DNA-delivered monoclonal antibodies targeting the p53 R175H mutant epitope inhibit tumor development in mice.

The tumor suppressor p53 is the most common mutated gene in cancer, with the R175H as the most frequent p53 missense mutant. However, there are currently no approved targeted therapies or immunotherapies against mutant p53. Here, we characterized and investigated a monoclonal antibody (mAb) that recognizes the mutant p53-R175H for its affinity, specificity, and activity against tumor cells in vitro. We then delivered DNA plasmids expressing the anti-R175H mAb or a bispecific antibody (BsAb) into mice to evaluate their therapeutic effects. Our results showed that the anti-R175H mAb specifically bound to the p53-R175H antigen with a high affinity and recognized the human mutant p53-R175H antigen expressed on HEK293T or MC38 cells, with no cross-reactivity with wild-type p53. In cultured cells, the anti-R175H mAb showed higher cytotoxicity than the control but did not induce antibody-dependent cellular cytotoxicity. We made a recombinant MC38 mouse cell line (MC38-p53-R175H) that overexpressed the human p53-R175H after knocking out the endogenous mutant p53 alleles. In vivo, administration of the anti-R175H mAb plasmid elicited a robust anti-tumor effect against MC38-p53-R175H in mice. The administration of the anti-R175H BsAb plasmid showed no therapeutic effects, yet potent anti-tumor activity was observed in combination with the anti-PD-1 antibody. These results indicate that targeting specific mutant epitopes using DNA-delivered mAbs or BsAbs presents a form of improved natural immunity derived from tumor-infiltrating B cells and plasma cells against intracellular tumor antigens.

Development of an anti-infective coating on the surface of intraosseous implants responsive to enzymes and bacteria.

BACKGROUND: Bacterial proliferation on the endosseous implants surface presents a new threat to the using of the bone implants. Unfortunately, there is no effective constructed antibacterial coating which is bacterial anti-adhesion substrate-independent or have long-term biofilm inhibition functions. METHODS: Drug release effect was tested in Chymotrypsin (CMS) solution and S. aureus. We used bacterial inhibition rate assays and protein leakage experiment to analyze the in vitro antibacterial effect of (Montmorillonite/Poly-L-lysine-Chlorhexidine)10 [(MMT/PLL-CHX)10] multilayer film. We used the CCK-8 assay to analyze the effect of (MMT/PLL-CHX)10 multilayer films on the growth and proliferation of rat osteoblasts. Rat orthopaedic implant-related infections model was constructed to test the antimicrobial activity effect of (MMT/PLL-CHX)10 multilayer films in vivo. RESULTS: In this study, the (MMT/PLL-CHX)10 multilayer films structure were progressively degraded and showed well concentration-dependent degradation characteristics following incubation with Staphylococcus aureus and CMS solution. Bacterial inhibition rate assays and protein leakage experiment showed high levels of bactericidal activity. While the CCK-8 analysis proved that the (MMT/PLL-CHX)10 multilayer films possess perfect biocompatibility. It is somewhat encouraging that in the in vivo antibacterial tests, the K-wires coated with (MMT/PLL-CHX)10 multilayer films showed lower infections incidence and inflammation than the unmodified group, and all parameters are close to SHAM group. CONCLUSION: (MMT/PLL-CHX)10 multilayer films provides a potential therapeutic method for orthopaedic implant-related infections.

Erratum: Microporous polysaccharide multilayer coated BCP composite scaffolds with immobilised calcitriol promote osteoporotic bone regeneration both in vitro and in vivo: Erratum.

[This corrects the article DOI: 10.7150/thno.29566.].

Antibiotic-Loaded MMT/PLL-Based Coating on the Surface of Endosseous Implants to Suppress Bacterial Infections.

