In the present study a combination of Transforming Growth Factor Beta 3 (TGF-β3) and Bone Morphogenetic Protein-2 (BMP-2) loaded gelatin films sandwiched between poly (L-lactide) (PLLA)/poly (ε-caprolactone) (PCL) matrices were produced to enhance bone formation in alveolar bone defects. Osteogenic properties of tissue constructs were tested in alveolar bone defect model in rats. Bone healing was assessed by osteogenic gene expression levels of bone sialoprotein (BSP), alkaline phosphatase (ALP), osteonectin (ON, SPARC), osteocalcin (OC), runt-related transcription factor 2 (RUNX2), bone specific alkaline phosphatase (BALP) activity, histomorphometry and microtomography. Increase in osteogenic gene expression levels and BALP activity results showed that new bone formation was significantly accelerated in TGF-β3 + BMP-2 loaded scaffold group compared to growth factor free and only BMP-2 loaded groups. The micro-computed tomography (μ-CT) data from the 4^th months revealed that (TGF-β3+ BMP-2) loaded scaffolds displayed increased bone formation and was able to fulfill 84% of the defect area (p < 0.05). Accelerated bone formation in the S-GF-B-T group compared to that of the S-GF group at the end of the 4th month was further verified via histomorphometric analysis (p = 0.008). Gene expression, BALP activity, microtomography and histomorphometry analysis indicated that (TGF-β3 + BMP-2) loaded PLLA/PCL scaffolds increased the new bone formation. BMP-2 loaded scaffolds were less effective than combination of TGF-β3 and BMP-2 loaded scaffolds. These findings demonstrated that focusing on the PLLA/PCL hybrid scaffolds combined with (TGF-β3 + BMP-2) may lay the groundwork for future therapy-oriented efforts to enhance bone formation in alveolar defects.

We aimed to investigate the role of the miR-29b and its effect on TGF-β3 pathway in vascular and valvular calcification in a rat model of calcific aortic valve diseases (CAVD). A rat model of CAVD was established by administration of warfarin plus vitamin K. The expression levels of miR-29b, osteogenic markers and other genes were determined by qRT-PCR, Western blot and/or immunofluorescence and immunohistochemistry. The calcium content and alkaline phosphatase (ALP) activity were measured. The calcium content, ALP activity and osteogenic markers levels in calcified aorta and aortic valve were augmented compared to controls. The expression of miR-29b, p-Smad3, and Wnt3 and β-catenin was significantly up-regulated, whereas TGF-β3 was markedly down-regulated. However, compared with the CAVD model group, the calcium content and ALP activity in rats treated with antagomiR-29b were significantly decreased, and antagomiR-29b administration reversed the effects of CAVD model on the expression of miR-29b and osteogenic markers. Inhibition of miR-29b in CAVD rats prevented from vascular and valvular calcification and induced TGF-β3 expression, suggesting that the miR-29b/TGF-β3 axis may play a regulatory role in the pathogenesis of vascular and valvular calcification and could play a significant role in the treatment of CAVD and other cardiovascular diseases.

Keywords: TGF-β3; antagomiR-29b; miR-29b; vascular calcification.

OBJECTIVE

To observe the effect of β3 adrenergic receptor (β3-AR) on fibrosis in cardiac fibroblasts(CFBs) and explore the related mechanisms.

METHODS

Neonatal CFBs were divided into negative control group (N-CFC): CFBs without any intervention; group treated with β3 adrenergic receptor agonist (AngⅡ-CFC-β3-AR BRL): CFBs treated with 10(-6) mol/L angiotensin Ⅱ(AngⅡ), 1 hour later treated with 10(-5) mol/L β3 adrenergic receptor agonist (β3-AR BRL37344); group treated with β3 adrenergic receptor antagonist (AngⅡ-CFC-β3-AR SR): CFBs treated with 10(-6) mol/L AngⅡ, 1 hour later treated with 10(-5) mol/L β3 adrenergic receptor antagonist (β3-AR SR59230A); and positive control group (AngⅡ-CFC): CFBs treated with 10(-6) mol/L AngⅡonly. Proliferation of CFBs was detected by the method of WST-1. Protein expression of β3-AR, transforming growth factor β1 receptor (TGF-β1-R), transforming growth factor β1(TGF-β1), Smad-2, phospho-Smad-2 (p-Smad-2), collagen-Ⅰ (COL-Ⅰ) and collagen-Ⅲ(COL-Ⅲ) was determined by Western blot assay.

RESULTS

(1) The proliferation of CFBs was the highest in AngⅡ-CFC-β3-AR BRL, followed by AngⅡ-CFC-β3-AR SR and AngⅡ-CFC group (all P<0.05 vs. N-CFC group). (2) The protein expression level of β3-AR, TGF-β1-R, TGF-β1 and p-Smad-2 was in the same order as proliferation of CFBs. (3) The expression level of COL-Ⅰ and COL-Ⅲ protein was also in the same order as proliferation of CFBs.

CONCLUSIONS

Activation of β3-AR may promote fibrosis of CFBs through the TGF-β/Smad signaling pathway and thus aggravate myocardial remodeling.

