Chlorogenic Acid Stimulates Glucose Transport in Skeletal Muscle via AMPK Activation: A Contributor to the Beneficial Effects of Coffee on Diabetes

Chlorogenic acid (CGA) has been shown to delay intestinal glucose absorption and inhibit gluconeogenesis. Our aim was to investigate the role of CGA in the regulation of glucose transport in skeletal muscle isolated from db/db mice and L6 skeletal muscle cells. Oral glucose tolerance test was performed on db/db mice treated with CGA and soleus muscle was isolated for 2-deoxyglucose transport study. 2DG transport was also examined in L6 myotubes with or without inhibitors such as wortmannin or compound c. AMPK was knocked down with AMPKα1/2 siRNA to study its effect on CGA-stimulated glucose transport. GLUT 4 translocation, phosphorylation of AMPK and Akt, AMPK activity, and association of IRS-1 and PI3K were investigated in the presence of CGA. In db/db mice, a significant decrease in fasting blood sugar was observed 10 minutes after the intraperitoneal administration of 250 mg/kg CGA and the effect persisted for another 30 minutes after the glucose challenge. Besides, CGA stimulated and enhanced both basal and insulin-mediated 2DG transports in soleus muscle. In L6 myotubes, CGA caused a dose- and time-dependent increase in glucose transport. Compound c and AMPKα1/2 siRNA abrogated the CGA-stimulated glucose transport. Consistent with these results, CGA was found to phosphorylate AMPK and ACC, consistent with the result of increased AMPK activities. CGA did not appear to enhance association of IRS-1 with p85. However, we observed activation of Akt by CGA. These parallel activations in turn increased translocation of GLUT 4 to plasma membrane. At 2 mmol/l, CGA did not cause any significant changes in viability or proliferation of L6 myotubes. Our data demonstrated for the first time that CGA stimulates glucose transport in skeletal muscle via the activation of AMPK. It appears that CGA may contribute to the beneficial effects of coffee on Type 2 diabetes mellitus

The antibody-based targeted delivery of TNF in combination with doxorubicin eradicates sarcomas in mice and confers protective immunity

Background: Soft-tissue sarcomas are a group of malignancies of mesenchymal origin, which typically have a dismal prognosis if they reach the metastatic stage. The observation of rare spontaneous remissions in patients suffering from concomitant bacterial infections had triggered the clinical investigation of the use of heat-killed bacteria as therapeutic agents (Coley’s toxin), which induced complete responses in patients in the pre-chemotherapy era and is now known to mediate substantial elevations in serum TNF levels. Methods: We designed and developed a novel immunocytokine based on murine TNF sequentially fused to the antibody fragment F8 (specific to extra-domain A of fibronectin). The antitumor activity was studied in two syngeneic murine sarcoma models. Results: The L19 antibody (specific to extra-domain B of fibronectin) has shown by SPECT imaging procedures to selectively localise on sarcoma in a patient with a peripheral nerve sheath tumour, and immunohistochemical analysis of human soft-tissue sarcoma samples showed comparable antigen expression of EDA and EDB. The antibody-based pharmacodelivery of TNF by the fusion protein ‘F8–TNF’ to oncofetal fibronectin in sarcoma-bearing mice leads to complete and long-lasting tumour eradications when administered in combination with doxorubicin, the first-line drug for the treatment of sarcomas in humans. Doxorubicin alone did not display any therapeutic effect in both tested models of this study. The cured mice had acquired protective immunity against the tumour, as they rejected subsequent challenges with sarcoma cells. Conclusion: The findings of this study provide a rationale for the clinical study of the fully human immunocytokine L19-TNF in combination with doxorubicin in patients with soft-tissue sarcoma.ISSN:0007-0920ISSN:1532-182

Targeted IL‐4 therapy synergizes with dexamethasone to induce a state of tolerance by promoting Treg cells and macrophages in mice with arthritis

