Keratose hydrogel for tissue regeneration and drug delivery.

Keratin (KRT), a natural fibrous structural protein, can be classified into two categories: "soft" cytosolic KRT that is primarily found in the epithelia tissues (e.g., skin, the inner lining of digestive tract) and "hard" KRT that is mainly found in the protective tissues (e.g., hair, horn). The latter is the predominant form of KRT widely used in biomedical research. The oxidized form of extracted KRT is exclusively denoted as keratose (KOS) while the reduced form of KRT is termed as kerateine (KRTN). KOS can be processed into various forms (e.g., hydrogel, films, fibers, and coatings) for different biomedical applications. KRT/KOS offers numerous advantages over other types of biomaterials, such as bioactivity, biocompatibility, degradability, immune/inflammatory privileges, mechanical resilience, chemical manipulability, and easy accessibility. As a result, KRT/KOS has attracted considerable attention and led to a large number of publications associated with this biomaterial over the past few decades; however, most (if not all) of the published review articles focus on KRT regarding its molecular structure, biochemical/biophysical properties, bioactivity, biocompatibility, drug/cell delivery, and in vivo transplantation, as well as its applications in biotechnical products and medical devices. Current progress that is directly associated with KOS applications in tissue regeneration and drug delivery appears an important topic that merits a commentary. To this end, the present review aims to summarize the current progress of KOS-associated biomedical applications, especially focusing on the in vitro and in vivo effects of KOS hydrogel on cultured cells and tissue regeneration following skin injury, skeletal muscle loss, peripheral nerve injury, and cardiac infarction.

Inhibiting ferroptosis in brain microvascular endothelial cells: A potential strategy to mitigate polystyrene nanoplastics‒induced blood‒brain barrier dysfunction.

Polystyrene nanoplastics (PS-NPs), a group of ubiquitous pollutants, may injure the central nervous system through the blood‒brain barrier (BBB). However, whether exposure to PS-NPs contributes to BBB disruption and the underlying mechanisms are still unclear. In vivo, we found that PS-NPs (25 mg/kg BW) could significantly increase BBB permeability in mice and downregulate the distribution of the tight junction-associated protein zona occludens 1 (ZO-1) in brain microvascular endothelial cells (BMECs). Using an in vitro BBB model, exposure to PS-NPs significantly reduced the transendothelial electrical resistance and altered ZO-1 expression and distribution in a dose-dependent manner. RNA-seq analysis and functional investigations were used to investigate the molecular pathways involved in the response to PS-NPs. The results revealed that the ferroptosis and glutathione metabolism signaling pathways were related to the disruption of the BBB model caused by the PS-NPs. PS-NPs treatment promoted ferroptosis in bEnd.3 cells by inducing disordered glutathione metabolism in addition to Fe2+ and lipid peroxide accumulation, while suppressing ferroptosis with ferrostatin-1 (Fer-1) suppressed ferroptosis-related changes in bEnd.3 cells subjected to PS-NPs. Importantly, Fer-1 alleviated the decrease in ZO-1 expression in bEnd.3 cells and the exacerbation of BBB damage induced by PS-NPs. Collectively, our findings suggest that inhibiting ferroptosis in BMECs may serve as a potential therapeutic target against BBB disruption induced by PS-NPs exposure.

In Vitro and In Vivo Inhibition of the Infectivity of Human Enterovirus 71 by a Sulfonated Food Azo Dye, Brilliant Black BN.