There is an urgent need to identify immunological markers of tuberculosis (TB) risk in HIV co-infected individuals. Previously we have shown that TB recurrence in HIV co-infected individuals on ART was associated with markers of systemic inflammation (IL-6, IL1β and IL-1Rα). Here we examined the effect of additional acute inflammation and microbial translocation marker expression on risk of TB recurrence. Stored plasma samples were drawn from the TB Recurrence upon Treatment with HAART (TRuTH) study, in which individuals with previously treated pulmonary TB were screened for recurrence quarterly for up to 4 years. Recurrent TB cases (n = 37) were matched to controls (n = 102) by original trial study arm assignment and ART start date. Additional subsets of HIV infected (n = 41) and HIV uninfected (n = 37) individuals from Improving Recurrence Success (IMPRESS) study were sampled at active TB and post successful treatment completion. Plasma concentrations of soluble adhesion molecules (sMAdCAM, sICAM and sVCAM), lipopolysaccharide binding protein (LBP) and transforming growth factor-beta (TGF-β1, TGF-β2, TGF-β3) were measured by multiplex immunoassays and ELISA. Cytokine data was square root transformed in order to reduce variability. Multivariable analysis adjusted for a number of potential confounders measured at sample time-point: age, BMI, CD4 count, viral load (VL) and measured at baseline: presence or absence of lung cavities, previous history of TB, and WHO disease stage (4 vs 3). The following analytes were associated with increased risk of TB recurrence in the multivariable model: sICAM (aOR 1.06, 95% CI: 1.02-1.12, p = 0.009), LBP (aOR 8.78, 95% CI: 1.23-62.66, p = 0.030) and TGF-β3 (aOR 1.44, 95% CI 1.01-2.05, p = 0.044). Additionally, we observed a positive correlation between LBP and sICAM (r= 0.347, p<0.0001), and LBP and IL-6, identified to be one of the strongest predictors of TB risk in our previous study (r=0.623, p=0.03). These data show that increased risk of TB recurrence in HIV infected individuals on ART is likely associated with HIV mediated translocation of microbial products and the resulting chronic immune activation.

Keywords: HIV; antiretroviral (ARV) therapy; inflammation; microbial translocation; tuberculosis - pulmonary.

To investigate the association between immune-cell-related cytokines and the development of chronic hepatitis B (CHB), patients with chronic hepatitis B virus (HBV) infection in the immunotolerant (IT) phase (n = 30) or hepatitis B envelope antigen (HBeAg)-positive CHB (n = 250) were enrolled in this study. Serological indicators and plasma cytokine levels were measured at the time of enrollment. The results showed that there were significant differences in the median age of the patients (27 vs. 31 years), alanine aminotransferase levels (ALT, 29.85 vs. 234.70 U/L), alanine aminotransferase levels (AST, 23.40 vs. 114.90 U/L), HBsAg levels (4.79 vs. 3.88 log₁₀ IU/ml), HBeAg levels (1606.36 vs. 862.47 S/CO), and the HBV DNA load (8.17 vs. 6.71 log₁₀ IU/ml) between the IT and CHB groups (all P < 0.01). The median values of Fms-like tyrosine kinase 3 ligand (FLT3-L), interferon-gamma (IFN-γ), interleukin- 17A (IL-17A), and transforming growth factor beta (TGF-β1) were significantly higher in the IT group than in the CHB group (FLT3-L, 41.62 vs. 27.47 pg/ml; IFN-γ, 42.48 vs. 33.18 pg/ml; IL-17A, 15.66 vs. 8.90 pg/ml; TGF-β1, 4921.50 vs. 2234 pg/ml; all P < 0.01). The median IFN-α2, TGF-β3 and IL-10 levels in the IT group were significantly lower than those in the CHB group (IFN-α2, 15.24 vs. 35.78 pg/ml, P = 0.000; TGF-β3, 131.69 vs. 162.61 pg/ml, P = 0.025; IL-10, 5.02 vs. 7.9 pg/ml, P = 0.012). Multivariate logistic regression analysis indicated that TGF-β 1 (OR = 0.999, 95% CI 0.999-1.000, P < 0.001) and TGF-β2 levels (OR = 1.008, 95%CI 1.004-1.012, P < 0.001) were modestly but significantly associated with the incidence of CHB. The results suggest that TGF-β level might be an independent factor related to the occurrence of CHB.

This study aimed to evaluate the effects of radiofrequency (RF) on patellar ligament repair through the analysis of type I and III collagens and immunostaining for TGF-β3. To evaluate the effect of RF on patellar ligament repair of Wistar rats, cross-sectional incision (60% of the width - grade I) was performed in patellar ligaments of the groups: lesion (L, n = 7), treated with RF on the 5-day (5RF, n = 7) and 7-day (7RF, n = 7) post injury were compared to control group (C, n = 7). Histological evaluation, immunohistochemistry, morphometry and statistical analysis were performed. At 10 days post injury, ligament rupture were observed only in L. Active fibroblasts, type 3 collagen and TGF-β3 in L, 5RF and 7RF was significantly (p < 0.05) higher than control (C). Type 1 collagen was significantly (p < 0.05) higher in C than L, 5RF and 7RF. A positive correlation (p < 0.05) was observed: TGF-β3 vs active fibroblasts and TGF-β3 vs type 3 collagen; otherwise, negative correlation (p < 0.05): type I collagen vs TGF-β3. These results suggest that RF seemed to accelerate the wound healing process of the patellar ligament and may be used as a non-invasive treatment of partial ligament injuries.

Keywords: Collagen; Radiofrequency; TGF-β3; Tendon; Tissue repair.

