Antibody agonists trigger immune receptor signaling through local exclusion of receptor-type protein tyrosine phosphatases.

Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.

Detection of Cell Surface Ligands for Human Synovial γδ T Cells.

Lack of understanding of the nature and physiological regulation of γδ T cell ligands has considerably hampered full understanding of the function of these cells. We developed an unbiased approach to identify human γδ T cells ligands by the production of a soluble TCR-γδ (sTCR-γδ) tetramer from a synovial Vδ1 γδ T cell clone from a Lyme arthritis patient. The sTCR-γδ was used in flow cytometry to initially define the spectrum of ligand expression by both human tumor cell lines and certain human primary cells. Analysis of diverse tumor cell lines revealed high ligand expression on several of epithelial or fibroblast origin, whereas those of hematopoietic origin were largely devoid of ligand. This allowed a bioinformatics-based identification of candidate ligands using RNAseq data from each tumor line. We further observed that whereas fresh monocytes and T cells expressed low to negligible levels of TCR-γδ ligands, activation of these cells resulted in upregulation of surface ligand expression. Ligand upregulation on monocytes was partly dependent upon IL-1ß. The sTCR-γδ tetramer was then used to bind candidate ligands from lysates of activated monocytes and analyzed by mass spectrometry. Surface TCR-γδ ligand was eliminated by treatment with trypsin or removal of glycosaminoglycans, and also suppressed by inhibition of endoplasmic reticulum-Golgi transport. Of particular interest was that inhibition of glycolysis also blocked TCR-γδ ligand expression. These findings demonstrate the spectrum of ligand(s) expression for human synovial Vδ1 γδ T cells as well as the physiology that regulates their expression.

One-step solid-oil-water emulsion for sustained bioactive ranibizumab release.

BACKGROUND: The advent of therapeutic proteins highlights the need for delivery systems that protect and extend the duration of its action. Ranibizumab-VEGF is one such drug used for treating wet AMD. This paper describes a facile method to sustain bioactive ranibizumab release from PLGA-based particles. METHODS: Two emulsion techniques were explored namely: water-in-oil-in-water (WOW) and solid-in-oil-in-water (SOW) emulsion. The bioactivity of ranibizumab was evaluated by comparing its binding capability to VEGF, measured with ELISA to total protein measured by microBCA. RESULTS: During the emulsion process, contact of ranibizumab with the water-oil interface is the main destabilizing factor and this can be prevented with the use of amphiphilic PVA and solid-state protein in WOW and SOW emulsion respectively. In vitro release of the ranibizumab-loaded particles indicated that a 15-day release could be achieved with SOW particles while the WOW particles generally suffered from a burst release. Released ranibizumab was capable of inhibiting endothelial cell growth indicating its retention of bioactivity. The suppression of burst release from the SOW particles was attributed to the relatively smooth surface morphology of the SOW microparticles. CONCLUSIONS: The use of SOW encapsulation in modulating ranibizumab release while maintaining their bioactivity has been highlighted.

Curcumin mediates polyamine metabolism and sensitizes gastrointestinal cancer cells to antitumor polyamine-targeted therapies.

Curcumin, a natural polyphenol that contributes to the flavor and yellow pigment of the spice turmeric, is known for its antioxidant, anti-inflammatory, and anticarcinogenic properties. Capable of affecting the initiation, promotion, and progression of carcinogenesis through multiple mechanisms, curcumin has potential utility for both chemoprevention and chemotherapy. Previous studies demonstrated that curcumin can inhibit ornithine decarboxylase (ODC) activity in human leukemia and breast cancer cells, and pretreatment with dietary curcumin blocks carcinogen-induced ODC activity in rodent models of skin, colon, and renal cancer. The current study investigated the regulation of polyamine metabolism in human gastric and colon carcinoma cell lines in response to curcumin. Curcumin treatment significantly induced spermine oxidase (SMOX) mRNA and activity, which results in the generation of hydrogen peroxide, a source of ROS. Simultaneously, curcumin down regulated spermidine/spermine N1-acetyltransferase (SSAT) activity and the biosynthetic enzymes ODC and S-adenosylmethionine decarboxylase (SAMDC), thereby diminishing intracellular polyamine pools. Combination treatments using curcumin with the ODC inhibitor 2-difluoromethylornithine (DFMO), an agent currently in clinical chemoprevention trials, significantly enhanced inhibition of ODC activity and decreased growth of GI cancer cell lines beyond that observed with either agent alone. Similarly, combining curcumin with the polyamine analogue bis(ethyl)norspermine enhanced growth inhibition that was accompanied by enhanced accumulation of the analogue and decreased intracellular polyamine levels beyond those observed with either agent alone. Importantly, cotreatment with curcumin permitted the lowering of the effective dose of ODC inhibitor or polyamine analogue. These studies provide insight into the polyamine-related mechanisms involved in the cancer cell response to curcumin and its potential as a chemopreventive or chemotherapeutic agent in the GI tract.

