ICG-Dimeric Her2-Specific Affibody Conjugates for Tumor Imaging and Photothermal Therapy for Her2-Positive Tumors.

Human epidermal growth factor receptor 2 (Her2) is abundantly expressed in various solid tumors. The Her2-specific Affibody (ZHer2:2891) has been clinically tested in patients with Her2-positive breast cancer and is regarded as an ideal drug carrier for tumor diagnosis and targeted treatment. Indocyanine green (ICG) can be used as a photosensitizer for photothermal therapy (PTT), in addition to fluorescent dyes for tumor imaging. In this study, a dimeric Her2-specific Affibody (ZHer2) based on ZHer2:2891 was prepared using the E. coli expression system and then coupled to ICG through an N-hydroxysuccinimide (NHS) ester reactive group to construct a novel bifunctional protein drug (named ICG-ZHer2) for tumor diagnosis and PTT. In vitro, ICG-ZHer2-mediated PTT selectively and efficiently killed Her2-positive BT-474 and SKOV-3 tumor cells rather than Her2-negative HeLa tumor cells. In vivo, ICG-ZHer2 specifically accumulated in Her2-positive SKOV-3 tumor grafts rather than Her2-negative HeLa tumor grafts; high-contrast tumor optical images were obtained. However, Her2-negative HeLa tumor grafts were not detected. More importantly, ICG-ZHer2-mediated PTT exhibited a significantly enhanced antitumor effect in mice bearing SKOV-3 tumor grafts owing to the good photothermal properties of ICG-ZHer2. Of note, ICG-ZHer2 did not exhibit acute toxicity in mice during short-term treatment. Overall, our findings indicate that ICG-ZHer2 is a promising bifunctional drug for Her2-positive tumor diagnosis and PTT.

Codelivery of TRAIL and Mitomycin C via Liposomes Shows Improved Antitumor Effect on TRAIL-Resistant Tumors.

Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) constitutes a promising antitumor drug, tumor resistance to TRAIL has become a major obstacle in its clinical application. Mitomycin C (MMC) is an effective TRAIL-resistant tumor sensitizer, which indicates a potential utility of combination therapy. However, the efficacy of this combination therapy is limited owing to its short half-life and the cumulative toxicity of MMC. To address these issues, we successfully developed a multifunctional liposome (MTLPs) with human TRAIL protein on the surface and MMC encapsulated in the internal aqueous phase to codeliver TRAIL and MMC. MTLPs are uniform spherical particles that exhibit efficient cellular uptake by HT-29 TRAIL-resistant tumor cells, thereby inducing a stronger killing effect compared with control groups. In vivo assays revealed that MTLPs efficiently accumulated in tumors and safely achieved 97.8% tumor suppression via the synergistic effect of TRAIL and MMC in an HT-29 tumor xenograft model while ensuring biosafety. These results suggest that the liposomal codelivery of TRAIL and MMC provides a novel approach to overcome TRAIL-resistant tumors.

Dimeric Her2-specific affibody mediated cisplatin-loaded nanoparticles for tumor enhanced chemo-radiotherapy.

BACKGROUND: Solid tumor hypoxic conditions prevent the generation of reactive oxygen species (ROS) and the formation of DNA double-strand breaks (DSBs) induced by ionizing radiation, which ultimately contributes to radiotherapy (RT) resistance. Recently, there have been significant technical advances in nanomedicine to reduce hypoxia by facilitating in situ O2 production, which in turn serves as a "radiosensitizer" to increase the sensitivity of tumor cells to ionizing radiation. However, off-target damage to the tumor-surrounding healthy tissue by high-energy radiation is often unavoidable, and tumor cells that are further away from the focal point of ionizing radiation may avoid damage. Therefore, there is an urgent need to develop an intelligent targeted nanoplatform to enable precise enhanced RT-induced DNA damage and combined therapy. RESULTS: Human epidermal growth factor receptor 2 (Her2)-specific dimeric affibody (ZHer2) mediated cisplatin-loaded mesoporous polydopamine/MnO2/polydopamine nanoparticles (Pt@mPDA/MnO2/PDA-ZHer2 NPs) for MRI and enhanced chemo-radiotherapy of Her2-positive ovarian tumors is reported. These NPs are biodegradable under a simulated tumor microenvironment, resulting in accelerated cisplatin release, as well as localized production of O2. ZHer2, produced using the E. coli expression system, endowed NPs with Her2-dependent binding ability in Her2-positive SKOV-3 cells. An in vivo MRI revealed obvious T1 contrast enhancement at the tumor site. Moreover, these NPs achieved efficient tumor homing and penetration via the efficient internalization and penetrability of ZHer2. These NPs exhibited excellent inhibition of tumor growth with X-ray irradiation. An immunofluorescence assay showed that these NPs significantly reduced the expression of HIF-1α and improved ROS levels, resulting in radiosensitization. CONCLUSIONS: The nanocarriers described in the present study integrated Her2 targeting, diagnosis and RT sensitization into a single platform, thus providing a novel approach for translational tumor theranostics.