Hand, foot, and mouth disease (HFMD), a highly contagious disease in children, is caused by human enteroviruses, including enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and coxsackievirus A6 (CVA6). Although HFMD is usually mild and self-limiting, EV71 infection occasionally leads to fatal neurological disorders. Currently, no commercial antiviral drugs for HFMD treatment are available. Here, numerous sulfonated azo dyes, widely used as food additives, were identified as having potent antiviral activities against human enteroviruses. Among them, brilliant black BN (E151) was able to inhibit all EV71, CVA16, and CVA6 strains tested. In rhabdomyosarcoma cells, the 50% inhibitory concentrations of the dye E151 for various strains of EV71 ranged from 2.39 µM to 28.12 µM, whereas its 50% cytotoxic concentration was 1,870 µM. Food azo dyes, including E151, interacted with the vertex of the 5-fold axis of EV71 and prevented viral entry. Their efficacy in viral inhibition was regulated by amino acids at VP1-98, VP1-145, and/or VP1-246. Dye E151 not only prevented EV71 attachment but also eluted attached viruses in a concentration-dependent manner. Moreover, E151 inhibited the interaction between EV71 and its cellular uncoating factor cyclophilin A. In vivo studies demonstrated that E151 at a dose of 200 mg/kg of body weight/day given on the initial 4 days of challenge protected AG129 mice challenged with 10× the 50% lethal dose of wild-type EV71 isolates. Taken together, these data highlight E151 as a promising antiviral agent against EV71 infection.IMPORTANCE Human enterovirus 71 (EV71) is one of the causative agents of hand, foot, and mouth disease in children and is responsible for thousands of deaths in the past 20 years. Food azo dyes have been widely used since the nineteenth century; however, their biological effects on humans and microbes residing in humans are poorly understood. Here, we discovered that one of these dyes, brilliant black BN (E151), was particularly effective in inhibiting the infectivity of EV71 in both cell culture and mouse model studies. Mechanistic studies demonstrated that these sulfonated dyes mainly competed with EV71 attachment factors for viral binding to block viral attachment/entry to host cells. As no commercial antiviral drugs against EV71 are currently available, our findings open an avenue to exploit the development of permitted food dye E151 as a potential anti-EV71 agent.

Polymer microfiber meshes facilitate cardiac differentiation of c-kit(+) human cardiac stem cells.

Electrospun microfiber meshes have been shown to support the proliferation and differentiation of many types of stem cells, but the phenotypic fate of c-kit(+) human cardiac stem cells (hCSCs) have not been explored. To this end, we utilized thin (~5µm) elastomeric meshes consisting of aligned 1.7µm diameter poly (ester-urethane urea) microfibers as substrates to examine their effect on hCSC viability, morphology, proliferation, and differentiation relative to cells cultured on tissue culture polystyrene (TCPS). The results showed that cells on microfiber meshes displayed an elongated morphology aligned in the direction of fiber orientation, lower proliferation rates, but increased expressions of genes and proteins majorly associated with cardiomyocyte phenotype. The early (NK2 homeobox 5, Nkx2.5) and late (cardiac troponin I, cTnI) cardiomyocyte genes were significantly increased on meshes (Nkx=2.5 56.2±13.0, cTnl=2.9±0.56,) over TCPS (Nkx2.5=4.2±0.9, cTnl=1.6±0.5, n=9, p<0.05 for both groups) after differentiation. In contrast, expressions of smooth muscle markers, Gata6 and myosin heavy chain (SM-MHC), were decreased on meshes. Immunocytochemical analysis with cardiac antibody exhibited the similar pattern of above cardiac differentiation. We conclude that aligned microfiber meshes are suitable for guiding cardiac differentiation of hCSCs and may facilitate stem cell-based therapies for treatment of cardiac diseases.

5-Hydroxytryptamine levels in the pulmonary arterioles of broilers with induced pulmonary hypertension and its relationship to pulmonary vascular remodelling.

