Tumor-Targeting Ability of Novel Anti-Prostate-Specific Membrane Antigen Antibodies.

Patients with prostate-specific membrane antigen (PSMA)-positive tumors can benefit from PSMA-targeted therapy; thus, we have constructed a phage-displayed synthetic antibody library for the production of novel PSMA antibodies with superior PSMA-targeting ability, favoring clinical management. The binding affinities of anti-PSMA antibodies were verified by an enzyme-linked immunosorbent assay (ELISA). Several in vitro and in vivo experiments, including cellular uptake, internalization, and cytotoxicity studies, micro single photon emission computed tomography (microSPECT)/CT, and biodistribution studies, were performed to select the most promising antibody among six different antibodies. The results showed the target affinities of our antibodies in the ELISA assays (7A, 8C, 8E, and 11A) were comparable to the existing antibodies (J591). The half-maximal effective concentrations of 7A, 8C, 8E, 11A, and J591 were 2.95, 6.64, 5.50, 2.08, and 4.79, respectively. The radiochemical yield of 111In-labeled antibodies ranged from 30% to 50% with high radiochemical purity (>90%). In the cellular uptake studies, the accumulated radioactivity of 111In-J591, 111In-7A, and 111In-11A increased over time. The internalized percentage of 111In-11A was the highest (32.14% ± 2.06%) at 48 h after incubation, whereas that of 111In-J591 peaked at 22.43% ± 4.38% at 24 h and dropped to 13.52% ± 3.03% at 48 h postincubation. Twenty-four hours after injection, radioactivity accumulation appeared in the LNCaP xenografts of the mice injected with 111In-11A, 111In-8E, 111In-7A, and 111In-J591 but not in the xenografts of the 111In-8C-injected group. Marked liver uptake was noticed in all groups except the 111In-11A-injected group. Moreover, the killing effect of 177Lu-11A was superior to that of 177Lu-J591 at low concentrations. In conclusion, we successfully demonstrated that 11A IgG owned the most optimal biological characteristics among several new anti-PSMA antibodies and it can be an excellent PSMA-targeting component for the clinical use.

Eradicating mesothelin-positive human gastric and pancreatic tumors in xenograft models with optimized anti-mesothelin antibody-drug conjugates from synthetic antibody libraries.

Mesothelin (MSLN) is an attractive candidate of targeted therapy for several cancers, and hence there are increasing needs to develop MSLN-targeting strategies for cancer therapeutics. Antibody-drug conjugates (ADCs) targeting MSLN have been demonstrated to be a viable strategy in treating MSLN-positive cancers. However, developing antibodies as targeting modules in ADCs for toxic payload delivery to the tumor site but not to normal tissues is not a straightforward task with many potential hurdles. In this work, we established a high throughput engineering platform to develop and optimize anti-MSLN ADCs by characterizing more than 300 scFv CDR-variants and more than 50 IgG CDR-variants of a parent anti-MSLN antibody as candidates for ADCs. The results indicate that only a small portion of the complementarity determining region (CDR) residues are indispensable in the MSLN-specific targeting. Also, the enhancement of the hydrophilicity of the rest of the CDR residues could drastically increase the overall solubility of the optimized anti-MSLN antibodies, and thus substantially improve the efficacies of the ADCs in treating human gastric and pancreatic tumor xenograft models in mice. We demonstrated that the in vivo treatments with the optimized ADCs resulted in almost complete eradication of the xenograft tumors at the treatment endpoints, without detectable off-target toxicity because of the ADCs' high specificity targeting the cell surface tumor-associated MSLN. The technological platform can be applied to optimize the antibody sequences for more effective targeting modules of ADCs, even when the candidate antibodies are not necessarily feasible for the ADC development due to the antibodies' inferior solubility or affinity/specificity to the target antigen.

Structure-based Development of Human Interleukin-1ß-Specific Antibody That Simultaneously Inhibits Binding to Both IL-1RI and IL-1RAcP.