OBJECTIVE

To observe the effect of overdose fluoride on the expression of TGF-β3 in rat incisor and to explore the possible mechanism of dental fluorosis． METHODS: Twenty Wistar rats were randomly divided into 2 groups. The animals were maintained in standard environmental conditions with free access to food and water (control group) or water added with 100mg/L F (experimental group). The rats were killed at the end of 8th week. The expression of TGF-β3 was detected by immunohistochemical staining. Statistical analysis was performed using SPSS12.0 software package.

RESULTS

The expression of TGF-β3 in ameloblasts was significantly inhibited in the experimental of group II（P<0.01）. The gray value of the control group and the fluorine group were 85.89±7.90, 116.76±8.04, respectively.

CONCLUSIONS

Fluoride might disturb the signal transduction between the epithelia and mesenchyma by inhibiting the expression of TGF-β3 in ameloblasts, which in turn may inhibit the differentiation and function of the tooth-forming cells.

Objective: To investigate the impact of TGF-β3 on the chondrogenesis of bone marrow mesenchymal stem cells (BM-MSCs) under hypoxia environment. Methods: BM-MSCs were obtained from SD rat tibias and femora and cultured with whole bone marrow adherent method. Cell surface antigens were analyzed by flow cytometry and the multiple-directional differentiation capabilities were detected with special differentiation agents to affirm the reality of BM-MSCs. Under normoxia or hypoxia condition, BM-MSCs were induced with TGF-β3 or not. Then, alcian blue and immunofluorescence staining were performed to evaluate the expression level of aggrecan, collagen Ⅱ. qRT-PCR analysis were performed to analyze the expression of aggrecan, collagen Ⅱ and collagen Ⅹ. qRT-PCR and Western blot analysis was performed to detect the mRNA and protein level of HIF-1α, collagenⅡ and β-catenin. Results: BM-MSCs were fibroblast-like shape and had ablities of osteogeic, adipogenic and chondrogenic differentiation, with the expression of CD(29, )CD(44) and CD(90) but not CD(45). Alcian blue and immunofluorescence staining showed that BM-MSCs strongly expressed the aggrecan and collagen Ⅱ with the presence of TGF-β3 under hypoxia condition. qRT-PCR analysis showed the mRNA expression levels of collagen Ⅱ, aggrecan and collagen Ⅹ were up-regulated at 2.46, 2.20 and 1.80 folds, comparing with control group (all P<0.05). Western blot analysis showed that the protein levels of HIF-1α, collagenⅡ in BM-MSCs were up-regulated with the presence of TGF-β3 under hypoxia condition, but β-catenin level was down-regulated. Conclusion: TGF-β3 promotes the chondrogenic differentiation ability of BM-MSCs under hypoxia condition, which may be relative with the inhibition of Wnt/β-catenin signaling pathway.

OBJECTIVE

To investigate the mobilization of peripheral blood mesenchymal stem cells (PBMSCs) and whether a combination of PBMSCs and modified demineralized bone matrix (DBM) promoted the repair of cartilage lesions in a pig model.

METHODS

Pig PBMSCs were mobilized by the combined administration of granulocyte colony-stimulating factor (G-CSF) and the CXCR4 antagonist AMD3100. Colony formation was detected by the fibroblast colony-forming unit (CFU-F) count and the percentage of the CD45-CD90+ cell population by flow cytometry. The mobilized cells were identified as MSCs by their morphological characteristics, surface markers, and differentiation potentials. The composite scaffolds carrying BMP-2 and TGF-β3 chitosan sustained-release microspheres/DBM were prepared by emulsion cross-linking and the Urist method, and scanning electron microscopy (SEM) observation was performed. The model of pig cartilage defect was prepared, and gross observation, histological examination, immunohistochemistry, and O'Driscoll scoring were performed 4, 8, and 12 weeks postoperation.

RESULTS

After mobilization, the number of CFU-Fs in the peripheral blood in the experimental group (G-CSF + AMD3100) was significantly increased compared with the control group (p < 0.05). The proportion and total number of CD45-CD90+ cells were increased (p < 0.05). The mobilized stem cells had MSC characteristics. SEM of the new tissue-engineered cartilage showed that PBMSCs were evenly grown on the surface of the scaffold and microsphere morphology had no obvious change. Gross observation, histological examination, immunohistochemistry, and O'Driscoll score were better in the experimental group than in the other groups (p < 0.05).

CONCLUSIONS

G-CSF + AMD3100 is an effective mobilization agent for PBMSCs. The new tissue-engineering cartilage constructed by two-factor sustained-release microspheres/DBM composite PBMSCs effected good repair of the cartilage defect in pigs.

OBJECTIVE

To construct and identify the recombinant adenovirus vector expressing bone morphogenetic protein 2 (BMP-2) and transforming growth factor β3 (TGF-β3) genes, to observe the expressions of BMP-2 and TGF-β3 after transfected into bone marrow mesenchymal stem cells (BMSCs) of the Diannan small-ear pigs.