This experiment was performed to explore the relationship between 5-hydroxytryptamine (5-HT) levels in pulmonary arterioles and in pulmonary vascular remodelling in broilers. Pulmonary arterial hypertension was induced by injecting cellulose microparticles intravenously. Pulmonary hypertension syndrome (PHS) morbidity, right ventricle/total ventricle weight ratio (RV/TV), packed cell volume (PCV), haemoglobin concentration (HB), vessel wall area to vessel total area ratio (WA/TA) and mean tunica media thickness in pulmonary arterioles (mMTPA) were measured. Proliferating cell nuclear antigen (PCNA), argyrophilic nucleolar organizer region proteins (Ag-NORs) and 5-HT content in pulmonary arterioles were determined. The results showed that injecting cellulose microparticles intravenously in broilers could successfully increase the PHS morbidity, significantly elevate RV/TV, PCV and HB, significantly increase mMTPA and WA/TA, and significantly increase the argyrophilic particles in smooth muscle cell nucleoli, PCNA-positive cells in the medial layer, and the 5-HT content in pulmonary arterioles. Correlation analysis showed that the level of 5-HT was strongly positively correlated with PCNA and Ag-NORs. The results indicated that the increase of 5-HT in the tunica media could possibly promote the proliferation of smooth muscle cells in pulmonary arterioles and thus the occurrence of pulmonary vascular remodelling.

[Morphological changes of upper airway in patients with skeletal Class â ¢ malocclusion after bimaxillary surgery and correlation analysis].

PURPOSE: To analyze the morphological changes of the upper airway and related influencing factors in patients with skeletal Class Ⅲ malocclusion after bimaxillary surgery. METHODS: Twenty skeletal Class Ⅲ patients who underwent Le Fort I osteotomy and bilateral sagittal split ramus osteotomy(BSSRO) for maxillary advancement and mandibular setback were selected. The patients received CT scans before(T0) and 3-6 months after surgery, and the images were reconstructed three-dimensionally with Dolphin Imaging 11.9 software. Changes in the volume, cross-sectional area, and landmarks of each soft and hard tissue of the airway were measured. Statistical analysis of the data was performed using SPSS 25.0 software package. RESULTS: The volume of nasopharyngeal airway increased after operation (P＜0.05), and the volume of oropharyngeal airway decreased significantly(P＜0.01). The cross-sectional area of the airway at the plane of the second cervical vertebra was significantly decreased (P＜0.01), and the coronal and sagittal diameters were decreased(P＜0.05). The change of nasopharyngeal airway volume was moderately positively correlated with the sagittal change of the posterior nasal spine (r=0.460, P＜0.05), and the change of oropharynx and laryngopharyngeal airway volume was positively correlated with the vertical change of the midpoint of the soft palate(r=0.496, 0.696, P＜0.05). The airway cross-sectional area in the second and third cervical vertebra planes and the sagittal diameter of the airway in the third cervical vertebra plane were positively correlated with the vertical changes of the midpoint of the soft palate(r=0.474, 0.629, 0.547, P＜0.05). The change of airway cross-sectional area at the third cervical vertebra plane was moderately negatively correlated with the change of mandibular plane angle(r=-0.536, P＜0.05). CONCLUSIONS: The volume and cross-sectional area of oropharyngeal airway in skeletal Class Ⅲ patients after bimaxillary surgery will decrease. However, the total upper airway volume doesn't change significantly. The changes in the upper airway are correlated with the changes in some soft and hard tissue landmarks.

Characterization and specificity of the linear epitope of the enterovirus 71 VP2 protein.