F8‐IL‐4 is a recently developed immunocytokine that delivers IL‐4 to sites of inflammation by targeting the neovasculature. We previously reported that F8‐IL‐4, in combination with dexamethasone (DXM), provides a durable therapy in mice with collagen‐induced arthritis (CIA). Therefore, the objective of this study was to identify the mechanism by which IL‐4 and DXM combination therapy provides long‐lasting disease remission. F8‐IL‐4 alone attenuated inflammation in CIA and this was associated with increased TH2 and decreased TH17 cell numbers in the joints. Similarly, DXM alone had an antiinflammatory effect associated with lower TH17 cell numbers. In both cases, these therapeutic benefits were reversed once treatment was stopped. On the other hand, combination therapy with F8‐IL‐4 plus DXM led to a synergistic increase in the percentage of regulatory T (Treg) cells and antiinflammatory macrophages in the arthritic joint and spleen as well as IL‐10 levels in serum and spleen. The net result of this was a more pronounced attenuation of inflammation and, more importantly, protection from arthritis relapse post therapy retraction. In conclusion, F8‐IL‐4 plus DXM is a durable treatment for arthritis that acts by promoting Treg cells in a synergistic manner, and by producing a sustained increase in antiinflammatory macrophages

The antibody-mediated targeted delivery of interleukin-13 to syngeneic murine tumors mediates a potent anticancer activity

We describe the expression and in vivo characterization of an antibody–cytokine fusion protein, based on murine Interleukin-13 (IL13) and the monoclonal antibody F8, specific to the alternatively spliced extra domain A of fibronectin, a marker of neo-angiogenesis. The IL13 moiety was fused at the C-terminal extremity of the F8 antibody in diabody format. The resulting F8-IL13 immunocytokine retained the full binding properties of the parental antibody and cytokine bioactivity. The fusion protein could be expressed in mammalian cells, purified to homogeneity and showed a preferential accumulation at the tumor site. When used as single agent at doses of 200 μg, F8-IL13 exhibited a strong inhibition of tumor growth rate in two models of cancer (F9 teratocarcinoma and Wehi-164), promoting an infiltration of various types of leukocytes into the neoplastic mass. This anticancer activity could be potentiated by combination with an immunocytokine based on the F8 antibody and murine IL12, leading to complete and long-lasting tumor eradications. Mice cured from Wehi-164 sarcomas acquired a durable protective antitumor immunity, and selective depletion of immune cells revealed that the antitumor activity was mainly mediated by cluster of differentiation 4-positive T cells. This study indicates that IL13 can be efficiently delivered to the tumor neo-vasculature and that it mediates a potent anticancer activity in the two models of cancer investigated in this study. The observed mechanism of action for F8-IL13 was surprising, since immunocytokines based on other payloads (e.g., IL2, IL4, IL12 and TNF) eradicate cancer by the combined contribution of natural killer cells and cluster of differentiation 8-positive T cells.ISSN:0340-7004ISSN:1432-085

Antibody-mediated delivery of VEGF-C potently reduces chronic skin inflammation.

VEGF-C is an important mediator of lymphangiogenesis and has been shown to alleviate chronic inflammation in a variety of disease models. In this study, we investigated whether targeted delivery of VEGF-C to sites of inflammation and site-specific activation of lymphatic vessels would represent a clinically feasible strategy for treating chronic skin inflammation. To this end, we generated a fusion protein consisting of human VEGF-C fused to the F8 antibody (F8-VEGF-C), which is specific for the alternatively spliced, angiogenesis-marking extradomain A (EDA) of fibronectin. In two mouse models of psoriasis-like skin inflammation, mediated by transgenic VEGF-A overexpression or repeated application of imiquimod, intravenous treatment with F8-VEGF-C but not with untargeted VEGF-C significantly reduced ear skin edema and was as effective as the clinically used TNF-α receptor-Fc fusion protein (TNFR-Fc). Treatment with F8-VEGF-C led to a marked expansion of lymphatic vessels in the inflamed skin and significantly improved lymphatic drainage function. At the same time, treatment with F8-VEGF-C significantly reduced leukocyte numbers, including CD4+ and γδ T cells. In sum, our results reveal that targeted delivery of VEGF-C and site-specific induction of lymphatic vessels represent a potentially new and promising approach for the treatment of chronic inflammatory diseases