Generation of novel anti-apoE monoclonal antibodies that selectively recognize apoE isoforms.

Apolipoprotein E (apoE) is a regulator of lipid metabolism, cholesterol transport, and the clearance and aggregation of amyloid ß in the brain. The three human apoE isoforms apoE2, apoE3, and apoE4 only differ in one or two residues. Nevertheless, the functions highly depend on the isoform types and lipidated states. Here, we generated novel anti-apoE monoclonal antibodies (mAbs) and obtained an apoE4-selective mAb whose epitope is within residues 110-117. ELISA and bio-layer interferometry measurements demonstrated that the dissociation constants of mAbs are within the nanomolar range. Using the generated antibodies, we successfully constructed sandwich ELISA systems, which can detect all apoE isoforms or selectively detect apoE4. These results suggest the usability of the generated anti-apoE mAbs for selective detection of apoE isoforms.

Practice differences and knowledge gaps in complex and high-risk interventions between Japan and the USA: A case-based discussion.

Advances in percutaneous coronary intervention (PCI) devices and techniques have expanded the pool of eligible patients for revascularization, including those with comorbidities, reduced left ventricular function, or anatomical complexity (defined as CHIP: complex and high-risk interventions in indicated patients). CHIP interventions are typically performed by selected operators who specialize in complex PCI. This review presents two cases performed in the USA, to discuss the similarities and differences in practice patterns between CHIP operators in Japan and the USA. The first case involves a 58-year-old male presenting with myocardial infarction and cardiogenic shock, and the second case involves a 51-year-old female with a history of coronary artery bypass grafting presenting with a chronic total occlusion and PCI complicated by vessel perforation. The discussion focuses on appropriate patient selection, the role of the heart team approach for decision-making, the use of hemodynamic support devices, and other relevant factors. By comparing practices in Japan and the USA, this review highlights opportunities for knowledge exchange and potential areas for improving patient outcomes.

Framework-Directed Amino-Acid Insertions Generated over 55-Fold Affinity-Matured Antibody Fragments That Enabled Sensitive Luminescent Immunoassays of Cortisol.

We generated three single-chain Fv fragments (scFvs) specific to cortisol according to our original affinity-maturation strategy and verified their utility in developing immunoassays. These scFv mutants (m-scFvs) had insertion of one, four, or six amino acid(s) in the framework region 1 of the VH-domain and showed >55-fold higher affinity (Ka, 2.0 - 2.2 × 1010 M-1) than the unmodified scFv (wt-scFv). Each m-scFv was fused with NanoLuc luciferase (NLuc) for the use in enzyme-linked immunosorbent assays (ELISAs). In these ELISA, the m-scFv-NLuc fusions were competitively reacted with immobilized cortisol residues and cortisol standards, and then the bound NLuc activity was monitored luminometrically. The luminescent ELISAs generated dose-response curves with extremely low midpoints (approx. 3 pg/assay) and were >150-fold more sensitive than the colorimetric ELISAs using wt-scFv and >8000-fold more sensitive than the ELISA using the parental native antibody. The luminescent ELISAs showed acceptable cross-reactivity patterns with related steroids, and the determination of control sera afforded cortisol levels in the reference range with satisfactory parallelism.

Two-year clinical outcomes of drug-coated balloon angioplasty and angiographic predictors of restenosis among patients with de novo femoropopliteal lesions.