Interleukin-1ß (IL-1ß) is a potent pleiotropic cytokine playing a central role in protecting cells from microbial pathogen infection or endogenous stress. After it binds to IL-1RI and recruits IL-1 receptor accessory protein (IL-1RAcP), signaling culminates in activation of NF-κB. Many pathophysiological diseases have been attributed to the derailment of IL-1ß regulation. Several blocking reagents have been developed based on two mechanisms: blocking the binding of IL-1ß to IL-1RI or inhibiting the recruitment of IL-1RAcP to the IL-1ß initial complex. In order to simultaneously fulfill these two actions, a human anti-IL-1ß neutralizing antibody IgG26 was screened from human genetic phage-display library and furthered structure-optimized to final version, IgG26AW. IgG26AW has a sub-nanomolar binding affinity for human IL-1ß. We validated IgG26AW-neutralizing antibodies specific for IL-1ß in vivo to prevent human IL-1ß-driving IL-6 elevation in C56BL/6 mice. Mice underwent treatments with IgG26AW in A549 and MDA-MB-231 xenograft mouse cancer models have also been observed with tumor shrank and inhibition of tumor metastasis. The region where IgG26 binds to IL-1ß also overlaps with the position where IL-1RI and IL-1RAcP bind, as revealed by the 26-Fab/IL-1ß complex structure. Meanwhile, SPR experiments showed that IL-1ß bound by IgG26AW prevented the further binding of IL-1RI and IL-1RAcP, which confirmed our inference from the result of protein structure. Therefore, the inhibitory mechanism of IgG26AW is to block the assembly of the IL-1ß/IL-1RI/IL-1RAcP ternary complex which further inhibits downstream signaling. Based on its high affinity, high neutralizing potency, and novel binding epitope simultaneously occupying both IL-1RI and IL-1RAcP residues that bind to IL-1ß, IgG26AW may be a new candidate for treatments of inflammation-related diseases or for complementary treatments of cancers in which the role of IL-1ß is critical to pathogenesis.

Preparation and characterization of antibody-drug conjugates acting on HER2-positive cancer cells.

Two systems of antibody-drug conjugates (ADCs), noncleavable H32-DM1 and cleavable H32-VCMMAE, were developed by using different linkers and drugs attached to the anti-HER2 antibody H32, which is capable of cell internalization. Activated functional groups, including an N-hydroxysuccinimidyl (NHS) ester and a maleimide, were utilized to make the ADCs. Mass spectrometry, hydrophobic interaction chromatography, polyacrylamide gel electrophoresis, and in vitro cell assays were performed to analyze and optimize the ADCs. Several H32-VCMMAE ADCs were established with higher DARs and greater synthetic yields without compromising potency. The anticancer efficacy of H32-DM1 was 2- to 8-fold greater than that of Kadcyla®. The efficacy of H32-VCMMAE was in turn better than that of H32-DM1. The anticancer efficacy of these ADCs against N87, SK-BR-3 and BT474 cells was in the following order: H32-VCMMAE series > H32-DM1 series > Kadcyla®. The optimal DAR for H32-VCMMAE was found to be 6.6, with desirable attributes including good cell penetration, a releasable payload in cancer cells, and high potency. Our results demonstrated the potential of H32-VCMMAE as a good ADC candidate.

A panel of anti-influenza virus nucleoprotein antibodies selected from phage-displayed synthetic antibody libraries with rapid diagnostic capability to distinguish diverse influenza virus subtypes.

Immunoassays based on sandwich immuno-complexes of capture and detection antibodies simultaneously binding to the target analytes have been powerful technologies in molecular analyses. Recent developments in single molecule detection technologies enable the detection limit of the sandwich immunoassays approaching femtomolar (10-15 M), driving the needs of developing sensitive and specific antibodies for ever-increasingly broad applications in detecting and quantifying biomarkers. The key components underlying the sandwich immunoassays are antibody-based affinity reagents, for which the conventional sources are mono- or poly-clonal antibodies from immunized animals. The downsides of the animal-based antibodies as affinity reagents arise from the requirement of months of development timespan and limited choices of antibody candidates due to immunodominance of humoral immune responses in animals. Hence, developing animal antibodies capable of distinguishing highly related antigens could be challenging. To overcome the limitation imposed by the animal immune systems, we developed an in vitro methodology based on phage-displayed synthetic antibody libraries for diverse antibodies as affinity reagents against closely related influenza virus nucleoprotein (NP) subtypes, aiming to differentiating avian influenza virus (H5N1) from seasonal influenza viruses (H1N1 and H3N2), for which the NPs are closely related by 90-94% in terms of pairwise amino acid sequence identity. We applied the methodology to attain, within four weeks, a panel of IgGs with distinguishable specificities against a group of representative NPs with pairwise amino acid sequence identities up to more than 90%, and the antibodies derived from the antibody libraries without further affinity refinement had comparable affinity of mouse antibodies to the NPs with the detection limit less than 1 nM of viral NP from lysed virus with sandwich ELISA. The panel of IgGs were capable of rapidly distinguishing infections due to virulent avian influenza virus from infections of seasonal flu, in responding to a probable emergency scenario where avian influenza virus would be transmissible among humans overlapping with the seasonal influenza infections. The results indicate that the in vitro antibody development methodology enables developing diagnostic antibodies that would not otherwise be available from animal-based antibody technologies.

