Fundamental Characterization of Antibody Fusion-Single-Chain TNF Recombinant Proteins Directed against Costimulatory TNF Receptors Expressed by T-Lymphocytes.

The costimulatory signal regulated by the members of the tumor necrosis factor receptor (TNFR) superfamily expressed by T cells plays essential roles for T cell responses and has emerged as a promising target for cancer immunotherapy. However, it is unclear how the difference in TNFR costimulation contributes to T cell responses. In this study, to clarify the functional significance of four different TNFRs, OX40, 4-1BB, CD27 and GITR, we prepared corresponding single-chain TNF ligand proteins (scTNFLs) connected to IgG Fc domain with beneficial characteristics, i.e., Fc-scOX40L, Fc-sc4-1BBL, Fc-scCD27L (CD70) and Fc-scGITRL. Without intentional cross-linking, these soluble Fc-scTNFL proteins bound to corresponding TNFRs induced NF-kB signaling and promoted proliferative and cytokine responses in CD4+ and CD8+ T cells with different dose-dependencies in vitro. Mice injected with one of the Fc-scTNFL proteins displayed significantly augmented delayed-type hypersensitivity responses, showing in vivo activity. The results demonstrate that each individual Fc-scTNFL protein provides a critical costimulatory signal and exhibits quantitatively distinct activity toward T cells. Our findings provide important insights into the TNFR costimulation that would be valuable for investigators conducting basic research in cancer immunology and also have implications for T cell-mediated immune regulation by designer TNFL proteins.

Generation of two induced pluripotent stem cell lines from individuals without auditory disorders.

We report the establishment of two human induced pluripotent stem cell (iPSC) lines from individuals without auditory disorders. Extensive audiometry tests were performed to confirm normal hearing. The generated iPSC lines expressed pluripotency genes and showed differentiation capability into the three germ layers. The iPSC lines will be used as controls for pathological analysis and drug screening for ear disorders.

Two O-Methyltransferases from Phylogenetically Unrelated Cow Parsley (Anthriscus sylvestris) and Hinoki-Asunaro (Thujopsis dolabrata var. hondae) as a Signature of Lineage-Specific Evolution in Lignan Biosynthesis.

O-Methyltransferases (OMTs) play important roles in antitumor lignan biosynthesis. To date, six OMTs catalyzing the methylation of dibenzylbutyrolactone lignans as biosynthetic precursors of antitumor lignans have been identified. However, there is still no systematic understanding of the diversity and regularity of the biosynthetic mechanisms among various plant lineages. Herein, we report the characterization of two OMTs from Anthriscus sylvestris and Thujopsis dolabrata var. hondae [designated as AsSecoNorYatein (SNY) OMT and TdSNYOMT] together with the six known OMTs to evaluate their diversity and regularity. Although A. sylvestris 5-O-methylthujaplicatin (SecoNorYatein) and 4-O-demethylyatein (NorYatein) OMT (AsSNYOMT) and TdSNYOMT accept 5-O-methylthujaplicatin and 4-O-demethylyatein as substrates, phylogenetic analysis indicated that these two OMTs shared low amino acid sequence identity, 33.8%, indicating a signature of parallel evolution. The OMTs and the six previously identified OMTs were found to be diverse in terms of their substrate specificity, regioselectivity and amino acid sequence identity, indicating independent evolution in each plant species. Meanwhile, two-entropy analysis detected four amino acid residues as being specifically acquired by dibenzylbutyrolactone lignan OMTs. Site-directed mutation of AsSNYOMT indicated that two of them contributed specifically to 5-O-methylthujaplicatin methylation. The results provide a new example of parallel evolution and the diversity and regularity of OMTs in plant secondary (specialized) metabolism.

Selective Synthesis of the Aminobutadiene Intermediate and Mechanistic Analysis of 1,4-Dihydropyridine Formation Reaction in Water.

