Coupling Tumor Necrosis Factor-α with αV Integrin Ligands Improves Its Antineoplastic Activity

Despite the impressive results obtained in animal models, the clinical use of tumor necrosis factor-α (TNF) as an anticancer drug is limited by severe toxicity. We have shown previously that targeted delivery of TNF to aminopeptidase N (CD13), a marker of angiogenic vessels, improved the therapeutic index of this cytokine in tumor-bearing mice. To assess whether the vascular-targeting approach could be extended to other markers of tumor blood vessels, in this work, we have fused TNF with the ACDCRGDCFCG peptide, a ligand of αV integrins by recombinant DNA technology. We have found that subnanogram doses of this conjugate are sufficient to induce antitumor effects in tumor-bearing mice when combined with melphalan, a chemotherapeutic drug. Cell adhesion assays and competitive binding experiments with anti-integrin antibodies showed that the Arg-Gly-Asp moiety interacts with cell adhesion receptors, including αVβ3 integrin, as originally postulated. In addition, ACGDRGDCFCG-mouse TNF conjugate induced cytotoxic effects in standard cytolytic assays, implying that ACGDRGDCFCG-mouse TNF conjugate can also bind TNF receptors and trigger death signals. These results indicate that coupling TNF with αV integrin ligands improves its antineoplastic activity and supports the concept that vascular targeting is a strategy potentially applicable to different endothelial markers, not limited to CD13

The Modulatory Effect of Ellagic Acid and Rosmarinic Acid on Ultraviolet-B-Induced Cytokine/Chemokine Gene Expression in Skin Keratinocyte (HaCaT) Cells

Ultraviolet radiation (UV) induces an increase in multiple cutaneous inflammatory mediators. Ellagic acid (EA) and rosmarinic acid (RA) are natural anti-inflammatory and immunomodulatory compounds found in many plants, fruits, and nuts. We assessed the ability of EA and RA to modulate IL-1β, IL-6, IL-8, IL-10, MCP-1, and TNF-α gene expression in HaCaT cells after UVB irradiation. Cells were treated with UVB (100 mJ/cm(2)) and simultaneously with EA (5 μM in 0.1% DMSO) or RA (2.7 μM in 0.5% DMSO). Moreover, these substances were added to the UVB-irradiated cells 1 h or 6 h before harvesting, depending on the established UVB-induced cytokine expression peak. Cytokine gene expression was examined using quantitative real time polymerase chain reaction. RA produced a significant reduction in UVB-induced expression of IL-6, IL-8, MCP-1, and TNF-α when applied at the same time as irradiation. EA showed milder effects compared with RA, except for TNF-α. Both substances decreased IL-6 expression, also when applied 5 h after irradiation, and always produced a significant increase in UVB-induced IL-10 expression. Our findings suggest that EA and RA are able to prevent and/or limit the UVB-induced inflammatory cascade, through a reduction in proinflammatory mediators and the enhancement of IL-10, with its protective function

Chromogranin A fragments modulate cell adhesion. Identification and characterization of a pro-adhesive domain.

Although several functions have been suggested for chromogranin A, a glycoprotein secreted by many neuroendocrine cells, the physiological role of this protein and of its proteolytic fragments has not been established. We have found that mixtures of chromogranin A fragments can inhibit fibroblast adhesion. The anti-adhesive activity was converted into pro-adhesive activity by limited trypsin treatment. Pro-adhesive effects were observed also with recombinant N-terminal fragments corresponding to residues 1–78 and 1–115 and with a synthetic peptide encompassing the residues 7–57. These fragments induced adhesion and spreading of fibroblasts on plates coated with collagen I or IV, laminin, fetal calf serum (FCS) but not on bovine serum albumin. The long incubation time required for adhesion assays (4 h) and the FCS requirements for optimal adhesion suggest that the adhesive activity is likely indirect and requires other proteins present in the FCS or made by the cells. These findings suggest that chromogranin A and its fragments could play a role in the regulation of cell adhesion. Since chromogranin A is concentrated and stored within granules and rapidly released by neuroendocrine cells and neurons after an appropriate stimulus, this protein could be important for the local control of cell adhesion by stimulated cells

Boosting Interleukin-12 Antitumor Activity and Synergism with Immunotherapy by Targeted Delivery with isoDGR-Tagged Nanogold.