Development of theranostic active-targeting boron-containing gold nanoparticles for boron neutron capture therapy (BNCT).

Successful boron neutron capture therapy (BNCT) requires sufficient and specific delivery of boron atoms to malignant cells. Gold nanoparticles (AuNPs) have been used as a useful delivery system for selectively releasing cytotoxic payloads in the tumor. However, studies demonstrating the in vivo distribution or pharmacokinetics of boron-containing AuNPs via noninvasive imaging are lacking. This study aims to develop theranostic AuNP-boron cage assemblies (B-AuNPs) and evaluate its feasibility for BNCT. The commercial citrate-coated AuNPs were subjected to PEGylation, azide addition, and carborane modification on the surface. To further arm the AuNPs, we conjugated anti-HER2 antibody (61 IgG) with boron-containing PEGylated AuNPs to form 61-B-AuNPs. The diameter and radiolabeling efficiency of boron-containing AuNPs were determined by dynamic light scattering (DLS) and radio thin-layer chromatography (radio TLC), respectively. Noninvasive single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging was performed to determine the pharmacokinetics of radioiodinated AuNPs in N87 gastric cancer xenografts, and the content of boron in tumor and muscle was assessed by inductively coupled plasma mass spectrometry (ICP-MS). After the 3-step modification, the diameter of B-AuNPs increased by ˜25 nm, and antibody conjugation did not affect the diameter of AuNPs. Radioactive iodine (I-123) was introduced in AuNPs by Click chemistry under copper catalysis. The radiolabeling efficiency of 123I-B-AuNPs and 123I-61-B-AuNPs was approximately 60 ± 5%. After purification, the radiochemical purity (RCP) of these NPs was greater than 90%. MicroSPECT/CT imaging showed that the tumor-to-muscle (T/M) ratio of 123I-B-AuNP-injected mice reached 1.91 ± 0.17 at 12 h post-injection, while that of 123I-61-B-AuNP-injected mice was 12.02 ± 0.94. However, the increased uptake of AuNPs by the thyroid was observed at 36 h after the administration of 123I-61-B-AuNPs, indicating antibody-mediated phagocytosis. The T/M ratio, assessed by ICP-MS, of B-AuNP- and 61-B-AuNP-injected mice was 4.91 ± 2.75 and 41.05 ± 11.15, respectively. We successfully developed detectable HER2-targeting boron-containing AuNPs with high RCP and an acceptable yield. Noninvasive imaging could be a valuable tool for the noninvasive determination of the pharmacokinetics of AuNPs and measurement of boron concentration in the tumor.

Effective binding to protein antigens by antibodies from antibody libraries designed with enhanced protein recognition propensities.

Antibodies provide immune protection by recognizing antigens of diverse chemical properties, but elucidating the amino acid sequence-function relationships underlying the specificity and affinity of antibody-antigen interactions remains challenging. We designed and constructed phage-displayed synthetic antibody libraries with enriched protein antigen-recognition propensities calculated with machine learning predictors, which indicated that the designed single-chain variable fragment variants were encoded with enhanced distributions of complementarity-determining region (CDR) hot spot residues with high protein antigen recognition propensities in comparison with those in the human antibody germline sequences. Antibodies derived directly from the synthetic antibody libraries, without affinity maturation cycles comparable to those in in vivo immune systems, bound to the corresponding protein antigen through diverse conformational or linear epitopes with specificity and affinity comparable to those of the affinity-matured antibodies from in vivo immune systems. The results indicated that more densely populated CDR hot spot residues were sustainable by the antibody structural frameworks and could be accompanied by enhanced functionalities in recognizing protein antigens. Our study results suggest that synthetic antibody libraries, which are not limited by the sequences found in antibodies in nature, could be designed with the guidance of the computational machine learning algorithms that are programmed to predict interaction propensities to molecules of diverse chemical properties, leading to antibodies with optimal characteristics pertinent to their medical applications.

