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The Astragalus membranaceus polysaccharides (APS) can improve immunity and enhance treatment reactions. This study analyzed the effects of effective antivascular endothelial growth factor (anti-VEGF) antibody production in mice treated with APS. After APS treatment, the serum of mice produced the antibody reactions that can cross-validate VEGF. The isolated single-chain fragment variable (scFv) antibodies could neutralize VEGF and inhibit in vivo tumor growth. Of the scFvs, scFv 4E can significantly compete the interaction of bevacizumab with VEGF. In cell experiments, scFv 4E effectively inhibited human umbilical vein endothelial cells induced by VEGF in vitro. In a matrix gel-assisted angiogenesis model, scFv 4E significantly inhibited angiogenesis reactions. In addition, in a xenograft model established in the colorectal cancer cell strain HCT116, scFv 4E treatment inhibited tumor growth by up to 52.7%. Finally, molecule docking was performed to simulate the complex interactions of scFv 4E and VEGF, the main driving forces of which involve the hydrophobic interactions and hydrogen bonds of Tyr108 and Tyr 109 of the complementarity-determining region H3 loop with VEGF. The results help in establishing antibody library with high diversity for selecting antibodies with specificity. In addition, this study indirectly expounded the correlations of APS enhancing immunity regulation in vivo.

This study constructs a simultaneous confidence region for two combinations of coefficients of linear models and their ratios based on the concept of generalized pivotal quantities. Many biological studies, such as those on genetics, assessment of drug effectiveness, and health economics, are interested in a comparison of several dose groups with a placebo group and the group ratios. The Bonferroni correction and the plug-in method based on the multivariate-t distribution have been proposed for the simultaneous region estimation. However, the two methods are asymptotic procedures, and their performance in finite sample sizes has not been thoroughly investigated. Based on the concept of generalized pivotal quantity, we propose a Bonferroni correction procedure and a generalized variable (GV) procedure to construct the simultaneous confidence regions. To address a genetic concern of the dominance ratio, we conduct a simulation study to empirically investigate the probability coverage and expected le...

The human cluster of differentiation 19 (CD19) is highly expressed in most leukemia, rendering is a promising therapeutic target. In this study, we generated anti-CD19 single-chain variable fragments (scFv) from immunized chickens by phage display technology. After constructing a scFv antibody library with 2.5 × 10

To understand the mechanism for inhibition of hepatitis B virus (HBV) infection is important. In this study, single-chain variable fragment (scFv) antibodies were generated and directed to the pre-S2 epitope of HBV surface antigen (HBsAg). These human scFvs were isolated from a person with history of HBV infection by phage display technology. An evaluation of panning efficiency revealed that the eluted phage titer was increased, indicating that specific clones were enriched after panning. Selected scFvs were characterized with the recombinant HBsAg through Western blotting and enzyme-linked immunosorbent assay to confirm the binding ability. Flow cytometry analysis and immunocytochemical staining revealed that one scFv, S17, could recognize endogenous HBsAg expressed on the HepG2215 cell membrane. Moreover, the binding affinity of scFv S17 to the pre-S2 epitope was determined to be 4.2 × 10-8 M. Two ion interactions were observed as the major driving forces for scFv S17 interacting with pre-S2 by performing a rational molecular docking analysis. This study provides insights into the structural basis to understand the interactions between an antibody and the pre-S2 epitope. The functional scFv format can potentially be used in future immunotherapeutic applications.