AbstractThe clinical use of interleukin‐12 (IL12), a cytokine endowed with potent immunotherapeutic anticancer activity, is limited by systemic toxicity. The hypothesis is addressed that gold nanoparticles tagged with a tumor‐homing peptide containing isoDGR, an αvβ3‐integrin binding motif, can be exploited for delivering IL12 to tumors and improving its therapeutic index. To this aim, gold nanospheres are functionalized with the head‐to‐tail cyclized‐peptide CGisoDGRG (Iso1) and murine IL12. The resulting nanodrug (Iso1/Au/IL12) is monodispersed, stable, and bifunctional in terms of αvβ3 and IL12‐receptor recognition. Low‐dose Iso1/Au/IL12, equivalent to 18–75 pg of IL12, induces antitumor effects in murine models of fibrosarcomas and mammary adenocarcinomas, with no evidence of toxicity. Equivalent doses of Au/IL12 (a nanodrug lacking Iso1) fail to delay tumor growth, whereas 15 000 pg of free IL12 is necessary to achieve similar effects. Iso1/Au/IL12 significantly increases tumor infiltration by innate immune cells, such as NK and iNKT cells, monocytes, and neutrophils. NK cell depletion completely inhibits its antitumor effects. Low‐dose Iso1/Au/IL12 can also increase the therapeutic efficacy of adoptive T‐cell therapy in mice with autochthonous prostate cancer. These findings indicate that coupling IL12 to isoDGR‐tagged nanogold is a valid strategy for enhancing its therapeutic index and sustaining adoptive T‐cell therapy

Structure-activity relationships of chromogranin A in cell adhesion. Identification of an adhesion site for fibroblasts and smooth muscle cells.

Previous studies showed that chromogranin A (CgA), a glycoprotein stored and co-released with various hormones by neuroendocrine cells and neurons, can modulate cell adhesion. We have investigated the structure-activity relationships of CgA using fibroblasts and coronary artery smooth muscle cells in adhesion assays. A recombinant CgA fragment 1-78 and a peptide 7-57 containing reduced and alkylated cysteines (Cys(17) and Cys(38)) induced cell adhesion after adsorption onto solid phases at 50-100 nm. Peptides lacking the disulfide loop region, including residues 47-68, 39-59, and 39-68, induced cell adhesion, either bound to solid phases at 200-400 nm or added to the liquid phase at 5-10 microm, whereas peptide 60-68 was inactive, suggesting that residues 47-57 are important for activity. The effect of CgA-(1-78) was blocked by anti-CgA antibodies against epitopes including residues Arg(53), His(54), and Leu(57). Substitutions of residues His(54), Gln(55), and Asn(56) with alanine decreased the cell adhesion activity of peptide 47-68. These results suggest that the region 47-57 (RILSILRHQNL) contains a cell adhesion site and that the disulfide bridge is not necessary for the proadhesive activity. The ability of soluble peptides to elicit proadhesive effects suggests an indirect mechanism. The high sequence conservation and accessibility to antibodies suggest that this region is important for the physiological role of CgA