Antibody-drug conjugates with HER2-targeting antibodies from synthetic antibody libraries are highly potent against HER2-positive human gastric tumor in xenograft models.

HER2-ECD (human epidermal growth factor receptor 2 - extracellular domain) is a prominent therapeutic target validated for treating HER2-positive breast and gastric cancer, but HER2-specific therapeutic options for treating advanced gastric cancer remain limited. We have developed antibody-drug conjugates (ADCs), comprising IgG1 linked via valine-citrulline to monomethyl auristatin E, with potential to treat HER2-positive gastric cancer in humans. The antibodies optimally selected from the ADC discovery platform, which was developed to discover antibody candidates suitable for immunoconjugates from synthetic antibody libraries designed using antibody-antigen interaction principles, were demonstrated to be superior immunoconjugate targeting modules in terms of efficacy and off-target toxicity. In comparison with the two control humanized antibodies (trastuzumab and H32) derived from murine antibody repertoires, the antibodies derived from the synthetic antibody libraries had enhanced receptor-mediated internalization rate, which could result in ADCs with optimal efficacies. Along with the ADCs, two other forms of immunoconjugates (scFv-PE38KDEL and IgG1-AL1-PE38KDEL) were used to test the antibodies for delivering cytotoxic payloads to xenograft tumor models in vivo and to cultured cells in vitro. The in vivo experiments with the three forms of immunoconjugates revealed minimal off-target toxicities of the selected antibodies from the synthetic antibody libraries; the off-target toxicities of the control antibodies could have resulted from the antibodies' propensity to target the liver in the animal models. Our ADC discovery platform and the knowledge gained from our in vivo tests on xenograft models with the three forms of immunoconjugates could be useful to anyone developing optimal ADC cancer therapeutics.

Noninvasive assessment of characteristics of novel anti-HER2 antibodies by molecular imaging in a human gastric cancer xenograft-bearing mouse model.

Human epidermal growth factor receptor 2 (HER2) overexpression occurs in various types of cancers. Regarding the anti-HER2 targeted therapies showed superior treatment outcomes in several (pre)clinical studies, we used multimodality image to rapidly select novel HER2-targeting antibodies for further therapeutics development. The four anti-HER2 antibodies (H32 IgG, 75 IgG, 61 IgG, and trastuzumab) labeled with either In-111 or a DyLight680 fluorescent dye were applied to perform cellular uptake, endocytosis, optical/microSPECT/CT imaging and biodistribution studies. In vitro and in vivo relative effectiveness of these antibodies were also compared in an N87 gastric cancer xenograft model. The internalized radioactivity of [111In]61 IgG in N87 cells increased from 33% at 12 hr to 56% at 48 hr after incubation, while the majority of other antibodies stayed on the cell membranes. Among these antibodies, 61 IgG showed the highest accumulation in tumors with the tumor-to-muscle ratio (T/M) of 131 ± 61.4 and 19.13 ± 3.42 conducted by IVIS and microSPECT/CT, respectively. We demonstrated that multimodality imaging is a reliable approach for selecting potential antibodies and found that 61 IgG manifested significant tumor accumulation with elevated internalization rate thus could be a suitable candidate for further development of new HER2-targeted therapies.

High throughput discovery of influenza virus neutralizing antibodies from phage-displayed synthetic antibody libraries.

Pandemic and epidemic outbreaks of influenza A virus (IAV) infection pose severe challenges to human society. Passive immunotherapy with recombinant neutralizing antibodies can potentially mitigate the threats of IAV infection. With a high throughput neutralizing antibody discovery platform, we produced artificial anti-hemagglutinin (HA) IAV-neutralizing IgGs from phage-displayed synthetic scFv libraries without necessitating prior memory of antibody-antigen interactions or relying on affinity maturation essential for in vivo immune systems to generate highly specific neutralizing antibodies. At least two thirds of the epitope groups of the artificial anti-HA antibodies resemble those of natural protective anti-HA antibodies, providing alternatives to neutralizing antibodies from natural antibody repertoires. With continuing advancement in designing and constructing synthetic scFv libraries, this technological platform is useful in mitigating not only the threats of IAV pandemics but also those from other newly emerging viral infections.