Iloprost, a stable prostaglandin I2 (PGI2) analog, can inhibit allergic inflammation in an ovalbumin (OVA)-induced asthma model via inhibition of airway dendritic cell (DC) function. However, the underlying mechanism of PGI2 signaling-mediated immunosuppression remains unclear. This study explored whether iloprost-treated DCs can suppress inflammation by promoting antigen-specific regulatory T cell (Treg) differentiation through PGI2-G-protein-coupled receptor (IP). We established an allergic lung inflammation model using a hydrogel biomaterial delivery system and observed that iloprost significantly suppressed OVA-induced Th2 lung inflammation and increased the frequency of OVA-specific Tregs in vivo. We further observed that iloprost-treated DCs displayed tolerogenic characteristics, including low inflammatory cytokine (IL-12, TNF-α, IL-6, IL-23) expression levels, high anti-inflammatory cytokine (IL-10) production, and a semimature phenotype. In addition, iloprost-treated DCs increased OVA-specific CD4+Foxp3+ T cell differentiation from naive T cells in an IP-dependent pathway in vitro and in vivo. Blocking experiments showed that iloprost-treated DCs promoted Treg differentiation, at least in part, through programmed death ligand 1 (PD-L1), whereas iloprost-induced PD-L1 expression in DCs was through the IP receptor. Furthermore, iloprost treatment suppressed DC-mediated airway inflammation and increased the frequency of OVA-specific Tregs through PD-L1 in vivo. Taken together, these results show that PGI2-IP signaling mediated by iloprost in DCs may lead to immune tolerance, suggesting that the PGI2 analog has the potential to be applied therapeutically for tolerogenic DC immunotherapy in autoimmune diseases or allergic asthma.

Chondrocytes (CH) and bone marrow stem cells (BMSCs) are sources that can be used in cartilage tissue engineering. Co-culture of CHs and BMSCs is a promising strategy for promoting chondrogenic differentiation. In this study, articular CHs and BMSCs were encapsulated in PCL-PEG-PCL photocrosslinked hydrogels for 4 weeks. Various ratios of CH:BMSC co-cultures were investigated to identify the optimal ratio for cartilage formation. The results thus obtained revealed that co-culturing CHs and BMSCs in hydrogels provides an appropriate in vitro microenvironment for chondrogenic differentiation and cartilage matrix production. Co-culture with a 1:4 CH:BMSC ratio significantly increased the synthesis of GAGs and collagen. In vivo cartilage regeneration was evaluated using a co-culture system in rabbit models. The co-culture system exhibited a hyaline chondrocyte phenotype with excellent regeneration, resembling the morphology of native cartilage. This finding suggests that the co-culture of these two cell types promotes cartilage regeneration and that the system, including the hydrogel scaffold, has potential in cartilage tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.

Photopolymerization techniques have been widely used to create hydrogels for biomedical applications. Visible light-based photopolymerizations are commonly initiated by type II (i.e., noncleavage-type) photoinitiator in conjunction with a coinitiator. On the other hand, type I photoinitiators (i.e., cleavage type) are rarely compatible with visible light-based initiation due to their limited molar absorbability in the visible light wavelengths. Here, we report visible light initiated orthogonal photoclick crosslinking to fabricate gelatin-norbornene and poly(ethylene glycol)-tetra-thiol hydrogels using either cleavage-type (i.e., lithium acylphosphinate, LAP) or noncleavage-type photoinitiator (i.e., eosin-Y, EY) without the use of a coinitiator. Regardless of the initiator type, the step-growth gelatin-PEG hybrid hydrogels crosslinked and degraded similarly. While both systems exhibited similar cytocompatibility for hepatic progenitor HepaRG cells, gelation initiated by noncleavage-type initiator EY afforded slightly higher degree of hepatic gene expression. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 44585.

Chikungunya virus (CHIKV) is an alphavirus that has emerged as a global health burden. There are 3 CHIKV genotypes: Asian, West African, and Eastern/Central/South African. No licensed CHIKV vaccine is available, and whether the antibody response elicited by one genotype can neutralize heterologous genotypes is unclear. We assessed neutralizing antibody (NAb) responses of volunteers in a phase 1 study of a CHIKV vaccine against 9 viral strains representing all 3 genotypes. Minimal differences in vaccine-elicited NAb responses were observed among genotypes, suggesting that vaccination with a single CHIKV strain can elicit cross-protective NAbs against all 3 genotypes.

