High efficacy and minimal peptide required for the anti‐angiogenic and anti‐hepatocarcinoma activities of plasminogen K5

Kringle 5(K5) is the fifth kringle domain of human plasminogen and its anti‐angiogenic activity is more potent than angiostatin that includes the first four kringle fragment of plasminogen. Our recent study demonstrated that K5 suppressed hepatocarcinoma growth by anti‐angiogenesis. To find high efficacy and minimal peptide sequence required for the anti‐angiogenic and anti‐tumour activities of K5, two deletion mutants of K5 were generated. The amino acid residues outside kringle domain of intact K5 (Pro452‐Ala542) were deleted to form K5mut1(Cys462‐Cys541). The residue Cys462 was deleted again to form K5mut2(Met463‐Cys541). K5mut1 specifically inhibited proliferation, migration and induced apoptosis of endothelial cells, with an apparent two‐fold enhanced activity than K5. Intraperitoneal injection of K5mut1 resulted in more potent tumour growth inhibition and microvessel density reduction than K5 both in HepA‐grafted and Bel7402‐xenografted hepatocarcinoma mouse models. These results suggested that K5mut1 has more potent anti‐angiogenic activity than intact K5. K5mut2, which lacks only the amino terminal cysteine of K5mut1, completely lost the activity, suggesting that the kringle domain is essential for the activity of K5. The activity was enhanced to K5mut1 level when five acidic amino acids of K5 in NH₂ terminal outside kringle domain were replaced by five serine residues (K5mut3). The shielding effect of acidic amino acids may explain why K5mut1 has higher activity. K5, K5mut1 and K5mut3 held characteristic β‐sheet spectrum while K5mut2 adopted random coil structure. These results suggest that K5mut1 with high efficacy is the minimal active peptide sequence of K5 and may have therapeutic potential in liver cancer.     

Direct Asymmetric anti‐Mannich‐Type Reactions Catalyzed by Cinchona Alkaloid Derivatives

A series of cinchona alkaloid derivatives were used to catalyze the asymmetric anti‐Mannich‐type reaction of 3‐methyl‐2‐oxindole with N‐tosyl aryl aldimines. The resulting anti‐3,3‐disubstituted 2‐oxindole products were obtained in good yields (up to 92%) with high diastereo‐ and enantioselectivities (anti/syn up to 97:3 and 91% ee). Chirality 26:801–805, 2014. © 2014 Wiley Periodicals, Inc.     

Molecular biomarker‐guided anti‐angiogenic targeted therapy for malignant glioma

Despite aggressive multimodality treatment, the prognosis of glioma, especially malignant glioma, remains very poor. After decades of effort, anti‐angiogenic therapy has become an important method of cancer treatment in addition to surgery, radiotherapy and chemotherapy. Although the performance of anti‐angiogenic therapy in colorectal cancer is good, its performance in malignant glioma remains unsatisfactory. Several phase III clinical trials showed no overall survival benefits. To solve this problem, the division of patients into groups based on their molecular biomarkers is an important step. This paper provides current insights into anti‐angiogenic drugs undergoing clinical trials and discusses the potential of molecular biomarkers to guide glioma diagnosis.     

Structural insights of Mycobacterium GTPase‐Obg and anti‐sigma‐F factor Usfx interaction

An essential protein for bacterial growth, GTPase‐Obg (Obg), is known to play an unknown but crucial role in stress response as its expression increases in Mycobacterium under stress conditions. It is well reported that Obg interacts with anti‐sigma‐F factor Usfx; however, a detailed analysis and structural characterization of their physical interaction remain undone. In view of above‐mentioned points, this study was conceptualized for performing binding analysis and structural characterization of Obg‐Usfx interaction. The binding studies were performed by surface plasmon resonance, while in silico docking analysis was done to identify crucial residues responsible for Obg‐Usfx interaction. Surface plasmon resonance results clearly suggest that N‐terminal and G domains of Obg mainly contribute to Usfx binding. Also, binding constants display strong affinity that was further evident by intermolecular hydrogen bonds and hydrophobic interactions in the predicted complex. Strong interaction between Obg and Usfx supports the view that Obg plays an important role in stress response, essentially required for Mycobacterium survival. As concluded by various studies that Obg is crucial for Mycobacterium survival under stress, this structural information may help us in designing novel and potential inhibitors against resistant Mycobacterium strains.     