METHODS

BMP-2 cDNA and TGF-β3 cDNA were amplified by PCR, and were subcloned into the pEC3.1 (+) plasmid to obtain pEC-GIE 3.1-BMP-2 and pEC-GIE3.1-TGF-β3 plasmid respectively. They were subcloned into pGSadeno vector by homologous recombination reaction and HEK293 cells were transfected after linearization to obtain Ad-BMP-2 and Ad-TGF-β3. The BMSCs were isolated from the bone marrow of Diannan small-ear pig and cultured. The 3rd passage BMSCs were transfered with Ad-BMP-2 (group A), Ad-TGF-β3 (group B), Ad-BMP-2+ Ad-TGF-β3 (group C), and untransfected cells served as a control (group D). The expressions of BMP-2 and TGF-β3 genes and proteins were detected by PCR, immunofluorescence, and Western blot. The chondrogenic differentiation of BMSCs was evaluated by immunohistochemical of collagen type II.

RESULTS

The Ad-BMP-2 and Ad-TGF-β3 were constructed successfully and confirmed by PCR and sequencing. The expression clones of Ad-BMP-2 and Ad-TGF-β3 were packaged into maturated adenovirus successfully, the titer was 5.6 x 10(8) and 1.6 x 10(8) pfu/mL respectively. The PCR results showed a light band at 310 bp in group A and at 114 bp in group B, and both 310 bp and 114 bp bands in group C, but no band in group D. The image of immunofluorescence showed that there were red fluorescence and green fluorescence expressions in the cytoplasm of BMSCs at 72 hours after transfection in groups A and B, respectively; in group C, both red and green fluorescence expressions were detected, and no red or green fluorescence was detected in group D. The results of Western blot showed that there was a light band at 18 x 10(3) in group A and at 50 x 10(3) in group B; both 18 x 10(3) and 50 x 10(3) bands were detected in group C; but no band was detected in group D. The cells were positive for collagen type II in groups A, B, and C; group C acquired strong collagen type II staining when compared with group A and group B; in group D, the cells were negative for collagen type II staining.

CONCLUSIONS

The recombinant adenovirus vector expressing BMP-2 and TGF-β3 are constructed successfully. The BMP-2 and TGF-β3 genes could be expressed effectively in BMSCs of Diannan small-ear pig after transfection, which could afford modified seeding cells for cartilage tissue engineering.

OBJECTIVE

In this study, folic acid (FA) was tested for antiteratogenic effects on Tetrachlorodibenzo-p-dioxin (TCDD)-induced cleft palate in fetal mice.

METHODS

In the present study, pregnant mice were dosed with TCDD 24 µg/kg and with or without FA 5 mg/kg body weight on gestation day 10. Control group mice received sesame oil 50 ml/kg body weight on gestation day(GD)10. The mice were sacrificed on GD12.5, GD13.5, GD14.5, GD15.5 and GD16.5. From each pregnant mouse on GD16.5, embryos were obtained to examine under a dissecting microscope, and routine histology was performed for detection and classification of palatal clefts. The fetuses were prepared for histologic examination, scanning electron microscope and TdT-mediated X-dUTP nick and labeling (TUNEL). On GD12.5, GD13.5, GD14.5 and GD15.5. Meanwhile, real-time (RT)-PCR was employed to detect the mRNA expression levels about arylhydrocarbon receptor (AHR) and transforming growth factor (TGF)β3 in this animal model.

RESULTS

Total frequencies of clefts were 70.2% in TCDD group(group B) and 66.3% in TCDD+FA group(group C) in relation to control fetuses(group A). Filopodia disappeared completely at the medial edge epithelia surface on GD15.5 (group A), GD12.5 (group B) and GD14.5 (group C). the RT-PCR results showed that TGF -β3 expression was down-regulated on GD13.5 and GD14.5 compared to the control.

CONCLUSIONS

It is found that folic acid has no protects agaist 2.3.7.8-TCDD-indued cleft palate in the experiment. Meanwhile, TCDD repressed the TGF-β3 expression during the palatal development. Anormal apoptosis was induced by 2, 3, 7, 8-TCDD at the medial edge epithelia (MEE) during the early development stage.

In vivo an ecological network of polysaccharides utilization by gut microbiota is not only an intense competition but also an impressive cooperation pattern. The present study evaluated the in vivo protective effect of combined fungal polysaccharides (CFP) from Cordyceps sinensis and Ganoderma atrum on colon immune dysfunction, induced by 150mg/kg cyclophosphamide (CP). The results showed that C. sinensis polysaccharides (CSP) significantly promoted microbial-derived butyrate to improve histone h3 acetylation mediating regulatory T (Treg) cell specific Foxp3, as well as significantly restored CP-induced elevation of interleukin (IL)-17 and IL-21. Additionally, G. atrum polysaccharides (PSG) significantly down-regulated MyD88, as well as significantly increased IL-10 and TGF-β3. Furthermore, CFP balanced the disequilibrium of cytokines secretion and Foxp3/RORγt ratio related Treg/T helper 17 (Th17) balance, as well as down-regulated the TLR-mediated inflammatory signaling pathway and promoted secretory immunoglobulin A (sIgA) secretion to suppress colonic inflammation. Therefore, our results typically contribute to understand the in vivo immunoregulatory function of fungal polysaccharides compounds, involving microbial-associated inflammatory signals and specific metabolic products.

OBJECTIVE

To study the expression of connective tissue growth factor (CTGF) in the myocardial tissue of mice with viral myocarditis (VMC).