Cellular messenger RNA (mRNA) of higher eukaryotes and many viral RNAs are methylated at the N-7 and 2'-O positions of the 5' guanosine cap by specific nuclear and cytoplasmic methyltransferases (MTases), respectively. Whereas N-7 methylation is essential for RNA translation and stability, the function of 2'-O methylation has remained uncertain since its discovery 35 years ago. Here we show that a West Nile virus (WNV) mutant (E218A) that lacks 2'-O MTase activity was attenuated in wild-type primary cells and mice but was pathogenic in the absence of type I interferon (IFN) signalling. 2'-O methylation of viral RNA did not affect IFN induction in WNV-infected fibroblasts but instead modulated the antiviral effects of IFN-induced proteins with tetratricopeptide repeats (IFIT), which are interferon-stimulated genes (ISGs) implicated in regulation of protein translation. Poxvirus and coronavirus mutants that lacked 2'-O MTase activity similarly showed enhanced sensitivity to the antiviral actions of IFN and, specifically, IFIT proteins. Our results demonstrate that the 2'-O methylation of the 5' cap of viral RNA functions to subvert innate host antiviral responses through escape of IFIT-mediated suppression, and suggest an evolutionary explanation for 2'-O methylation of cellular mRNA: to distinguish self from non-self RNA. Differential methylation of cytoplasmic RNA probably serves as an example for pattern recognition and restriction of propagation of foreign viral RNA in host cells.

Summary An intact complement system is crucial for limiting West Nile virus (WNV) dissemination. Herein, we define how complement directly restricts flavivirus infection in an antibody-independent fashion. Mannose-binding lectin (MBL) recognized N-linked glycans on the structural proteins of WNV and Dengue virus (DENV), resulting in neutralization through a C3- and C4-dependent mechanism that utilized both the canonical and bypass lectin activation pathways. For WNV, neutralization occurred with virus produced in insect cells, whereas for DENV, neutralization of insect and mammalian cell-derived virus was observed. Mechanism of action studies suggested that the MBL-dependent neutralization occurred, in part, by blocking viral fusion. Experiments in mice showed an MBL-dependent accelerated intravascular clearance of DENV or a WNV mutant with two N-linked glycans on its E protein, but not with wild-type WNV. Our studies show that MBL recognizes terminal mannose-containing carbohydrates on flaviviruses, resulting in neutralization and efficient clearance in vivo.

This study evaluates the sustained analgesic effect of ketorolac-eluting thermosensitive biodegradable hydrogel in the plantar incisional pain model of the rat hind-paw. A ketorolac-embedded 2, 2'-Bis (2-oxazolin) (BOX) linking methoxy-poly(ethylene glycol) and poly(lactide-co-glycolide) (mPEG-PLGA) diblock copolymer (BOX copolymer) was synthesized as keto-hydrogel based on optimal sol-gel phase transition and in vitro drug release profile. The effect of keto-hydrogel on postoperative pain (POP) was assessed using the established plantar incisional pain model in hind-paw of rats and compared to that of ketorolac solution. Pain and sensory threshold, as well as pain scoring, were evaluated with behavioral tests by means of anesthesiometer and incapacitance apparatus, respectively. Pro-inflammatory cytokine levels (TNF-α, IL-6, VEGF, and IL-1β) around incisional wounds were measured by ELISA. Tissue histology was assessed using hematoxylin and eosin and Masson's trichrome staining. Ten mg/mL (25 wt%) keto-hydrogel showed a sol-gel transition at 26.4°C with a 10-day sustained drug release profile in vitro. Compared to ketorolac solution group, the concentration of ketorolac in tissue fluid was higher in the keto-hydrogel group during the first 18 h of application. Keto-hydrogel elevated pain and sensory threshold, increased weight-bearing capacity, and significantly reduced the levels of TNF-α, IL-6, and IL-1β while enhanced VEGF in tissue fluid. Histologic analysis reveals greater epithelialization and collagen deposition around wound treated with keto-hydrogel. In conclusion, our study suggests that keto-hydrogel is an ideal compound to treat POP with a secondary gain of improved incisional wound healing.