BACKGROUND: Enterovirus 71 (EV71) has emerged as a major causative agent of hand, foot and mouth disease in the Asia-Pacific region over the last decade. Hand, foot and mouth disease can be caused by different etiological agents from the enterovirus family, mainly EV71 and coxsackieviruses, which are genetically closely related. Nevertheless, infection with EV71 may occasionally lead to high fever, neurologic complications and the emergence of a rapidly fatal syndrome of pulmonary edema associated with brainstem encephalitis. The rapid progression and high mortality of severe EV71 infection has highlighted the need for EV71-specific diagnostic and therapeutic tools. Monoclonal antibodies are urgently needed to specifically detect EV71 antigens from patient specimens early in the infection process. Furthermore, the elucidation of viral epitopes will contribute to the development of targeted therapeutics and vaccines. RESULTS: We have identified the monoclonal antibody 7C7 from a screen of hybridoma cells derived from mice immunized with the EV71-B5 strain. The linear epitope of 7C7 was mapped to amino acids 142-146 (EDSHP) of the VP2 capsid protein and was characterized in detail. Mutational analysis of the epitope showed that the aspartic acid to asparagine mutation of the EV71 subgenogroup A (BrCr strain) did not interfere with antibody recognition. In contrast, the serine to threonine mutation at position 144 of VP2, present in recently emerged EV71-C4 China strains, abolished antigenicity. Mice injected with this virus strain did not produce any antibodies against the VP2 protein. Immunofluorescence and Western blotting confirmed that 7C7 specifically recognized EV71 subgenogroups and did not cross-react to Coxsackieviruses 4, 6, 10, and 16. 7C7 was successfully used as a detection antibody in an antigen-capture ELISA assay. CONCLUSIONS: Detailed mapping showed that the VP2 protein of Enterovirus 71 contains a single, linear, non-neutralizing epitope, spanning amino acids 142-146 which are located in the VP2 protein's E-F loop. The S/T(144) mutation in this epitope confers a loss of VP2 antigenicity to some newly emerged EV71-C4 strains from China. The corresponding monoclonal antibody 7C7 was used successfully in an AC-ELISA and did not cross-react to coxsackieviruses 4, 6, 10, and 16 in immunofluorescence assay and Western blots. 7C7 is the first monoclonal antibody described, that can differentiate Coxsackievirus 16 from Enterovirus 71.

In vivo degradation forms, anti-degradation strategies, and clinical applications of therapeutic peptides in non-infectious chronic diseases.

Current medicinal treatments for diseases comprise largely of two categories: small molecular (chemical) (e.g., aspirin) and larger molecular (peptides/proteins, e.g., insulin) drugs. Whilst both types of therapeutics can effectively treat different diseases, ranging from well-understood (in view of pathogenesis and treatment) examples (e.g., flu), to less-understood chronic diseases (e.g., diabetes), classical small molecule drugs often possess significant side-effects (a major cause of drug withdrawal from market) due to their low- or non-specific targeting. By contrast, therapeutic peptides, which comprise short sequences from naturally occurring peptides/proteins, commonly demonstrate high target specificity, well-characterized modes-of-action, and low or non-toxicity in vivo. Unfortunately, due to their small size, linear permutation, and lack of tertiary structure, peptidic drugs are easily subject to rapid degradation or loss in vivo through chemical and physical routines, thus resulting in a short half-life and reduced therapeutic efficacy, a major drawback that can reduce therapeutic efficiency. However, recent studies demonstrate that the short half-life of peptidic drugs can be significantly extended by various means, including use of enantiomeric or non-natural amino acids (AAs) (e.g., L-AAs replacement with D-AAs), chemical conjugation [e.g., with polyethylene glycol], and encapsulation (e.g., in exosomes). In this context, we provide an overview of the major in vivo degradation forms of small therapeutic peptides in the plasma and anti-degradation strategies. We also update on the progress of small peptide therapeutics that are either currently in clinical trials or are being successfully used in clinical therapies for patients with non-infectious diseases, such as diabetes, multiple sclerosis, and cancer.

[Cone-beam CT measurement of morphological changes of the root and alveolar bone of the central incisor during orthodontic treatment with extraction].

PURPOSE: To study the effect of orthodontic treatment with extraction on root resorption and alveolar bone morphology of the central incisor in adult patients. METHODS: Eleven adult patients receiving orthodontic treatment were enrolled, and asked to take cone-beam CT(CBCT) scanning before and after treatment. Root resorption of the upper and lower central incisors after treatment, changes in alveolar bone thickness and height of alveolar bone were measured and compared. Statistical analysis was performed using SPSS 23.0 software package. RESULTS: The length of the tooth and root was reduced to a certain degree. The change in root length of the maxillary incisor was larger than that of the mandibular incisor. The alveolar bone width of the lingual and palatal neck of the central incisor showed some reduction, and alveolar bone width of the palatal neck of the upper central incisor and the middle lingual side of the mandibular central incisor changed to a certain extent. The width of the alveolar bone in the middle labial side of the mandibular central incisor increased, but the alveolar bone on the lingual and palatal side increased after orthodontic treatment, which was more obvious than that of the maxillary central incisor. CONCLUSIONS: Orthodontic treatment with tooth extraction is accompanied by a certain degree of root resorption of the central incisor and alveolar bone on the lingual and palatal side. However it is also accompanied by an increase in the amount of alveolar bone on the labial side. More fenestration and dehiscence are observed in the mandible.

