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FGF-2 accumulates in many tumor tissues and is closely related to the development of tumor angiogenesis and the immunosuppressive microenvironment. This study aimed to investigate whether active immunization against FGF-2 could modify antitumor immunity and enhance the efficacy of an HPV16 E7-specific therapeutic vaccine. Combined immunization targeting both FGF-2 and E7 significantly suppressed tumor growth, which was accompanied by significantly increased levels of IFN-γ-expressing splenocytes and effector CD8 T cells and decreased levels of immunosuppressive cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells(MDSCs) in both the spleen and tumor; in addition, the levels of FGF-2 and neovascularization in tumors were decreased in the mice receiving the combined immunization, and tumor cell apoptosis was promoted. The combination of an HPV16 E7-specific vaccine and active immunization against FGF-2 significantly enhances antitumor immune responses in mice with TC-1 tumors, indicating a promising strategy for tumor immunotherapy., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cultured peritoneal macrophages from intact (control) and BCG-infected (experiment) male BALB/c mice were studied 90 days after infection. Polarization of macrophages by M1 (expression of GM-CSF, IFN γ , and CD16/32) and M2 (expression of bFGF and CD36) differentiation pathways was studied with consideration for their the nuclearity class. Mononuclear cells predominated (90% and higher) in macrophage cultures of both groups and presumably, were presented by mainly epithelioid cells. The results indicated polarization of mononuclear and multinuclear macrophages in the M2 direction under conditions of BCG granulomatosis and a higher initial M2 polarization of binuclear macrophages. In control cultures, the ratio of M2 to M1 macrophages was 0.57, in experimental cultures this ratio was 1.6. It seems that long persistence of Mycobacterium tuberculosis in macrophages served as a factor stimulating the plastic processes and transformation of macrophages into epithelioid cells that form the "core" of granulomas and their enlargement upon incorporation of macrophages.

Mesenchymal stem cells (MSCs) have become a promising tool in cellular therapy for restoring immune system haemostasis; however, the success of clinical trials has been impaired by the lack of standardized manufacturing processes. This study aims to determine the suitability of source tissues and culture media for the production of MSC-based advanced therapy medicinal products (ATMPs) and to define parameters to extend the set of release criteria. MSCs were isolated from umbilical cord (UC), bone marrow and lipoaspirate and expanded in three different culture media. MSC phenotype, proliferation capacity and immunosuppressive parameters were evaluated in normal MSCs compared to primed MSCs treated with cytokines mimicking an inflammatory environment. Compared to bone marrow and lipoaspirate, UC-derived MSCs (UC-MSCs) showed the highest proliferative capacity, which was further enhanced by media supplemented with bFGF, while the cells maintained their immunosuppressive characteristics. Moreover, UC-MSCs expanded in the bFGF-enriched medium were the least sensitive to undesirable priming-induced changes in the MSC phenotype. Surface markers and secreted factors were identified to reflect the cell response to inflammatory priming and to be variable among MSCs from different source tissues. This study demonstrates that UC is a favorable cell source for manufacturing MSC-based ATMPs for immunosuppressive applications. UC-MSCs are able to use the bFGF-enriched medium for higher cell yields without the impairment of immunosuppressive parameters and undesirable phenotype changes after inflammatory preconditioning of MSCs before transplantation. Additionally, immunosuppressive parameters were identified to help finding predictors of clinically efficient MSCs in the following clinical trials.

Background: Fibroblast growth factor (FGF)-2 is overexpressed in various tumor tissues. It affects tumor cell proliferation, invasion and survival, promotes tumor angiogenesis and is tightly involved in the development of systemic and local immunosuppressive tumor mechanisms., Purpose: This study aimed to develop an effective vaccine against FGF-2 and to investigate the effects of anti-FGF-2 immunization on tumor growth and antitumor immune responses., Methods: A set of thirteen synthesized overlapping peptides covering all possible linear B-cell epitopes of murine FGF-2 and a recombinant FGF-2 protein were conjugated to virus-like particles (VLPs) of recombinant hepatitis B core antigen (HBcAg). The VLPs were immunized through a preventive or therapeutic strategy in a TC-1 or 4T1 grafted tumor model., Results: Immunization with FGF-2 peptides or full-length protein-coupled VLPs produced FGF-2-specific antibodies with a high titer. Peptide 12, which is located in the heparin-binding site of FGF-2, or protein-conjugated VLPs presented the most significant effects on the suppression of TC-1 tumor growth. The levels of IFN-γ-expressing splenocytes and serum IFN-γ were significantly elevated; further, the immune effector cells CD8 + IFN-γ + cytotoxic T lymphocytes (CTLs) and CD4 + IFN-γ + Th1 cells were significantly increased, whereas the immunosuppressive cells CD4 + CD25 + FOXP3 + Treg cells and Gr-1 + CD11b + myeloid-derived suppressor cells (MDSCs) were decreased in the immunized mice. In addition, VLP immunization significantly suppressed tumor vascularization and promoted tumor cell apoptosis. In mice bearing 4T1 breast tumor, preventive immunization with FGF-2-conjugated VLPs suppressed tumor growth and lung metastasis, and increased effector cell responses., Conclusion: Active immunization against FGF-2 is a new possible strategy for tumor immunotherapy., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Shu et al.)

Administration of RC28-E, a VEGF/bFGF dual decoy receptor (IgG1 Fc-fusion protein), have shown relative therapeutic value in ocular in vivo models, including laser-induced choroidal neovascularization (CNV) in monkeys and streptozotocin (STZ)-induced diabetic retinopathy (DR) in rats. In the present study, we have elucidated the pharmacokinetics profiles of RC28-E at the systemic, vitreous and aqueous humor after administration in a primate model (Macaca fascicularis). Moreover, here we tease out the ocular tissue distribution of RC28-E after intravitreal administration, and we also determine the systemic bioavailability after both intravitreal and intravenous administration. Our results show that RC28-E is rapidly and well-distributed into ocular tissues after intravitreal administration. Drug exposure in choroid and retina was approximately one-quarter and one-twelfth of that in vitreous humor, while its half-life in vitreous and aqueous humor were well-sustained (3.3 and 3.0 days). Remarkably, RC28-E could cross the blood-ocular barrier, and the systemic bioavailability of RC28-E was ~25%. No drug accumulation after multiple administration was noticed, but low titers of antibody produce against RC28-E were detected. Overall, RC28-E exhibited high clinical value due to adequate pharmacokinetic profiling, safety and efficacy., (Copyright © 2019. Published by Elsevier Ltd.)