Residual FDG‐PET metabolic activity in metastatic melanoma patients with prolonged response to anti‐PD‐1 therapy

18‐Fluorodeoxyglucose positron emission tomography (FDG‐PET) scans were performed on 27 patients with unresectable stage IIIC or IV melanoma after prolonged treatment with anti‐PD‐1 antibodies to examine the hypothesis that patients with prolonged response to treatment may have metabolically inactive lesions by FDG‐PET. Scans were performed at a median of 15.2 months (range 12–29 months) after starting treatment. Overall, 15 of 27 (56%) patients had a positive FDG‐PET scan. Eight patients with positive scans underwent biopsy; 5 of 8 (62%) were melanoma and 3 of 8 (38%) were immune cell infiltrates. Of the 12 patients with negative FDG‐PET scans, six had residual computerized tomography‐visible lesions, five have ceased treatment, and none have recurred with follow‐up of 6–10 months. Patients with residual metastases after a prolonged period without progression on anti‐PD‐1 therapy may have metabolically inactive lesions. Isolated metabolically active lesions in clinically well patients may reveal immune cell infiltrates rather than melanoma.     

Non‐invasive imaging of allogeneic transplanted skin graft by 131I‐anti‐TLR5 mAb

Although ¹⁸F‐fluorodeoxyglucose (¹⁸F‐FDG) uptake can be used for the non‐invasive detection and monitoring of allograft rejection by activated leucocytes, this non‐specific accumulation is easily impaired by immunosuppressants. Our aim was to evaluate a ¹³¹I‐radiolabelled anti‐Toll‐like receptor 5 (TLR5) mAb for non‐invasive in vivo graft visualization and quantification in allogeneic transplantation mice model, compared with the non‐specific radiotracer ¹⁸F‐FDG under using of immunosuppressant. Labelling, binding, and stability studies were performed. BALB/c mice transplanted with C57BL/6 skin grafts, with or without rapamycin treatment (named as allo‐treated group or allo‐rejection group), were injected with ¹³¹I‐anti‐TLR5 mAb, ¹⁸F‐FDG, or mouse isotype ¹³¹I‐IgG, respectively. Whole‐body phosphor‐autoradiography and ex vivo biodistribution studies were obtained. Whole‐body phosphor‐autoradiography showed ¹³¹I‐anti‐TLR5 mAb uptake into organs that were well perfused with blood at 1 hr and showed clear graft images from 12 hrs onwards. The ¹³¹I‐anti‐TLR5 mAb had significantly higher graft uptake and target‐to‐non‐target ratio in the allo‐treated group, as determined by semi‐quantification of phosphor‐autoradiography images; these results were consistent with ex vivo biodistribution studies. However, high ¹⁸F‐FDG uptake was not observed in the allo‐treated group. The highest allograft‐skin‐to‐native‐skin ratio (A:N) of ¹³¹I‐anti‐TLR5 mAb uptake was significantly higher than the ratio for ¹⁸F‐FDG (7.68 versus 1.16, respectively). ¹³¹I‐anti‐TLR5 mAb uptake in the grafts significantly correlated with TLR5 expression in the allograft area. The accumulation of ¹³¹I‐IgG was comparable in both groups. We conclude that radiolabelled anti‐TLR5 mAb is capable of detecting allograft with high target specificity after treatment with the immunosuppressive drug rapamycin.     

