Multilayered pore-closed PLGA microsphere delivering OGP and BMP-2 in sequential release patterns for the facilitation of BMSCs osteogenic differentiation.

Bone tissue regeneration may be more effectively administrated by controlled release of multiple biofactors, given that bone healing comprises a cascade of biological events controlled by numerous cytokines and growth factors (GFs). Here, we propose a novel microcarrier with the capability to sequentially deliver dual biofactors for better controlling the bone regeneration process. First, osteogenic growth peptide (OGP) was incorporated in porous poly(lactic-co-glycolic) acid (PLGA) microspheres by a simple solution dipping method and subsequent pore-closing treatment. Then, a multilayered polyelectrolyte coating ((HA-CS)2 -Hep-BMP-2-Hep-(CS-HA)2 ) was prepared on the surface of such OGP-loaded pore-closed PLGA microspheres by layer-by-layer assembly. Results showed that the OGP release was minimal (<17.1%) in the first 15 days but accelerated remarkably thereafter, while at least 60.3% of the bone morphogenetic protein-2 (BMP-2) load was released in the first 15 days and only very slow release was observed subsequently. Further in vitro cell experiments showed that the dual-biomolecule-loaded microspheres elicited more cells with extremely elongated cellular morphology, much higher alkaline phosphatase level and upregulated expression of osteocalcin. Such a dual loading of OGP and BMP-2 had a more positive impact on bone marrow mesenchymal stem cells proliferation and osteogenic differentiation compared with either OGP or BMP-2 alone, suggesting potential synergistic benefit of the sequential release of multiple peptide-based biofactors in a coordinated manner. Overall, this dual delivery system may provide a therapeutic strategy sequentially targeting multiple events (or mechanisms) during bone healing, which is believed to benefit the regenerative repair of bone defects. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 95-105, 2018.

89Zr-anti-γH2AX-TAT but not 18F-FDG Allows Early Monitoring of Response to Chemotherapy in a Mouse Model of Pancreatic Ductal Adenocarcinoma.

Purpose: Late-stage, unresectable pancreatic ductal adenocarcinoma (PDAC) is largely resistant to chemotherapy and consequently has a very poor 5-year survival rate of <5%. The ability to assess the efficacy of a treatment soon after its initiation would enable rapid switching to potentially more effective therapies if the current treatment is found to be futile. We have evaluated the ability of the PET imaging agent, 89Zr-anti-γH2AX-TAT, to monitor DNA damage in response to fluorouracil (5-FU), gemcitabine, or capecitabine treatment in a mouse model of pancreatic cancer. We have also compared the utility of this approach against the standard clinical PET radiotracer, 18F-FDG.Experimental Design: C57BL/6 mice bearing subcutaneous pancreatic cancer (KPC; B8484) allografts were treated with 5-FU, gemcitabine, or capecitabine. Therapeutic response was monitored by PET and ex vivo biodistribution experiments using either 89Zr-anti-γH2AX-TAT or 18F-FDG as imaging agents. To further examine the effect of therapeutic response upon uptake of these imaging agents, IHC analysis of harvested tumor allograft tissue was also performed.Results: Accumulation of 89Zr-anti-γH2AX-TAT in the tumors of mice that received chemotherapy was higher compared with vehicle-treated mice and was shown to be specifically mediated by γH2AX. In contrast, 18F-FDG did not provide useful indications of therapeutic response.Conclusions:89Zr-anti-γH2AX-TAT has shown a superior ability to monitor early therapeutic responses to chemotherapy by PET imaging compared with 18F-FDG in an allograft model of PDAC in mice. Clin Cancer Res; 23(21); 6498-504. ©2017 AACR.

Enhanced osteogenic activity of poly(ester urea) scaffolds using facile post-3D printing peptide functionalization strategies.

Additive manufacturing has the potential to revolutionize regenerative medicine, but the harsh thermal or photochemical conditions during the 3D printing process limit the inclusion of drugs, growth factors and other biologics within the resulting scaffolds. Functionalization strategies that enable specific placement of bioactive species on the surface of 3D printed structures following the printing process afford a promising approach to sidestep the harsh conditions and incorporate these valuable bioactive molecules with precise control over concentration. Herein, resorbable polymer scaffolds were prepared from propargyl functionalized L-phenylalanine-based poly(ester urea)s (PEUs). Osteogenic growth peptide (OGP) or bone morphogenic protein-2 (BMP-2) peptides were immobilized on PEU scaffolds through surface available propargyl groups via copper-catalyzed azide alkyne cycloaddition (CuAAC) post 3D printing. The presence of either OGP or BMP-2 significantly enhanced hMSCs osteogenic differentiation compared to unfunctionalized scaffolds.