METHODS

Balb/c mice were infected with coxsackie virus B3 (CVB3) to establish VMC model. The mice were divided into control group (n = 50) and VMC group (n = 50). on days 4, 7, 14 and 21 after infection, heart specimens of 8 mice were randomly taken and examined after HE staining for myocardial necrosis and cellular infiltration. The area of positive Masson stained myocardium collagen fibers was measured, and collagen volume fraction (CVF) was measured. Then the level of serum creatine phosphokinase-MB (CKMB) was determined. The levels of CTGF and TGF-β₁ were detected by streptavidin peroxidase immunoperoxidase technique. Expression of CTGF and TGF-β₁ were detected with reverse transcription-polymerase chain reaction (RT-PCR). At the same time, the correlations were analyzed.

RESULTS

(1) The level of CKMB peaked on day 7, and decreased afterwards (455.45 ± 37.95, 606.95 ± 35.64, 573.62 ± 42.90, 308.60 ± 20.49, respectively, 4 - 21 d points), in which 4, 7, 14 d points, there was significant difference compared with control group (t = 6.144, 12.558, 11.182, respectively, P < 0.01). (2) CVF increased significantly on day 14 (8.22 ± 1.95, t = 4.486, P < 0.01) and day 21 (9.46 ± 1.87, t = 4.486, P < 0.01) in VMC group. (3) Measured by streptavidin peroxidase immunoperoxidase technique, the levels of CTGF (171.50 ± 10.25, 141.70 ± 10.863, 110.35 ± 11.051, 81.05 ± 10.190, respectively, 4 - 21 d points) and TGF-β₁ (184.90 ± 11.480, 150.25 ± 9.915, 103.50 ± 10.455, 84.15 ± 9.848, respectively, 4 - 21 d points) increased after day 4 in VMC (P < 0.01). (4) Measured by RT-PCR, the expression of CTGF mRNA and TGF-β₁ increased in VMC group, and the increase was enhanced with the disease development (P < 0.01). (5) The expression of CTGF and TGF-β₁ was positively linearly correlated (r = 0.987, P < 0.01), the expression of CTGF was negatively correlated with CVF (r = -0.901, P < 0.01), but the expression of CTGF was detected earlier than myocardial fibrosis.

CONCLUSIONS

The increase of CTGF expression was associated with the severity of myocardial fibrosis in VMC. These results suggest that abnormal expression of CTGF may take part in the development of fibrosis in VMC.

The severity of tissue injury in burn wounds from associated inflammatory and immune sequelae presents a significant clinical management challenge. Among various biophysical wound management approaches, low dose biophotonics treatments, termed Photobiomodulation (PBM) therapy, has gained recent attention. One of the PBM molecular mechanisms of PBM treatments involves photoactivation of latent TGF-β1 that is capable of promoting tissue healing and regeneration. This work examined the efficacy of PBM treatments in a full-thickness burn wound healing in C57BL/6 mice. We first optimized the PBM protocol by monitoring tissue surface temperature and histology. We noted this dynamic irradiance surface temperature-monitored PBM protocol improved burn wound healing in mice with elevated TGF-β signaling (phospho-Smad2) and reduced inflammation-associated gene expression. Next, we investigated the roles of individual cell types involved in burn wound healing following PBM treatments and noted discrete effects on epithelieum, fibroblasts, and macrophage functions. These responses appear to be mediated via both TGF-β dependent and independent signaling pathways. Finally, to investigate specific contributions of TGF-β1 signaling in these PBM-burn wound healing, we utilized a chimeric TGF-β1/β3 knock-in (TGF-β1^Lβ3/Lβ3) mice. PBM treatments failed to activate the chimeric TGF-β1^Lβ3/Lβ3 complex and failed to improve burn wound healing in these mice. These results suggest activation of endogenous latent TGF-β1 following PBM treatments plays a key role in burn wound healing. These mechanistic insights can improve the safety and efficacy of clinical translation of PBM treatments for tissue healing and regeneration.

BACKGROUND

Diseases of adulthood, such as diabetes and hypertension, may be related to changes during pregnancy, particularly in kidney. We hypothesized that acute kidney injury progresses more rapidly in cases of fetal programming.

METHODS

Diabetic dams' offspring were divided into: CC (controls, receiving vehicle); DC (diabetics, receiving vehicle); CA (controls receiving folic Acid solution, 250 mg/kg); and DA (diabetics receiving folic acid solution). Renal function tests, morphometry, gene, and protein expression of epithelial-mesenchymal transition (EMT) markers were analyzed by qPCR and immunohistochemistry, respectively.

RESULTS

Creatinine, urea, Bowman's space, and EMT markers were increased in CA and DA groups. TGF-β3, actin, and fibronectin expression was higher in CA and DA, with significant increase in DA compared to CA 2-mo offspring. There was higher expression level of TGF-β1, TGF-β3, fibronectin, and vimentin in the offspring of diabetic dams at 5 mo. Increases in TGF-β1 and TGF-β3 were more evident in the offspring of diabetic dams.

CONCLUSIONS

Fetal programming promotes remarkable changes in kidney morphology, and function in offspring and renal failure progression may be faster in younger offspring of diabetic dams subjected to an additional injury.

Objective: To investigate the changes in the cytokine profiles of chronic hepatitis B (CHB) patients undergoing antiviral treatment.

Methods: Hepatitis B e antigen (HBeAg)-positive patients were treated with Pegylated interferon (PEG-IFN) and entecavir (ETV). Clinical biochemistry and cytokines were detected at baseline and every 3 months.