The human fibroblast growth factor-2 (FGF-2) highly expressed in tumours is an important factor to promote tumour angiogenesis and lymphangiogenesis. A disulphide-stabilized diabody (ds-Diabody) could specifically target FGF-2 and show its advantages in inhibition of tumour angiogenesis and growth. It is very important for antibody drugs to confirm the fine epitope. Here, theoretical structure models of FGF-2 and antibody were built by homology modelling. The amino acid residues in the interaction interface of antigen and antibody were analysed by molecular docking. The potential epitope was predicted by homology modelling and molecular docking of antigen-antibody and site-directed mutation assays of alanine scanning. The predicted epitope was verified by antigen mutagenesis and enzyme-linked immunosorbent assay (ELISA). The epitope mapping assay showed that the epitope of ds-Diabody against FGF-2 was defined by the discontinuous sites including six amino acid residues (P23, Q65, R69, G70, Y82 and R118). The results showed that the epitope was localized in the interaction interface of FGF-2 and ds-Diabody. The fine epitope mapping provided the important information for understanding the inhibition activity of ds-Diabody against FGF-2 and helping in the further development of ds-Diabody against FGF-2 as a potentially promising antibody drug for future cancer therapy., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Objective: The study aimed to investigate the correlations of insulin resistance and hemoglobin A1c (HbA1c) with cytokines [insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF) and interleukin-6 (IL-6)] in the aqueous humor of patients with diabetic cataract., Patients and Methods: 59 patients with diabetic cataract and 58 patients with simple cataract treated in Jining No. 1 People´s Hospital (Jining, China) from January 2017 to February 2018, were selected randomly. The levels of homeostasis model assessment of insulin resistance (HOMA-IR) and HbAlc, as well as IGF-1, bFGF and IL-6 in the aqueous humor were compared between the two groups. The correlations of HOMA-IR and HbAlc with IGF-1, bFGF and IL-6 were analyzed. In control group, the levels of HOMA-IR and HbAlc, as well as IGF-1, bFGF and IL-6 in the aqueous humor were significantly lower than those in observation group (p<0.05)., Results: Compared with the group with HbAlc ≤ 7%, the groups with HbAlc ≥ 9% and 7%

Influenza virus (IAV) infection is a major cause of severe respiratory illness that affects almost every country in the world. IAV infections result in respiratory illness and even acute lung injury and death, but the underlying mechanisms responsible for IAV pathogenesis have not yet been fully elucidated. In this study, the basic fibroblast growth factor 2 (FGF2) level was markedly increased in H1N1 virus-infected humans and mice. FGF2, which is predominately derived from epithelial cells, recruits and activates neutrophils via the FGFR2-PI3K-AKT-NFκB signaling pathway. FGF2 depletion or knockout exacerbated influenza-associated disease by impairing neutrophil recruitment and activation. More importantly, administration of the recombinant FGF2 protein significantly alleviated the severity of IAV-induced lung injury and promoted the survival of IAV-infected mice. Based on the results from experiments in which neutrophils were depleted and adoptively transferred, FGF2 protected mice against IAV infection by recruiting neutrophils. Thus, FGF2 plays a critical role in preventing IAV-induced lung injury, and FGF2 is a promising potential therapeutic target during IAV infection.

Fibroblast growth factor receptor-1 (FGFR1) activity at the plasma membrane is tightly controlled by the availability of co-receptors and competing receptor isoforms. We have previously shown that FGFR1 activity in pancreatic beta-cells modulates a wide range of processes, including lipid metabolism, insulin processing, and cell survival. More recently, we have revealed that co-expression of FGFR5, a receptor isoform that lacks a tyrosine-kinase domain, influences FGFR1 responses. We therefore hypothesized that FGFR5 is a co-receptor to FGFR1 that modulates responses to ligands by forming a receptor heterocomplex with FGFR1. We first show here increased FGFR5 expression in the pancreatic islets of nonobese diabetic (NOD) mice and also in mouse and human islets treated with proinflammatory cytokines. Using siRNA knockdown, we further report that FGFR5 and FGFR1 expression improves beta-cell survival. Co-immunoprecipitation and quantitative live-cell imaging to measure the molecular interaction between FGFR5 and FGFR1 revealed that FGFR5 forms a mixture of ligand-independent homodimers (∼25%) and homotrimers (∼75%) at the plasma membrane. Interestingly, co-expressed FGFR5 and FGFR1 formed heterocomplexes with a 2:1 ratio and subsequently responded to FGF2 by forming FGFR5/FGFR1 signaling complexes with a 4:2 ratio. Taken together, our findings identify FGFR5 as a co-receptor that is up-regulated by inflammation and promotes FGFR1-induced survival, insights that reveal a potential target for intervention during beta-cell pathogenesis., (© 2018 Regeenes et al.)

Tumor growth and metastasis are closely related to angiogenesis. Basic fibroblast growth factor(bFGF) is an angiogenic factor, and up-regulated expression of bFGF plays a crucial role in the development and metastasis of melanoma. Therefore, in this study, we sought to achieve antitumor activity by immunity targeting bFGF which would inhibit tumor angiogenesis and simultaneously induce bFGF specific cytotoxic T lymphocytes to kill melanoma cells. A human bFGF protein was used as exogenous antigen, coupled with a saponin-liposome adjuvant formulation to enhance CTL response. The results showed that the immunity induced strong immune response and produced prominent anti-cancer activities. CD31 immunohistochemistry and alginate-encapsulated tumor cell assay displayed that tumor angiogenesis was effectively inhibited. Further, the higher production of IFN-γ and cytotoxic T lymphocyte killing assay suggested that the anti-cancer activities may mainly depend on cellular immune response, which could cause the inhibition of tumor angiogenesis and specific killing of tumor cells by bFGF-specific cytotoxic T lymphocytes. We concluded that immunotherapy targeting bFGF may be a prominent strategy for melanoma, and that the adjuvant formulation of saponin-liposome is very desirable in enhancing cytotoxic T lymphocytes response.

The incidence of mono- and multinuclear cells and their expression of pro- and antifibrotic factors were studied in cultured peritoneal macrophages from intact and BCG-infected mice. Generally, the expression of factors increased with an increase in the number of nuclei per cell. However, the expression was higher in macrophages from BCG infected mice, except the cells with 3 and more nuclei, extremely rarely expressing IL-1α in cultures from intact and BCG-infected animals. The number of macrophages with 3 and more nuclei, expressing CatD, was comparable with the number of mono- and binuclear macrophages. Presumably, this was determined by various mechanisms of formation of multinuclear (3-5 and more nuclei) macrophages, for example, by amitosis.

Objective: We aimed at exploring the positive feedback loop in eutopic and ectopic endometrial glandular epithelial cells (EuECs and EECs) in endometriosis., Materials and Methods: Normal epithelial cells (NECs), EuECs and EECs were treated with fibroblast growth factor (FGF)2, FGF2 neutralizing antibody, mitogen-activated protein kinases (MAPKs) inhibitors U0126 and PD98059. FGF2 protein level was detected by enzyme-linked immunosorbent assay (ELISA). The expressions of FGF2, FGF receptor 1 (FGFR1), extracellular signal-regulated kinase (ERK)1/2/pERK1/2 and Sproutys (SPRYs) (Sprouty1, Sprouty2, Sprouty4) and dual specificity phosphatase 6 (DUSP6) were detected by Western blot. The mRNA levels of FGF2, FGFR1 (FGF receptor 1), SPRYs (Sprouty1, Sprouty2, Sprouty4) and DUSP6 mRNA were detected by RT-PCR., Results: Among treatment groups, the content of FGF2 in EuECs and EECs was significantly higher than that in NECs (p < 0.05). The mRNA and protein levels of FGF2, FGFR1, SPRYs (Sprouty1, Sprouty2, Sprouty4) and DUSP6 in EuECs and EECs were increased after adding FGF2 (p < 0.05), but decreased after adding FGF2 neutralizing antibody, no significant change was found in NECs (p > 0.05). The inhibitory effect of PD9805 on NECs was not significantly different from that of U0126 (p > 0.05); however, the inhibitory effects of PD9805 on EuECs and EECs were significantly lower than those of U0126 (p< 0.05)., Conclusions: The positive feedback loop existed in EuECs and EECs, but maybe not in NECs. The results may provide the guideline to treat endometriosis patients.

