Osteogenic growth peptide (OGP)-loaded amphiphilic peptide (NapFFY) supramolecular hydrogel promotes osteogenesis and bone tissue reconstruction.

Efficient bone reconstruction after bone injury remains a great challenge. Injectable supramolecular hydrogels based on amphiphilic peptide have been widely used due to their good biocompatability, non-immunogenicity, and manipulable physicochemical properties by sequence design. Herein, we used a well-studied hydrogelator, NapFFY, to coassemble with osteogenic growth peptide (OGP) to prepare a supramolecular hydrogel, NapFFY-OGP. Both in vitro and in vivo studies demonstrate that OGP was ideally synchronously, and continuously released from the hydrogel to effectively promote the regeneration and reconstruction of skull bone defects. More specifically, after the embedding the rat skull defect area with NapFFY-OGP hydrogels, a bone regeneration rate of 37.54% bone volume fraction (BV/TV) was achieved compared to that of NapFFY hydrogel group (25.09%). NapFFY-OGP hydrogel shows great promise in the clinic repair of bone defects in the future.

Site-specific opening of the blood-brain barrier by extracellular histones.

BACKGROUND: Increased extracellular histones in the bloodstream are known as a biomarker for vascular dysfunction associated with severe trauma or sepsis. There is limited information regarding the pathogenic role of circulating histones in neuroinflammation and cerebrovascular endothelial injury. Particularly, it remains unclear whether histones affect the blood-brain barrier (BBB) permeability function. METHODS: The direct effects of unfractionated histones on endothelial barrier properties were first assessed in brain microvascular endothelial cell monolayers by measuring transendothelial electrical resistance and solute flux. This was followed by in vivo mouse experiments, where BBB function was assessed by quantifying brain tissue accumulation of intravenously injected tracers of different molecular sizes, and comparison was made in mice receiving a sublethal dose of histones versus sterile saline. In parallel, the endothelial barrier ultrastructure was examined in histone- and saline-injected animals under transmission electron microscopy, corresponding to the expression of tight junction and adherens junction proteins. RESULTS: Histones increased paracellular permeability to sodium fluorescein and reduced barrier resistance at 100 µg/mL; these responses were accompanied by discontinuous staining of the tight junction proteins claudin-5 and zona ocludens-1. Interestingly, the effects of histones did not seem to result from cytotoxicity, as evidenced by negative propidium iodide staining. In vivo, histones increased the paracellular permeability of the BBB to small tracers of < 1-kDa, whereas tracers larger than 3-kDa remained impermeable across brain microvessels. Further analysis of different brain regions showed that histone-induced tracer leakage and loss of tight junction protein expression mainly occurred in the hippocampus, but not in the cerebral cortex. Consistently, opening of tight junctions was found in hippocampal capillaries from histone-injected animals. Protein expression levels of GFAP and iBA1 remained unchanged in histone-injected mice indicating that histones did not affect reactive gliosis. Moreover, cell membrane surface charge alterations are involved in histone-induced barrier dysfunction and tight junction disruption. CONCLUSIONS: Extracellular histones cause a reversible, region-specific increase in BBB permeability to small molecules by disrupting tight junctions in the hippocampus. We suggest that circulating histones may contribute to cerebrovascular injury or brain dysfunction by altering BBB structure and function.

Histone H2B induces retinal ganglion cell death through toll-like receptor 4 in the vitreous of acute primary angle closure patients.

Acute primary angle closure (APAC) is a disease of ophthalmic urgency; lack of treatment can lead to blindness. Even after adequate treatment for APAC, subsequent elevated acute intraocular pressure induces severe neuronal damage which can result in secondary glaucomatous optic neuropathy (GON). Damage-associated molecular patterns (DAMPs) are released from damaged and dead neuronal cells, which induce secondary inflammatory changes and further tissue damage. Our hypothesis is that histone H2B (H2B), which is one of the DAMPs, is released from damaged cells in the development of GON after APAC treatment. Intravitreal injection of H2B induces neuronal cell death through toll-like receptor 4 (TLR4) expression, following the upregulation of inflammatory cytokine mRNAs and phosphorylation of mitogen activated protein kinases (MAPKs). Knockdown of TLR4 caused a reduction of H2B neurotoxicity in damaged cells through TLR4 signaling. Significantly increased H2B was observed in the vitreous cells of APAC patients. In addition, enhanced H2B protein correlated with decreased ganglion cell analysis and retinal ganglion cell (RGC) layer thinning, which indicates the effect of H2B on RGCs. Our data from clinical and animal studies show the involvement of H2B-TLR4 pathways in the development of GON after APAC treatment providing new insight for the mechanism of RGC degeneration.