Food Pyramid for Subjects with Chronic Obstructive Pulmonary Diseases

Nutritional problems are an important part of rehabilitation for chronic obstructive pulmonary disease (COPD) patients. COPD patients often present with malnutrition, sarcopenia, and osteoporosis with possible onset of cachexia, with an inadequate dietary intake and a poor quality of life. Moreover, diet plays a pivotal role in patients with COPD through three mechanisms: regulation of carbon dioxide produced/oxygen consumed, inflammation, and oxidative stress. A narrative review based on 99 eligible studies was performed to evaluate current evidence regarding optimum diet therapy for the management of COPD, and then a food pyramid was built accordingly. The food pyramid proposal will serve to guide energy and dietary intake in order to prevent and treat nutritionally related COPD complications and to manage progression and COPD-related symptoms. The nutrition pyramid described in our narrative review is hypothetical, even in light of several limitations of the present review; the main limitation is the fact that to date there are no randomized controlled trials in the literature clearly showing that improved nutrition, via the regulation of carbon dioxide produced/oxygen consumed, inflammation and oxidative stress, improves symptoms and/or progression of COPD. Even if this nutritional pyramid is hypothetical, we hope that it can serve the valuable purpose of helping researchers focus on the often-ignored possible connections between body composition, nutrition, and COPD

Where to Find Leucine in Food and How to Feed Elderly With Sarcopenia in Order to Counteract Loss of Muscle Mass: Practical Advice

The term sarcopenia refers to the loss of skeletal muscle mass and strength that generally occurs during aging. The interventions that have proved most effective in reducing the severity and preventing the worsening of sarcopenia include physical exercise, especially resistance, and the administration of dietary supplements in association with a targeted diet; nutritional intervention is the main therapeutic approach for elderly people, since they are very often sedentary (also due to possible disabilities). Among the various nutrients, high biological value proteins and leucine are of particular interest for their demonstrated effects on the health of skeletal muscle. The intake of food containing proteins and leucine during meals stimulates muscle protein synthesis. Lower blood levels of leucine were associated with lower values of the skeletal muscle index, grip strength and performance. The international guidelines recommended that a leucine intake of 3 g at three main meals together with 25-30 g of protein is the goal to be achieved to counteract loss of lean mass in elderly. Food composition databases rarely show the amounts of leucine contained in foods and therefore it becomes difficult to build a diet that follows these guidelines. A table was therefore created for the first time in the literature to collect all the foods richest in leucine, thanks to the union of the most important Italian food databases. Moreover, in order to implement a diet that follows the right recommendations, another tables shows nutritional composition of breakfast, lunch and dinner (that each provide 3 grams of leucine and 25 grams of protein) for seven days

The HCN domain couples voltage gating andcAMP response in hyperpolarization-activatedcyclic nucleotide-gated channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control spontaneous electrical activity in heart and brain. Binding of cAMP to the cyclic nucleotide-binding domain (CNBD) facilitates channel opening by relieving a tonic inhibition exerted by the CNBD. Despite high resolution structures of the HCN1 channel in the cAMP bound and unbound states, the structural mechanism coupling ligand binding to channel gating is unknown. Here we show that the recently identified helical HCN-domain (HCND) mechanically couples the CNBD and channel voltage sensing domain (VSD), possibly acting as a sliding crank that converts the planar rotational movement of the CNBD into a rotational upward displacement of the VSD. This mode of operation and its impact on channel gating are confirmed by computational and experimental data showing that disruption of critical contacts between the three domains affects cAMP- and voltage-dependent gating in three HCN isoforms

The HCN domain couples voltage gating and cAMP response in hyperpolarization-activated cyclic nucleotide-gated channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control spontaneous electrical activity in heart and brain. Binding of cAMP to the cyclic nucleotide-binding domain (CNBD) facilitates channel opening by relieving a tonic inhibition exerted by the CNBD. Despite high resolution structures of the HCN1 channel in the cAMP bound and unbound states, the structural mechanism coupling ligand binding to channel gating is unknown. Here we show that the recently identified helical HCN-domain (HCND) mechanically couples the CNBD and channel voltage sensing domain (VSD), possibly acting as a sliding crank that converts the planar rotational movement of the CNBD into a rotational upward displacement of the VSD. This mode of operation and its impact on channel gating are confirmed by computational and experimental data showing that disruption of critical contacts between the three domains affects cAMP- and voltagedependent gating in three HCN isoforms