Personalized neoantigen-pulsed dendritic cell vaccines show superior immunogenicity to neoantigen-adjuvant vaccines in mouse tumor models.

Development of personalized cancer vaccines based on neoantigens has become a new direction in cancer immunotherapy. Two forms of cancer vaccines have been widely studied: tumor-associated antigen (including proteins, peptides, or tumor lysates)-pulsed dendritic cell (DC) vaccines and protein- or peptide-adjuvant vaccines. However, different immune modalities may produce different therapeutic effects and immune responses when the same antigen is used. Therefore, it is necessary to choose a more effective neoantigen vaccination method. In this study, we compared the differences in immune and anti-tumor effects between neoantigen-pulsed DC vaccines and neoantigen-adjuvant vaccines using murine lung carcinoma (LL2) candidate neoantigens. The enzyme-linked immunospot (ELISPOT) assay showed that 4/6 of the neoantigen-adjuvant vaccines and 6/6 of the neoantigen-pulsed DC vaccines induced strong T-cell immune responses. Also, 2/6 of the neoantigen-adjuvant vaccines and 5/6 of the neoantigen-pulsed DC vaccines exhibited potent anti-tumor effects. The results indicated that the neoantigen-pulsed DC vaccines were superior to the neoantigen-adjuvant vaccines in both activating immune responses and inhibiting tumor growth. Our fundings provide an experimental basis for the selection of immune modalities for the use of neoantigens in individualized tumor immunotherapies.

CXCL16 exacerbates Pseudomonas aeruginosa keratitis by promoting neutrophil activation.

Pseudomonas aeruginosa (PA) keratitis is a major cause of blindness characterized by corneal inflammation. In a murine model of PA keratitis, we assessed the detrimental effects of CXC chemokine ligand 16 (CXCL16). Quantitative PCR (qPCR), western blotting (WB) and immunofluorescence were used to measure the expression and localization of CXCL16 and its receptor, CXC chemokine receptor 6 (CXCR6). Clinical scores, plate counting, and hematoxylin-eosin staining were used to assess infection severity and its exacerbation by CXCL16. Immunofluorescence, myeloperoxidase assays, and flow cytometry were used to detect neutrophil activity and colocalization with CXCR6. WB and immunofluorescence were used to measure levels of reactive oxygen species (ROS) and matrix metalloproteinases (MMPs). These methods also were used to measure the activation of downstream NF-κB signaling and its positive feedback on CXCL16 expression. ELISA, flow cytometry, and qPCR were used to measure the expression of CXCL2 and T helper 17 (Th17) cell-related genes. CXCL16 and CXCR6 expression was increased in infected corneas. Topical application of CXCL16 exacerbated keratitis by increasing corneal bacterial load and promoting neutrophil infiltration, whereas neutralizing antibody against CXCL16 had the opposite effect. CXCL16 also increased ROS and MMP levels. This neutrophil activation may be caused by its positive feedback with the NF-κB pathway and the upregulation of CXCL2 and Th17 cell related-genes. These data suggest that CXCL16 is an attractive therapeutic target for PA keratitis.

Regulation of the exopolysaccharide from an anamorph of Cordyceps sinensis on dendritic cell sarcoma (DCS) cell line.