Effects of gripping volume in the mechanical strengths of orthodontic mini-implant.

The objective of study was to investigate the correlation between the mechanical strengths [insertion torque (IT); resonance frequency (RF); and horizontal pullout strength (HPS)] and gripping volume (GV) of mini-implants. Thirty mini-implants of three types (Type A: 2 mm × 10 mm, cylindrical, titanium alloy; Type B: 2 mm × 10 mm, tapered, stainless steel; and Type C: 2 mm × 11 mm, cylindrical, titanium alloy) were inserted 7 mm into artificial bones. One-way analysis of variance and Spearman's test were applied to assess intergroup comparisons and intragroup correlations. The null hypothesis was that no statistically significant correlations exist between the GV and mechanical strengths (IT, RF, and HPS). In the IT test, Type C (14.2 Ncm) had significantly (p=0.016) greater values than did Type A (12.4 Ncm). In the RF analysis, no significant difference was observed among the three types of mini-implants. In the HPS test, Type C (388.9 Ncm) was significantly larger than both Type B (294.5 Ncm) and Type A (286 Ncm). In the GV measurement, Type C (14.4 mm3) was significantly larger than Type B (11.4 mm3) and Type A (9.2 mm3). Type A and Type B exhibited no significant correlations among the tests. Therefore, the null hypothesis was accepted. Although no significant correlation was noted between the GV and mechanical strengths (IT, RF, and HPS), we observed a trend that the mechanical strengths (IT, RF, and HPS) of the mini-implants corresponded to the order and values of GV (Type C > Type B > Type A).

Effect of Anchor Length on the Pullout Strength of Palatal Mini Implants.

PURPOSE: The aim of this study was to evaluate the pullout strength of 3 different orthodontic mini implants. MATERIALS AND METHODS: Twenty-seven mini implants (diameter: 2 mm, length: 7 mm) were implanted into artificial bone (Sawbones; Pacific Research Laboratories Inc.) at depths of 3, 4, and 5 mm. The insertion torque (IT), resonance frequency (RF), pullout strength (PS), and anchor length (AL) were measured. One-way analysis of variance with Tukey honest significant difference (HSD) postcomparison were used to detect intergroup differences. The null hypothesis was that IT, RF, and PS would significantly correlate in the same brand. RESULTS: In the implantation depths (ID) (5 and 4 mm), IT of Types C (16.67 and 14.33 N·cm) and Type B (14 and 13.33 N·cm) were significantly higher than Type A (10.33 and 9.33 N·cm). Type A had a largest AL and PS at the IDs (5 and 4 mm). In the IDs (3 mm), PS was no different. Type C had no correlation among the RF, IT, and PS. Therefore, null hypothesis was rejected. CONCLUSION: AL exerted crucial effects on the PS of the mini implants.

Evaluation of mechanical strengths of three types of mini-implants in artificial bones.

We investigates the effect of the anchor area on the mechanical strengths of infrazygomatic mini-implants. Thirty mini-implants were divided into three types based on the material and shape: Type A (titanium alloy, 2.0×12 mm), Type B (stainless steel, 2.0×12 mm), and Type C (titanium alloy, 2.0×11 mm).The mini-implants were inserted at 90° and 45° into the artificial bone to a depth of 7 mm, without predrilling. The mechanical strengths [insertion torque (IT), resonance frequency (RF), and removal torque (RT)] and the anchor area were measured. We hypothesized that no correlation exists among the mechanical forces of each brand. In the 90° tests, the IT, RF, and RT of Type C (8.5 N cm, 10.2 kHz, and 6.1 N cm, respectively) were significantly higher than those of Type A (5.0 N cm, 7.7 kHz, and 4.7 N cm, respectively). In the 45° test, the RFs of Type C (9.2 kHz) was significantly higher than those of Type A (7.0 kHz) and Type B (6.7 kHz). The anchor area of the mini-implants was in the order of Type C (706 mm2)>Type B (648 mm2)>Type A (621 mm2). Type C exhibited no significant correlation in intragroup comparisons, and the hypothesis was accepted. In the 90° and 45° tests, Type C exhibited the largest anchor area and the highest mechanical strengths (IT, RF, and RT) among the three types of mini-implants. The anchor area plays a crucial role in the mechanical strength of mini-implants.