BACKGROUND: Bone infections remain one of the most common and serious complications of orthopedic surgery, posing a tremendous economic burden to society and patients. This is because bacteria colonize and multiply on the surface of the implant. The (MMT/PLL)8 multilayer films have been shown to effectively release antibiotics depending on the changes in the microenvironment. Here, vancomycin was loaded into the (MMT/PLL)8 multilayer films, which were prepared to be used as a local delivery system for the treatment of bone infections. METHODS: We used the layer-by-layer self-assembly method to prepare VA-loaded coatings (MMT/PLL-VA)8 consisting of montmorillonite (MMT), poly-L-lysine (PLL), and VA. The thickness and surface morphology of coatings were characterized using spectroscopic ellipsometry and scanning electron microscopy (SEM). In order to evaluate the drug release behavior from coatings in different media, we measured the size of the zone of inhibition. Additionally, in vitro antibacterial activity was assessed using the shake-flask culture method and SEM images, while that of in vivo was evaluated by establishing an animal model of bone infection. RESULTS: Our findings revealed that small-molecule antibiotics were successfully loaded into the (MMT/PLL-VA)8 multilayer film structure during the hierarchical self-assembly process and subsequently the multilayer film structure depicted linear growth behavior. The PLL in the multilayer films was progressively degraded which triggered the VA release when contacted with CMS or bacterial infections. The release of VA from multilayer film structure depends on the concentration changes of CMS. Notably, the multilayer films presented great in vitro cell compatibility. Moreover, the prepared antibacterial multilayer films showed excellent antibacterial property by killing more than 99.99% of S. aureus in 24 h. More importantly, we found that multilayer film exhibits good sterilization effect and biocompatibility under the stimulation of bacterial liquid both in vitro and in vivo antibacterial ability tests. CONCLUSION: Altogether, this study shows that (MMT/PLL-VA)8 multilayer films containing CMS and bacteria-responsive drug release properties posess high bactericidal activity and good biocompatibility. This finding provides a novel strategy for the treatment of bone infections.

Asiatic acid ameliorates obesity-related osteoarthritis by inhibiting myeloid differentiation protein-2.

Obesity is related to osteoarthritis (OA). Aberrant lipid metabolism results in increased levels of free fatty acids, such as palmitate (PA), leading to inflammatory responses and excess catabolism of chondrocytes. Asiatic acid (AA), a plant anti-inflammatory compound, has been reported to exert protective effects for several diseases, but its effect on obesity-related OA is still unclear. The aim of this study is to evaluate the chondro-protective effect of AA on PA-induced human chondrocytes and a high fat diet (HFD)-fed mouse cartilage degeneration model. In vitro, the levels of the inflammatory and extracellular matrix (ECM) markers of chondrocytes after being treated with PA (500 µM) and AA (2.5-10 µM) were determined using western blotting and immunofluorescence enzyme-linked immunosorbent assay (ELISA). In vivo, after the oral administration of HFD and AA, X-ray examination, safranin O staining, and ELISA assay were conducted to evaluate cartilage calcification and degeneration and cytokine and adipokine levels in the serum of mice. AA treatment eliminated the inflammation caused by PA and extracellular matrix degradation. Mechanistically, AA blocked the stimulation of the NF-κB pathway. Analysis with co-immunoprecipitation and molecular docking indicated that the MD-2/TLR4 complex was a target of AA. In vivo, AA treatment not only prevented HFD-induced OA changes but also reduced proinflammatory cytokine and adipokine production in obese mice. AA exerted a chondroprotective effect by inhibiting the TLR4/MD-2 axis, thus showing promise for treating obesity-related OA.

Microporous polysaccharide multilayer coated BCP composite scaffolds with immobilised calcitriol promote osteoporotic bone regeneration both in vitro and in vivo.

Incorporating a biomimetic coating and integrating osteoinductive biomolecules into basic bone substitutes are two common strategies to improve osteogenic capabilities in bone tissue engineering. Currently, the underlying mechanism of osteoporosis (OP)-related deficiency of osteogenesis remains unclear, and few treatments target at OP-related bone regeneration. Herein, we describe a self-assembling polyelectrolyte multilayered (PEM) film coating with local immobilisation of calcitriol (Cal) in biphasic calcium phosphate (BCP) scaffolds to promote osteoporotic bone regeneration by targeting the calcium sensing receptor (CaSR). Methods: The ovariectomy-induced functional changes in bone marrow mesenchymal stem cells (BMSCs), protective effects of Cal, and the potential mechanism were all verified. A PEM film composed of hyaluronic acid (HA) and chitosan (Chi) was prepared through layer-by-layer self-assembly. The morphology, growth behaviour, and drug retention capability of the composite scaffolds were characterised, and their biocompatibility and therapeutic efficacy for bone regeneration were systematically explored in vitro and in vivo.Results: The osteogenic differentiation, adhesion, and proliferation abilities of ovariectomised rat BMSCs (OVX-rBMSCs) decreased, in accordance with the deficiency of CaSR. Cal effectively activated osteogenesis in these OVX-rBMSCs by binding specifically to the active pocket of the CaSR structure, while the biomimetic PEM coating augmented OVX-rBMSCs proliferation and adhesion due to its porous surface structure. The PEM-coated scaffolds showed advantages in Cal loading and retention, especially at lower drug concentrations. HA/Chi PEM synergised with Cal to improve the proliferation, adhesion, and osteogenesis of OVX-rBMSCs and promote bone regeneration and BCP degradation in the critical-size calvarial bone defect model of OVX rats. Conclusion: A composite scaffold based on BCP, created by simply combining a biomimetic PEM coating and Cal immobilisation, could be clinically useful and has marked advantages as a targeted, off-the-shelf, cell-free treatment option for osteoporotic bone regeneration.