PURPOSE: Cordyceps sinensis has been regarded as a precious tonic food and herbal medicine in China for thousands of years. The exopolysaccharide (EPS) from an anamorph of Cordyceps sinensis was found to have antitumor immunomodulatory activity. Mature dendritic cells play a role in initiating antitumor immunity, so we try to investigate the effects of EPS on the murine dendritic cell line DCS. METHODS: Flow cytometry was used to assay the expression levels of cell surface molecules including major histocompatibility complex (MHC)-II, CD40, CD80, and CD86 of DCS cells and their ability to take up antigens. The ability of DCS cells to activate the proliferation of CTLL-2 T cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. IL-12 and TNF-α levels were detected using ELISA. Western blotting was performed to estimate the levels of phosphorylated Janus kinase 2 (p-JAK2), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), nuclear factor-κB (NF-κB) p65 and p105. RESULTS: EPS increased the expressions of MHC-II, CD40, CD80, and CD86 of DCS cells and up-regulated their ability to take up antigens. EPS also enhanced their ability to activate the proliferation of CTLL-2 T cells. IL-12 and TNF-α secreted from DCS cells were up-regulated after EPS treatment. Furthermore, EPS significantly caused the decline of p-JAK2 and p-STAT3, significantly increased levels of NF-κB p65 in the nucleus and decreased levels of NF-κB p105 in the cytoplasm. CONCLUSIONS: EPS may induce DCS cells to exhibit mature characteristics, and the mechanism involved is probably related to the inhibition of the JAK2/STAT3 signal pathway and promotion of the NF-κB signal pathway.

Protective effect of sesaminol from Sesamum indicum Linn. against oxidative damage in PC12 cells.

Sesaminol is one component of sesame oil and has been widely used as the stabilizer to extend the storage period of food oil in China. In this study, we tried to investigate the antioxidant activity of sesaminol on rat pheochromocytoma (PC12) cells oxidative damaged by H2 O2 . Cell viability, LDH level and apoptosis of the PC12 cells were assayed after treatment with sesaminol for 3 h and exposure to H2 O2 . Furthermore, superoxide (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and intracellular ROS were assayed after exposure of the PC12 cells to H2 O2 . The results showed that pre-treatment with sesaminol prior to H2 O2 exposure significantly elevated cell survival rate and SOD, CAT and GSH-Px activity. Meanwhile, sesaminol declined the secreted LDH level, apoptosis rate and ROS level of H2 O2 exposed cells. Thus, sesaminol may protect PC12 against oxidative injury.

SLAMF7 Promotes Foam Cell Formation of Macrophage by Suppressing NR4A1 Expression During Carotid Atherosclerosis.

Macrophage-derived lipid-laden foam cells from the subendothelium play a crucial role in the initiation and progression of atherosclerosis. However, the molecule mechanism that regulates the formation of foam cells is not completely understood. Here, we found that SLAMF7 was upregulated in mice bone marrow-derived macrophages and RAW264.7 cells stimulated with oxidized low-density lipoprotein (ox-LDL). SLAMF7 promoted ox-LDL-mediated macrophage lipid accumulation and M1-type polarization. SLAMF7 deficiency reduced serum lipid levels and improved the lesions area of carotid plaque and aortic arch in high-fat diet-fed ApoE-/- mice. In response to ox-LDL, SLAMF7 downregulated NR4A1 and upregulated RUNX3 through transcriptome sequencing analysis. Overexpression NR4A1 reversed SLAMF7-induced lipid uptake and M1 polarization via inhibiting RUNX3 expression. Furthermore, RUNX3 enhanced foam cell formation and M1-type polarization. Taken together, the study suggested that SLAMF7 play contributing roles in the pro-atherogenic effects by regulating NR4A1-RUNX3.

Targeted co-delivery of resiquimod and a SIRPα variant by liposomes to activate macrophage immune responses for tumor immunotherapy.

Tumor-associated macrophages (TAMs) are the major immune cells infiltrating the tumor microenvironment (TME) and typically exhibit an immunosuppressive M2-like phenotype, which facilitates tumor growth and promotes resistance to immunotherapy. Additionally, tumor cells tend to express high levels of CD47, a "don't eat me" signal, that obstructs macrophage phagocytosis. Consequently, re-educating TAMs in combination with CD47 blockage is promising to trigger intense macrophage immune responses against tumors. As a toll-like receptor 7/8 agonist, resiquimod (R848) possesses the capacity to re-educate TAMs from M2 type to M1 type. We found that intratumoral administration of R848 synergistically improved the antitumor immunotherapeutic effect of CV1 protein (a SIRPα variant with high antagonism to CD47). However, the poor bioavailability and potential toxicity of this combo strategy remain a challenge. Here, a TAMs-targeted liposome (named: R-LS/M/CV1) co-delivering R848 and CV1 protein was constructed via decorating mannose on the liposomal surface. R-LS/M/CV1 exhibited high abilities of targeting, re-education and pro-phagocytosis of tumor cells to M2 macrophages in vitro. Intratumoral administration of R-LS/M/CV1 remarkedly eliminated tumor burden in the MC38 tumor model via repolarization of TAMs to M1 type, pro-phagocytosis of TAMs against tumors, and recruitment of tumor-infiltrating T cells. More encouragingly, due to the double targeting to TAMs and tumor cells of mannose and CV1 protein, R-LS/M/CV1 effectively accumulated at the tumor site, thereby not only remarkedly inhibiting tumors, but also exerting no hematological and histopathological toxicity when administered systemically. Our integrated strategy based on re-educating TAMs and CD47 blockade provides a promising approach to trigger macrophage immune responses against tumors for immunotherapy.