Safety and Immunogenicity of a Stable, Cold-Adapted, Temperature-Sensitive/Conditional Lethal Enterovirus A71 in Monkey Study.

Enterovirus A71 (EV-A71) and coxsackievirus A16 (CA16) are major etiological agents of hand foot and mouth disease (HFMD) in children, which may result in fatal neurological complications. The development of safe, cost effective vaccines against HFMD, especially for use in developing countries, is still a top public health priority. We have successfully generated a stable, cold-adapted, temperature sensitive/conditional lethal EV-A71 through adaptive culturing in Vero cells at incrementally lower cultivation temperatures. An additional 40 passages at an incubation temperature of 28 °C, and a temperature reversion study at an incubation temperature of 37 °C and 39.5 °C, reveals the virus's phenotypic and genetic stability at the predefined culture conditions. Six unique mutations (two in noncoding regions and four in nonstructural protein-coding genes) in combination may have contributed to its stable phenotype and inability to fully revert to its original wild phenotype. The safety and immunogenicity of this stable, cold-adapted, temperature sensitive/conditional lethal EV-A71 was performed in six monkeys. None of the inoculated monkeys developed any obvious clinical illness except one which developed a transient spike of fever. No gross postmortem lesion or abnormal histological finding was noted for all monkeys at autopsy. No virus was reisolated although EV-A71 specific RNA was detected in serum samples collected on both day 4 and day 8 postinoculation. Only EV-A71 RNA and viral antigen were detected in the spleen homogenate and peripheral blood mononuclear cells, respectively, collected on day 4. The two remaining monkeys developed good humoral immune response on day 14 and day 30 post-inoculation.

A novel possible strategy based on self-assembly approach to achieve complete periodontal regeneration.

Limitations of current regeneration modalities underscore the importance of restoring the three-dimensional (3D) microenvironment of periodontal development, which is able to elicit the intrinsic capacity of mesenchymal stem cells to proceed to engage in a redevelopment-like program. With increased attention for the potential therapeutic applications of periodontal ligament stem cells (PDLSCs) in periodontal regeneration, it has been proposed that bone marrow mesenchymal stem cells (BMMSCs) are very likely another cell source of physiological repair of periodontal tissues. With this in mind, enlightened from the research targeting the fabrication of laminar structures such as liver and kidney with heterotypic stratification of cell sheets, we proposed a novel possible strategy based on self-assembly approach, which is akin to the physiological phenomenon that occurs during organogenesis, to enhance complete reconstruction of functional complex periodontium-organ systems. We assumed that in this strategy, using the intrinsic capacity of monodispersed cells to self-assemble into a microtissue such as a 3D spheroid, bilayered cell pellet constructs comprising calcified bone-forming cell pellets (i.e., BMMSCs) and cementum/PDL-forming cell pellets (i.e., PDLSCs) would be fabricated in vitro in a tissue-mimicking way and then implanted into periodontal defects. We hypothesize that this novel strategy might open new options to reconstruct extended periodontal defects and then achieve the ultimate goal of predictable and complete regeneration of the periodontium.

Self-assembled L-alanine derivative organogel as in situ drug delivery implant: characterization, biodegradability, and biocompatibility.