OBJECTIVES: We analyzed the 2-year clinical outcomes of patients with de novo femoropopliteal (FP) lesions who underwent drug-coated balloon (DCB) angioplasty and the angiographic predictors of restenosis. METHODS: This single-center, retrospective, and observational study evaluated 129 de novo FP lesions treated with DCB angioplasty without bailout stenting. Clinical outcomes and risk factors for loss of primary patency were analyzed using univariate and multivariate Cox proportional hazards regression models. RESULTS: The participants were aged 48-93 (mean: 73.6 ± 9.8) years, and 31% were women. Approximately 33% of the patients were receiving regular dialysis, and 35% of the affected limbs had critical ischemia. The mean lesion length was 132 ± 96 mm, and the mean reference vessel diameter (RVD) was 4.7 ± 0.8 mm. Forty-three (33%) limbs had chronic total occlusion of the target artery segment. Fifty-seven (44%) and 72 (56%) lesions were treated with DCB angioplasty using IN.PACT Admiral and Lutonix, respectively. The primary patency and amputation-free survival at 2 years were 59.3% and 89.5%, respectively. RVD was found to be an independent predictor of loss of primary patency. Based on the receiver operating characteristic analysis, an RVD of 4.2 mm was the best predictor of loss of primary patency at 2 years. CONCLUSIONS: The short-term clinical outcome of DCB angioplasty for de novo FP lesions was acceptable. Moreover, an RVD of <4.2 mm was an independent predictor of restenosis after DCB angioplasty.

Intramolecular interaction kinetically regulates fibril formation by human and mouse α-synuclein.

Regulation of α-synuclein (αS) fibril formation is a potent therapeutic strategy for αS-related neurodegenerative disorders. αS, an intrinsically disordered 140-residue intraneural protein, comprises positively charged N-terminal, hydrophobic non-amyloid ß component (NAC), and negatively charged C-terminal regions. Although mouse and human αS share 95% sequence identity, mouse αS forms amyloid fibrils faster than human αS. To evaluate the kinetic regulation of αS fibrillation, we examined the effects of mismatched residues in human and mouse αS on fibril formation and intramolecular interactions. Thioflavin T fluorescence assay using domain-swapped or C-terminal-truncated αS variants revealed that mouse αS exhibited higher nucleation and fibril elongation than human αS. In mouse αS, S87N substitution in the NAC region rather than A53T substitution is dominant for enhanced fibril formation. FÓ§rester resonance energy transfer analysis demonstrated that the intramolecular interaction of the C-terminal region with the N-terminal and NAC regions observed in human αS is perturbed in mouse αS. In mouse αS, S87N substitution is responsible for the perturbed interaction. These results indicate that the interaction of the C-terminal region with the N-terminal and NAC regions suppresses αS fibril formation and that the human-to-mouse S87N substitution in the NAC region accelerates αS fibril formation by perturbing intramolecular interaction.

Impact of "black rock" on clinical outcomes after endovascular therapy for de novo calcified femoropopliteal lesions.

The relationship between severity of calcification and clinical outcomes after endovascular therapy (EVT) for femoropopliteal lesions is well known. We often encounter dense calcifications in our daily practice, which are darker than normal calcifications on angiography. Accordingly, we named it "black rock" (BR), and investigated its impact on clinical outcomes after EVT. We retrospectively analyzed 677 lesions in 495 patients who underwent EVT for de novo calcified femoropopliteal lesions at our hospital between April 2007 and June 2020. BR is defined as a calcification which is 1 cm or more in length, occupies more than half of the vessel diameter, and appears darker than the body of the femur on angiography. Propensity score matching analysis was performed to compare clinical outcomes between lesions with BR [BR (+) group] and without BR [BR (-) group]. A total of 119 matched pairs of lesions were analyzed. Primary patency at 2 years was significantly lower in the BR (+) group than in the BR (-) group (48% vs. 75%, p = .0007). Multivariate analysis revealed that the presence of BR [hazard ratio (HR) = 2.23, 95% confidence interval (CI); 1.48-3.38, p = .0001], lesion length (HR = 1.03, 95%CI; 1.00-1.06, p = .0244), and no scaffold use (HR = 1.58, 95%CI; 1.06-2.36, p = .0246) were predictors of restenosis. The presence of BR is independently associated with clinical outcomes after EVT for de novo calcified femoropopliteal lesions.

Visual learning in tethered bees modifies flight orientation and is impaired by epinastine.