NTN4 as a prognostic marker and a hallmark for immune infiltration in breast cancer.

Netrin-4 (NTN4), a member of neurite guidance factor family, can promote neurite growth and elongation. This study aims to investigate if NTN4 correlates with prognosis and immune infiltration in breast cancer. The prognostic landscape of NTN4 and its relationship with immune infiltration in breast cancer were deciphered with public databases and immunohistochemistry (IHC) in tissue samples. The expression profiling and prognostic value of NTN4 were explored using UALCAN, TIMER, Kaplan-Meier Plotter and Prognoscan databases. Based on TIMER, relationships of NTN4 expression with tumor immune invasion and immune cell surface markers were evaluated. Transcription and survival analyses of NTN4 in breast cancer were investigated with cBioPortal database. The STRING database was explored to identify molecular functions and signaling pathways downstream of NTN4. NTN4 expression was significantly lower in invasive breast carcinoma compared with adjacent non-malignant tissues. Promoter methylation of NTN4 exhibited different patterns in breast cancer. Low expression of NTN4 was associated with poorer survival. NTN4 was significantly positively related to infiltration of CD8+ T cells, macrophages and neutrophils, whereas significantly negatively related to B cells and tumor purity. Association patterns varied with different subtypes. Various associations between NTN4 levels and immune cell surface markers were revealed. Different subtypes of breast cancer carried different genetic alterations. Mechanistically, NTN4 was involved in mediating multiple biological processes including morphogenesis and migration.

Her3-specific affibody mediated tumor targeting delivery of ICG enhanced the photothermal therapy against Her3-positive tumors.

Photothermal therapy (PTT), mediated by tumor-targeted drug delivery of indocyanine green (ICG), is a promising strategy for cancer therapy. Human epidermal growth factor receptor 3 (Her3) is highly expressed in several solid tumors and is an ideal target for tumor diagnosis and therapy. This study prepared a Her3-specific dimeric affibody (ZHer3) using an Escherichia coli expression system. The affibody could bind explicitly to Her3-positive MCF7 and LS174T cells, rather than to Her3-negative SKOV-3 cells in vitro. ICG was coupled with the ZHer3 affibody (ICG-ZHer3) through an N-hydroxysuccinimide (NHS) ester reactive group for tumor-targeted delivery. As expected, Her3-positive cells were selectively and efficiently killed by ICG-ZHer3-mediated PTT in vitro. In vivo, ICG-ZHer3 preferentially accumulated in Her3-positive LS174T tumor grafts because of the tumor-targeting ability of the ZHer3 affibody. As a result of the local generation of cytotoxic reactive oxygen species and hyperthermia, the growth rates of LS174T tumor grafts were significantly inhibited by ICG-ZHer3-mediated PTT, and ICG-ZHer3 showed good safety performance during short-term treatment. In conclusion, these results demonstrated that ICG-ZHer3 is a promising photosensitizer for PTT against Her3-positive tumors.

Fusion of an EGFR-antagonistic affibody enhances the anti-tumor effect of TRAIL to EGFR positive tumors.