OBJECTIVE: The purpose of this work is to prepare and characterize the novel in situ forming implants, obtained through self-assembling of N-stearoyl-L-alanine methyl ester (SAM) in pharmaceutical oils, and to evaluate the biodegradability and biocompatibility of this organogel system. METHODS: Minimum gelation concentration was used to measure the gelling ability of gelator SAM in different oils to select the optimal oil for further research. Phase transition temperatures of SAM/soybean oil organogels were determined by differential scanning calorimetry. Comparative studies on the in vitro degradation and in vivo degradation of SAM/soybean oil organogels in mice were investigated. Cytotoxicity tests and histological analysis of SAM/soybean oil organogels were studied by using mouse fibrosarcoma cells and mouse, respectively. RESULTS: As an organogelator, SAM could gel a variety of oils at different minimum gelation concentration. Among them, it had the best-gelling ability in soybean oil, and the SAM/soybean oil organogel could be turned into gels abruptly at body temperature when the concentration of SAM was higher than 5% (w/v) to be used as an injectable system. The in vitro degradation rate of organogel was inversely proportional to the organogelator concentration, whereas the degradation rate in vivo was much higher than in vitro, and gels were almost disappeared after 6 weeks. The selected formulation showed excellent biocompatibility as tested by in vitro cytotoxicity and in vivo histological evaluation. CONCLUSION: SAM/soybean oil organogel has excellent biodegradability and biocompatibility, which indicates that it has a great potential for safe in situ forming drug delivery.

[Study on absorption mechanism of puerarin and its nanoparticles across Caco-2 cell model].

OBJECTIVE: To study the absorption mechanism of puerarin and its nanoparticles across Caco-2 cell model. METHODS: The Caco-2 monolayer model was used to explore the effect of time and drug concentration on the absorption of puerarin and its nanoparticeles. The drug concentration was determined by high-performance liquid chromatography and the Papp was caculated. RESULTS: The puerarin-PLGA-NP could significantly improve the absorption of puerarin. Puerarin was not only absorbed simply through passive diffusion, but also had relations with high dispersion of nanoparticles, increase of affinity and interaction with intestinal mucous membrane. CONCLUSION: The main mechanism of puerarin and its nanoparticles across Caco-2 monolayer model is passive transference. Puerarin-NP could increase the absorption of the drug in intestinal epithlial.

Effective in vivo therapeutic IgG antibody against VP3 of enterovirus 71 with receptor-competing activity.

Passive immunization is an effective option for treatment against hand, foot and mouth disease caused by EV71, especially with cross-neutralizing IgG monoclonal antibodies. In this study, an EV71-specific IgG2a antibody designated 5H7 was identified and characterized. 5H7 efficiently neutralizes the major EV71 genogroups (A, B4, C2, C4). The conformational epitope of 5H7 was mapped to the highly conserved amino acid position 74 on VP3 capsid protein using escape mutants. Neutralization with 5H7 is mediated by the inhibition of viral attachment, as revealed by virus-binding and post-attachment assays. In a competitive pull-down assay with SCARB2, 5H7 blocks the receptor-binding site on EV71 for virus neutralization. Passive immunization of chimeric 5H7 protected 100% of two-week-old AG129 mice from lethal challenge with an EV71 B4 strain for both prophylactic and therapeutic treatments. In contrast, 10D3, a previously reported neutralizing antibody that takes effect after virus attachment, could only confer prophylactic protection. These results indicate that efficient interruption of viral attachment is critical for effective therapeutic activity with 5H7. This report documents a novel universal neutralizing IgG antibody for EV71 therapeutics and reveals the underlying mechanism.

Effects of first radioiodine ablation on functions of salivary glands in patients with differentiated thyroid cancer.