Visual-orientation learning of a tethered flying bee was investigated using a flight simulator and a novel protocol in which orientation preference toward trained visual targets was assessed in tests performed before and after appetitive conditioning. Either a blue or a green rectangle (conditioned stimulus, CS) was associated with 30% sucrose solution (unconditioned stimulus, US), whereas the other rectangle was not paired with US. Bees were tested in a closed-looped flight simulator 5 min after ten pairings of the US and CS. Conditioned bees were preferentially oriented to the CS after such training. This increase in preference for CS was maintained for 24 h, indicating the presence of long-term memory. Because the total orienting time was not altered by conditioning, conditioning did not enhance orientation activity itself but increased the relative time for orientation to CS. When 0.4 or 4 mM epinastine (an antagonist of octopamine receptors) was injected into the bee's head 30 min prior to the experiment, both short- and long-term memory formation were significantly impaired, suggesting that octopamine, which is crucial for appetitive olfactory learning in insects, is also involved in visual orientation learning.

Comparison Between Clinical Outcomes of Low- and High-Dose Paclitaxel Drug-Coated Balloon in Endovascular Therapy for Femoropopliteal Lesion.

PURPOSE: There is a little datum about the impact of paclitaxel dosage in patients undergoing drug-coated balloons (DCB) in endovascular therapy (EVT) for femoropopliteal lesions. In the current study, the authors sought to compare the clinical outcomes of low-dose (LD) and high-dose (HD) paclitaxel DCBs for patients undergoing EVT for femoropopliteal lesions in a real-world setting. MATERIALS AND METHODS: The study population was derived from a multicenter registry named "Evaluation of clinical outcome after endovascular therapy for femoropopliteal artery disease in Kanagawa" (LANDMARK registry). This registry consists of patients from 5 hospitals in Kanagawa, Japan. Overall, 1,378 patients with 1,777 lesions received treatment between July 2017 and June 2020. Among these, DCB angioplasty was performed in 477 patients (516 lesions). Propensity score matching analysis was performed to compare the clinical outcomes of LD-DCB (Lutonix; Becton Dickinson and Company, Franklin Lakes, New Jersey) and HD-DCB (IN.PACT Admiral; Medtronic Vascular, Santa Clara, CA, USA). RESULTS: A total of 160 matched pairs of lesions were analyzed. Primary patency and freedom from target lesion revascularization at 2 years were similar between the two groups (LD-DCB vs. HD-DCB: 72% vs. 70%, p = 0.53; and 75% vs. 73%, p = 0.59, respectively). CONCLUSION: No significant differences were found in the clinical outcomes between LD-DCB and HD-DCB angioplasty for femoropopliteal lesions. LEVEL OF EVIDENCE: Level 3.

The Patrol Yeast: A new biosensor armed with antibody-receptor chimera detecting a range of toxic substances associated with food poisoning.

Baker's yeast is an attractive host with established safety and stability characteristics. Many yeast-based biosensors have been developed, but transmembrane signal transduction has not been used to detect membrane-impermeable substances using antigen-antibody interactions. Therefore, we created Patrol Yeast, a novel yeast-based immunosensor of various targets, particularly toxic substances in food. A membrane-based yeast two-hybrid system using split-ubiquitin was successfully used to detect practically important concentration ranges of caffeine and aflatoxins using separated variable regions of an antibody. Moreover, enterohemorrhagic Escherichia coli O157 was detected using a specific single-chain antibody, in which Zymolyase was added to partially destroy the cell wall. The incorporation of secreted Cypridina luciferase reporter further simplified the signal detection procedures without cell lysis. The methodology is more cost-effective and faster than using mammalian cells. The ability to detect various targets renders Patrol Yeast a valuable tool for ensuring food and beverage safety and addressing other environmental and technological issues.

Prediction of Wound Recurrence in Patients With Chronic Limb-Threatening Ischemia Undergoing Endovascular Treatment.