We previously isolated an aminobutadiene derivative as a by-product in the synthesis of a 1,4-dihydropyridine (1,4-DHP) derivative by the reaction of methyl propiolate with excess ammonium acetate in water, and we proposed that it is an intermediate in the formation of 1,4-DHP. Here, to test this idea and to investigate the reaction mechanism, we selectively synthesized the aminobutadiene derivative in EtOH and examined its reactivity. The yield of the aminobutadiene derivative was increased in the presence of excess ammonium salt. X-Ray crystal structure analysis indicated the presence of an intramolecular hydrogen bond between the terminal amine and ester carbonyl oxygen, together with a short C-N bond length consistent with enamine-imine equilibrium. Direct cyclization of the aminobutadiene derivative with methyl propiolate to afford the 1,4-DHP derivative did not proceed well, but the yield was increased in the presence of morpholine salt as an additive. These results suggest that the predominant reaction pathway from the intermediate to 1,4-DHP in water involves Michael addition of a second amine molecule and reaction with methyl propiolate, followed by intramolecular cyclization and elimination of amine.

[Bilateral Medial Medulla Infarction Mimicking Guillain-Barré Syndrome and its Variants].

A 70-year-old man presented with dizziness and unsteadiness when standing and was hospitalized in another hospital. Magnetic resonance imaging (MRI) of the brain on Day 1 showed no abnormalities. The patient developed respiratory failure on Day 1and flaccid tetraplegia on Day 3, and was transferred to our hospital. Progressive upper and lower limb weakness and bulbar symptoms suggested Guillain-Barré syndrome or its variant. Diffusion-weighted MRI on Day 6 disclosed high signal intensities in the bilateral medial portion of the medulla, and the patient was diagnosed with bilateral medial medulla infarction. Bilateral medial medulla infarction should be considered when a patient shows progressive tetraplegia, and bulbar palsy and follow-up MRI is important to confirm the diagnosis. (Received January 23, 2020; Accepted April 21, 2020; Published August 1, 2020).

Sexual dimorphism in external morphology of the American bullfrog Rana (Aquarana) catesbeiana and the possibility of sex determination based on tympanic membrane/eye size ratio.

The American bullfrog Rana (Aquarana) catesbeiana has been reported to show significant sexual dimorphism based on the size ratio between the tympanic membrane and the eye. In males the tympanic membrane is much larger than the eye, but not in females. The ratio has been used as a convenient criterion to discriminate sexes (sexing) in the American bullfrog, though its reliability is unknown. In this study, we examined 86 adult American bullfrogs to clarify whether the tympanic membrane long diameter/eye long diameter (Dtm/De) ratio is a reliable index to discriminate sexes in this species. In addition, we examined the growth of this sexually dimorphic trait. Results indicated that there is a significant difference but there is a small overlap in this ratio Dtm/De between sexes. The allometric comparisons showed the sexual dimorphism of the Dtm/De ratio was increased during growth and the dimorphism is attributable to the difference in the growth rate of the tympanic membrane (Dtm). Therefore, sex determination of American bullfrogs cannot be wholly reliably achieved by the Dtm/De ratio alone; other external morphological features are required in addition.

Stable Immune Response Induced by Intradermal DNA Vaccination by a Novel Needleless Pyro-Drive Jet Injector.

DNA vaccination can be applied to the treatment of various infectious diseases and cancers; however, technical difficulties have hindered the development of an effective delivery method. The efficacy of a DNA vaccine depends on optimal antigen expression by the injected plasmid DNA. The pyro-drive jet injector (PJI) is a novel system that allows for adjustment of injection depth and may, thus, provide a targeted delivery approach for various therapeutic or preventative compounds. Herein, we investigated its potential for use in delivering DNA vaccines. This study evaluated the optimal ignition powder dosage, as well as its delivery effectiveness in both rat and mouse models, while comparing the results of the PJI with that of a needle syringe delivery system. We found that the PJI effectively delivered plasmid DNA to intradermal regions in both rats and mice. Further, it efficiently transfected plasmid DNA directly into the nuclei, resulting in higher protein expression than that achieved via needle syringe injection. Moreover, results from animal ovalbumin (OVA) antigen induction models revealed that animals receiving OVA expression plasmids (pOVA) via PJI exhibited dose-dependent (10 µg, 60 µg, and 120 µg) production of anti-OVA antibodies; while only low titers (< 1/100) of OVA antibodies were detected when 120 µg of pOVA was injected via needle syringe. Thus, PJI is an effective, novel method for delivery of plasmid DNA into epidermal and dermal cells suggesting its promise as a tool for DNA vaccination.