Objective: To study the synergistic inhibitory effects of basic fibroblast growth factor (bFGF) monoclonal antibody (bFGF mAb) and irinotecan on the proliferation of small cell lung cancer H223 cells., Methods: CCK-8 assay and flow cytometry were used to assess the effects of bFGF mAb combined with irinotecan on the proliferation and apoptosis of H223 cells, respectively. Western blotting was performed to analyze the effect of bFGF-mAb combined with irinotecan on AKT and ERK1/2 phosphorylation in the cells., Results: Both bFGF mAb and irinotecan alone inhibited H223 cell proliferation in a dose-dependent manner (P<0.05). The inhibitory rate was significantly higher in H223 cells treated with bFGF mAb + irinotecan (54.30%) than in cell treated with bFGF mAb (18.73%) or irinotecan (21.96%) alone (P<0.05). Both bFGF mAb and irinotecan induced H223 cell apoptosis in a dose-dependent manner (P<0.05), and the combined treatment resulted in a significantly higher early apoptosis rates (6.5%) than treatment with bFGF mAb (2.7%) or irinotecan (4.3%) alone (P<0.05). bFGF mAb and irinotecan, either alone or in combination, significantly inhibited the levels of p-AKT protein and p-ERK1/2 protein without obviously affecting AKT and ERK1/2 protein levels., Conclusion: bFGF mAb and irinotecan produce synergistic inhibitory effects on small cell lung cancer H223 cells by suppressing proliferation and promoting apoptosis of the cells, and can effectively block the MAPK/ERK and PI3K/AKT signaling pathways associated with bFGF.

Activated CD4 T cells connect to airway smooth muscle cells (ASMCs) in vitro via lymphocyte-derived membrane conduits (LMCs) structurally similar to membrane nanotubes with unknown intercellular signals triggering their formation. We examined the structure and function of CD4 T cell-derived LMCs, and we established a role for ASMC-derived basic fibroblast growth factor 2 (FGF2b) and FGF receptor (FGFR)1 in LMC formation. Blocking FGF2b's synthesis and FGFR1 function reduced LMC formation. Mitochondrial flux from ASMCs to T cells was partially FGF2b and FGFR1 dependent. LMC formation by CD4 T cells and mitochondrial transfer from ASMCs was increased in the presence of asthmatic ASMCs that expressed more mRNA for FGF2b compared with normal ASMCs. These observations identify ASMC-derived FGF2b as a factor needed for LMC formation by CD4 T cells, affecting intercellular communication., (Copyright © 2017 by The American Association of Immunologists, Inc.)

The over-expression of basic fibroblast growth factor (bFGF) plays a crucial role in the development, invasion and metastasis of lung cancer. Therefore, neutralizing antibodies against bFGF may inhibit the growth of lung cancer. In this study, a Disulfide-stabilized diabody (ds-Diabody) against bFGF was constructed by site-directed mutation and overlap extension PCR (SOE-PCR) at the position of VH44 and VL100 in the scFv. The ds-Diabody was constructed and expressed in Pichia pastoris. We found that the ds-Diabody against bFGF could efficiently suppress the proliferation, migration and invasion of human lung cancer A549 cells in vitro. Moreover, in A549 cells, the ds-Diabody against bFGF could inhibit bFGF-induced activation of downstream signaling regulators, such as phospho-Akt and phospho-MAPK. In the nude mouse xenograft model of lung cancer, the ds-Diabody against bFGF could significantly inhibit tumor growth and decrease the densities of micro-vessels and lymphatic vessels in tumor tissue. Our data indicate that the ds-Diabody against bFGF could effectively suppress the lung cancer growth through blockade of bFGF signaling pathway and inhibition of tumor angiogenesis, which may make it a potential therapeutic candidate antibody drug for human lung cancer therapy.

A subset of patients with depression have elevated levels of inflammatory cytokines, and some studies demonstrate interaction between inflammatory factors and treatment outcome. However, most studies focus on only a narrow subset of factors in a patient sample. In the current study, we analyzed broad immune profiles in blood from patients with treatment-resistant depression (TRD) at baseline and following treatment with the glutamate modulator ketamine. Serum was analyzed from 26 healthy control and 33 actively depressed TRD patients free of antidepressant medication, and matched for age, sex and body mass index. All subjects provided baseline blood samples, and TRD subjects had additional blood draw at 4 and 24 h following intravenous infusion of ketamine (0.5 mg kg -1 ). Samples underwent multiplex analysis of 41 cytokines, chemokines and growth factors using quantitative immunoassay technology. Our a priori hypothesis was that TRD patients would show elevations in canonical pro-inflammatory cytokines; analyses demonstrated significant elevation of the pro-inflammatory cytokine interleukin-6. Further exploratory analyses revealed significant regulation of four additional soluble factors in patients with TRD. Several cytokines showed transient changes in level after ketamine, but none correlated with treatment response. Low pretreatment levels of fibroblast growth factor 2 were associated with ketamine treatment response. In sum, we found that patients with TRD demonstrate a unique pattern of increased inflammatory mediators, chemokines and colony-stimulating factors, providing support for the immune hypothesis of TRD. These patterns suggest novel treatment targets for the subset of patients with TRD who evidence dysregulated immune functioning.

Fibroblast growth factor-2 (FGF-2) is one of the most important angiogenic factors to promote tumor growth, progression and metastasis. Neutralizing antibodies against FGF-2 may suppress the growth of tumor cells by blocking the FGF-2 signaling pathway. In this study, a disulfide-stabilized diabody (ds-Diabody) that specifically targets FGF-2 was designed. Compared to its parent antibody, the introduction of disulphide bonds in the diabody could significantly increase the stability of ds-Diabody and maintain its antigen binding activity. The ds-Diabody against FGF-2 could effectively inhibit the tube formation and migration of vascular endothelial cells and block the proliferation and invasion of human breast cancer cells. In the mouse model of breast cancer xenograft tumors, the ds-Diabody against FGF-2 could significantly inhibit the growth of tumor cells. Moreover, the densities of microvessels stained with CD31 and lymphatic vessels stained with LYVE1 in tumors showed a significant decrease following treatment with the ds-Diabody against FGF-2. Our data indicated that the ds-Diabody against FGF-2 could inhibit tumor angiogenesis, lymphangiogenesis and tumor growth., (© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Aim: To investigate the inhibitory efficacy of (125)I-labeled anti-basic fibroblast growth factor (bFGF) monoclonal antibody (mAb) in hepatocellular carcinoma (HCC)., Methods: bFGF mAb was prepared by using the 1G9B9 hybridoma cell line with hybridization technology and extracted from ascites fluid through a Protein G Sepharose affinity column. After labeling with (125)I through the chloramine-T method, bFGF mAb was further purified by a Sephadex G-25 column. Gamma radiation counter GC-1200 detected radioactivity of (125)I-bFGF mAb. The murine H22 HCC xenograft model was established and randomized to interventions with control (phosphate-buffered saline), (125)I-bFGF mAb, (125)I plus bFGF mAb, bFGF mAb, or (125)I. The ratios of tumor inhibition were then calculated. Expression of bFGF, fibroblast growth factor receptor (FGFR), platelet-derived growth factor, and vascular endothelial growth factor (VEGF) mRNA was determined by quantitative reverse transcriptase real-time polymerase chain reaction., Results: The purified bFGF mAb solution was 8.145 mg/mL with a titer of 1:2560000 and was stored at -20 °C. After coupling, (125)I-bFGF mAb was used at a 1: 1280000 dilution, stored at 4 °C, and its specific radioactivity was 37 MBq/mg. The corresponding tumor weight in the control, (125)I, bFGF mAb, (125)I plus bFGF mAb, and (125)I-bFGF mAb groups was 1.88 ± 0.25, 1.625 ± 0.21, 1.5 ± 0.18, 1.41 ± 0.16, and 0.98 ± 0.11 g, respectively. The tumor inhibition ratio in the (125)I, bFGF mAb, (125)I plus bFGF mAb, and (125)I-bFGF mAb groups was 13.6%, 20.2%, 25.1%, and 47.9%, respectively. Growth of HCC xenografts was inhibited significantly more in the (125)I-bFGF mAb group than in the other groups (P < 0.05). Expression of bFGF and FGFR mRNA in the (125)I-bFGF mAb group was significantly decreased in comparison with other groups (P < 0.05). Groups under interventions revealed increased expression of VEGF mRNA (except for (125)I group) compared with the control group., Conclusion: (125)I-bFGF mAb inhibits growth of HCC xenografts. The coupling effect of (125)I-bFGF mAb is more effective than the concomitant use of (125)I and bFGF mAb.