Tumor necrosis factor-related apoptosis ligand (TRAIL) is a promising antitumor agent candidate for its selective proapoptotic activity to various tumor cells. However, TRAIL showed limited efficacy in clinical trials despite of good tolerability. One important reason might be attributed to the poor tumor-homing ability of TRAIL. Herein, we designed an EGFR-targeting TRAIL (Z-TRAIL) by genetically fusing an EGFR-antagonistic affibody (ZEGFR:1907) to the N-terminus of TRAIL. Z-TRAIL was produced as a soluble protein with high yield in E. coli and it maintained the trimeric state of active TRAIL. Under the EGFR-binding mediated by ZEGFR:1907, Z-TRAIL showed a âˆ¼5 to 20-fold enhancement of cytotoxicity compared to TRAIL on tumor cells in vitro. Furthermore, fusion to ZEGFR:1907 endowed TRAIL with a âˆ¼1.8-fold increase of tumor uptake and a dramatical stronger apoptosis-inducing ability in the mice bearing EGFR-overexpressing A431 tumor xenografts. More importantly, Z-TRAIL exhibited significantly enhanced antitumor efficacy against whether EGFR high-expressing or low-expressing tumors than TRAIL in vivo. In addition, repeated injection of high-dose Z-TRAIL did not show obvious acute toxicity in mice. These results demonstrated that the newly engineered Z-TRAIL might be a promising agent for targeted therapy of EGFR-expressing tumors.

Coupling EGFR-Antagonistic Affibody Enhanced Therapeutic Effects of Cisplatin Liposomes in EGFR-expressing Tumor Models.

Epidermal growth factor receptor (EGFR) is an efficient target for cancer therapy. In this study, a high-affinity EGFR-antagonistic affibody (ZEGFR) molecule coupled with cisplatin-loaded PEGylated liposomes (LS-DDP) was applied to actively target EGFR+ A431 tumor cells in vitro and in vivo. The LS-DDP coupled with ZEGFR (AS-DDP) had an average size of 140.01 ± 0.84 nm, low polydispersity, a zeta potential of -13.40 ± 0.8 mV, an acceptable encapsulation efficiency of 17.30 ± 1.35%, and released cisplatin in a slow-controlled manner. In vitro, AS-DDP demonstrated a higher amount of platinum intracellular uptake by A431 cells than LS-DDP. The IC50 value of AS-DDP (9.02 ± 1.55 µg/ml) was much lower than that of LS-DDP (16.44 ± 0.87 µg/ml), indicating that the anti-tumor effects of AS-DDP were remarkable due to the modification of ZEGFR. In vivo, the concentration of AS-DDP in the tumor site increased more than 1.76-fold, while an increase in apoptotic cells at 48 h compared to the LS-DDP was also observed, illustrating that AS-DDP possessed excellent tumor-targeting efficiency. As a result, the targeted nano-liposomes achieved greater tumor suppression. Therefore, selective targeting of LS-DDP coupled with ZEGFR enhanced the anti-tumor effects and appeared to be a promising strategy for the treatment of EGFR+ tumors.

Ex vivo stimulation of murine dendritic cells by an exopolysaccharide from one of the anamorph of Cordyceps sinensis.

Dendritic cells (DCs) play an important role in initiating antitumour immune response. Tumour progression usually induces defects in DC maturation and thus tumour-bearing hosts exhibit immunosuppression and tumour escape. The previous studies showed that an exopolysaccharide (EPS) from a fungus, one anamorph of Cordyceps sinensis, inhibited tumour growth via activating immune response in the hosts. In view of the crucial actions of DCs in antitumour immunity, the present study aims to explore the effects of EPS on murine DCs. Murine DCs were derived from the bone marrow of C57BL/6 mice, and the effects of EPS on phenotype molecules and ingestion function of DCs were assayed using flow cytometry. Cytokine expressions of DCs were assayed by reverse transcriptase-PCR. Additionally, the level of phosphorylated signal transducers and activators of transcription 3 (p-STAT3) of DCs was evaluated using Western blotting. The results showed that EPS promoted the levels of surface molecules MHC II, CD40, CD80 and CD86 of DCs and decreased their ingestion ability. The mRNA expressions of cytokines (IL-12p40 and TNF-α) and inducible nitric oxide synthase were up-regulated by EPS. We also found that EPS significantly down-regulated p-STAT3 level of DCs. The results suggested that the promotion of DC's maturation and activation by EPS is probably related to the inhibition of STAT3 phosphorylation.

Increasing the antitumor efficacy of doxorubicin liposomes with coupling an anti-EGFR affibody in EGFR-expressing tumor models.