Stress-related over-enhancement of the hypothalamic-pituitary-adrenal axis causes experimental neurolathyrism in rats.

Neurolathyrism is a motor neuron disease that is caused by the overconsumption of grass peas (Lathyrus sativus L.) under stressful conditions. The neuro-excitatory ß-N-oxalyl-L-α,ß-diaminopropionic acid present in grass peas was proposed the causative agent of spastic paraparesis of the legs. Historical reports of neurolathyrism epidemics, studies of neurolathyrism animal models, and in vitro studies on the mechanism of ß-N-oxalyl-L-α,ß-diaminopropionic acid toxicity support the hypothesis that stress increases susceptibility to neurolathyrism. To elucidate the role of stress in neurolathyrism-induced motor dysfunction, we focused on the hypothalamic-pituitary-adrenal axis in a rodent model of neurolathyrism. Our results implicated increased glucocorticoid and neuroinflammation in the motor dysfunction (paraparesis) exhibited by the stress loaded rat models of neurolathyrism.

Usefulness of acceleration time ratio in diagnosis of internal carotid artery origin stenosis.

PURPOSE: The acceleration time (AcT) ratio of the internal carotid artery (ICA) is increased in ICA stenosis. However, there are few reports that have directly compared the AcT ratio to digital subtraction angiography (DSA) findings. METHODS: We evaluated 177 vessels with DSA and carotid artery ultrasonography. The AcT ratio was calculated as AcT of the ICA (ICA-AcT)/AcT of the ipsilateral common carotid artery (CCA). We evaluated the correlation of DSA-NASCET stenosis with the origin of the ICA or the peak systolic velocity (ICApsv) in the stenotic region, ICApsv/peak systolic velocity of the CCA (CCApsv), ICA-AcT, and AcT ratio. Sensitivity and specificity for stenosis ≥ 70% were calculated based on the ICApsv, ICApsv/CCApsv, ICA-AcT, and AcT ratio. RESULTS: Using NASCET criteria, 34 vessels had 70% or greater stenosis. DSA-NASCET showed a significant positive correlation with ICApsv, ICApsv/CCApsv, ICA-AcT, and AcT ratio (p < 0.0001). When the cut-off value for ICApsv was set at 176 cm/s, ICApsv/CCApsv at 2.42, ICA-AcT at 0.095 s, and the AcT ratio at 1.35, the sensitivity was 97.1, 97.1, 82.4, and 97.1%, and the specificity was 94.4, 91.0, 83.2, and 83.2%, for DSA-NASCET ≥ 70%, respectively. CONCLUSION: The AcT ratio is a beneficial parameter for evaluating ICA stenosis as well as ICApsv and ICApsv/CCApsv.

Comparison of carotid artery ultrasonography findings between acute coronary syndrome and atherothrombotic cerebral infarction.

BACKGROUND AND PURPOSE: Although risk factors for acute coronary syndrome (ACS) and atherothrombotic cerebral infarction (ACI) are common, it is unknown if the risk factors for these two conditions are similar. The purpose of our study was to elucidate differences in carotid artery atherosclerotic features between ACS and ACI. METHODS: We measured carotid artery ultrasound-based atherosclerotic parameters in 61 ACS and 33 ACI patients. In the ACS patients, 31 had single-vessel coronary artery disease (SVD) and 30 had multivessel coronary artery disease (MVD). RESULTS: The maximum intima-media thickness (IMT) of the common carotid artery was higher in ACS patients with MVD than in ACS patients with SVD (P < 0.05), and tended to be higher than that in ACI patients (P = 0.085). The values in ACS patients with SVD and ACI patients were similar. The maximum IMT of the carotid artery bulb in ACS patients with MVD was similar to that in both ACS patients with SVD and ACI patients. The plaque score was higher in ACS patients with MVD than in ACS patients with SVD (P < 0.01), but similar to that in ACI patients. CONCLUSION: ACS and ACI show common atherosclerotic features as assessed by carotid artery ultrasonography.

Prevalence of Right to Left Shunts in Japanese Patients with Migraine: A Single-center Study.