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.

Extracellular histones induce erythrocyte fragility and anemia.

Extracellular histones have been shown to play an important pathogenic role in many diseases, primarily through their cytotoxicity toward nucleated cells and their ability to promote platelet activation with resultant thrombosis and thrombocytopenia. In contrast, little is known about the effect of extracellular histones on erythrocyte function. We demonstrate in this study that histones promote erythrocyte aggregation, sedimentation, and using a novel in vitro shear stress model, we show that histones induce erythrocyte fragility and lysis in a concentration-dependent manner. Furthermore, histones impair erythrocyte deformability based on reduced passage of erythrocytes through an artificial spleen. These in vitro results were mirrored in vivo with the injection of histones inducing anemia within minutes of administration, with a concomitant increase in splenic hemoglobin content. Thrombocytopenia and leukopenia were also observed. These findings suggest that histones binding to erythrocytes may contribute to the elevated erythrocyte sedimentation rates observed in inflammatory conditions. Furthermore, histone-induced increases in red blood cell lysis and splenic clearance may be a significant factor in the unexplained anemias seen in critically ill patients.

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.

Extracellular histones induce tissue factor expression in vascular endothelial cells via TLR and activation of NF-κB and AP-1.

Extracellular histones have been recognized recently as proinflammatory mediators; they are released from dying cells in response to inflammatory challenge, contributing to endothelial cell dysfunction, thrombin formation, organ failure, and death during sepsis. Clinical studies suggest that the plasma concentration of the histone-DNA complex is correlated with the severity of DIC and is a poor independent prognostic marker in sepsis. In addition, platelet activation stimulates thrombus formation. Whether histones contribute to procoagulant activity in other ways remains elusive. In this study, we confirmed that histones induce tissue factor (TF) expression in a concentration- and time-dependent manner in vascular endothelial cells (ECs) and macrophages. However, histones did not affect TF pathway inhibitor expression. Moreover, blocking the cell surface receptors TLR4 and TLR2 with specific neutralizing antibodies significantly reduced histone-induced TF expression. Furthermore, histones enhanced the nuclear translocation of NF-κB (c-Rel/p65) and AP-1 expression in a time-dependent manner in ECs. Mutating NF-κB and AP-1 significantly reduced histone-induced TF expression. Altogether, our experiments suggest that histone induces TF expression in ECs via cell surface receptors TLR4 and TLR2, simultaneously depending on the activation of the transcription factors NF-κB and AP-1.

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.

[Proteolytic activity of IgG-antibodies of mice, immunized by calf thymus histones].

The main goal of the study was to determine the ability of histones to induce production of the proteolytically active IgG-antibodies in BALB/c mice. In order to perform this study 8 mice were immunized with the fraction of total calf thymus histones. IgGs were isolated from the serum of the immunized and not immunized animals by means of precipitation with 33% ammonium sulfate, followed by affinity chromatography on protein G-Sepharose column. Histones, myelin basic protein (MBP), lysozyme, BSA, ovalbumin, macroglobulin, casein and cytochrome c served as substrates for determining the proteolytic activity. It was found that IgGs from the blood serum of immunized mice are capable of hydrolyzing histone H1, core histone and MBP. On the contrary, the proteolytic activity of IgGs from the blood serum of not immunized mice was not detected. The absence of proteolytical enzymes in the fraction of IgGs was proven by HPLC chromatography. High levels of proteolytic activity toward histones have been also detected in affinity purified IgGs from blood serum of patients with rheumatoid arthritis, but not in healthy donors. These data indicate that eukaryotic histones may induce production of protabzymes in mammals. The possible origin of these protabzymes and their potential biological role in mammalians is discussed.

Studies on peroxynitrite-modified H1 histone: implications in systemic lupus erythematosus.