Compelling evidence suggests that fibroblast growth factor 2 (FGF2), overexpressed in melanomas, plays an important role in tumor growth, angiogenesis and metastasis. In this study, we evaluated the therapeutic use of a new anti-FGF2 monoclonal antibody (mAb), 3F12E7, using for that the B16-F10 melanoma model. The FGF2 neutralizing effect of this antibody was certified by in vitro assays, which allowed the further track of its possible in vivo application. 3F12E7 mAb could be retained in B16-F10 tumors, as shown by antibody low-pH elution and nuclear medicine studies, and also led to reduction in number and size of metastatic foci in lungs, when treatment starts one day after intravenous injection of B16-F10 cells. Such data were accompanied by decreased CD34(+) tumor vascular density and impaired subcutaneous tumor outgrowth. Treatments starting one week after melanoma cell intravenous injection did not reduce tumor burden, remaining the therapeutic effectiveness restricted to early-adopted regimens. Altogether, the presented anti-FGF2 3F12E7 mAb stands as a promising agent to treat metastatic melanoma tumors in adjuvant settings., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Fibroblast growth factor (FGF)2,which is one of the 22 members of the FGF family, functions as an extracellular molecule involved in canonical receptor tyrosine kinase signaling. It has been implicated in angiogenesis and the development of the CNS. In this article, we reveal that cytosolic low m.w. isoform (LMW) FGF2 (18 kDa), not its secreted form, plays an unexpected role in the innate immune response. Cytosolic LMW FGF2 directly associated with inactivated RIG-I under physiological conditions, which enhanced RIG-I protein stability, thereby maintaining basal RIG-I levels. However, during RIG-I activation induced by viral RNA, cytosolic FGF2 bound to the caspase recruitment domains of activated RIG-I, which blocked RIG-I-MAVS complex formation. LMW FGF2 deficiency increased type I IFN production, whereas the overexpression of LMW FGF2 exerted the opposite effect. Cytosolic LMW FGF2 functions as a negative regulator in RIG-I-mediated antiviral signaling. This work provides insight into the role of FGF2 in innate immune response., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Background: Glial derived neurotrophic factor (GDNF) and basic fibroblast growth factor (FGF-2) protect nigrostriatal dopaminergic (DA) neurons and their projections in animal models of Parkinson's disease (PD). Recent data indicate neuroprotective effects of estrogen in PD animal models through its anti-inflammatory and anti-oxidative effects, yet the hormonal effects on GDNF and FGF-2 expression in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice remain uninvestigated., Objective: To determine the effects of 17 beta-estradiol (E2) on DA innervation and the expression ofGDNFandFGF-2 in the striatum of MPTP-treated mice., Material and Method: Adult male mice were treated with E2 or vehicle for 11 days during which they were injected with MPTP or saline on the sixth day. The striatum was collected on day 11 and processedfor tyrosine hydroxylase (TH), GDNF and FGF-2 immunohistochemistry. Extent ofDA innervation and the expression of GDNF and FGF-2 in the striatum were assessed by measuring optical density of TH, GDNF and FGF-2 immunoreactivity, respectively., Results: MPTP induced loss of DA axons and upregulation of FGF-2 expression, but did not alter GDNF level. E2 alleviated loss of DA axons, increased GDNF level, yet caused no change in FGF-2 level ofthe MPTP-intoxicated animals., Conclusion: One possible mechanism by which E2 protects nigrostriatal DA axons against MPTP is through upregulation ofstriatal GDNF.

The intestinal epithelial barrier plays a critical role in the mucosal immunity. However, it remains largely unknown how the epithelial barrier is maintained after damage. Here we show that growth factor FGF2 synergized with interleukin-17 (IL-17) to induce genes for repairing of damaged epithelium. FGF2 or IL-17 deficiency resulted in impaired epithelial proliferation, increased pro-inflammatory microbiota outgrowth, and consequently worse pathology in a DSS-induced colitis model. The dysregulated microbiota in the model induced transforming growth factor beta 1 (TGFβ1) expression, which in turn induced FGF2 expression mainly in regulatory T cells. Act1, an essential adaptor in IL-17 signaling, suppressed FGF2-induced ERK activation through binding to adaptor molecule GRB2 to interfere with its association with guanine nucleotide exchange factor SOS1. Act1 preferentially bound to IL-17 receptor complex, releasing its suppressive effect on FGF2 signaling. Thus, microbiota-driven FGF2 and IL-17 cooperate to repair the damaged intestinal epithelium through Act1-mediated direct signaling cross-talk., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: Human bone marrow mesenchymal stromal cells (hBM-MSC) are being applied in tissue regeneration and treatment of autoimmune diseases (AD). Their cellular and immunophenotype depend on isolation and culture conditions which may influence their therapeutic application and reflect their in vivo biological functions. We have further characterised the phenotype induced by fibroblast growth factor 2 (FGF2) on healthy donor hBM-MSC focusing on the osteoimmunological markers osteoprotegerin (OPG), receptor activator of nuclear factor kB (RANK), RANK ligand (RANKL) and HLA-DR and their regulation of expression by the inflammatory cytokines IL1β and IFNγ., Methods: RANK, RANKL, OPG and HLA-DR expression in hBM-MSC expanded under specific culture conditions, were measured by RT-PCR and flow cytometry. MAPKs induction by FGF2, IL1β and IFNγ in hBM-MSC was analysed by immunoblotting and RT-PCR., Results: In hBM-MSC, OPG expression is constitutive and FGF2 independent. RANKL expression depends on FGF2 and ERK1/2 activation. IL1β and IFNγ activate ERK1/2 but fail to induce RANKL. Only IL1β induces P38MAPK. The previously described HLA-DR induced by FGF2 through ERK1/2 on hBM-MSC, is suppressed by IL1β through inhibition of CIITA transcription. HLA-DR induced by IFNγ is not affected by IL1β in hBM-MSC, but is suppressed in articular chondrocytes and lung fibroblasts., Conclusions: RANKL expression and IL1β regulated MHC-class II, both induced via activation of the ERK1/2 signalling pathway, are specific for progenitor hBM-MSC expanded in the presence of FGF2. HLA-DR regulated by IL1β and ERK1/2 is observed on hBM-MSC during early expansion without FGF2 suggesting previous in vivo acquisition. Stromal progenitor cells with this phenotype could have an osteoimmunological role during bone regeneration., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are important proangiogenic factors in tumor procession. The autocrine and paracrine bFGF and the VEGF in tumor tissue can promote tumor angiogenesis, tumor growth, and metastasis. A VEGF/bFGF Complex Peptide (VBP3) was designed on the basis of epitope peptides from both VEGF and bFGF to elicit in vivo production of anti-bFGF and anti-VEGF antibodies. In this study, we reported on the production of recombinant VBP3 using high cell density fermentation. Fed-batch fermentation for recombinant VBP3 production was conducted, and the production procedure was optimized in a 10-L fermentor. The fraction of soluble VBP3 protein obtained reached 78% of total recombinant protein output under fed-batch fermentation. Purified recombinant VBP3 could inhibit tumor cell proliferation in vitro and stimulate C57BL/6 mice to produce high titer anti-VEGF and anti-bFGF antibodies in vivo. A melanoma-grafted mouse model and an immunohistochemistry assay showed that tumor growth and tumor angiogenesis were significantly inhibited in VBP3-vaccinated mice. These results demonstrated that soluble recombinant VBP3 could be produced by large-scale fermentation, and the product, with good immunogenicity, elicited production of high-titer anti-bFGF and anti-VEGF antibodies, which could be used as a therapeutic tumor vaccine to inhibit tumor angiogenesis and tumor growth., (© 2014 American Institute of Chemical Engineers.)