Objective Several studies have shown an increased prevalence of right-to-left shunt (RLs) in migraine patients, particularly those with aura. However, the prevalence of RLs and its relation to Japanese patients with migraine are unknown. We investigated the prevalence of RLs in Japanese patients with migraine. Methods In total, 112 consecutive patients with migraine were recruited from our headache outpatient clinic. Migraine with aura (MA) and migraine without aura (MWOA) were diagnosed according to the International Classification of Headache Disorders, 3rd edition (beta-version). Contrast transcranial Doppler ultrasound was used to detect RLs, including patent foramen ovale (PFO). Then, the associations between RLs and patients' backgrounds and presence of aura were assessed. Results The overall prevalence of RLs and PFO in migraine patients was 54.5% and 43.8%, respectively. The prevalence of RLs and PFO in the MA group were significantly higher than in the MWOA group (RLs, 62.9% vs. 44.0%, p=0.046; PFO, 54.8% vs. 30.0%, p=0.008). There were no marked differences in the prevalence of large, middle and small shunts between MA and MWOA patients. Compared with the MWOA patients, the MA patients were younger (p=0.013) and had early onset age (p=0.013) and increased prevalence of photophobia (p=0.008). Conclusion RLs were found in over half of the Japanese patients with migraine. Our study suggests a possible link between RLs and MA.

Increased maximum common carotid intima-media thickness is associated with smoking and hypertension in Tochigi Prefecture residents.

OBJECTIVE: We investigated maximum intima-media thickness of the common carotid artery (IMT-Cmax) in residents of Tochigi Prefecture, who have been reported to have high stroke mortality. METHOD: Our study included 840 individuals. All participants underwent carotid ultrasonography and answered a questionnaire during participation in a health festival in Tochigi Prefecture. The questionnaire was designed to collect information on age, gender, and risk factors for stroke. IMT-Cmax was measured. Statistical analyses were performed to identify factors contributing to IMT-Cmax values ≥1.1 mm. RESULTS: In total, 117 subjects had an IMT-Cmax value ≥1.1 mm. IMT-Cmax correlated significantly with age, current smoking, hypertension, diabetes mellitus, heart disease, and previous symptomatic stroke (p < 0.05) in univariate analysis. Current smoking (p < 0.001, odds ratio 3.88) and hypertension (p = 0.0070, odds ratio 1.83) were seen as significant contributing factors to IMT-Cmax ≥1.1 mm in logistic regression analysis adjusted by age, gender, and previous symptomatic stroke. CONCLUSION: We identified current smoking and hypertension as the most significant contributing factors to increased IMT-Cmax in residents of Tochigi Prefecture, emphasizing the importance of routine blood pressure monitoring and anti-smoking education in this population.

The relationship between right-to-left shunt and brain white matter lesions in Japanese patients with migraine: a single center study.

BACKGROUND: There may be a link between right-to-left shunt (RLs) and brain white matter lesions (WMLs) in patients with migraine. In this study, we assessed the relationship between WMLs and RLs in Japanese migraine patients. METHODS: A total of 107 consecutive patients with migraine with (MA) and without aura (MWOA) were included in this study. Contrast transcranial Doppler ultrasound was used to detect RLs. WMLs were graded using brain magnetic resonance imaging based on well-established criteria. FINDINGS: The prevalence of RLs was significantly increased in the WMLs positive group (n = 24) compared with the WMLs negative group (n = 83) (75.0% vs. 47.0%, p = 0.015). In prevalence of WMLs between MA and MWOA patients, there were no statistical differences (p = 0.410). Logistic regression analysis adjusted by age and disease duration of migraine identified an RLs-positive status as the sole determinant for the presence of WMLs (OR = 6.15; 95% CI 1.82-20.8; p = 0.003) CONCLUSION: Our study suggests a possible link between RLs and WMLs in Japanese patients with migraine.

Endothelial cell-specific expression of roundabout 4 is regulated by differential DNA methylation of the proximal promoter.