Isofraxidin targets the TLR4/MD-2 axis to prevent osteoarthritis development.

Osteoarthritis (OA) is a major cause of joint pain and disability, resulting in large socioeconomic costs worldwide. Isofraxidin (ISO), a bioactive coumarin compound isolated from the functional foods Siberian ginseng and Apium graveolens, exerts anti-inflammatory effects in a variety of diseases. However, no studies have reported the protective effects of ISO against OA development. Accordingly, this study aimed to assess the therapeutic effect of ISO in human OA chondrocytes, and in a mouse model of OA induced by destabilisation of the medial meniscus (DMM). In vitro, lipopolysaccharide (LPS)-induced overproduction of nitric oxide (NO), prostaglandin E2 (PGE2), tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) was decreased by ISO pre-treatment. Furthermore, ISO attenuated the increased expression of inflammatory enzymes, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in response to LPS stimulation. Meanwhile, LPS-induced extracellular matrix (ECM) degradation was also reversed by ISO treatment. Mechanistically, ISO competitively inhibited Toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2) complex formation, and thus TLR4/nuclear factor kappa B (NF-κB) signalling cascades. In vivo, ISO treatment not only prevented the calcification and erosion of cartilage, as well as the thickening of subchondral bone, but also reduced the serum levels of inflammatory cytokines in the mouse OA model. Taken together, these data suggest that ISO has potential in the treatment of OA.

Paeonol Inhibits IL-1ß-Induced Inflammation via PI3K/Akt/NF-κB Pathways: In Vivo and Vitro Studies.

Paeonol, the main active component isolated from the root of Paeonia suffruticosa, has been reported to have anti-inflammatory properties. However, the effects of paeonol on osteoarthritis (OA) remain unclear. The aim of this study was to investigate the anti-inflammatory effects and mechanism of paeonol in IL-1ß-induced human OA chondrocytes as well as mice OA models. Human OA chondrocytes were pretreated with different concentrations of paeonol 2 h prior to IL-1ß (10 ng/mL) stimulation for 24 h. Nitric oxide (NO) production was determined by Griess method. The levels of prostaglandin E2 (PGE2), matrix metalloproteinase 1 (MMP-1), MMP-3, and MMP-13 were assessed by ELISA. Inducible nitric oxide synthase (INOS), COX-2, and PI3K/Akt/NF-κB-related signaling molecules production were measured by Western blot. In vivo, mice OA models were established by destabilization of the medial meniscus. One month after surgery, mice in paeonol-treated group were given intraperitoneal injection of paeonol in 30 mg/kg every day, while mice of vehicle-treated group were injected with DMSO under the same conditions. Hematoxylin and eosin as well as Safranin-O staining were applied to assess the severity of cartilage lesions. The results showed that pretreatment with paeonol could inhibit IL-1ß-induced NO and PGE2 production. Meanwhile, the overproduction of INOS, COX-2, MMP-1, MMP-3, and MMP-13 were also reversed by paeonol. Moreover, paeonol was found to inhibit IL-1ß-induced NF-κB activation, PI3K, and AKT phosphorylation. In vivo, treatment with paeonol exhibited less cartilage degradation and lower Osteoarthritis Research Society International scores in mice OA models. In conclusion, these results suggest that paeonol may be a potential therapeutic agent in the treatment of OA.