Epidermal growth factor receptor (EGFR) is overexpressed in a wide range of solid tumors. In this study, we exploited a high-affinity EGFR-antagonistic affibody (ZEGFR) coupled to a doxorubicin loaded pegylated liposome (LS-Dox) for concurrent passive and active targeting of EGFR+ A431 tumor cells in vitro and in vivo. The in vitro studies revealed that the Dox liposomes coupled with ZEGFR (AS-Dox) showed a higher Dox uptake than LS-Dox in EGFR+ A431 cells but not in EGFR- B16F10 cells, resulting in a selectively enhanced cytotoxicity. In vivo, AS-Dox confirmed its long circulation time and efficient accumulation in tumors. This targeted chemotherapy achieved greater tumor suppression. Further, this low-dose but effective targeted treatment reduced systemic toxicity such as body weight loss and organ injury demonstrated by H&E staining. Thus, selective targeting of LS-Dox coupled with ZEGFR enhanced antitumor effects and improved systemic safety. These results demonstrated that LS-Dox coupled with ZEGFR might be developed as a potential tool for therapy of EGFR+ tumors.

Hydrogen peroxide-inactivated bacteria induces potent humoral and cellular immune responses and releases nucleic acids.

The problem of nosocomial infection is seriously escalating. Bacterial vaccines are indispensable for preventing infections caused by multi-drug resistant organisms. Some researchers have put forward the use of hydrogen peroxide (H2O2) as a new technology platform for virus deactivation. This deactivated virus can induce the number of CD8+ T lymphocytes, which can enhance antiviral responses. Although, H2O2 treatment has been rarely reported on the exploration of bacterial deactivation, H2O2 deactivation of whole-cell bacteria could be a potential novel approach for bacterial vaccine development. Here we present a strategy for H2O2-deactivated bacterial whole-cell vaccines, for two major pathgens, Pseudomonas aeruginosa and Staphylococcus aureus. The proactive effects of vaccination were assessed in vitro and in vivo. H2O2-deactivation of bacterial vaccines retains more complete epitopes and exhibits lower toxicity, as compared to formaldehyde, a conventional deactivator that was investigated in this study. Furthermore, H2O2-deactivated bacterial vaccines induce anti-infection responses through enhancement of humoral immunity and cellular immunity. Vaccination with H2O2-deactivated whole-cell bacteria in mice mainly elicits whole-cell specific antibody titers and balances the IgG2a and IgG1 response, predominantly with IgG3 induction at the later stages, meanwhile provides opsonic protection against challenge with pathogens. Finally, H2O2 deactivation of bacteria has been found to cause the release of bacterial DNA which is followed by NF-κB activation. These findings demonstrate that the deactivation of whole-cell bacteria with H2O2 is potentially advantageous for immune responses. Considering the prevention of drug-resistant infections, this deactivation method could be simultaneously applied as an innovative strategy for bacterial vaccine development.

Discovery of a self-assembling and self-adjuvant lipopeptide as a saccharide-free peptide vaccine targeting EGFRvIII positive cutaneous melanoma.

Recently, tumor immunotherapy has achieved great progress in the treatment of hematological and solid neoplasms. The DC vaccines, KLH-conjugated vaccines or glycosylated peptide vaccines can efficiently induce immune responses against tumors. In the current study, we have discovered cholesteryl PADRE-EGFRvIII epitope-conjugated lipopeptide self-assembled micelles as a potential self-adjuvant vaccine against cutaneous melanoma. The lipopeptide vaccines were synthesized using a standard solid phase peptide synthesis method, and these vaccines could elicit both a humoral and a cellular immune response to EGFRvIII positive melanoma cells. Their high humoral immunoreaction stimulation properties in combination with their cytotoxic T-cell eliciting properties provide them with potent tumor inhibitory capacity. In therapeutic and preventive xenograft models of B16-EGFRvIII melanoma cells, the self-adjuvant lipopeptide vaccine micelles efficiently prevented tumor growth as well as tumorigenesis. Our results provide a novel platform for eliciting immune responses to non-antigenic cancer-related epitopes in peptide cancer vaccine discovery and development.

Directed modification of l-LcLDH1, an l-lactate dehydrogenase from Lactobacillus casei, to improve its specific activity and catalytic efficiency towards phenylpyruvic acid.