Obese Zucker diabetic fatty (ZDF) rats are used as a type-2 diabetes model for microvascular complications. In order to study retinopathy in this model, changes in retinal vasculature were analyzed by quantitative morphometry and related to retinal expression of 46 selected genes that were analyzed by microfluidic card PCR technology. At 3 months of age, obese animals had developed stable hyperglycemia (20.7 ± 1.3 mmol/L plasma glucose vs. 6.5 ± 0.1 mmol/L in lean). Hyperinsulinemia initially presented in obese rats at 2 months (10.5 ± 0.7 μg/L plasma insulin vs. 0.2 ± 0.04 μg/L in lean) and decreased at 3 months (3.9 ± 0.6 vs. 0.5 ± 0.09 μg/ml in lean). At 8 months of age, animals had developed microvascular complications. An increased number of acellular capillaries in obese (24 ± 5/mm(2)) versus lean (15 ± 4/mm(2)) and a decreased number of retinal pericytes in obese (2,270 ± 250/mm(2)) versus lean animals (1,620 ± 243/mm(2)) could be observed. VEGFa, MIF, and HIF-1α were the most abundantly expressed and inflammatory genes such as TNFα and IL-6 are the least abundantly expressed genes. None of these genes were differentially regulated. Surprisingly, specific growth factors such as bFGF (FGF2) and placental growth factor, and adhesion molecules such as ICAM-1 were abundantly expressed and up-regulated in diabetic versus non-diabetic ZDF rats. In summary, we observed in type-2 diabetic ZDF rats retinopathy with retinal vasoregression along with a simultaneous up-regulation of specific growth factors such as bFGF and adhesion molecules, but only minor changes in key inflammatory genes.

Fibroblast growth factor-2 (FGF-2) plays a critical role in endothelial survival, proliferation, and angiogenesis and is localized on the cell membrane by binding to heparan sulfate proteoglycans. Here we established a neutralizing monoclonal antibody, 1B9B9, against FGF-2 using the rat medial iliac lymph node method. 1B9B9 blocked the binding of FGF-2 to its receptor, inhibiting FGF-2-induced proliferation and corresponding downstream signaling in endothelial cells. Treatment of human umbilical vein endothelial cells with 1B9B9 reduced the basal phosphorylation levels of Akt and MAPK. Furthermore, continued treatment with 1B9B9 induced cell death by apoptosis. Compared with FGF-2 knockdown, 1B9B9 significantly reduced cell survival. In addition, the combination of FGF-2 siRNA and 1B9B9 showed a synergistic effect. The data indicate that 1B9B9 established by the rat iliac lymph node method is a fully compatible neutralizing antibody.

Identifying immune escape mechanisms used by tumors may define strategies to sensitize them to immunotherapies to which they are otherwise resistant. In this study, we show that the antiapoptotic gene API5 acts as an immune escape gene in tumors by rendering them resistant to apoptosis triggered by tumor antigen-specific T cells. Its RNAi-mediated silencing in tumor cells expressing high levels of API5 restored antigen-specific immune sensitivity. Conversely, introducing API5 into API5(low) cells conferred immune resistance. Mechanistic investigations revealed that API5 mediated resistance by upregulating FGF2 signaling through a FGFR1/PKCδ/ERK effector pathway that triggered degradation of the proapoptotic molecule BIM. Blockade of FGF2, PKCδ, or ERK phenocopied the effect of API5 silencing in tumor cells expressing high levels of API5 to either murine or human antigen-specific T cells. Our results identify a novel mechanism of immune escape that can be inhibited to potentiate the efficacy of targeted active immunotherapies., (©2014 American Association for Cancer Research.)

Background: Dendritic cells (DCs) have a pivotal role in the onset and regulation of innate and adaptive immune responses. Moreover, DCs can interact with angiogenic modulators, resulting in modification of their biology and participation in angiogenesis., Objectives: This study was designed to evaluate the relationship between the density of DCs, vascularity and expression of angiogenic factors [vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β and basic fibroblast growth factor (bFGF)] in the central airways of chronic obstructive pulmonary disease (COPD) patients., Methods: The study included 20 patients with moderate/severe COPD and 8 healthy control subjects. Bronchial biopsies were evaluated by immunohistochemistry. Specimens were examined for CD83 and CD207 to mark mature and immature DCs, respectively, for collagen IV to evaluate vascularity, and for VEGF, TGF-β and bFGF., Results: Compared to controls, COPD patients had a significant reduction of CD83+ cells and an increased CD207/CD83 ratio (p < 0.05). Vascularity, VEGF, TGF-β and bFGF were also significantly increased in COPD patients as compared to controls (p < 0.01). In COPD patients, CD83+ cells were inversely related to VEGF and TGF-β expression (p < 0.05). Moreover, the CD207/CD83 ratio was positively related to VEGF, TGF-β and vascularity (p < 0.05). Finally, CD207+ cells were inversely related to FEV1 (p < 0.05)., Conclusion: Our results show a reduced maturation of DCs in COPD that was related to airway vascularity and angiogenic factors (VEGF and TGF-β). Additionally, immature DCs were significantly related to disease severity. We propose that the interplay between airway vascular changes, on one hand, and DCs maturation on the other, may play a key role in the pathogenetic mechanisms of COPD., (© 2013 S. Karger AG, Basel.)

Limited neuronal migration into host brain tissue is a key challenge in neural transplantation. We found that one important mechanism underlying this phenomenon is an intrinsic chemotactic interaction between the grafted neural precursor cells (NPCs) and their neuronal progeny. NPCs secrete the receptor tyrosine kinase ligands FGF2 and VEGF, which act as chemoattractants for neurons. Interference with these signaling pathways resulted in enhanced migration of human neurons from neural clusters.

MICA are major histocompatibility complex class I-related molecules, expressed by endothelial cells (ECs), that may be targets for alloantibodies and NKG2D-expressing natural killer (NK) and T effector cells in organ allografts. This study shows that basal levels of MICA expressed on vascular ECs is sufficient to functionally modulate the expression and activity of the immunoreceptor NKG2D in allogeneic NK cells. We found that MICA expression is differentially regulated at the EC surface in response to cytokines. TNFα upregulates MICA while IFNγ significantly decreases MICA at the EC surface. Both cytokines induce the release of soluble MICA by ECs. Modulation of NKG2D correlates with the MICA level on the EC surface. Glycosylation and metalloproteinase activities account for major post-transcriptional mechanisms controlling MICA level and the function in ECs. Our results indicate that, in addition to the NFκB pathway, the mitogen-activated protein kinase pathways JNK, ERK1/2 and p38 are key signaling pathways in the control of MICA by the cytokines. Finally, we show that EC proliferation mediated by FGF-2 or wound healing increases the MICA level. Together, our data suggest that inflammation and proliferation regulate endothelial MICA expression and shedding, enabling ECs to modulate NKG2D activity on effector NK and T cells, and provide further evidence of a role for ECs in immunoregulation., (© 2013 S. Karger AG, Basel.)