Insertion torque, resonance frequency, and removal torque analysis of microimplants.

This study aimed to compare the insertion torque (IT), resonance frequency (RF), and removal torque (RT) among three microimplant brands. Thirty microimplants of the three brands were used as follows: Type A (titanium alloy, 1.5-mm × 8-mm), Type B (stainless steel, 1.5-mm × 8-mm), and Type C (titanium alloy, 1.5-mm × 9-mm). A synthetic bone with a 2-mm cortical bone and bone marrow was used. Each microimplant was inserted into the synthetic bone, without predrilling, to a 7 mm depth. The IT, RF, and RT were measured in both vertical and horizontal directions. One-way analysis of variance and Spearman's rank correlation coefficient tests were used for intergroup and intragroup comparisons, respectively. In the vertical test, the ITs of Type C (7.8 Ncm) and Type B (7.5 Ncm) were significantly higher than that of Type A (4.4 Ncm). The RFs of Type C (11.5 kHz) and Type A (10.2 kHz) were significantly higher than that of Type B (7.5 kHz). Type C (7.4 Ncm) and Type B (7.3 Ncm) had significantly higher RTs than did Type A (4.1 Ncm). In the horizontal test, both the ITs and RTs were significantly higher for Type C, compared with Type A. No significant differences were found among the groups, and the study hypothesis was accepted. Type A had the lowest inner/outer diameter ratio and widest apical facing angle, engendering the lowest IT and highest RF values. However, no significant correlations in the IT, RF, and RT were observed among the three groups.

High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with antibody fragments from phage-displayed synthetic antibody libraries.

Immunotoxins are an important class of antibody-based therapeutics. The potency of the immunotoxins depends on the antibody fragments as the guiding modules targeting designated molecules on cell surfaces. Phage-displayed synthetic antibody scFv libraries provide abundant antibody fragment candidates as targeting modules for the immunoconjugates, but the discovery of optimally functional immunoconjugates is limited by the scFv-payload conjugation procedure. In this work, cytotoxicity screening of non-covalently assembled immunotoxins was developed in high throughput format to discover highly functional synthetic antibody fragments for delivering toxin payloads. The principles governing the efficiency of the antibodies as targeting modules have been elucidated from large volume of cytotoxicity data: (a) epitope and paratope of the antibody-based targeting module are major determinants for the potency of the immunotoxins; (b) immunotoxins with bivalent antibody-based targeting modules are generally superior in cytotoxic potency to those with corresponding monovalent targeting module; and (c) the potency of the immunotoxins is positively correlated with the densities of the cell surface antigen. These findings suggest that screening against the target cells with a large pool of antibodies from synthetic antibody libraries without the limitations of natural antibody responses can lead to optimal potency and minimal off-target toxicity of the immunoconjugates.

Correlation Between Blood Loss and Patient-Related Factors in the Bilateral Parasymphyseal Osteotomy.

The aim of this study was to determine the correlation between pre- and postsurgical loss of blood and blood components among patients undergoing treatment of facial deformities by bilateral parasymphyseal osteotomy (BPsO).The pre- and postoperative values of blood components were determined in 30 facial deformity patients who underwent orthognathic surgery by hypotensive anesthesia. Correlations among the blood loss, sex, age, operation time, and reduced values of blood components were assessed by a correlation matrix. The mean blood loss and operation time were 437.5 (± 52.5) mL and 355.8 (± 209.42) minutes, respectively. Two patients included in this study had required blood transfusion. The mean reduced red blood cell (× 10/µL), hemoglobin (g/dL), and hematocrit (%) were -1.02, -2.98, and -9.18, respectively. There was no significant correlation between blood loss and other related factors (eg, age, operation time, and reduced blood components). All patients, however, showed significantly lower values of blood components after surgery. In conclusion, no significant factor was associated with blood loss and reduced blood components among patients undergoing BPsO. Furthermore, hypotensive anesthesia is a well-accepted method to reduce blood loss during orthognathic surgery.

Predominant structural configuration of natural antibody repertoires enables potent antibody responses against protein antigens.