Results: In all, 200 patients completed 48 weeks of treatment, 100 in the PEG-IFN group and 100 in the ETV group. During 3-6 months of treatment, compared with baseline, the PEG-IFN group showed a significant decrease in interferon-gamma (IFN-γ), interleukin-17A (IL-17A), interleukin-6(IL-6), interleukin-10(IL-10), and transforming growth factor beta (TGF-β) ( P < 0.001) and a significant increase in interferon-alpha 2(IFN-α2) ( P < 0.001). In the ETV group, IL-10 and TGF-β1 decreased significantly ( P < 0.001). After 3 months, the levels of IFN-α2, IL-17A, and tumor necrosis factor-alpha(TNF-α) in the PEG-IFN group were significantly higher than those in the ETV group ( P < 0.01). The levels of IL-6 and TGF-β3 were significantly lower than those in the ETV group ( P < 0.01). After 6 months, the levels of IFN-α2, IFN-γ, and TNF-α in the PEG-IFN group were significantly higher than those in the ETV group ( P < 0.01), while the levels of IL-6 and TGF-β3 were significantly lower than those in the ETV group ( P < 0.01). Compared with ETV, PEG-IFN had higher HBeAg and HBsAg disappearance rates.

Conclusion: During antiviral therapy, a change in the cytokine profile occurred; in the aspect of immune control and functional cure, PEG-IFN was significantly better than ETV.

Keywords: Chronic hepatitis B; Cytokine; Interferon; Nucleoside (nucleotide) analog.

Mesenchymal cell proliferation is critical for the growth of the palate shelf. All-trans retinoic acid (atRA), as well as pathways associated with TGF-β/Smad signaling, play crucial roles in the proliferation of mouse embryonic palate mesenchymal (MEPM) cells. We have found that MEPM-cell proliferation was regulated by atRA and exogenous TGF-β3 could significantly antagonize the atRA-mediated suppression of MEPM cell proliferation, which is closely associated with the regulation of TGF-β/Smad signaling pathway. The long non-coding RNA (lncRNA) MEG3 has been reported to activate TGF-β/Smad signaling, thereby regulating cellular proliferation, differentiation, and related processes. Here, we found that Meg3 expression increased significantly in atRA-treated MEPM cells while TGF-β3 treatment markedly inhibited Meg3 expression and antagonized the effect of atRA on Meg3. Moreover, Smad2 was found to interact directly with Meg3, and atRA treatment significantly enriched Meg3 in Smad2-immunoprecipitated samples. After Meg3 deletion, the effects of atRA on the proliferation of MEPM cells and TGF-β3-dependent protein expression were lost. Hence, we speculate that Meg3 has a role in the RA-induced suppression of MEPM cell proliferation by targeting Smad2 and thereby mediating TGF-β/Smad signaling inhibition.

Keywords: Cleft palate; MEPM; Meg3; TGF-β/Smad signaling pathway; atRA.

The most appropriate surface treatment to enhance gingival connective tissue formation on the abutment of dental implants remains undefined, with healing associated with a scar-like response. We have previously shown that topographies with an arithmetic average of the absolute profile height deviations (R_a) = 4.0 induces an anti-fibrotic phenotype in human gingival fibroblasts (HGFs) by causing nascent adhesion formation. With bacterial colonization considerations, we hypothesized that a lower R_a could be identified that would alter adhesion stability and promote a matrix remodeling phenotype. Focal adhesions (FAs) area decreased with increasing roughness, although no differences in cell attachment or proliferation were observed. Alpha smooth muscle actin (α-SMA) protein levels were significantly reduced on R_a = 3.0 and 4.0 vs. 0.1 (p < 0.05), with incorporation of α-SMA into stress fibers most prominent on R_a = 0.1. Fibronectin protein levels were reduced on 3.0 and 4.0 vs. 0.1 (p < 0.05), and R_a = 1.5 and deeper significantly altered fibronectin deposition. Addition of exogenous TGF-β3 increased HGF adhesion size on 0.1 surfaces, but not on any other topography. We conclude that R_a = 1.5 is sufficient to reduce adhesion size and inhibit α-SMA incorporation into stress fibers in HGFs, but 3.0 is required in the presence of exogenous TGF-β3. Our findings have implications for inhibiting fibrotic tissue formation surrounding percutaneous devices such as dental implants.

Keywords: adhesion stability; fibrosis; gingiva; myofibroblasts; titanium surface roughness.

Background: In recent years, there has been a significant decline in the demand for cord blood units (CBUs). This trend has led to cord blood banks (CBBs) exploring complementary uses of CBUs in order to exploit the full potential of this unique, valuable, and readily available product. The aim of this study was to establish standardized protocols for the preparation of four cord blood (CB) derivatives: plasma (CB-P), serum (CB-S), induced-serum (CB-IS) and growth factor-rich plasma (GFRP); to measure their respective growth-factor content and their potential as cell culture supplements, and finally, to identify the characteristics of each derivative beyond their growth-factor composition, in the context of optimizing CBBs resources.

Study design and method: To this end, CB plasma and serum were prepared and their concentrations for ten growth factors (TGF-β1, TGF-β2, TGF-β3, EGF, HGF, PDGF-BB, VEGF-A, VEGF-D, IGF-I, IGF-II) were determined using a multiplex bead array, and compared to adult plasma and serum. Protocols for the preparation of CB-IS and GFRP with reverse anticoagulation using CaCl₂·2H₂O were also established, and all four derivatives were compared for their EGF and TGF-β1 content by enzyme-linked immunosorbent assay (ELISA), and also for their cell culture supplement capacity.