Angiogenesis plays a pivotal role in the progression and metastasis of hepatocellular carcinoma (HCC). However, the expression of a wide range of angiogenic factors remains obscure in HCC. The purpose of the present study was to determine the expression of various angiogenic factors related to hepatocarcinogenesis. We examined the expression of 19 angiogenic factors using antibody arrays in human tissues of various liver diseases, including HCC. We also studied the expression of 19 angiogenic factors in the human HCC cell lines PLC/PRF/5, Hep 3B, HuH7, HLE, HLF and Li-7 and the normal hepatocyte cell line ACBRI3716. In human tissues, although the expression of acidic fibroblast growth factor (aFGF) was found to increase from normal liver to chronic hepatitis, its expression remained unchanged in the transition from chronic hepatitis to HCC. Vascular endothelial growth factor (VEGF) was elevated in liver cirrhosis, but the amounts remained unchanged in the transition from liver cirrhosis to HCC. In contrast, either interleukin-8 (IL-8) or basic fibroblast growth factor (bFGF) was upregulated in HCC. In the HCC cell lines PLC/PRF/5, Hep 3B and HuH-7, the expression of IL-8 was elevated. Although IL-8 was not elevated, bFGF was upregulated in the other HCC cell lines HLE, HLF and Li-7. Thus, either IL-8 or bFGF was upregulated in HCC cell lines and in HCC tissue samples. These data suggest that the upregulation of either IL-8 or bFGF is closely related to the transition from liver cirrhosis into HCC. Therefore, the analysis of the expression of these cytokines using protein arrays may identify novel therapies for individual patients with HCC.

Background: Unlike other IL-17 family members, the Th2-derived cytokine IL-25 (IL-17E) induces (promotes) Th2 responses. One or both of the two receptors for IL-25 (IL-17RA, IL-17RB) is expressed on inflammatory cells and tissue structural cells, suggesting that in addition to promoting Th2-type inflammation IL-25 may also act on structural cells at sites of Th2-type inflammation such as in the asthmatic bronchial mucosa to promote remodelling changes., Objective: Our previous studies showed elevated expression of IL-25 and IL-17RB immunoreactivity in asthmatic airways with co-localization of the latter to endothelial cells. We therefore hypothesized that IL-25 acts on endothelial cells through this receptor to induce production of the key angiogenic and remodelling cytokine basic fibroblast growth factor (bFGF)., Methods: Polymerase chain reaction (PCR) immunocytochemistry/immunohistochemistry and ELISA were employed to detect expression of IL-17RB, IL-17RA and bFGF by human vascular endothelial cells (HUVEC) and immunoreactivity for IL-25 and bFGF in asthmatic bronchial biopsies. Receptor-blocking antibodies, PCR and an in vitro angiogenesis assay were used to investigate whether IL-25 acts on IL-17RB or IL-17RA to induce bFGF expression and angiogenesis. PCR was also employed to investigate the signalling pathways involved in IL-25-mediated bFGF expression., Results: HUVEC constitutively expressed IL-17RB, IL-17RA and bFGF. Production of the latter was further increased by IL-25, but attenuated after blockade of the IL-17RB, but not the IL-17RA receptor. Neutralization of endogenous VEGF and bFGF completely abrogated IL-25-induced angiogenesis which was also inhibited by blocking IL-17RB, but not IL-17RA. The PI3K-specific inhibitor LY294002 also completely attenuated IL-25-induced bFGF expression. Immunoreactivity for IL-25 and bFGF was elevated in the asthmatic bronchial mucosa and the expression of each correlated with the other., Conclusions and Clinical Relevance: Our data support the hypothesis that IL-25 contributes to elevated bFGF in asthmatic airways by acting on the endothelial cell IL-17RB receptor through PI3K-signalling pathways. Targeting the pathways might benefit therapy of airways remodelling., (© 2012 Blackwell Publishing Ltd.)

Background: Fibroblast growth factor-2 promotes in vitro heart valve interstitial cell repair. Fibroblast growth factor-2 acts through betaglycan which is known to bind both transforming growth factor-β and fibroblast growth factor-2 at different locations on the molecule. When fibroblast growth factor-2 binds to betaglycan, transforming growth factor-β binding to betaglycan is reduced, allowing for more transforming growth factor-β to be available to activate pSmad2/3 which then promotes repair. This study investigates another pathway through which fibroblast growth factor-2 regulates valve interstitial cell repair., Methods: We used an in vitro model of cell culture disruption. Confluent valve interstitial cell monolayers were disrupted, creating an experimental wound in the confluent monolayer, and incubated in treatments of exogenous fibroblast growth factor-2, anti-fibroblast growth factor receptor antibody, active Akt1, and Akt inhibitor. Valve interstitial cell monolayers were immunohistochemically stained and quantified for nuclear pSmad2/3 at the wound edge. The extent of closure was measured up to 96 h after disruption., Results: Anti-fibroblast growth factor receptor antibody significantly increased both nuclear pSmad2/3 staining at the wound edge and wound closure compared to nontreated control. This increase was less than that seen when valve interstitial cells were treated with fibroblast growth factor-2 and combined treatments of fibroblast growth factor-2 and anti-fibroblast growth factor receptor antibody did not further increase nuclear pSmad2/3 staining compared to fibroblast growth factor-2 alone. This suggested that the regulation of wound closure by fibroblast growth factor-2 also involved pathways other than transforming growth factor-β/Smad signaling. Treatment with Akt1 significantly increased wound closure, while Akt inhibitor reduced closure as compared to nontreated valve interstitial cells. Fibroblast growth factor-2 and fibroblast growth factor-2 neutralizing antibody up-regulated and down-regulated phosphorylated Akt1 expression in valve interstitial cells, respectively., Conclusion: Fibroblast growth factor-2 promotes valve interstitial cell repair in two ways: the fibroblast growth factor-2/fibroblast growth factor-2 receptor interaction through the activation of Akt1 independent of the transforming growth factor-β/Smad2/3 signaling pathway and the previously described transforming growth factor-β/Smad signaling., (Copyright © 2012. Published by Elsevier Inc.)

Expression of fibroblast growth factor 2 (FGF2) is believed to be a contributing factor to the growth of a number of tumor types, including hepatocellular carcinoma (HCC). However, the potential of monoclonal antibodies that neutralize FGF2 for treatment of patients with cancer has not yet been explored in clinical trials. We therefore generated a novel monoclonal antibody (mAb), GAL-F2, specific for FGF2 and characterized its properties in vitro and in vivo. GAL-F2 binds to a different epitope than several previous anti-FGF2 mAbs tested. This novel epitope was defined using chimeric FGF1/FGF2 proteins and alanine scanning mutagenesis and was shown to comprise amino acids in both the amino and carboxy regions of FGF2. GAL-F2 blocked binding of FGF2 to each of its four cellular receptors, strongly inhibited FGF2-induced proliferation and downstream signaling in human umbilical vein endothelial cells, and inhibited proliferation and downstream signaling in two HCC cell lines. Moreover, GAL-F2, administered at 5 mg/kg i.p. twice weekly, potently inhibited growth of xenografts of the SMMC-7721, HEP-G2, and SK-HEP-1 human HCC cell lines in nude mice, and in some models, had a strong additive effect with an anti-VEGF mAb or sorafenib. Treatment with GAL-F2 also blocked angiogenesis and inhibited downstream cellular signaling in xenografts, indicating its antitumor mechanism of action. Our report supports clinical testing of a humanized form of the GAL-F2 mAb for treatment of HCC and potentially other cancers., (©2012 AACR.)

