogenesis in human melanocytes cocultured with UV-irradiated skin constituent cells, and UV-irradiated human skin.

Methods: UVB irradiation or treatment with stem cell factor (SCF) and endothelin-1 (ET-1) was applied to human melanocytes cocultured with keratinocytes and/or fibroblasts and ex vivo human skin. Mechanistic pathways were further explored after treatment with TGF-β3.

Results: While UVB irradiation or SCF/ET-1 enhanced melanogenesis, TGF-β3 effectively inhibited melanin accumulation and tyrosinase activity via downregulation of the extracellular signal-regulated kinase (ERK)/microphthalmia-associated transcription factor (MITF) pathway. TGF-β3 increased the expression of differentiation markers of keratinocytes.

Conclusion: TGF-β3 effectively suppressed UVR-stimulated melanogenesis indicating that topical TGF-β3 may be a suitable candidate for the treatment of UV-associated hyperpigmentation disorders.

Keywords: Human skin; Melanogenesis; TGF-β3; UV radiation.

CD4⁺ T cells regulate inflammation and metabolism in obesity. An imbalance of CD4⁺ T regulatory cells (Tregs) is critical in the development of insulin resistance and diabetes. Although cytokine control of this process is well understood, transcriptional regulation is not. KLF10, a member of the Kruppel-like transcription factor family, is an emerging regulator of immune cell function. We generated CD4⁺-T-cell-specific KLF10 knockout (TKO) mice and identified a predisposition to obesity, insulin resistance, and fatty liver due to defects of CD4⁺ Treg mobilization to liver and adipose tissue depots and decreased transforming growth factor β3 (TGF-β3) release in vitro and in vivo. Adoptive transfer of wild-type CD4⁺ Tregs fully rescued obesity, insulin resistance, and fatty liver. Mechanistically, TKO Tregs exhibit reduced mitochondrial respiration and glycolysis, phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR signaling, and consequently impaired chemotactic properties. Collectively, our study identifies CD4⁺ T cell KLF10 as an essential regulator of obesity and insulin resistance by altering Treg metabolism and mobilization.

Keywords: CD4(+) T cell; KLF10; PI3K-Akt-mTOR pathway; Treg; glycolysis; metabolic disorders; mitochondria; obesity; oxidative phosphorylation.

Background In sarcoidosis, the direction and intensity of immunological reactions involved in disease pathophysiology is affected by variation in the genes coding for effector and regulatory molecules with immune functions. This study, therefore, investigates polymorphic variants in genes involved in inflammation, immune reactions, and granuloma formation in context of their plausible association with sarcoidosis, with specific focus on Greek population. Methods A total of 18 single-nucleotide polymorphisms (SNPs) were genotyped in Greek patients with pulmonary sarcoidosis (n = 103) and in healthy Greek control subjects (n = 100) using multiplexed MassARRAY (MassARRAY ®) iPLEX assay based on MALDI-TOF mass spectrometry. Results TGF-β3 rs3917200*G variant was associated with sarcoidosis (OR: 3.04 [95% CI: 1.98-4.69], p = 2.76*10^-7). Further, ANXA11 rs1049550*A variant was associated with sarcoidosis (OR: 0.59 [0.39-0.89], p = 0.01). Conclusions This first study of genetic variation of immune-related genes in Greek patients with sarcoidosis brings to attention a novel disease 'susceptibility' factor: TGF-β3 rs3917200*G allele. It also confirms previously reported 'protective' association between sarcoidosis and functional variant ANXA11 rs1049550*A. Further work is required to validate these findings and to expand investigation of their plausible relationship with clinical course of the disease.

Keywords: ANXA11; TGF-β3; Greek; genetic factors; polymorphisms; sarcoidosis.