The aim of this study was to evaluate the effects of the first radioactive iodine (I) therapy on functions of salivary glands in patients with differentiated thyroid carcinoma (DTC).There were 36 consented patients with DTC enrolled in this study, who received 3.7 GBq (100mCi) I for ablation after total thyroidectomy. Salivary gland function was assessed using salivary gland scintigraphy in two phases, one 4 hours before and the other 6 months after I therapy (both under thyrotropin stimulation condition). Quantitative parameters including uptake fraction (UF), uptake index (UI), excretion fraction (EF), and excretion ratio (ER) were measured and compared. Blood parameters were also compared. Associations between sex and outcome of the first I therapy as well as individual salivary gland function were measured. Wilcoxon Signed Rank Sum test and χ test were used for statistical analysis.When compared between pre-ablation and post-ablation, UF of bilateral parotid and submandibular glands were significantly increased (all P < .01). UI of both submandibular glands were significantly increased (P < .05). This seemingly increased uptake function after the first I therapy was actually compensatory mechanism of salivary gland, which indicated a possible intermediate state after radiation. But salivary glands' secretory function had not changed significantly except for left submandibular gland; we demonstrated that only left submandibular gland showed significantly decreased ER (P < .05). Thyroglobulin and thyroglobulin antibody significantly decreased after I therapy (P < .05). There were no sex differences on therapeutic outcome and salivary gland dysfunctions after the first I therapy. Salivary gland of both males and females could be affected by I therapy.The first I ablative therapy may impair the salivary uptake and secretory function of patients with DTC. There was no association between sex and salivary gland dysfunction.

[Condylar morphological changes before and after orthodontic treatment for angle Class I malocclusion adult patients].

PURPOSE: To evaluate the diagnostic sensitivity and specificity of panoramic radiography in detection of maxillary teeth roots projecting into the maxillary sinus. METHODS: Paired panoramic radiographs and cone-beam CT (CBCT) images of maxilla from 110 subjects were analyzed. 42 males and 68 females (15~36 years old) with the second molars erupted completely were included. The 2 radiographic techniques were used to observe if the roots of maxillary teeth project into the sinus, including canine, the first premolar, the second premolar, the first molar and the second molar. With CBCT as a gold standard, the diagnostic sensitivity and specificity of panoramic radiography were evaluated, as well as the accuracy, prevalence, positive likehood ratio, and negtive likehood ratio. RESULTS: The sensitivity for canine, the first premolar, the second premolar, the first molar, and the second molar was 100%, 100%, 96%, 99%, and 96%, respectively. The specificity for canine, the first premolar the second premolar, the first molar, and the second molar was 94%, 92%, 84%, 67%, and 67%, respectively. CONCLUSIONS: Panoramic radiography has a high diagnostic sensitivity in detection of the teeth roots projecting into the maxillary sinus for maxillary teeth. However, it can't afford sufficient proof for orthodontic anchorage design because of low specificity, especially for the second premolar, the first molar and the second molar.

A novel universal neutralizing monoclonal antibody against enterovirus 71 that targets the highly conserved "knob" region of VP3 protein.

Hand, foot and mouth disease caused by enterovirus 71(EV71) leads to the majority of neurological complications and death in young children. While putative inactivated vaccines are only now undergoing clinical trials, no specific treatment options exist yet. Ideally, EV71 specific intravenous immunoglobulins could be developed for targeted treatment of severe cases. To date, only a single universally neutralizing monoclonal antibody against a conserved linear epitope of VP1 has been identified. Other enteroviruses have been shown to possess major conformational neutralizing epitopes on both the VP2 and VP3 capsid proteins. Hence, we attempted to isolate such neutralizing antibodies against conformational epitopes for their potential in the treatment of infection as well as differential diagnosis and vaccine optimization. Here we describe a universal neutralizing monoclonal antibody that recognizes a conserved conformational epitope of EV71 which was mapped using escape mutants. Eight escape mutants from different subgenogroups (A, B2, B4, C2, C4) were rescued; they harbored three essential mutations either at amino acid positions 59, 62 or 67 of the VP3 protein which are all situated in the "knob" region. The escape mutant phenotype could be mimicked by incorporating these mutations into reverse genetically engineered viruses showing that P59L, A62D, A62P and E67D abolish both monoclonal antibody binding and neutralization activity. This is the first conformational neutralization epitope mapped on VP3 for EV71.