Results: CB plasma was shown to be richer than adult plasma for TGF-β2 and TGF-β3, with a lower concentration of IGF-I and IGF-II. CB serum was shown to be richer than adult serum for TGF-β2, TGF-β3, EGF, HGF, PDGF-BB, and VEGF-A and D, and poorer in IGF-I and II. The measure of EGF and TGF-β1 concentrations has shown that CB serum is the most concentrated (1874 pg/ml and 41 094 pg/ml) of the CB derivatives, followed by induced-serum (405 pg/ml and 16 735 pg/ml), growth factor-rich plasma (131 and 7947 pg/ml) and plasma (8 and 2845 pg/ml). All four CB derivatives were shown to be superior to FBS in sustaining cell growth at low doses.

Conclusion: Our study shows that four CB derivatives can be easily prepared and pooled to provide significant volume of products that vary in their growth factor composition. A cord blood bank interested in introducing such manufacturing will need to evaluate the financial and processing characteristics of each derivative. The use of standardized manufacturing protocols such as the ones we suggest could help research initiatives exploring the potential therapeutic uses of such rich and high-quality starting material.

Keywords: Cord blood; Growth factors; Manufacturing; Plasma; Serum.

Objective: To prepare Pluronic F-127 composite gel loaded with transforming growth factor β ₃ (TGF-β ₃) and bone marrow mesenchymal stem cells (BMSCs) and observe its osteogenesis and angiogenesis effects in vivo and in vitro.

Methods: BMSCs were isolated from the tibial and femoral bone marrow of New Zealand white rabbits and passaged, and the 3rd generation cells were used for subsequent experiments after identification of osteogenic and adipogenic induction. Pluronic F-127 powder and TGF-β ₃ were dissolved in L-DMEM medium to prepare Pluronic F-127 gel, TGF-β ₃+Pluronic F-127 gel, BMSCs+Pluronic F-127 gel, and TGF-β ₃+BMSCs+Pluronic F-127 gel. The 3rd generation of BMSCs were cultured with L-DMEM medium (group A), osteogenic induction medium (group B), osteogenic induction medium containing Pluronic F-127 gel (group C), and osteogenic induction medium containing TGF-β ₃+Pluronic F-127 gel (group D), respectively. After 14 days of culturing, alkaline phosphatase (ALP) staining and Alizarin red staining were used to observe the osteogenesis. In addition, the BMSCs were cultured with L-DMEM medium containing Pluronic F-127 gel (experimental group) and L-DMEM medium (control group) for 1, 2, 3, and 4 days, respectively. And the cell proliferation was detected by MTT assay. Ten New Zealand white rabbits were taken to prepare the maxillary sinus lift models, and Pluronic F-127 gel (group A), TGF-β ₃+Pluronic F-127 gel (group B), BMSCs+Pluronic F-127 gel (group C), and TGF-β ₃+BMSCs+Pluronic F-127 gel (group D) were injected into the bone defects, respectively. On the 8th week, imaging examination and HE staining were used to observe the formation of new bone, immunohistochemical staining was used to observe the expression of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2) in bone tissue, and Western blot was used to detect the relative expressions of VEGF, oncostatin M (OSM), and BMP-4 proteins in bone tissue.

Results: Osteogenic and adipogenic induction identified the isolated and cultured cells as BMSCs. In vitro staining showed that ALP activity and Alizarin red concentration in group D were significantly higher than those in other groups ( P<0.05). MTT assay showed that the absorbency ( A) value of the two groups increased gradually, and there was no significant difference between the groups at each time point ( P>0.05). In vivo experimental imaging examination showed that the bone mineral density and osteogenic continuity of group D were the best, and the proportion of new bone volume was superior to other groups ( P<0.05). HE staining showed that compared with other groups, bone trabeculae in group D were dense and arranged regularly, on which a large number of osteoblasts and osteoclasts were distributed, and a large number of new bone formation could be seen. Immunohistochemical staining showed the strong positive expressions of BMP-2 and VEGF in group D ( P<0.05); Western blot detection showed that the relative expressions of VEGF, OSM, and BMP-4 proteins in group D were significantly higher than those in other groups ( P<0.05).

Conclusion: The BMSCs in Pluronic F-127 composite gel loaded with TGF-β ₃ and BMSCs can be induced to differentiate into osteoblasts, and the composite gel has no toxic effect on cells, and has obvious osteogenesis and angiogenesis in the maxillary sinus of rabbits.

目的: 制备负载TGF-β ₃及BMSCs的Pluronic F-127复合凝胶，观察其体内、外成骨及成血管作用。.