PURPOSE: Chronic limb-threatening ischemia due to isolated below-the-knee lesions is a factor associated with wound recurrence. However, there is a lack of data regarding wound recurrence in such cases. This study aimed to determine the predictors of wound recurrence in patients with chronic limb-threatening ischemia undergoing endovascular treatment. PATIENTS AND METHODS: This was a single-center, retrospective, observational study. We enrolled 152 consecutive patients with chronic limb-threatening ischemia (172 limbs) who achieved complete wound healing after undergoing endovascular treatment for isolated below-the-knee lesions between February 2008 and December 2017. Of these, the wound had recurred in 56 limbs (33%), and we divided the patients into 2 groups based on wound recurrence. We evaluated the recurrence rate of chronic limb-threatening ischemia and predictors of wound recurrence. Wound recurrence was defined as recurrence of the wound within 2 years of complete wound healing. RESULTS: Patients' backgrounds were similar in both groups, including mean age (72±9 vs 72±11; p=0.76) and hemodialysis (43% vs 40%; p=0.66). Pre-pedal arch type 2 (52% vs 8%; p<0.01), infrapopliteal grade 4 of the Global Limb Anatomic Staging System (77% vs 59%; p=0.02), and Wound, Ischemia, and foot Infection criteria stage 4 (43% vs 28%; p=0.04) were more common in the wound recurrence group. Multivariate Cox proportional hazard analysis identified pre-pedal arch type 2 (hazard ratio, 5.28; 95% confidence interval, 3.08-9.10; p<0.01) and Wound, Ischemia, and foot Infection criteria stage 4 (hazard ratio, 1.98; 95% confidence interval, 1.15-3.36; p=0.01) as predictors of wound recurrence after complete wound healing. CONCLUSION: Pre-pedal arch type 2 and Wound, Ischemia, and foot Infection classification system stage 4 were associated with wound recurrence in patients with chronic limb-threatening ischemia who achieved complete wound healing after undergoing endovascular treatment for isolated below-the-knee lesions.

In vivo genotoxicity assessment of a multiwalled carbon nanotube in a mouse ex vivo culture.

BACKGROUND: Multiwalled carbon nanotubes (MWCNTs) are suspected lung carcinogens because their shape and size are similar to asbestos. Various MWCNT types are manufactured; however, only MWNT-7 is classified into Group 2B by The International Agency for Research on Cancer. MWNT-7's carcinogenicity is strongly related to inflammatory reactions. On the other hand, inconsistent results on MWNT-7 genotoxicity have been reported. We previously observed no significant differences in both Pig-a (blood) and gpt (lung) mutant frequencies between MWNT-7-intratracheally treated and negative control rats. In this study, to investigate in vivo MWNT-7 genotoxicity on various endpoints, we attempted to develop a lung micronucleus assay through ex vivo culture targeting the cellular fraction of Clara cells and alveolar Type II (AT-II) cells, known as the initiating cells of lung cancer. Using this system, we analyzed the in vivo MWNT-7 genotoxicity induced by both whole-body inhalation exposure and intratracheal instillation. We also conducted an erythrocyte micronucleus assay using the samples obtained from animals under intratracheal instillation to investigate the tissue specificity of MWNT-7 induced genotoxicities. RESULTS:  We detected a significant increase in the incidence of micronucleated cells derived from the cellular fraction of Clara cells and AT-II cells in both MWNT-7-treated and positive control groups compared to the negative control group under both whole-body inhalation exposures and intratracheal instillation. Additionally, the erythrocyte micronucleus assay detected a significant increase in the incidence of micronucleated reticulocytes only in the positive control group. CONCLUSIONS: Our findings indicated that MWNT-7 was genotoxic in the lungs directly exposed by both the body inhalation and intratracheal instillation but not in the hematopoietic tissue.

Retrieving Dissociation-Resistant Antibody Mutants: An Efficient Strategy for Developing Immunoassays with Improved Sensitivities.

