Centrifugal Field-Flow Fractionation Enables Detection of Slight Aggregation of Nanoparticles That Impacts Their Biomedical Applications.

Nanoparticles (NPs) are anticipated to be used for various biomedical applications in which their aggregation has been an important issue. However, concerns regarding slightly aggregated but apparently monodispersed NPs have been difficult to address because of a lack of appropriate evaluation methods. Here, we report centrifugal field-flow fractionation (CF3) as a powerful method for analyzing the slight aggregation of NPs, using antibody-modified gold NPs (Ab-AuNPs) prepared by a conventional protocol with centrifugal purification as a model. While common evaluation methods such as dynamic light scattering cannot detect significant signs of aggregation, CF3 successfully detects distinct peaks of slightly aggregated NPs, including dimers and trimers. Their impact on biological interactions was also demonstrated by a cellular uptake study: slightly aggregated Ab-AuNPs exhibited 1.8 times higher cellular uptake than monodispersed Ab-AuNPs. These results suggest the importance of aggregate evaluation via CF3 as well as the need for careful attention to the bioconjugation procedures for NPs.

Intraocular Water Movement Visualization Using 1 H-MRI With Eye Drops of O-17-Labeled Saline: First-in-Human Study.

BACKGROUND: Visualization of aqueous humor flow in MR contrast images using gadolinium is challenging because of the delayed contrast effects associated with the blood-retinal and blood-aqueous humor barriers. However, oxygen-17 water (H2 17 O) might be used as an ocular contrast agent. PURPOSE: To observe the distribution of H2 17 O in the human eye, and its flow in and out of the anterior chamber, using dynamic T2-weighted MRI. STUDY TYPE: Prospective. POPULATION: Six ophthalmologically normal volunteers (20-37 years, six females). FIELD STRENGTH/SEQUENCE: A 3 T/dynamic T2-weighted MRI. ASSESSMENT: H2 17 O eye drops were administered to the right eye. Time-series images were created by subtracting the image before the eye drops from each of the images obtained after the eye drops. The normalized signal intensity of the right anterior chamber (nAC) was obtained by dividing the signal intensity of the right anterior chamber region by that of the left. The inflow and outflow constants of H2 17 O and H2 17 O concentration were calculated from the nAC. STATISTICAL TESTS: A paired t-test was used to compare the flow-related values and temporal changes in signal intensity. P-values < 0.05 were considered statistically significant. RESULTS: Significantly decreased signal intensity was observed in the right anterior chamber but not the right vitreous body (P = 0.39). The nAC signal intensity decreased significantly and then recovered. The inflow and outflow constants were 0.36-0.94 min-1 and 0.023-0.13 min-1 , respectively. The maximum H2 17 O concentration was 0.078%-0.24%. DATA CONCLUSION: H2 17 O were distributed in the anterior chamber. The H2 17 O inflow into the anterior chamber was significantly faster than that of the outflow. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 2.

Effective Epilepsy Surgery for Post-Traumatic West Syndrome Following Abusive Head Trauma.

West syndrome, an infantile developmental and epileptic encephalopathy with a deleterious impact on long-term development, requires early treatment to minimize developmental abnormality; in such cases, epilepsy surgery should be considered a powerful therapeutic option. We describe a 10-month-old female admitted with West syndrome associated with a hemispheric lesion following abusive head trauma. Her seizures were suppressed by hemispherotomy at 12 months of age, leading to developmental improvement. Surgical treatment of West syndrome following traumatic brain injury has not been reported previously but is worth considering as a treatment option, depending on patient age and brain plasticity.

Artificial Intelligence-based Detection of Epileptic Discharges from Pediatric Scalp Electroencephalograms: A Pilot Study.

We developed an artificial intelligence (AI) technique to identify epileptic discharges (spikes) in pediatric scalp electroencephalograms (EEGs). We built a convolutional neural network (CNN) model to automatically classify steep potential images into spikes and background activity. For the CNN model' training and validation, we examined 100 children with spikes in EEGs and another 100 without spikes. A different group of 20 children with spikes and 20 without spikes were the actual test subjects. All subjects were ≥ 3 to < 18 years old. The accuracy, sensitivity, and specificity of the analysis were >0.97 when referential and combination EEG montages were used, and < 0.97 with a bipolar montage. The correct classification of background activity in individual patients was significantly better with a referential montage than with a bipolar montage (p=0.0107). Receiver operating characteristic curves yielded an area under the curve > 0.99, indicating high performance of the classification method. EEG patterns that interfered with correct classification included vertex sharp transients, sleep spindles, alpha rhythm, and low-amplitude ill-formed spikes in a run. Our results demonstrate that AI is a promising tool for automatically interpreting pediatric EEGs. Some avenues for improving the technique were also indicated by our findings.

Long-Term Outcomes in Patients with Not-Retrieval Inferior Vena Cava Filter Under Anticoagulation.