Peroxynitrite is a powerful nitrating and oxidizing molecule and capable of modifying proteins' structure. Hyper-nitration of tyrosine residues has been seen in various pathological states, including autoimmune disorders like systemic lupus erythematosus (SLE) and rheumatoid arthritis. SLE, a chronic autoimmune disease, is primarily characterized by increased levels of autoantibodies, predominantly against ds-DNA. However, the initial antigenic stimulus for the disease etiopathogenesis has remained elusive. Carbonyl and nitrotyrosine have been extensively used as a biomarker of oxidative and nitrosative stress. In this study, commercially available H1 histone was exposed to increasing concentrations of peroxynitrite for 30 min. The peroxynitrite-mediated structural changes in histone were studied by ultraviolet & fluorescence spectroscopy, CD, HPLC, 1-anilinonaphthalene-8-sulfonic acid binding and polyacrylamide gel electrophoresis. Analysis of results revealed that carbonyl and nitrotyrosine contents were significantly increased in peroxynitrite-modified H1 compared to native H1. In experimental animal, peroxynitrite-modified H1 induced high titre antibodies as compared to native H1, and the immunogenicity was found to be directly proportional to nitrotyrosine content. Further, the induced antibodies showed specificity for the immunogen and appreciable cross-reactions with tyrosine rich nitrated proteins. Formation of high molecular weight immune complex with retarded mobility further supports the specificity of induced anti-peroxynitrite-modified H1 antibodies for the immunogen. Binding of SLE anti-DNA autoantibodies with peroxynitrite-modified H1 was analyzed by direct binding and competition ELISA. The data show preferential binding of SLE autoantibodies to peroxynitrite-modified H1 as compared to native H1 histone and native DNA. The results point towards the possible role of peroxynitrite-modified H1 histone in SLE etiopathogenesis.

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.

Histone exchange activity and its correlation with histone acetylation status in porcine oocytes.

In mammalian oocytes, histone H3 and histone H4 (H4) in the chromatin are highly acetylated at the germinal vesicle (GV) stage, and become globally deacetylated after GV breakdown (GVBD). Although nuclear core histones can be exchanged by cytoplasmic free histones in somatic cells, it remains unknown whether this is also the case in mammalian oocytes. In this study, we examined the histone exchange activity in maturing porcine oocytes before and after GVBD, and investigated the correlations between this activity and both the acetylation profile of the H4 N-terminal tail and the global histone acetylation level in the chromatin. We injected Flag-tagged H4 (H4-Flag) mRNA into GV oocytes, and found that the Flag signal was localized to the chromatin. We next injected mRNAs of mutated H4-Flag, which lack all acetylation sites and the whole N-terminal tail, and found that the H4 N-terminal tail and its modification were not necessary for histone incorporation into chromatin. Despite the lack of acetylation sites, the mutated H4-Flag mRNA injection did not decrease the acetylation level on the chromatin, indicating that the histone exchange occurs partially in the GV chromatin. In contrast to GV oocytes, the Flag signal was not detected on the chromatin after the injection of H4-Flag protein into the second meiotic metaphase oocytes. These results suggest that histone exchange activity changes during meiotic maturation in porcine oocytes, and that the acetylation profile of the H4 N-terminal tail has no effect on histone incorporation into chromatin and does not affect the global level of histone acetylation in it.

Transgene expression efficiency from plasmid DNA delivered as a complex with histone H3.

The intranuclear disposition of plasmid DNA is extremely important for transgene expression. Exogenous histones have been used as carriers of plasmid DNA in histone-mediated gene delivery. In this study, the effects of exogenous histone H3 complexed with plasmid DNA on transgene expression efficiency were examined. The plasmid-histone complexes in various ratios were transfected into HeLa cells by osmotic pressure. Histone H3 suppressed transgene expression in the nucleus in a dose-dependent manner. Our results suggest that the histone-mediated gene delivery is unlikely to be useful, from the viewpoint of the intranuclear disposition.

Effects of carriers on transgene expression from plasmids containing a DNA sequence with high histone affinity.

The intranuclear disposition of plasmid DNA is highly important for transgene expression. The effects of a left-handedly curved sequence with high histone affinity on transgene expression were examined in COS-7 cells with two kinds of carriers (Lipofectamine Plus and TransIT-LT1). Three plasmids containing the curved sequence at different positions were transfected. The transgene expression was affected by the position of the left-handedly curved sequence, and the sequence at appropriate locations enhanced the expression from plasmid DNAs. However, the position effects on the expression differed from those obtained by electroporation of the same plasmid DNAs in a naked form. In addition, the degree of expression enhancement seemed to depend on the carriers. These results suggest that the left-handedly curved sequence with high histone affinity could increase the transgene expression from a plasmid delivered with carriers.