To improve the specific activity and catalytic efficiency of l-LcLDH1, an NADH-dependent allosteric l-lactate dehydrogenase from L. casei, towards phenylpyruvic acid (PPA), its directed modification was conducted based on the semi-rational design. The three variant genes, Lcldh1Q88R, Lcldh1I229A and Lcldh1T235G, were constructed by whole-plasmid PCR as designed theoretically, and expressed in E. coli BL21(DE3), respectively. The purified mutant, l-LcLDH1Q88R or l-LcLDH1I229A, displayed the specific activity of 451.5 or 512.4 U/mg towards PPA, by which the asymmetric reduction of PPA afforded l-phenyllactic acid (PLA) with an enantiomeric excess (eep) more than 99%. Their catalytic efficiencies (kcat/Km) without d-fructose-1,6-diphosphate (d-FDP) were 4.8- and 5.2-fold that of l-LcLDH1. Additionally, the kcat/Km values of l-LcLDH1Q88R and l-LcLDH1I229A with d-FDP were 168.4- and 8.5-fold higher than those of the same enzymes without d-FDP, respectively. The analysis of catalytic mechanisms by molecular docking (MD) simulation indicated that substituting I229 in l-LcLDH1 with Ala enlarges the space of substrate-binding pocket, and that the replacement of Q88 with Arg makes the inlet of pocket larger than that of l-LcLDH1.

Induction and Amelioration of Methotrexate-Induced Gastrointestinal Toxicity are Related to Immune Response and Gut Microbiota.

As a widely used anticancer and immunosuppressive agent, methotrexate (MTX) can induce multiple adverse drug reactions (ADRs), such as gastrointestinal toxicity, the mechanisms are poorly understood. Gut microbiota has been widely reported to be associated with the onset of multiple diseases as well as treatment outcomes of different drugs. In this study, mucosal injury was observed in MTX-treated mice, leading to significant changes in macrophages (i.e., M1/M2 ratio, P < 0.05) but not in dendritic cells. Moreover, the population, diversity and principal components of the gut microbiota in mice were dramatically altered after MTX treatment in a time-dependent manner, and Bacteroidales exhibited the most distinct variation among all the taxa (P < 0.05). Bacteroides fragilis was significantly decreased with MTX treatment (P < 0.01) and tended to decrease proportionately with increasing macrophage density. Gavage of mice with B. fragilis ameliorated MTX-induced inflammatory reactions and modulate macrophage polarization. In conclusion, our results delineate a strong impact of the gut microbiota on MTX-induced intestinal mucositis and provide a potential method for the prevention of such ADRs.

Engineering a family 27 carbohydrate-binding module into an Aspergillus usamii ß-mannanase to perfect its enzymatic properties.

A family 27 carbohydrate-binding module of a Thermotoga maritima ß-mannanase (TmCBM27) was chosen from the carbohydrate-active enzyme database by computer-aided design, possessing the lowest binding free energy with mannopentaose. To improve the enzymatic properties of a glycoside hydrolase family 5 ß-mannanase from Aspergillus usamii (AuMan5A), two fusion ß-mannanases, AuMan5A-F-M and AuMan5A-R-M, were designed by fusing a TmCBM27 into its C-terminus linked with a flexible peptide F (GGGGS)3 and rigid peptide R (EAAAK)3. Two fusion enzyme genes, Auman5A-F-m and Auman5A-R-m, were constructed as designed theoretically by overlapping PCR. Then, Auman5A and two fusion genes were expressed in Pichia pastoris GS115. Three recombinant ß-mannanases, reAuMan5A, reAuMan5A-F-M and reAuMan5A-R-M, were purified to homogeneity with specific activities of 230.6, 153.3 and 241.7 U/mg. The temperature optimum of reAuMan5A-R-M was 70°C, identical with that of reAuMan5A, while its thermostability and melting temperature (Tm) reached 68°C and 74.9°C, being 8.0°C and 8.4°C higher than those of the latter, respectively. Additionally, the Km values of reAuMan5A-R-M, towards locust bean gum, konjac gum and guar gum, significantly decreased to 0.9, 1.9 and 2.5 mg/mL from 1.7, 3.8 and 4.2 mg/mL of reAuMan5A, while its kcat/Km (catalytic efficiency) values increased to 287.8, 163.7 and 84.4 mL/mg⋅s from 171.2, 97.6 and 56.0 mL/mg⋅s of the latter, respectively. These results verified that the fusion of a TmCBM27 into the C-terminus of AuMan5A mediated by (EAAAK)3 linker contributed to its improved thermostability and catalytic efficiency.