Stable H3 peptide was delivered by gold nanorods to inhibit LSD1 activation and induce human mesenchymal stem cells differentiation.

Recently, lysine-specific demethylase 1 (LSD1), which is the first identified histone demethylase, regulates post-translational modifications and has great promise as new targets for cancer and other diseases. Moreover, the ability of LSD1 to induce the differentiation of stem cells has attracted great attention in biological fields. In this study, we designed LSD1 peptide inhibitor based on its substrate H3 peptide. Through introducing a disulfide bond to stabilize the native peptide into alpha helical structure, we get a peptide with higher cell permeability and stability compared to its parent form. Using gold nanorods (AuNRs) as delivery systems to deliver stable peptide into human MSCs, the delivery efficiency has been enhanced significantly by flow cytometry and cell fluorescent imaging. The intracellular delivery of stable peptide by AuNRs-PEI-based nanocarriers could inhibit the activation of LSD1, which together with hepatocyte growth factor (HGF) exhibits obviously synergistic effect to induce human MSCs differentiation. Furthermore, the hepatic marker genes AFP (alpha fetal protein) and ck19 are up-regulated by AuNRs-stable peptide (AuNRs- SP- PEI) with HGF. In conclusion, our study is the first time to use stable H3 peptide to inhibit LSD1 activation, which has been further delivered by AuNRs as nanocarriers into human MSCs.

Hydrogel depots for local co-delivery of osteoinductive peptides and mesenchymal stem cells.

The outcome of cell-based therapies can benefit from carefully designed cell carriers. A multifunctional injectable vehicle for the co-delivery of human mesenchymal stem cells (hMSCs) and osteoinductive peptides is proposed, to specifically direct hMSCs osteogenic differentiation. The osteogenic growth peptide (OGP) inspired the design of two peptides, where the bioactive portion of OGP was flanked by a protease-sensitive linker, or its scrambled sequence, to provide faster and slower release rates, respectively. Peptides were fully characterized and chemically grafted to alginate. Both OGP analogs released bioactive fragments in vitro, at different kinetics, which stimulated hMSCs proliferation and osteogenesis. hMSCs-laden OGP-alginate hydrogels were tested at an ectopic site in a xenograft mouse model. After 4weeks, OGP-alginate hydrogels were more degraded and colonized by vascularized connective tissue than the control (without OGP). hMSCs were able to proliferate, migrate outward the hydrogels, produce endogenous extracellular matrix and mineralize it. Moreover, OGP-groups stimulated hMSCs osteogenesis, as compared with the control. Overall, the ability of the proposed platform to direct the fate of transplanted hMSCs in loco was demonstrated, and OGP-releasing hydrogels emerged as a potentially useful system to promote bone regeneration.

Vaccination with Leishmania histone H1-pulsed dendritic cells confers protection in murine visceral leishmaniasis.

Visceral leishmaniasis is the most severe form of leishmaniases affecting millions of people worldwide often resulting in death despite optimal therapy. Thus, there is an urgent need for the development of effective anti-infective vaccine(s). In the present study, we evaluated the prophylactic value of bone marrow-derived dendritic cells (BM-DCs) pulsed with the Leishmania (L.) infantum histone H1. We developed fully mature BM-DCs characterized by enhanced capacity of IL-12 production after ex vivo pulsing with GST-LeishH1. Intravenous administration of these BM-DCs in naive BALB/c mice resulted in antigen-specific spleenocyte proliferation and IgG1 isotype antibody production and conferred protection against experimental challenge with L. infantum independently of CpG oligonucleotides (ODNs) co-administration. Protection was associated with a pronounced enhancement of parasite-specific IFNγ-producing cells and reduction of cells producing IL-10, whereas IL-4 production was comparable in protected and non-protected mice. The polarization of immune responses to Th1 type was further confirmed by the elevation of parasite-specific IgG2a/IgG1 ratio in protected mice. The above data indicate the immunostimulatory capacity of Leishmania histone H1 and further support its exploitation as a candidate protein for vaccine development against leishmaniasis.