Initiation of T cell signaling by CD45 segregation at 'close contacts'.

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.

Evolutionarily conserved primary TNF sequences relate to its primitive functions in cell death induction.

TNF is a primitive protein that has emerged from more than 550 million years of evolution. Our bioinformatics study of TNF from nine different taxa in vertebrates revealed several conserved regions in the TNF sequence. By screening overlapping peptides derived from human TNF to determine their role in three different TNF-induced processes--apoptosis, necrosis and NF-κB stimulation--we found that TNF conserved regions are mostly related to cell death rather than NF-κB stimulation. Among the most conserved regions, peptides (P)12, P13 and P1213 (comprising P12 and P13) induced apoptosis, whereas P14, P15, P16 and P1516 (comprising P15 and P16) induced necrosis. Cell death induced by these peptides was not through binding to the TNF receptor. P16-induced necrosis was mainly through disruption of the cell membrane, whereas P1213-induced apoptosis involved activation of TRADD followed by formation of complex II. Finally, using a monoclonal antibody and a mutant TNF protein, we show that TNF-induced apoptosis is determined by a conserved linear sequence that corresponds to that within P1213. Our results reveal the determinant sequence that is key to the TNF primitive function of inducing apoptosis.

Calcium phosphate coated Keratin-PCL scaffolds for potential bone tissue regeneration.

The incorporation of hydroxyapatite (HA) nanoparticles within or on the surface of electrospun polymeric scaffolds is a popular approach for bone tissue engineering. However, the fabrication of osteoconductive composite scaffolds via benign processing conditions still remains a major challenge to date. In this work, a new method was developed to achieve a uniform coating of calcium phosphate (CaP) onto electrospun keratin-polycaprolactone composites (Keratin-PCL). Keratin within PCL was crosslinked to decrease its solubility, before coating of CaP. A homogeneous coating was achieved within a short time frame (~10min) by immersing the scaffolds into Ca(2+) and (PO4)(3-) solutions separately. Results showed that the incorporation of keratin into PCL scaffolds not only provided nucleation sites for Ca(2+) adsorption and subsequent homogeneous CaP surface deposition, but also facilitated cell-matrix interactions. An improvement in the mechanical strength of the resultant composite scaffold, as compared to other conventional coating methods, was also observed. This approach of developing a biocompatible bone tissue engineering scaffold would be adopted for further in vitro osteogenic differentiation studies in the future.

Insights into the role of focal adhesion modulation in myogenic differentiation of human mesenchymal stem cells.

We report the establishment of a novel platform to induce myogenic differentiation of human mesenchymal stem cells (hMSCs) via focal adhesion (FA) modulation, giving insights into the role of FA on stem cell differentiation. Micropatterning of collagen type I on a polyacrylamide gel with a stiffness of 10.2 kPa efficiently modulated elongated FA. This elongated FA profile preferentially recruited the ß(3) integrin cluster and induced specific myogenic differentiation at both transcription and translation levels with expression of myosin heavy chain and α-sarcomeric actin. This was initiated with elongation of FA complexes that triggered the RhoA downstream signaling toward a myogenic lineage commitment. This study also illustrates how one could partially control myogenic differentiation outcomes of similar-shaped hMSCs by modulating FA morphology and distribution. This technology increases our toolkit choice for controlled differentiation in muscle engineering.

[Culture of human epidermal stem cells in different media and their biological characteristics].

OBJECTIVE: To select the appropriate media to culture the epidermal stem cells in vitro, and to observe the biological characteristics of the epidermal stem cells. METHODS: The epidermal stem cells were cultured in five different media, including FAD, FAD+1 ng/ml bFGF, FAD+5 ng/ml bFGF, FAD+10 ng/ml bFGF and K-SFM, and the same fetus fibroblasts were used as the nutrient cells. The proliferation ability was investigated by cell growth curve and MTT detection. Then the biological characteristics of epidermal stem cells were observed through phase-contrast microscope, cell growth curve, BrdU detection and FBM analysis. RESULTS: The epidermal stem cells grew best in FAD with bFGF and nutrient cells. And the epidermal stem cells retained proliferative capacity, and formed larger and more expandable clones in vitro. And 80.2% of the cells show a G0/G1 cycle, and the cells had long cell proliferation cycle. CONCLUSION: The above results demonstrate that the media with bFGF and the use of nutrient layer were appropriate to culture epidermal stem cell in vitro. And the epidermal stem cells have a slow cell cycle, characteristics of immaturity.