Macrophages largely contribute to heterologous anti‐Propionibacterium acnes antibody‐mediated protection from Actinobacillus pleuropneumoniae infection in mice

Actinobacillus pleuropneumoniae is the causative agent of acute and chronic pleuropneumonia. Propionibacterium acnes is a facultative anaerobic gram‐positive corynebacterium. We have previously found that anti‐P. acnes antibodies can prevent A. pleuropneumoniae infections in mice. To investigate the role of macrophages in this process, affinity‐purified anti‐P. acnes IgG and anti‐A. pleuropneumoniae IgG were used in opsonophagocytosis assays. Additionally, the efficacy of passive immunization with P. acnes serum against A. pleuropneumoniae was tested in macrophage‐depleted mice. It was found that anti‐P. acnes IgG had an effect similar to that of anti‐A. pleuropneumoniae IgG (P > 0.05), which significantly promotes phagocytosis of A. pleuropneumoniae by macrophages (P < 0.01). It was also demonstrated that, after passive immunization with anti‐P. acnes serum, macrophage‐replete mice had the highest survival rate (90%), whereas the survival rate of macrophage‐depleted mice was only 40% (P < 0.05). However, macrophage‐depleted mice that had been passively immunized with naïve serum had the lowest survival rate (20%), this rate being lower than that of macrophage‐replete mice that had been passively immunized with naïve serum. Overall, anti‐P. acnes antibodies did not prevent A. pleuropneumoniae infection under conditions of macrophage depletion (P > 0.05). Furthermore, in mice that had been passively immunized with anti‐P. acnes serum, macrophage depletion resulted in a greater A. pleuropneumoniae burden and more severe pathological features of pneumonia in lung tissues than occurred in macrophage‐replete mice. It was concluded that macrophages are essential for the process by which anti‐P. acnes antibody prevents A. pleuropneumoniae infection in mice.     

Features of anti‐inflammatory effects of modulated extremely high‐frequency electromagnetic radiation

Using a model of acute zymosan‐induced paw edema in NMRI mice, we test the hypothesis that anti‐inflammatory effects of extremely high‐frequency electromagnetic radiation (EHF EMR) can be essentially modified by application of pulse modulation with certain frequencies. It has been revealed that a single exposure of animals to continuous EHF EMR for 20 min reduced the exudative edema of inflamed paw on average by 19% at intensities of 0.1–0.7 mW/cm² and frequencies from the range of 42.2–42.6 GHz. At fixed effective carrier frequency of 42.2 GHz, the anti‐inflammatory effect of EHF EMR did not depend on modulation frequencies, that is, application of different modulation frequencies from the range of 0.03–100 Hz did not lead to considerable changes in the effect level. On the contrary, at “ineffective” carrier frequencies of 43.0 and 61.22 GHz, the use of modulation frequencies of 0.07–0.1 and 20–30 Hz has allowed us to restore the effect up to a maximal level. The results obtained show the critical dependence of anti‐inflammatory action of low‐intensity EHF EMR on carrier and modulation frequencies. Within the framework of this study, the possibility of changing the level of expected biological effect of modulated EMR by a special selection of combination of carrier and modulation frequencies is confirmed. Bioelectromagnetics 30:454–461, 2009. © 2009 Wiley‐Liss, Inc.     

Retinitis Pigmentosa: over‐expression of anti‐ageing protein Klotho in degenerating photoreceptors

Retinitis Pigmentosa involves a hereditary degeneration of photoreceptors by as yet unresolved mechanisms. The secretable protein α‐Klotho has a function related to ageing processes, and α‐Klotho‐deficient mice have reduced lifespan and declining functions in several tissues. Here, we studied Klotho in connection with inherited photoreceptor degeneration. Increased nuclear immunostaining for α‐Klotho protein was seen in degenerating photoreceptors in four different Retinitis Pigmentosa models (rd1, rd2 mice; P23H, S334ter rhodopsin mutant rats). Correspondingly, in rd1 retina α‐Klotho mRNA expression was significantly up‐regulated. Moreover, immunostaining for another Klotho family protein, β‐Klotho, also co‐localized with degenerating rd1 photoreceptors. The rd1 retina displayed reduced levels of fibroblast growth factor 15, a member of the fibroblast growth factor subfamily for which Klotho acts as a co‐receptor. Exogenous α‐Klotho protein added to retinal explant cultures did not affect cell death in rd1 retinae, but caused a severe layer disordering in wild‐type retinae. Our study suggests Klotho as a novel player in the retina, with a clear connection to photoreceptor cell death as well as with an influence on retinal organization.     