Previously, we generated high-affinity antibody mutants that enabled sensitive immunoassays by exploring diverse libraries of single-chain Fv fragments (scFvs) displayed on bacteriophage. To isolate rarely-occurring desirable clones, "panning" has commonly been performed but is often unsuccessful. Therefore, we previously developed a clonal array profiling (CAP) method, wherein scFv-displaying phage (scFv-Ph) clones in a library were examined individually regarding their ability to target antigens immobilized on microwells. Clones that showed strong reactivity were recovered via dissociation using an acidic treatment. The CAP successfully discovered cortisol-specific scFvs showing 17-31-fold improved Ka from libraries generated via site-directed insertions in a prototype anti-cortisol scFv (wt-scFv; Ka, 3.6 × 108 M-1), but their Ka did not exceed 1.1 × 1010 M-1. In this study, to break this possible affinity ceiling, we devised a new system employing a dissociation-independent recovery. scFv-Phs were individually reacted to target antigen (cortisol) immobilized on microwells via a linker containing a disulfide bond. Following acidic and basic treatments to eliminate scFv-Phs with "ordinary affinities," dissociation-resistant scFv-Phs remaining on the microwells were retrieved via reductive cleavage of the disulfide bonds. This system allowed for a straightforward and efficient discovery of scFv mutants with 33-56-fold increased Ka (1.2-2.0 × 1010 M-1), exceeding the previous affinity ceiling. These scFvs enabled an enzyme-linked immunosorbent assay for cortisol with 18-51-fold higher sensitivity than the assay performed using wt-scFv.

Predictive performance of J-Calc-CTO score for guidewire crossing through chronic total occlusion lesions within 30 minutes: J-CTO score modified by computed tomography coronary angiography.

The original J-CTO score predicts the difficulty of percutaneous coronary intervention (PCI) for chronic total occlusion (CTO) lesions, but the grade of calcification has not been fully evaluated. We examined 137 patients (141 CTO lesions) who underwent coronary computed tomography angiography (CTA) pre-PCI between October 2016 and October 2021. They were randomly divided into derivation (n = 94) and validation (n = 47) groups. The degree and distribution of calcification in the occluded segment were assessed using CTA. The calcified index was defined as calcium volume divided by the length of the occluded segment. We created the J-Calc-CTO score consisting of calcification parameters associated with 30-min wire crossing in the derivation group. The validity of the J-Calc-CTO score was compared with that of the original J-CTO score using c-statistics. The procedural success rate was 96%, and 30-min wire crossing during the procedure was achieved in 29%. Dense calcification (calcified-index >12) (odds ratio [OR]: 4.63; 95% confidence interval [CI]: 1.24-22.2; p = 0.04) and calcification in the center of the lumen (OR: 7.25; 95% CI: 1.48-32.1; p = 0.02) were independently associated with 30-min wire crossing as variables evaluated using CTA. The J-Calc-CTO score was created by adding 1 point to the two parameters in place of "calcification" in the original J-CTO score. The J-Calc-CTO score showed a higher predictive value of 30-min wire crossing than the J-CTO score in the derivation (c-statistics; 0.836 vs. 0.670; p > 0.01) and validation groups (c-statistics; 0.879 vs. 0.767, p > 0.01). The degree and distribution of calcification evaluated using CTA refined the predictive value of the original J-CTO score for 30-min wire crossing.

Impact of Chocolate percutaneous transluminal angioplasty balloon on vessel preparation in drug-coated balloon angioplasty for femoropopliteal lesion.