Wound healing is a sophisticated biologic process. In the case of hemithyroidectomy, the operation time is relatively short with small tissue damage and without skin excision, and bacterial contamination before, during, and after the operation is uncommon. Here, we explored which cytokine(s) affected the rates of healing of skin wounds after hemithyroidectomy of 29 patients. We assessed the amounts of cytokines (e.g., interleukin-6, platelet-derived growth factor, basic fibroblast growth factor, vascular endothelial growth factor, and tumor necrosis factor-α) in either the preoperative or postoperative lavage fluids, or in the drainage fluids on postoperative days (PODs) 1-8. All of these cytokines showed a similar pattern; after reaching a peak on POD1, the production fell sharply on POD2-8, revealing that wound healing commenced on POD1. The rates of wound healing were inversely related to the levels of histamine in six patients (i.e., those with the three largest and those with the three smallest total volumes of drainage fluid on POD1): high (or low) levels of histamine in the postoperative lavage fluids with low (or high) levels in the drainage fluids on POD1 caused earlier (or the delay of) wound healing, suggesting involvement of histamine in the acceleration and delay of wound healing., (© 2012 by the Wound Healing Society.)

Due to the poor immunogenicity of subunit protein antigens, there is a need to use adjuvants in order to generate effective immune responses. Basic fibroblast growth factor (bFGF) is one of the best characterized pro-angiogenic cytokine and is a candidate target for anticancer therapy. We used truncated bFGF (tbFGF) combined with engineered pVAX-nCpG as novel adjuvant to immunize mice in order to inhibit tumor angiogenesis and suppress tumor growth. In our study, the results demonstrated that the mice immunized with tbFGF-alum-pVAX-8CpG produced a better tumor-suppression effect compared with the other groups, apart from the group treated with tbFGF-alum-CpG. In addition, the function of immune modulation of pVAX-8CpG was similar to CpG ODNs. The vaccine composed of tbFGF, alum and pVAX-8CpG effectively inhibited tumor angiogenesis and induced strong antitumor immune responses. The antitumor activity induced by the vaccine tbFGF-alum-pVAX-8CpG was not only associated with the antigen-specific antibody, but also with the killing activity of cytotoxic cells. This indicates that alum-pVAX-8CpG may be an innovative adjuvant for cancer vaccines.

The roles of fibroblast growth factor-2 (FGF-2) in the hepatocellular carcinoma (HCC) development are still controversial. In this study, we investigated the expression of FGF-2 in chronic hepatitis (CH) type C patients with or without HCC and the immunoregulation of FGF-2 in NK sensitivity of HCC cells. The FGF-2 expressions were detected in the liver tissues of patients, but not in normal liver. The serum FGF-2 levels of the patients with CH, liver cirrhosis (LC) or HCC were significantly higher than those of healthy volunteers. The serum FGF-2 levels of patients decreased with the progression of chronic liver disease. HCC occurrence of LC patients with high levels of serum FGF-2 was significantly lower than that with low levels of serum FGF-2. Proinflammatory cytokines, such as IL-1β and IL-6, induced FGF-2 expressions in HCC cells and normal hepatocytes. FGF-2 stimulation resulted in increasing the expression of the membrane-bound major histocompatibility complex class I-related chain A (MICA), an NK activating molecule, and decreasing that of human leukocyte antigen (HLA) class I, an NK inhibitory molecule, on HCC cells. This did not occur with normal hepatocytes. Adding anti-FGF receptor-2 neutralizing antibody resulted in inhibiting the change of MICA and HLA class I expressions on FGF-2 stimulated HCC cells. FGF-2 stimulation on HCC cells resulted in increasing NK sensitivity against HCC cells. These findings indicate that FGF-2 produced by HCC cells or normal hepatocytes of chronic liver disease may play critical roles in eliminating HCC cells by innate immunity., (Copyright © 2011 UICC.)

Objective: To investigate the expression of NOD2 in human VSMCs, its role in the production of inflammatory cytokines in VSMC and the possible interaction of NOD2-mediated signaling pathway with those mediated by TLR2 and TLR4., Methods: Human coronary artery smooth muscle cells were stimulated with NOD2 agonist MDP alone or in combination with either TLR2 agonist PAM3 or TLR4 agonist LPSs. The mRNA expression of NOD2 and FGF-2 were measured by RT-PCR. The concentration of IL-8 and TNF-α in the culture supernatants was determined by ELISA. VSMC proliferation ability was analyzed by MTT assay., Results: MDP up regulated the expression of NOD2 mRNA in VSMC in a time-dependent manner, up regulated the expression of FGF-2 mRNA in VSMC, induced the production of IL-8 and TNF-α, and promoted the proliferation of VSMC. Additionally, MDP synergied with LPS and PAM3 to promote the proliferation of VSMC and induce the production of IL-8 and TNF-α., Conclusion: The activation of NOD2-mediated innate immune signaling pathway can increase the proliferation ability of VSMC and induce the production of inflammatory cytokines in VSMC. It is also shown a synergistic effect with TLR2- and TLR4-mediated signaling pathways in this process.

Long term survival of the human lung allografts are hindered by chronic rejection, manifested clinically as bronchiolitis obliterans syndrome (BOS). We previously demonstrated significant correlation between the development of antibodies (Abs) to K-α1-tubulin (Kα1T) and BOS. In this study, we investigated the molecular basis for fibrinogenesis mediated by ligation of Kα1T expressed on airway epithelial cells by its specific Abs. Using RT-PCR we demonstrate that normal human bronchial epithelial (NHBE) cells upon ligation of Kα1T with specific Abs caused upregulation of pro-fibrotic growth factors. Western blot analysis of NHBE incubated with Kα1T Abs increased hypoxia inducible factor (HIF-1α). Kα1T Ab-mediated growth factor expression is dependent on HIF-1α as inhibition of HIF-1α returned fibrotic growth factor expression to basal levels. In conclusion, we propose that HIF-1α -mediated upregulation of fibrogenic growth factors induced by ligation of Kα1T Abs is critical for development of fibrosis leading to chronic rejection of lung allograft., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In this work, a novel vaccine delivery system, biodegradable nanoparticles (NPs) in thermosensitive hydrogel, was investigated. Human basic fibroblast growth factor (bFGF)-loaded NPs (bFGF-NPs) were prepared, and then bFGF-NPs were incorporated into thermosensitive hydrogel to form bFGF-NPs in a hydrogel composite (bFGF-NPs/hydrogel). bFGF-NPs/hydrogel was an injectable sol at ambient temperature, but was converted into a non-flowing gel at body temperature. The in vitro release profile showed that bFGF could be released from bFGF-NPs or bFGF-NPs/hydrogel at an extended period, but the release rate of bFGF-NPs/hydrogel was much lower. In vivo experiments suggested that immunogenicity of bFGF improved significantly after being incorporated into the NPs/hydrogel composite, and strong humoral immunity was maintained for longer than 12 weeks. Furthermore, an in vivo protective anti-tumor immunity assay indicated that immunization with bFGF-NPs/hydrogel could induce significant suppression of the growth and metastases of tumors. Thus, the NPs/hydrogel composite may have great potential application as a novel vaccine delivery system.