方法: 取新西兰大白兔胫骨及股骨骨髓，分离培养BMSCs并传代，取第3代细胞经成骨、成脂诱导培养鉴定后用于后续实验。采用L-DMEM培养基溶解Pluronic F-127粉末、TGF-β ₃，分别制备 Pluronic F-127凝胶、TGF-β ₃+Pluronic F-127凝胶、BMSCs+Pluronic F-127凝胶、TGF-β ₃+BMSCs+Pluronic F-127凝胶。取第3代BMSCs，分别采用L-DMEM培养基（A组）、成骨诱导液（B组）、含Pluronic F-127凝胶的成骨诱导液（C组）、含TGF-β ₃+Pluronic F-127凝胶的成骨诱导液（D组）培养14 d后，ALP染色和茜素红染色观测成骨情况；另采用含Pluronic F-127凝胶的L-DMEM培养基（实验组）、L-DMEM培养基（对照组）培养1、2、3、4 d，MTT法检测细胞增殖情况。取10只新西兰兔制备上颌窦提升模型后，于每只兔骨缺损处注入Pluronic F-127凝胶（A组）、TGF-β ₃+Pluronic F-127凝胶（B组）、BMSCs+Pluronic F-127凝胶（C组）、TGF-β ₃+BMSCs+Pluronic F-127凝胶（D组），于第8周取材行影像学检查、HE染色观察新骨形成情况，免疫组织化学染色观察骨组织VEGF及BMP-2表达情况，Western blot检测骨组织VEGF、抑瘤素M（oncostatin M，OSM）及BMP-4蛋白表达。.

结果: 成骨、成脂诱导鉴定示分离培养细胞为BMSCs。体外实验染色显示D组ALP活性及茜素红浓度高于其他组（ P<0.05）；MTT法检测示随着时间延长，两组吸光度（ A）值均逐渐升高，各时间点组间比较差异均无统计学意义（ P>0.05）。体内实验影像学检查示D组成骨密度及成骨连续性最好，新骨体积占比优于其他组（ P<0.05）；HE染色示与其他组比较，D组骨小梁致密且排列规则，其上分布大量成骨细胞和破骨细胞，可见大量新骨形成；免疫组织化学染色示D组BMP-2、VEGF呈强阳性表达（ P<0.05）；Western blot检测D组VEGF、OSM及BMP-4蛋白相对表达量高于其他组（ P<0.05）。.

结论: 负载TGF-β ₃及BMSCs的Pluronic F-127复合凝胶中的BMSCs能被诱导分化为成骨细胞，并且复合凝胶对细胞无毒性，在兔上颌窦内有明显成骨及成血管效果。.

Keywords: Bone tissue engineering; Pluronic F-127 gel; angiogenesis; bone marrow mesenchymal stem cells; maxillary sinus lift; osteogenesis; rabbit; transforming growth factor β3.

Varicocele is an age-related disease with no current medical treatments positively impacting infertility. Toll-like receptor 4 (TLR4) expression is present in normal testis with an involvement in the immunological reactions. The role of peroxisome proliferator-activated receptor-α (PPAR-α), a nuclear receptor, in fertility is still unclear. N-Palmitoylethanolamide (PEA), an emerging nutraceutical compound present in plants and animal foods, is an endogenous PPAR-α agonist with well-demonstrated anti-inflammatory and analgesics characteristics. In this model of mice varicocele, PPAR-α and TLR4 receptors' roles were investigated through the administration of ultra-micronized PEA (PEA-um). Male wild-type (WT), PPAR-α knockout (KO), and TLR4 KO mice were used. A group underwent sham operation and administration of vehicle or PEA-um (10 mg/kg i.p.) for 21 days. Another group (WT, PPAR-α KO, and TLR4 KO) underwent surgical varicocele and was treated with vehicle or PEA-um (10 mg/kg i.p.) for 21 days. At the end of treatments, all animals were euthanized. Both operated and contralateral testes were processed for histological and morphometric assessment, for PPAR-α, TLR4, occludin, and claudin-11 immunohistochemistry and for PPAR-α, TLR4, transforming growth factor-beta3 (TGF-β3), phospho-extracellular signal-Regulated-Kinase (p-ERK) 1/2, and nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) Western blot analysis. Collectively, our data showed that administration of PEA-um revealed a key role of PPAR-α and TLR4 in varicocele pathophysiology, unmasking new nutraceutical therapeutic targets for future varicocele research and supporting surgical management of male infertility.

Keywords: NLRP3; PEA-um; PPAR; TGF-β3; TLR4; claudin-11; nutraceutical; occludin; p-ERK 1/2; testis; varicocele.

Transforming growth factor beta (TGF-β) plays an important role in the viral liver disease progression via controlling viral propagation and mediating inflammation-associated responses. However, the antiviral activities and mechanisms of TGF-β isoforms, including TGF-β1, TGF-β2 and TGF-β3, remain unclear. Here, we demonstrated that all of the three TGF-β isoforms were increased in Huh7.5 cells infected by hepatitis C virus (HCV), but in turn, the elevated TGF-β isoforms could inhibit HCV propagation with different potency in infectious HCV cell culture system. TGF-β isoforms suppressed HCV propagation through interrupting several different stages in the whole HCV life cycle, including virus entry and intracellular replication, in TGF-β/SMAD signalling pathway-dependent and TGF-β/SMAD signalling pathway-independent manners. TGF-β isoforms showed additional anti-HCV activities when combined with each other. However, the elevated TGF-β1 and TGF-β2, not TGF-β3, could also induce liver fibrosis with a high expression of type I collagen alpha-1 and α-smooth muscle actin in LX-2 cells. Our results showed a new insight into TGF-β isoforms in the HCV-related liver disease progression.

Keywords: TGF-β isoform; TGF-β signalling pathway; addition; hepatitis C virus; liver fibrosis.