Characterization of a novel monoclonal antibody reactive against the N-terminal region of Enterovirus 71 VP1 capsid protein.

Hand, foot and mouth disease (HFMD) is a viral infectious disease caused by human Enterovirus A, particularly Enterovirus 71 (EV71) and Coxsackievirus 16 (CA16) serotypes, with EV71 infection associated with severe neurological complications and mortality. Lots of attention has been placed on elucidating viral epitopes, which is useful for EV71 viral research. In this study, a murine monoclonal antibody (mAb 4) specific for EV71 was generated and mapped to target the N-terminal region of VP1 capsid protein, spanning amino acid residues 12-19 (IGDSVSRA). mAb 4 can cross-react with all the 11 representative EV71 subgenotypes (A, B1-5, C1-5), but not with the representative strain of CA16 as demonstrated by immunofluorescence assay (IFA). BLAST analyses of this epitope against all Enterovirus entries in Genbank also demonstrated that this epitope is unique in EV71, but not other Enterovirus such as CA16 It may be useful for structural study of VP1 morphogenesis during infection and also applications for identification of EV71 infection.

Characterization of an isotype-dependent monoclonal antibody against linear neutralizing epitope effective for prophylaxis of enterovirus 71 infection.

BACKGROUND: Enterovirus 71 (EV71) is the main causative agent of Hand, Foot and Mouth disease (HFMD) and is associated with severe neurologic complications and mortalities. At present, there is no vaccine or therapeutic available for treatment. METHODOLOGY/PRINCIPAL FINDING: In this study, we generated two mAbs, denoted as mAb 51 and 53, both targeting the same linear epitope on VP1 capsid protein, spanning amino acids 215-219. In comparison, mAb 51 belonging to isotype IgM possesses neutralizing activity in vitro, whereas, mAb 53 belonging to isotype IgG1 does not have any neutralizing ability, even towards its homologous strain. When mAb 51 at 10 µg/g of body weight was administered to the 2-week-old AG129 mice one day prior to lethal challenge, 100% in vivo passive protection was observed. In contrast, the isotype control group mice, injected with an irrelevant IgM antibody before the challenge, developed limb paralysis as early as day 6 post-infection. Histological examination demonstrated that mAb 51 was able to protect against pathologic changes such as neuropil vacuolation and neuronal loss in the spinal cord, which were typical in unprotected EV-71 infected mice. BLAST analyses of that epitope revealed that it was highly conserved among all EV71 strains, but not coxsachievirus 16 (CA16). CONCLUSION: We have defined a linear epitope within the VP1 protein and demonstrated its neutralizing ability to be isotype dependent. The neutralizing property and highly conserved sequence potentiated the application of mAb 51 and 53 for protection against EV71 infection and diagnosis respectively.

Induction of protective immune responses against EV71 in mice by baculovirus encoding a novel expression cassette for capsid protein VP1.

EV71 is a major causative agent of hand, foot and mouth disease (HFMD) and is responsible for large outbreaks in various Asian Pacific countries. In the present study, we generated the recombinant baculovirus (Bac-VP1) encoding VP1 in a novel expression cassette. The transmembrane domain of hemagglutinin of the H3N2 influenza virus was included in the cassette as a minimal membrane anchor for VP1. The protective immunity of Bac-VP1 was investigated in a mouse model. The results showed that mice vaccinated with live Bac-VP1 had strong VP1 specific antibody responses. In an in vitro neutralization assay Bac-VP1 sera exhibited cross-neutralization against homologous and heterologous EV71 strains with a maximum titer of 1:512. Passive immunization studies confirmed that these sera were able to provide 100% protection against 5 MLD(50) of mouse adapted EV71 (B4 strain). This study revealed that baculovirus displaying VP1 with a HA transmembrane domain efficiently induced cross-neutralizing antibody responses in mice.