Summary The IFITMs inhibit influenza A virus (IAV) replication in vitro and in vivo. Here, we establish that the antimycotic heptaen, amphotericin B (AmphoB), prevents IFITM3-mediated restriction of IAV, thereby increasing viral replication. Consistent with its neutralization of IFITM3, a clinical preparation of AmphoB, AmBisome, reduces the majority of interferon’s protective effect against IAV in vitro. Mechanistic studies reveal that IFITM1 decreases host-membrane fluidity, suggesting both a possible mechanism for IFITM-mediated restriction and its negation by AmphoB. Notably, we reveal that mice treated with AmBisome succumbed to a normally mild IAV infection, similar to animals deficient in Ifitm3. Therefore, patients receiving antifungal therapy with clinical preparations of AmphoB may be functionally immunocompromised and thus more vulnerable to influenza, as well as other IFITM3-restricted viral infections., Graphical Abstract, Highlights • Amphotericin B or AmBisome prevents IFITM3-mediated restriction of IAV • AmBisome overcomes the majority of IFN’s antiviral effects in vitro • IFITM1 decreases membrane fluidity and inhibits membrane fusion • AmBisome increases the morbidity and mortality of influenza, IFITM3 is a ubiquitously expressed antiviral protein that inhibits multiple human pathogenic viruses, including influenza A virus (IAV). Brass and colleagues now show that a widely used antifungal therapy, AmBisome, prevents IFITM3 from blocking IAV replication and that mice given AmBisome succumb to a normally mild influenza virus infection. Therefore, patients receiving antifungal therapy with AmBisome may be functionally immunocompromised and thus more vulnerable to influenza as well as other IFITM3-restricted viral infections.

Background The capsid (CA) protein of HIV-1 binds with high affinity to the host protein cyclophilin A (CypA). This binding positively affects some early stage of the viral life-cycle because prevention of binding either by drugs that occupy that active site of cyclophilin A, by mutation in HIV-1 CA, or RNAi that knocks down intracellular CypA level diminishes viral infectivity. The closely related lentivirus, SIVcpz also binds CypA, but it was thought that this interaction was limited to the HIV-1/SIVcpz lineage because other retroviruses failed to interact with CypA in a yeast two-hybrid assay. Results We find that diverse lentiviruses, FIV and SIVagmTAN also bind to CypA. Mutagenesis of FIV CA showed that an amino acid that is in a homologous position to the proline at amino acid 90 of HIV-1 CA is essential for FIV interactions with CypA. Conclusion These results demonstrate that CypA binding to lentiviruses is more widespread than previously thought and suggest that this interaction is evolutionarily important for lentiviral infection.

Background Reconstruction of a segmental fracture with massive bone loss is still a challenge for orthopaedic surgeons. The aim of our study was to develop a suitable biodegradable thermosensitive hydrogel system as a carrier for bone morphogenetic protein (BMP)-2 delivery in the treatment of critical-sized femoral defects. Methods A block copolymer composed of monomethoxypoly(ethylene glycol) (mPEG), poly(lactic-co-glycolic acid) (PLGA) and 2, 2’-Bis (2-oxazolin) (Box) was synthesized by ring opening polymerization. The synthesized block copolymer was characterized by 1H-NMR spectroscopy and gel permeation chromatography (GPC). Different biophysical and biochemical properties of the synthesized copolymer, including temperature-induced structure changes, degradation rate, pH changes during hydrolytic degradation, cell toxicity, and the release profile of BMP-2, were also evaluated and/or were compared with those of a well-characterized mPEG-PLGA copolymer. In animal testing, rabbits (n = 36) that received critically sized (10 mm) femoral defects were divided into 6 groups. These experimental groups included an untreated group, autograft, and groups treated with the synthesized copolymer carrying different concentrations of BMP-2 (0, 5, 10, and 20 μg/ml). Bone repair was evaluated using X-ray radiography, histological staining, micro-computed tomography (μCT), biomarker examination and biomechanical testing in a 12-week treatment period. Results A new thermosensitive mPEG-PLGA/Box/mPEG-PLGA block copolymer, or named as BOX copolymer, was successfully prepared. Compared to the reported mPEG-PLGA in vitro, the prepared BOX copolymer at the same weight percent concentrations exhibited wider temperature ranges of gelation, slower degradation rates, higher the pH values, as well as less cytotoxicity. Furthermore, the BMP-2 release from BOX hydrogel exhibited a near-linear release profile in vitro. In animal experiments, treatment of critical-sized bony defects with 25 wt% BOX hydrogel carrying BMP-2 effectively promoted fracture healing during the 12-week trial period and higher concentrations of BMP-2 treatment correlated with better bone quality. Most importantly, clinical outcome and bone healing in the BOX-hydrogel group with 20 μg/ml BMP-2 were nearly equivalent to those in the autograft group in a 12-week treatment course. Conclusion These data support that the use of BOX hydrogel (25 wt%) as a drug delivery system is a promising method in the treatment of large bone defects.