PURPOSE: To compare the impact of Chocolate and conventional balloons on vessel preparation in percutaneous transluminal angioplasty. MATERIALS AND METHODS: This single-center retrospective study included 111 endovascular therapy consecutive cases of femoropopliteal lesions using drug-coated balloon strategy with a 1:1 pre-dilation balloon diameter between February 2020 and August 2021, divided into the Chocolate percutaneous transluminal angioplasty (n = 48) and conventional (n = 63) groups. Before the availability of Chocolate balloons in Japan (December 2020), a standard semi-compliant or non-compliant balloon was used for vessel preparation. The primary endpoint was rate of severe dissection after pre-dilatation. Secondary endpoints were angiographic percent diameter stenosis, bailout stent rate, primary patency rate, and freedom from target-lesion-revascularization rate at six months. RESULTS: There was no significant difference in patient and lesion characteristics. The procedural characteristics comprised balloon length 90 ± 37 and 149 ± 95 mm (P = 0.004) and inflation pressure 11 ± 3 and 16 ± 7 atm (P < 0.001) in the Chocolate and conventional groups, respectively. Regarding primary endpoint, rates of severe dissection were 4.2% and 25% (P = 0.003); regarding secondary endpoints, percent diameter stenosis was 18 ± 15% and 20 ± 17% (P = 0.409), and the rate of bailout stenting was 2.1% and 15.9% (P = 0.016) in the Chocolate and conventional groups, respectively. The primary patency rates at six months were 89.1% and 85.2% (P = 0.670), and freedom from target-lesion-revascularization rate at six months was 100% and 92.8% (P = 0.691) in the Chocolate and conventional groups, respectively. CONCLUSION: Chocolate percutaneous transluminal angioplasty balloons reduce the rate of severe dissection while maintaining a sufficient dilatation effect during drug-coated balloon vessel preparation.

Impact of Extravascular Ultrasound-Guided Wiring on Achieving Optimal Vessel Preparation and Patency in Endovascular Therapy for Superficial Femoral Artery Chronic Total Occlusion.

PURPOSE: The study aim was to evaluate the impact of extravascular ultrasound-guided (EVUSG) wiring on achieving optimal vessel preparation and patency in endovascular therapy (EVT) for superficial femoral artery (SFA) chronic total occlusion (CTO). METHODS: Between April 2007 and January 2019, a total of 239 SFA-CTO limbs were successfully treated with EVT and bailout implantation of self-expandable nitinol stents at our hospital. The study subjects were divided into 2 groups according to the type of guidance strategy used during CTO wiring, ie, the EVUSG group and the conventional angiography guidance (AG) group. Immediately after the initial balloon angioplasty and successful passage of the wire through the SFA-CTO lesions, the EVUSG (65 limbs) and AG groups (174 limbs) were retrospectively evaluated for angiographic dissection patterns. The primary patency rate was also compared between the 2 groups. RESULTS: No significant difference was observed in the balloon diameter at the initial dilation immediately after successful wire passing (3.7 ± 0.5 mm in the EVUSG group vs 3.8 ± 0.5 mm in the AG group; P=.17). The incidence of severe dissection was significantly lower (P<.001) in the EVUSG group (28/65; 43%) than in the AG group (137/174; 79%). The 3-year primary patency rates in the EVUSG and AG groups were 84.5% and 68.4%, respectively (P<.001). CONCLUSIONS: EVUSG for SFA-CTO may achieve optimal vessel preparation, defined as an initial balloon angioplasty without severe dissection, and subsequent implantation of self-expandable stents may lead to a better patency rate.

Purification of Emu IgY for Therapeutic and Diagnostic Use Based on Monoclonal Secondary Antibodies Specific to Emu IgY.

The emu is the second largest ratite; thus, their sera and egg yolks, obtained after immunization, could provide therapeutic and diagnostically important immunoglobulins with improved production efficiency. Reliable purification tools are required to establish a pipeline for supplying practical emu-derived antibodies, the majority of which belongs to the immunoglobulin Y (IgY) class. Therefore, we generated a monoclonal secondary antibody specific to emu IgY. Initially, we immunized an emu with bovine serum albumin multiply haptenized with 2,4-dinitrophenyl (DNP) groups. Polyclonal emu anti-DNP antibodies were partially purified using conventional precipitation method and used as antigen for immunizing a BALB/c mouse. Splenocytes were fused with myeloma cells and a hybridoma clone secreting a desirable secondary antibody (mAb#2-16) was established. The secondary antibody bound specifically to emu-derived IgY, distinguishing IgYs from chicken, duck, ostrich, quail, and turkey, as well as human IgGs. Affinity columns immobilizing the mAb#2-16 antibodies enabled purification of emu IgY fractions from sera and egg yolks via simple protocols, with which we succeeded in producing IgYs specific to the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) spike protein with a practical binding ability. We expect that the presented purification method, and the secondary antibody produced in this study, will facilitate the utilization of emus as a novel source of therapeutic and diagnostic antibodies.