Introduction: The goal of this study was to evaluate the mechanism involved in the expression of fibroblast growth factor-2 (FGF-2) by odontoblast-like cells (ODs) stimulated by lipopolysaccharide (LPS) via p42/44, p38, and PI3K., Methods: ODs (MDPC-23) were stimulated with LPS for 1, 6, and 24 hours. The FGF-2 expression was evaluated by reverse transcriptase-polymerase chain reaction and protein production by Western blot analysis. Cells were pretreated with dexamethasone (DEX), MK886 (MK), p42/44 inhibitor (PD98059, PD), p38 inhibitor (SB202190, SB), or PI3K inhibitor (wortmannin, Wort) and then stimulated with LPS (0.1 μg/mL) for 1 hour., Results: LPS-stimulated ODs express FGF-2 in concentrations at 0.1, 1, 10, and 100 μg/mL after 1 hour. DEX and MK were able to inhibit FGF-2 mRNA expression. PD, SB, and Wort also decreased expression., Conclusions: LPS-induced FGF-2 mRNA expression on ODs occurs via leukotriene production or cytokine and/or chemokine production activating p42/44, p38, and PI3K pathway. The data suggest that FGF-2 released by ODs might act as modulators of immune response mainly in the tissue repair., (Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Human embryonic stem cells (hESCs) are typically cultured on fibroblast feeder cells or in fibroblast conditioned medium supplemented with fibroblast growth factor 2 (FGF2, also known as bFGF). FGF signaling appears to be important for hESC self-renewal and is required to enable the culture of hESCs in an undifferentiated state. In this study, we generated a transgenic fibroblast feeder line stably expressing a secretable FGF4 signal peptide tagged hFGF2 (4SP-hFGF2). The expression of this transgene functionally replaced the requirement for exogenous FGF2 when using these cells as feeders for the maintenance of hESCs. Under these conditions, hESCs maintained the typical marker of pluripotency assessed after long term culture, while still retaining the capacity for differentiation to all three germ layers. This transgene could be applied to mass produce 4SP-hFGF2 protein, serving to be an economical and effective strategy for culturing pluripotent stem cells as feeder cells., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Tumor growth is partly dependent on tumor-associated angiogenesis, which is regulated by angiogenic growth factors. As the first angiogenic growth factor to be identified, basic fibroblast growth factor (bFGF) plays a major role in angiogensis and tumor growth and has been an effective target for anti-tumor therapy. However, due to its low immunogenicity, injection with bFGF alone cannot stimulate the body to produce a strong immune response. In this study, we investigated the role of CF (containing bFGF and CRM197) assisted by CpG and alum in enhancing antigen-specific immune response and suppressing the growth of murine colon carcinoma. The results revealed that compared to bFGF, CF could not stimulate NIH-3T3 fibroblast proliferation even at a concentration of 10 µg/ml in vitro. In vivo, the CF-CpG-alum produced a stronger antigen-specific immune response and inhibited tumor growth. The anti-tumor activity was associated with generating antigen-specific antibody, suppressing angiogenesis, promoting the apoptosis of tumor cells and inducing the mixed Th1 and Th2 responses. This indicates that CRM197 may be an innovative intramolecular adjuvant and provides a rational preservation for mouse CT26 colon carcinoma.

Basic fibroblast growth factor (bFGF), an angiogenic factor, exhibits pro-angiogenic abilities by interacting with tyrosine kinase receptors and heparin-sulfated proteoglycan receptors. Here, we designed an N-, C-terminally truncated basic fibroblast growth factor (tbFGF) for immuno-therapy of murine lung carcinoma with PCEC hydrogel as adjuvant, comparing it with the wild-type bFGF. In vitro, tbFGF did not stimulate NIH-3T3 fibroblast proliferation. In vivo, after immunization, both tbFGF and bFGF were able to induce a robust bFGF-specific immune response. The protective anti-tumor investigation showed a significant inhibition of tumor growth and reduction of tumor vascularization detected by immunohistochemical staining and the alginate-encapsulated tumor cell assay in the tbFGF or the bFGF group. These data suggested that tbFGF can be used in the immunotherapy of tumors, without the risks associated with bFGF, which induces neovascularization in normal tissues.

One of the critical steps in the development of an analytical technique is to confirm that its experimental response correlates with predictions derived from the theoretical framework on which it is based. This validates the technique quantitatively and, in the case of a biosensor, facilitates a correlation of the sensor's output signal to the concentration of the analyte being tested. Herein we report studies demonstrating that the quantitative response of arrayed imaging reflectometry (AIR), a highly sensitive label-free biosensing method, is a predictable function of the probe and analyte properties. We first incorporated a standard one-site Langmuir binding model describing probe-analyte interactions at the surface into the theoretical model for thickness-dependent reflectance in AIR. This established a hypothetical correlation between the analyte concentration and the AIR response. Spectroscopic ellipsometry, surface plasmon resonance, and AIR were then used to validate this model for two biomedically important proteins, fibroblast growth factor-2 and vascular endothelial growth factor. While our studies demonstrated that the 1:1 one-site Langmuir model accurately described the observed response of macrospot AIR arrays, either a two-site Langmuir model or a Sips isotherm better described the behavior of AIR microarrays. These studies confirmed the quantitative performance of AIR across a range of probe-analyte affinities. Furthermore, the methodology developed here can be extended to other label-free biosensing platforms, thus facilitating a more accurate and quantitative interpretation of the sensor response.

Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.

Basic fibroblast growth factor (bFGF) is an important stimulator of angiogenesis involving in neovascularization progression. The aim of this study is to evaluate whether a liposomal vaccine (MLB) based on xenogeneic human bFGF plus monophosphoryl lipid A (MPLA) could effectively induce cross-reaction immunity in mice and increase antitumor activity. Sera of mice were analyzed and IgG antibody titer in MLB group was obviously higher than other groups including the mice immunized with liposomal bFGF vaccine, bFGF plus Freund's adjuvant, empty liposome and PBS. Furthermore, tumor metastasis was significantly inhibited in MLB group, compared with L and PBS group. The IFN-γ production of cultured splenocytes in vitro was evidently up-regulated meanwhile IL-4 production sustained in a low level, revealing that this vaccine stimulated Th1 immunity response preferentially. Taken together, these findings suggested that this novel bFGF vaccine could effectively induce humoral immunity through cross-reaction, mediate Th1 immune response preferentially and enhance antitumor activity in vivo., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Up-regulated basic fibroblast growth factor (bFGF or FGF-2) plays an important role in the development and metastasis of melanoma; therefore, neutralizing antibodies to bFGF may suppress melanoma growth. In this study, we have developed three monoclonal antibodies against bFGF (anti-bFGF mAbs), which display remarkable anti-tumor and anti-angiogenic effects in vitro and in vivo. Anti-bFGF mAbs significantly inhibit the proliferation and induce apoptosis of B16 cells, and show inhibitory effects on the migration of B16F10 cells and the tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Treatment of B16 melanoma spheroids with anti-bFGF mAbs in vivo results in significant reduction in tumor size and prolonged survival time of animals. Moreover, TUNEL (terminal transferase dUTP nick end labeling) assay and CD31 staining confirmed the increase of apoptosis and decrease of intratumoral microvessel density in tumor sections from animals treated with anti-bFGF mAbs. Our data indicate that anti-bFGF mAbs are potential therapeutic candidates for melanoma therapy by effectively suppressing the melanoma growth through inhibition of angiogenesis and induction of apoptosis in the tumor.