Humoral immunity against diverse pathogens is rapidly elicited from natural antibody repertoires of limited complexity. But the organizing principles underlying the antibody repertoires that facilitate this immunity are not well-understood. We used HER2 as a model immunogen and reverse-engineered murine antibody response through constructing an artificial antibody library encoded with rudimentary sequence and structural characteristics learned from high throughput sequencing of antibody variable domains. Antibodies selected in vitro from the phage-displayed synthetic antibody library bound to the model immunogen with high affinity and specificities, which reproduced the specificities of natural antibody responses. We conclude that natural antibody structural repertoires are shaped to allow functional antibodies to be encoded efficiently, within the complexity limit of an individual antibody repertoire, to bind to diverse protein antigens with high specificity and affinity. Phage-displayed synthetic antibody libraries, in conjunction with high-throughput sequencing, can thus be designed to replicate natural antibody responses and to generate novel antibodies against diverse antigens.

Effects of Ti surface treatments with silane and arginylglycylaspartic acid peptide on bone cell progenitors.

Achieving optimal aesthetic appearance is a major objective in dental implant design, and the interaction between the materials and the bone cell progenitors is an important factor in the attainment of this objective. In this study, a novel concept was evaluated by varying the surface modifications on titanium (Ti). Different levels of roughness can be attained by machine grinding (M), sand blasting, and acid etching (SLA) of the samples. The behavior of bone cell progenitors (D1) on the surfaces of Ti disks with different surface modifications was investigated. The surfaces of M or SLA disks were silanized (MS or SLAS group) through treatment with silane/Gly-Arg-Gly-Asp-Ser (GRGDS) peptide (MSP or SLASP group) and anchored particles of tetracalcium phosphate (TTCP) on the specimen surfaces (SLA-TTCP group). Physicochemical analysis was performed by metallographic microscopy, scanning electron microscopy, and contact angle analysis. The proliferation and the quantitative alkaline phosphatase (ALP) production of D1 cells on the surface of different sample groups were determined. The SLASP group had a significantly larger D1 cell proliferation than the other groups after 4 and 7 d of incubation (p < 0.05). ALP expression was a very early marker of differentiation, and was the first indication of the increasing number of cells at 7 d of culture. Among the groups in the M substrate series (i.e., M, MS, and MSP) and in the SLA series (i.e., SLA, SLAS, and SLASP), the MSP and SLASP specimens exhibited superior differentiation abilities on respective cultures until day 7 and day 10. A high number of hydrophilic surfaces dominated cell proliferation in the early stage of cell attachment. However, factors affecting the pore structure and the surface morphology can improve cell proliferation and differentiation. According to analyses of proliferation and ALP expression of bone cell progenitors D1, the original SLA implant surface can be improved with surface treatment methods, such as silanization and treatment with graft GRGDS pentapeptide. These methods can be potential candidates for the promotion of bone growth.

Analysis of maximum mouth opening and its related factors in 3- to 5-year-old Taiwanese children.

Maximum mouth opening (MMO) can reflect the function of the dentofacial musculature and joint system, and routine oral examinations should include its assessment. To diagnose abnormalities using MMO measurements, it is necessary to establish the normal range of MMO; however, few studies have investigated this subject in Taiwan. Therefore, the purposes of this study were to determine the normal MMO range in 3- to 5-year-old preschool children and to investigate the factors correlated with MMO. We examined the interincisal distance, defined as the distance between the edges of the upper and lower incisors, in 518 preschool children (age range 3-5 years; 271 boys and 247 girls) with a plastic sliding caliper. The MMO on both sides of the mouth and mouth width (MW) was measured 3 times. No differences in MMO were found between the genders. The interincisal distance was 37.47 (±4.11) mm for boys and 36.93 (±3.85) mm for girls, whereas the mean MMO was 37.21 (±3.99) mm. The MMO increased with the increasing age of the children, and the mean value of MMO in children aged 3, 4, and 5 was 35.31 (±4.03), 36.61 (±3.79), and 38.31 (±3.88) mm, respectively. Furthermore, MMO was found to correlate with weight and MW. MMO increased by 0.19 mm per increased weight and 0.37 mm per increased MW. The mean value of MMO in 3- to 5-year-old preschool children was 37.21 (±3.99) mm. MMO in 3- to 5-year-old preschool children increased with age and was correlated with weight and MW.