Adoptive transfer of dendritic cells pulsed with Leishmania infantum nucleosomal histones confers protection against cutaneous leishmaniosis in BALB/c mice.

The mechanisms underlying the protective effects induced by dendritic cells (DC)-based vaccines against Leishmania major in mice are not yet completely understood. In the present study, we investigated the potential of DC loaded with a mixture of the Leishmania infantum histones in the absence (HIS-pulsed DC) or presence of CpG motifs (HIS+CpG-pulsed DC) as a candidate vaccine against cutaneous leishmaniosis. Our data showed that a single intravenous administration of HIS-pulsed DC or HIS+CpG-pulsed DC confers control to L. major infection in BALB/c mice. Interestingly, all HIS-pulsed DC vaccinated mice remained susceptible to a second challenge. We found that the efficient immunity in BALB/c mice was associated to a Th1 response and a restriction of Th2 type of response upon challenge with L. major parasites. More importantly, the anti-leishmanial immunological mechanisms of protection were dependent on the ability to induce a low frequency of Foxp3(+) regulatory T cells at the site of infection. These results document that a vaccine based on a HIS+CpG-pulsed DC formulation may be as efficient for vaccination as one based on L. major antigen (Lm)+CpG-pulsed DC. Thus, HIS+CpG-pulsed DC may prove to be a new and further tool to add to those designed.

Vaccination with the divergent portion of the protein histone H2B of Leishmania protects susceptible BALB/c mice against a virulent challenge with Leishmania major.

To identify approaches for vaccination against leishmaniasis, we analyzed the protective effect of different constructions using recombinant peptides from the protein Leishmania (L.) major histone H2B. H2B sequence displays two distinct regions: an amino-terminal region divergent from mammalian H2B (27% identity) and a carboxy-terminal region highly conserved with mammalian H2B (55% identity). We tested the ability of the entire H2B protein, its divergent or conserved regions to provide protection against virulent L. major challenge. While the recombinant H2B protein adjuved with CpG induces potent cellular and antibody responses when injected to BALB/c mice, only the divergent amino-terminal region of H2B is able to confer potent protection against a virulent challenge. These findings indicate that different portions of the same parasite protein may express contrasting protective effects likely through the induction of different effector mechanisms. Due to its potent protective properties in the BALB/c mouse model, the amino-terminal region of Leishmania H2B could constitute a good vaccine candidate.

Activity of histone H1.2 in infected burn wounds.

OBJECTIVES: Infections with multidrug-resistant microorganisms (e.g. Pseudomonas aeruginosa and Staphylococcus aureus) cause immense complications in wound care and in the treatment of immunosuppressed patients. Like most antimicrobial peptides, histones are relatively small polycationic proteins located in each eukaryotic nucleus, which naturally supercoil DNA. The aim of this study was to investigate the in vitro and in vivo activity of histone H1.2 in infected burn wounds and its potential toxicity. METHODS: To characterize the antimicrobial properties of histone H1.2 against potential causative organisms of burn wound infections, the in vitro radial diffusion assay and modified NCCLS microbroth dilution MIC assay were carried out. Haemolytic and cytotoxic properties were determined in human red blood cells and primary human keratinocytes. In vivo antimicrobial activity was tested in an infected rat burn model with P. aeruginosa (ATCC 27853). All results were compared with the naturally occurring broad-spectrum antimicrobial peptide protegrin-1 and with antibiotics clinically used against the corresponding bacteria. RESULTS: Human histone H1.2 exerted good antimicrobial activity against all tested microorganisms without significant haemolytic activity. Surprisingly, histone H1.2 showed cytotoxicity with an LD50 of 7.91 mg/L in primary human keratinocytes. The in vivo burn model data revealed a significant three-fold higher reduction in bacterial counts within 4 h compared with carrier control. CONCLUSIONS: These findings indicate that histone H1.2 is a potential candidate for use as a local and, because of its low haemolytic activity, systemic antimicrobial agent. However, further investigations are needed to specify the cytotoxicity and the dose-response relationship for histone H1.2.