Anti‐inflammatory and anti‐endotoxin properties of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi

Antimicrobial peptides are small cationic peptides that possess a large spectrum of bioactivities, including antimicrobial, anti‐inflammatory and antioxidant activities. Several antimicrobial peptides are known to inhibit lipopolysaccharide (LPS)‐induced inflammation in vitro and to protect animals from sepsis. In this study, the cellular anti‐inflammatory and anti‐endotoxin activities of Os and Os‐C, peptides derived from the carboxy‐terminal of a tick defensin, were investigated. Both Os and Os‐C were found to bind LPS in vitro, albeit to a lesser extent than polymyxin B and melittin, known endotoxin‐binding peptides. Binding to LPS was found to reduce the bactericidal activity of Os and Os‐C against Escherichia coli confirming the affinity of both peptides for LPS. At a concentration of 25 µM, the nitric oxide (NO) scavenging activity of Os was higher than glutathione, a known NO scavenger. In contrast, Os‐C showed no scavenging activity. Os and Os‐C inhibited LPS/IFN‐γ induced NO and TNF‐α production in RAW 264.7 cells in a concentration‐dependent manner, with no cellular toxicity even at a concentration of 100 µM. Although inhibition of NO and TNF‐α secretion was more pronounced for melittin and polymyxin B, significant cytotoxicity was observed at concentrations of 1.56 µM and 25 µM for melittin and polymyxin B, respectively. In addition, Os, Os‐C and glutathione protected RAW 264.7 cells from oxidative damage at concentrations as low as 25 µM. This study identified that besides previously reported antibacterial activity of Os and Os‐C, both peptides have in addition anti‐inflammatory and anti‐endotoxin properties. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Correlation of interleukin‐6 and monocyte chemotactic protein‐1 concentrations with crescent formation and myeloperoxidase‐specific anti‐neutrophil cytoplasmic antibody titer in SCG/Kj mice by treatment with anti‐interleukin‐6 receptor antibody or mizoribine

Myeloperoxidase‐specific anti‐neutrophil cytoplasmic antibody (MPO–ANCA) is associated with rapidly progressive glomerulonephritis (RPGN) and glomerular crescent formation. Pathogenic factors in RPGN were analyzed by using SCG/Kj mice, which spontaneously develop MPO–ANCA‐associated RPGN. The serum concentration of soluble IL‐6R was determined by using ELISA and those of another 23 cytokines and chemokines by Bio‐Plex analysis. Sections of frozen kidney tissue were examined by fluorescence microscopy and the CD3⁺B220⁺ T cell subset in the spleen determined by a flow cytometry. Concentrations of IL‐6 and monocyte chemotactic protein‐1 were significantly correlated with the percentages of crescent formation. Anti‐IL‐6R antibody, which has been effective in patients with rheumatoid arthritis, was administered to SCG/Kj mice to elucidate the role of IL‐6 in the development of RPGN. MPO–ANCA titers decreased after administration of anti‐IL‐6R antibody, but not titers of mizoribine, which is effective in Kawasaki disease model mice. These results suggest that IL‐6‐mediated signaling is involved in the production of MPO–ANCA.     