OBJECTIVE: The molecular basis of endothelial cell (EC)-specific gene expression is poorly understood. Roundabout 4 (Robo4) is expressed exclusively in ECs. We previously reported that the 3-kb 5'-flanking region of the human Robo4 gene contains information for lineage-specific expression in the ECs. Our studies implicated a critical role for GA-binding protein and specificity protein 1 (SP1) in mediating overall expression levels. However, these transcription factors are also expressed in non-ECs. In this study, we tested the hypothesis that epigenetic mechanisms contribute to EC-specific Robo4 gene expression. METHODS AND RESULTS: Bisulfite sequencing analysis indicated that the proximal promoter of Robo4 is methylated in non-ECs but not in ECs. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine increased Robo4 gene expression in non-ECs but not in ECs. Proximal promoter methylation significantly decreased the promoter activity in ECs. Electrophoretic mobility shift assays showed that DNA methylation of the proximal promoter inhibited SP1 binding to the -42 SP1 site. In DNase hypersensitivity assays, chromatin condensation of the Robo4 promoter was observed in some but not all nonexpressing cell types. In Hprt (hypoxanthine phosphoribosyltransferase)-targeted mice, a 0.3-kb proximal promoter directed cell-type-specific expression in the endothelium. Bisulfite sequencing analysis using embryonic stem cell-derived mesodermal cells and ECs indicated that the EC-specific methylation pattern of the promoter is determined by demethylation during differentiation and that binding of GA-binding protein and SP1 to the proximal promoter is not essential for demethylation. CONCLUSIONS: The EC-specific DNA methylation pattern of the Robo4 proximal promoter is determined during cell differentiation and contributes to regulation of EC-specific Robo4 gene expression.

Cauliflower ear dissection.

Cauliflower ear (CE) is caused by repeated direct trauma to the external ear. Surgical correction of an established CE is one of the most challenging problems in ear reconstruction. However, no reports have clarified the dissection of an established CE in detail. In this report, the dissection of a CE is described based on macroscopic, microscopic and imaging features.

Mid-arm lymph nodes dissection for melanoma.

An interval node in the upper limb termed the mid-arm node was recently identified. However, its surgical anatomy remains unclear. We report a patient with metastatic melanoma of the mid-arm node and the epitrochlear node at 10 years after removal of the primary tumour from the forearm and therapeutic axillary lymph node dissection. The mid-arm node is located halfway up the upper arm on the medial intermuscular septum, at the site where the brachial vessels, the median nerve and the ulnar nerve run adjacent to each other. The mid-arm node lies adjacent to the basilic vein where lymphatic vessels ascend and converge. This is the first report regarding the surgical anatomy of the mid-arm node.

Prefabricated scalping forehead flap with skeletal support.

It is difficult to reconstruct a nose with adequate shape, color, and texture in patients who have full-thickness nasal defects with extensive loss of skeletal support. The scalping forehead flap is a reliable technique for nasal reconstruction. To our knowledge, however, there have been no reports about a prefabricated scalping forehead flap with a bone graft as skeletal support. In the case reported here, a prefabricated scalping forehead flap combined with an iliac bone graft as skeletal support was used to successfully reconstruct a full-thickness defect of the nose associated with partial frontal bone loss and complete loss of the nasal bones. Acceptable functional and aesthetic results were achieved. This method may be a good alternative for reconstruction of full-thickness nasal defects with extensive loss of skeletal support.

Sentinel lymph node detection in skin cancer patients using real-time fluorescence navigation with indocyanine green: preliminary experience.

BACKGROUND: Sentinel lymph node (SLN) biopsy has become the most widely used procedure to determine the regional lymph node status of patients with cutaneous malignant melanoma, and its application has spread rapidly to other malignancies such as squamous cell carcinoma of the skin, breast cancer and gastric cancer. OBJECTIVE: SLN detection was performed in skin cancer patients using a newly developed real-time fluorescence navigation system with indocyanine green (ICG). METHODS: SLN biopsy was performed by this method in 10 skin cancer patients (seven with melanoma and three with squamous cell carcinoma). After ICG was injected intradermally around the tumours, the lymphatic drainage was detected and the SLNs were identified by real-time fluorescence imaging. RESULTS: The SLNs and their associated subcutaneous lymphatics were successfully identified in all patients. Fluorescence from SLNs was detected by this system for at least 3 hours after the injection of ICG. CONCLUSION: SLN biopsy using ICG fluorescence achieves a high identification rate and allows effective observation for several hours. This method may become a useful option for the detection of SLNs in patients with skin cancer.