Increase of immunoglobulin productivity of human-human hybridoma HB4C5 cells by histone.

A histone mixture (H1, H2A, H2B, H3, and H4) derived from calf thymus stimulated IgM production by human-human hybridoma HB4C5 cells. On the contrary, the histone mixture did not increase IgM production by the human Burkitt's lymphoma cell line NAT-30, IgG production by the human B lymphoblastoid cell line HMy-2, and IgE production by the human myeloma cell line U266. The immunoglobulin production-stimulating activity of the histone mixture was inactivated by trypsin or chymotrypsin digestion. In addition, confocal laser microscopic analysis had shown that HB4C5 cells incorporated a lot of histone but other cell lines did not incorporate it as much. These facts strongly suggest that histone acts as an immunoglobulin production-stimulating factor (IPSF) after internalization into the human B cell lines and the native structure of histone is required for the IPSF activity.

Histones as extracellular messengers: effects on growth hormone secretion.

Histones display hormone-like properties when present in extracellular fluids. The authors report that histones H2A and H2B possess growth hormone (GH)-releasing activity in vitro and describe the specificity and signal transduction pathways involved in these effects. Perfused and incubated rat pituitary cells were used in different sets of experiments and GH release was measured by radio-immunoassay (RIA). Perfusion of cells with 30 microM histone H2A or H2B, generated significant GH secretory responses. Cells incubated with histone H2A showed a dose- and time-dependent stimulatory effect on GH release which was blocked by peptide MB35, a synthetic fragment of histone H2A. Incubation of pituitary cells with the GH secretagogue GHRP-6, and histones revealed an additive release of GH, whereas GHRH and histones revealed a synergistic effect. The basic peptide poly-Lys did not mimetize the action of histones. Both EGTA and the protein kinase C inhibitor trifluoperazine, but not the calcium ionophre A23187, were able to reduce significantly the GH response of somatotrophs to histones. Pituitary cell incubation with 30 microM forskolin alone or in the presence of H2A or H2B, stimulated GH release in the same magnitude. The results confirm previous evidence that histones may act as hypophysotropic signals and suggest, although do not prove, that this activity is receptor dependent. Calcium- and diacylglycerol-associated pathways participate in these effects.

New Zealand white rabbits immunized with RNA-complexed total histones develop an autoimmune-like response.

The antibody response of rabbits immunized with a total histone mixture containing randomly coiled H1/H5, H2A, H2B, H3 and H4 devoid of DNA was investigated in direct and competitive ELISA. The antisera were tested with isolated histones and chromatin and with a series of overlapping synthetic peptides covering the entire sequences of the four core histones and two peptides of H1. It was found that the New Zealand (NZ) white rabbits immunized with the total histone (TH) mixture complexed with RNA produced IgG antibodies reacting with histones and with a number of histone peptides but not with chromatin. The antisera also contained IgG antibodies which bound components that correspond to common target antigens in autoimmune diseases such as native dsDNA, peptides of Sm-D antigen, ubiquitin, branched peptides of ubiquitinated H2A and poly(ADP-ribose). By competition experiments, it was shown that these antibodies corresponded to non-crossreacting antibody populations. New Zealand rabbits immunized with TH in the absence of RNA or random outbred rabbits immunized with the RNA-complexed histone fraction produced antibodies reacting with histone, chromatin and very few histone peptides, while no activity with non-related antigens was observed. The pattern of reactivity of antisera raised in NZ rabbits with RNA-complexed TH was found to be very similar to that observed in sera of patients with systemic lupus erythematosus while, in contrast, the antibody response was very different in NZ or outbred rabbits immunized with various native nuclear particles and with individual histones. Altered nucleosome particles rather than native nucleosomes may represent the antigenic stimulus giving rise to autoantibodies.

Histones inhibit human chorionic gonadotrophin-stimulated but not atrial peptide-stimulated testosterone production and cyclic nucleotide formation by isolated mouse Leydig cells.