Co‐encapsulation of anti‐BCR‐ABL siRNA and imatinib mesylate in transferrin receptor‐targeted sterically stabilized liposomes for chronic myeloid leukemia treatment

Chronic myeloid leukemia (CML) is triggered by the BCR‐ABL oncogene. Imatinib is the first‐line treatment of CML; however imatinib resistance and intolerance have been detected in many patients. Therefore, new therapeutic approaches are required. The present work aimed at the development and application of transferrin receptor (TrfR) targeted liposomes co‐encapsulating anti‐BCR‐ABL siRNA and imatinib at different molar ratios. The encapsulation yields and drug loading of each molecule was evaluated. Anti‐leukemia activity of the developed formulations co‐encapsulating siRNA and imatinib and of the combination of Trf‐liposomes carrying siRNA and free imatinib under two different treatment schedules of pre‐sensitization was assessed. The results obtained demonstrate that the presence of imatinib significantly decreases the encapsulation yields of siRNA, whereas imatinib encapsulation yields are increased by the presence of siRNA. Cytotoxicity assays demonstrate that the formulations co‐encapsulating siRNA and imatinib promote a 3.84‐fold reduction on the imatinib IC₅₀ (from 3.49 to 0.91 µM), whereas a 8.71‐fold reduction was observed for the pre‐sensitization protocols (from 42.7 to 4.9 nM). It was also observed that the formulations with higher siRNA to imatinib molar ratios promote higher cell toxicity. Thus, the present work describes a novel triple targeting strategy with one single system: cellular targeting (through the targeting ligand, transferrin) and molecular targeting at the BCR‐ABL mRNA and Bcr‐Abl protein level. Biotechnol. Bioeng. 2010;107: 884–893. © 2010 Wiley Periodicals, Inc.     

Chemiluminescence of non‐differentiated THP‐1 promonocytes: developing an assay for screening anti‐inflammatory milk proteins and peptides

Human leukemic THP‐1 promonocytes are widely used as a model for peripheral blood monocytes. However, superoxide production during respiratory burst (RB) of non‐differentiated THP‐1 (nd‐THP‐1) cells is very low. Here we present a rapid and low‐cost method for measuring the chemiluminescence (CL) of opsonized zymosan (OZ) induced RB which allows detection of Escherichia coli lipopolysaccharide (LPS) induced priming of nd‐THP‐1 cells on the basis of CL reaction kinetics. Maximum CL intensity obtained was 2.20 ± 0.25 and 1.30 ± 0.11 relative light units, while CL peak time was achieved at 18.1 ± 2.6 and 28.7 ± 1.3 min in primed and non‐primed cells, respectively. The priming of nd‐THP‐1 cells with LPS evoked typical TNF‐α and IL‐6 production. We tested the effects of bovine lactoferrin and protein fractions from Lactobacillus helveticus BGRA43 fermented milk for potential anti‐inflammatory effects on LPS primed nd‐THP‐1 cells. Four fractions were found to inhibit the OZ‐induced CL in a dose‐dependent manner (IC₅₀ 3–30 µg/mL), while lactoferrin inhibited CL to a lesser extent (IC₅₀ 270 µg/mL). These results suggest that measuring CL response of nd‐THP‐1 cells can serve as a method for screening anti‐inflammatory compounds which could be used in reducing the risk of phagocyte‐mediated inflammatory diseases. Copyright © 2010 John Wiley & Sons, Ltd.     

Losing anti‐predatory behaviour: A cost of translocation

Translocation of endangered species to habitats where exotic predators have been removed is now a common conservation practice around the world. Many of these translocated populations have thrived, and they are often used as sources for the harvesting of individuals for translocations to sites where exotic predators still exist, albeit at reduced densities. This study investigates how isolation from exotic predators affects the ability of individuals to recognize such predators using the North Island robin (Petroica longipes) as a model. The study was carried out in three robin populations in the North Island, New Zealand: a translocated population on Tiritiri Matangi Island, where exotic mammalian predators are absent; a population reintroduced from Tiritiri Matangi Island to Wenderholm Regional Park, a mainland site where these mammals are controlled to low densities; and a mainland population at Benneydale where exotic predatory mammals are common. The response intensity of robins to a model stoat was high at Benneydale and low at Tiritiri Matangi and Wenderholm. This result indicates that isolation from mammalian predators on Tiritiri Matangi has suppressed the ability of North Island robins to recognize these predators. It is possible that the low predatory mammal densities at Wenderholm have reduced robin contact with stoats, therefore reduced the opportunity for robins to learn to recognize stoats. Thus, translocation of individuals from populations without predators to places where key predators still exist could be unsuccessful if translocated individuals fail to perform appropriate anti‐predator behaviours.     