Derivatization-assisted immunoassays: application for group-specific detection of potent methamphetamine and amphetamine enantiomers.

Reliable and feasible tools for detecting (S)-methamphetamine [(S)-MAP] and (S)-amphetamine [(S)-AP] are required for regulating their illicit circulation. Antibodies that react equally to these stimulants are desirable for this purpose, but have been difficult to generate because of the crucial difference between their characteristic structures: i.e., N-methylamino (MAP) and amino (AP) groups. Furthermore, their small molecular masses (Mr < 150) have hampered the generation of high-affinity antibodies. To overcome these problems, we converted (S)-MAP and -AP into their 2-(trimethylsilyl)ethyl carbamate forms, Teoc-(S)-MAP and -AP, respectively, as surrogate analytes. The Teoc-derivatization not only increases their molecular masses, but also masks their structural differences. We generated a novel monoclonal antibody that showed a satisfactory affinity to Teoc-(S)-MAP residues (Kd = 13 nM as the IgG form) and developed a competitive enzyme-linked immunosorbent assay (ELISA) using microplates containing immobilized Teoc-(S)-MAP residues. Almost overlapping dose-response curves were obtained for Teoc-(S)-MAP and -AP, with the limit of detection of 0.078 and 0.10 ng per assay, respectively. A fixed amount of test powder sample (1 mg) was derivatized with Teoc-O-succinimidyl for 5 min, and subjected to ELISA using Teoc-(S)-MAP as the calibration standard. Under this protocol, (S)-MAP and -AP were converted to their Teoc derivatives with 30% and 34% yield, respectively, determined using ELISA as "Teoc-(S)-MAP equivalent," being distinguished from the derivatization products of (R)-MAP, (R)-AP, ephedrine, (S)-methylenedioxymethamphetamine, tyramine, dopamine, and ß-alanine. This ELISA detected as little as 10 µg of (S)-MAP and -AP, and (S)-MAP in urine obtained from (S)-MAP-administered rats. Immunochromatography devices were also developed using gold nanoparticles coated with the monoclonal antibody, with which 0.10 mg of (S)-MAP and -AP was detected by the naked eye. We conclude that the present derivatization-assisted immunoassays may be useful for the detection of (S)-MAP and/or -AP in early stage screening of suspicious substances.

More than 370-Fold Increase in Antibody Affinity to Estradiol-17ß by Exploring Substitutions in the VH-CDR3.

Antibodies that specifically target biomarkers are essential in clinical diagnosis. Genetic engineering has assisted in designing novel antibodies that offer greater antigen-binding affinities, thus providing more sensitive immunoassays. We have succeeded in generating a single-chain Fv fragment (scFv) targeted estradiol-17ß (E2) with more than 370-fold improved affinity, based on a strategy focusing the complementarity-determining region 3 in the VH domain (VH-CDR3). Systematic exploration of amino acid substitutions therein, using a clonal array profiling, revealed a cluster of four substitutions, containing H99P and a serial substitution E100eN-I100fA-L100gQ that lead to a 90-fold increase in E2-binding affinity. This substitution quartet in the VH-CDR3, combined with the substitution cluster I29V/L36M/S77G in the VL domain, resulted in a scFv fragment with a further increase in the affinity (Ka, 3.2 × 1010 M-1). This enabled a highly sensitive enzyme-linked immunosorbent assay capable of detecting up to 0.78 pg/assay. The current study has, thus, focused on the significance of reevaluating the potential of mutagenesis targeting the VH-CDR3, and encouraging the production and use of engineered antibodies that enable enhanced sensitivities as next-generation diagnostic tools.

Procedural results and in-hospital outcomes of percutaneous coronary intervention for chronic total occlusion in patients with reduced left ventricular ejection fraction: Sub-analysis of the Japanese CTO-PCI Expert Registry.