Recently it has been reported that histone type H2A can inhibit gonadotrophin-stimulated cAMP formation and steroidogenesis by ovarian cells. In the present study we have investigated if similar antigonadotrophic effects of commercially available histones can also be demonstrated on testicular steroidogenic cells. Using percoll-purified mouse Leydig cells, we have demonstrated that several types of histones could almost completely inhibit hCG-stimulated testosterone production and cAMP formation. The inhibition was dose-dependent and could be reversed by the addition of excess of hCG. The most potent histone types were H2AS and H8S, both of which could inhibit hCG-stimulated cAMP formation half-maximally at concentrations of 4-5 micrograms/ml. Forskolin-stimulated cAMP formation was not affected by histones. When the cells were stimulated with either db-cAMP or rAP-II, histone H2AS and H8S failed to inhibit the testosterone production. In fact there was a marked increase in the amount of testosterone produced, the reason for which is not yet understood. The amount of cGMP accumulated in response to rAP-II was not affected by the presence of H2AS or H8S. In unstimulated cells, neither the cyclic nucleotide level nor the amount of steroid produced was affected by the histones. Based on the [125I]hCG binding data it is possible to conclude that histone H2AS inhibits the binding of hCG to its receptors on Leydig cells and thereby causes the inhibition of hCG-stimulated cAMP formation and steroidogenesis.

[Comparative study of the antiviral properties of histones of animal and plant origin]. / Sravnitel'noe izuchenie antivirusnykh svoistv gistonov zhivotnogo i rastitel'nogo proiskhozhdeniia.

The antiviral properties of histones of animal (thymus) and plant (French beans) origin were studied in plants and with a plant virus, tobacco mosaic virus (TMV). Histones of the thymus and French beans were shown to be able to inhibit TMV reproduction. The antiviral properties of histones were found to depend on their concentration, pH, and to be determined by the modes of their introduction into leaves. The manifestation of the antiviral properties of histones seems to require not only their direct contact with virus but also a certain exposure on the leaf. The similarities of antiviral protective mechanisms of plants and animals determined by substances of the protein nature are discussed.

The "stright-jacket" syndrome in chicks. II. Mechanism of development.

In a longitudinal study including measurement of the pressure in the amniotic cavity, amniotomy, and planimetric evaluation of the size of the amniotic sac, we investigated the development of the "strait jacket" syndrome in White Leghorn chicken embryos injected intraamniotically (ia) or paraamniotically on the fourth day of incubation with histone or embryotoxic serum, with the following results. Hyperlordosis and eventration developed as an outcome of tonic contraction of the amnion, which was observed only three hours after ia administration. Contraction of the amnion caused elevation of the intraamniotic pressure, which, 12 hours after ia injection, attained a mean value of 22.4 Pa (2.3 mm H2O). This value was not only significantly higher than the mean for control embryos (3.9 Pa), but it was critically close to the mean fluid pressure in the brain vesicles. Loss of the latter overpressure caused the vesicles to collapse, and the walls shriveled and exencephaly developed. Paraamniotic injection was not followed by either contraction of the amnion, or significant increase in intraamniotic pressure. This did not prevent heart malformations and cranioschisis of various extent. The majority of cardiovascular malformations were probably the hemodynamic consequence of overfilling of the intraembryonic vascular bed, which was one of the early signs of the effect question. Cranial-vault defects can be causally associated with the formation of amnionic adhesion and fusion with the epidermal ectoderm. This observation stresses the significance of the embryonic membranes and the fluid pressures within them for the development of certain congenital deformities and concentrates attention on teratological study of substances that induce protracted contraction of smooth muscle.

Changes in 3H-thymidine incorporation into the DNA by histones from normal and tumor tissues.

Attempts were made to isolate a factor from tumor tissue which, after injection, enhances the 3H-thymidine incorporation into the DNA of mice. This factor was in the cell nuclei and was extractable together with acid-soluble nuclear proteins (histones). Additional purification showed that this factor was either closely bound with the very lysine-rich histone (F1), or it was this histone itself. Acid-soluble nuclear proteins prepared from liver and thymus did not influence the incorporation of 3H-thymidine into DNA after injection. The very lysine-rich histone (F1) in these extracts was in an inactive form. It could be activated by separation from other acid-soluble nuclear proteins. Purified, the very lysine-rich histone (F1) enhanced the 3H-thymidine incorporation into DNA, regardless of its being prepared from tumor or nontumor tissues. By enhancing the incorporation of 3H-thymidine into DNA after injection, the very lysine-rich histone (F1) significantly differed from arginine-rich (F2aF3) and lysine-rich (F2b) histones which suppressed the 3H-thymidine incorporation.