A novel human anti‐VCAM‐1 monoclonal antibody ameliorates airway inflammation and remodelling

Asthma is a chronic inflammatory disease induced by Type 2 helper T cells and eosinophils. Vascular cell adhesion molecule‐1 (VCAM‐1) has been implicated in recruiting eosinophils and lymphocytes to pathological sites in asthma as a regulatory receptor. Accordingly, monoclonal antibody (mAb) against VCAM‐1 may attenuate allergic inflammation and pathophysiological features of asthma. We attempted to evaluate whether a recently developed human anti‐VCAM‐1 mAb can inhibit the pathophysiological features of asthma in a murine asthma model induced by ovalbumin (OVA). Leucocyte adhesion inhibition assay was performed to evaluate the in vitro blocking activity of human anti‐VCAM‐1 mAb. OVA‐sensitized BALB/c mice were treated with human anti‐VCAM‐1 mAb or isotype control Ab before intranasal OVA challenge. We evaluated airway hyperresponsiveness (AHR) and bronchoalveolar lavage fluid analysis, measured inflammatory cytokines and examined histopathological features. The human anti‐VCAM‐1 mAb bound to human and mouse VCAM‐1 molecules and inhibited adhesion of human leucocytes in vitro. AHR and inflammatory cell counts in bronchoalveolar lavage fluid were reduced in mice treated with human anti‐VCAM‐1 mAb as compared with a control Ab. The levels of interleukin (IL)‐5 and IL‐13, as well as transforming growth factor‐β, in lung tissue were decreased in treated mice. Human anti‐VCAM‐1 mAb reduced goblet cell hyperplasia and peribronchial fibrosis. In vivo VCAM‐1 expression decreased in the treated group. In conclusion, human anti‐VCAM‐1 mAb attenuated allergic inflammation and the pathophysiological features of asthma in OVA‐induced murine asthma model. The results suggested that human anti‐VCAM‐1 mAb could potentially be used as an additional anti‐asthma therapeutic medicine.     

Metformin's antitumour and anti‐angiogenic activities are mediated by skewing macrophage polarization

Beneficial effects of metformin on cancer risk and mortality have been proved by epidemiological and clinical studies, thus attracting research interest in elucidating the underlying mechanisms. Recently, tumour‐associated macrophages (TAMs) appeared to be implicated in metformin‐induced antitumour activities. However, how metformin inhibits TAMs‐induced tumour progression remains ill‐defined. Here, we report that metformin‐induced antitumour and anti‐angiogenic activities were not or only partially contributed by its direct inhibition of functions of tumour and endothelial cells. By skewing TAM polarization from M2‐ to M1‐like phenotype, metformin inhibited both tumour growth and angiogenesis. Depletion of TAMs by clodronate liposomes eliminated M2‐TAMs‐induced angiogenic promotion, while also abrogating M1‐TAMs‐mediated anti‐angiogenesis, thus promoting angiogenesis in tumours from metformin treatment mice. Further in vitro experiments using TAMs‐conditioned medium and a coculture system were performed, which demonstrated an inhibitory effect of metformin on endothelial sprouting and tumour cell proliferation promoted by M2‐polarized RAW264.7 macrophages. Based on these results, metformin‐induced inhibition of tumour growth and angiogenesis is greatly contributed by skewing of TAMs polarization in microenvironment, thus offering therapeutic opportunities for metformin in cancer treatment.