OBJECTIVES: To evaluate the procedural results and in-hospital outcomes of percutaneous coronary intervention (PCI) for chronic total occlusion (CTO) in patients with reduced left ventricular ejection fraction (LVEF). BACKGROUND: While the technical success of general CTO-PCI has improved, CTO-PCI patients with reduced LVEF remain at high-risk for adverse events. METHODS: The data of 820 patients with LVEF ≤ 35% (Group 1), 1816 patients with LVEF = 35%-50% (Group 2), and 5503 patients with LVEF ≥ 50% (Group 3), registered in the Japanese CTO-PCI Expert Registry from January 2014 to December 2019, were retrospectively analyzed. The primary endpoint was in-hospital major adverse cardiac or cerebrovascular events (MACCEs), including death, myocardial infarction, stent thrombosis, stroke, and emergent revascularization. Secondary endpoints included procedural details, guidewire success, and technical success. RESULTS: There were no differences in guidewire and technical success rates between the groups. In-hospital MACCEs was significantly higher in Group 1 (Group 1 vs. Group 2 vs. Group 3: 3.4% vs. 1.7% vs. 1.5%, p = 0.001) and was especially driven by death (1.3% vs. 0.3% vs. 0.1%, p < 0.001) and stroke (0.7% vs. 0.2% vs. 0.2%, p = 0.007). Multivariate analysis showed that LVEF ≤ 35% (odds ratio [OR]; 1.58, 95% confidence interval [CI]; 1.04-2.41, p = 0.03) and New York Heart Association (NYHA) class ≥ 3 (OR; 2.01, 95% CI; 1.03-3.93, p = 0.04) were predictors of in-hospital MACCEs. CONCLUSIONS: In-hospital MACCEs were significantly higher in patients with LVEF ≤ 35%. LVEF ≤;35% and NYHA class ≥ 3 were predictors of in-hospital MACCEs after CTO-PCI.

Two-year intermittent exposure of a multiwalled carbon nanotube by intratracheal instillation induces lung tumors and pleural mesotheliomas in F344 rats.

BACKGROUND: A mounting number of studies have been documenting the carcinogenic potential of multiwalled carbon nanotubes (MWCNTs); however, only a few studies have evaluated the pulmonary carcinogenicity of MWCNTs in vivo. A 2-year inhalation study demonstrated that MWNT-7, a widely used MWCNT, was a pulmonary carcinogen in rats. In another 2-year study, rats administered MWNT-7 by intratracheal instillation at the beginning of the experimental period developed pleural mesotheliomas but not lung tumors. To obtain data more comparable with rats exposed to MWNT-7 by inhalation, we administered MWNT-7 to F344 rats by intratracheal instillation once every 4-weeks over the course of 2 years at 0, 0.125, and 0.5 mg/kg body weight, allowing lung burdens of MWNT-7 to increase over the entire experimental period, similar to the inhalation study. RESULTS: Absolute and relative lung weights were significantly elevated in both MWNT-7-treated groups. Dose- and time-dependent toxic effects in the lung and pleura, such as inflammatory, fibrotic, and hyperplastic lesions, were found in both treated groups. The incidences of lung carcinomas, lung adenomas, and pleural mesotheliomas were significantly increased in the high-dose group compared with the control group. The pleural mesotheliomas developed mainly at the mediastinum. No MWNT-7-related neoplastic lesions were noted in the other organs. Cytological and biochemical parameters of the bronchoalveolar lavage fluid (BALF) were elevated in both treated groups. The lung burden of MWNT-7 was dose- and time-dependent, and at the terminal necropsy, the average value was 0.9 and 3.6 mg/lung in the low-dose and high-dose groups, respectively. The number of fibers in the pleural cavity was also dose- and time-dependent. CONCLUSIONS: Repeated administration of MWNT-7 by intratracheal instillation over the 2 years indicates that MWNT-7 is carcinogenic to both the lung and pleura of rats, which differs from the results of the 2 carcinogenicity tests by inhalation or intratracheal instillation.