Since permanent inferior vena cava (IVC) filters increase deep vein thrombosis (DVT), filter retrieval should be performed as possible. Despite the guideline recommendation, IVC filters are not always retrieved in clinical practice. To date, many patients with not-retrieval IVC filters have been prescribed anticoagulant therapy, but the long-term prognosis, including venous thromboembolism (VTE) and bleeding events, remains unknown. In this study, 195 patients who underwent IVC filter implantation between 2006 and 2017 at 3 institutions in Niigata City have been investigated about their deaths, VTE recurrence, and bleeding events. After peaking 2009, the number of IVC filter implantation gradually decreased. During observational period, there were 158 patients with not-retrieval IVC filters (the overall retrieval rate of 19.0%). The not-retrieval group included significantly older and more patients with cancer compared to the retrieval group. Anticoagulation therapy was continued in 88% of the not-retrieval group. During a mean follow-up of 5.0 years, 6 symptomatic DVT events associated with inadequate control of anticoagulation and 13 bleeding events were observed. A total of 52 patients died and only the presence of cancer was prognostic risk factor. Although long-term anticoagulation therapy may be associated with bleeding events, there were few recurrent VTE under optimal anticoagulation. It is anticipated that even if the IVC filter cannot be retrieved, appropriate anticoagulation is useful for prevention of DVT recurrence despite the risk of bleeding.

A Case of Chronic Heart Failure Complicated by Primary Biliary Cholangitis and Skeletal Myopathy.

Several autoantigens related to inflammatory myopathy have been identified. Antimitochondrial antibody M2 (AMA-M2) is known as one of the serologic hallmarks of primary biliary cholangitis (PBC). There have been several reports on the association between AMA-M2 and various types of inflammatory myopathy, including cardiomyopathy. We report a case of a 58-year-old man with decompensated heart failure who also had PBC and skeletal inflammatory myopathy. Endomyocardial biopsy revealed severe fibrotic replacement of the myocardium without massive inflammatory infiltration, which was pathologically similar to what happens in dilated cardiomyopathy (DCM). Although the potential relationship between chronic autoimmune inflammation and DCM has been discussed, the concept of the inflammatory DCM has not yet been established. When we see elevated liver enzymes, and which is not simply due to congestive hepatopathy, we should consider the coexisting disease such as PBC.

Diffusion-tensor-based method for robust and practical estimation of axial and radial diffusional kurtosis.

OBJECTIVES: A new method that can estimate diffusional kurtosis image (DKI), estimated DKI (eDKI), parallel and perpendicular to neuronal fibres from greatly limited image data was designed to enable quick and practical assessment of DKI in clinics. The purpose of this study was to discuss the potential of this method for clinical use. METHODS: Fourteen healthy volunteers were examined with a 3-Tesla MRI. The diffusion-weighting parameters included five different b-values (0, 500, 1,500, 2,000 and 2,500 s/mm(2)) with 64 different encoding directions for each of the b-values. K values were calculated by both conventional DKI (convDKI) and eDKI from these complete data, and also from the data that the encoding directions were abstracted to 32, 21, 15, 12 and 6. Error-pixel ratio and the root mean square error (RMSE) compared with the standard were compared between the methods (Wilcoxon signed-rank test: P < 0.05 was considered significant). RESULTS: Error-pixel ratio was smaller in eDKI than in convDKI and the difference was significant. In addition, RMSE was significantly smaller in eDKI than in convDKI, or otherwise the differences were not significant when they were obtained from the same data set. CONCLUSION: eDKI might be useful for assessing DKI in clinical settings. KEY POINTS: • A method to practically estimate axial/radial DKI from limited data was developed. • The high robustness of the proposed method can greatly improve map images. • The accuracy of the proposed method was high. • Axial/radial K maps can be calculated from limited diffusion-encoding directions. • The proposed method might be useful for assessing DKI in clinical settings.

Possible involvement of convergent nociceptive input to medullary dorsal horn neurons in intraoral hyperalgesia following peripheral nerve injury.

Previous studies demonstrated that the number of c-Fos protein-like immunoreactive (c-Fos-IR) neurons in the medullary dorsal horn (MDH) evoked by noxious stimulation was increased after peripheral nerve injury, and such increase has been proposed to reflect the development of neuropathic pain state. The aim of this study was to examine the MDH for convergent collateral primary afferent input to second order neurons deafferented by peripheral nerve injury, and to explore a possibility of its contribution to the c-Fos hyperinducibility. Double immunofluorescence labeling for c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK) was performed to detect convergent synaptic input. c-Fos expression and the phosphorylation of ERK were induced by the intraoral application of capsaicin and by electrical stimulation of the inferior alveolar nerve (IAN), respectively. The number of c-Fos-IR neurons in the MDH induced by the intraoral application of capsaicin was increased after IAN injury, whereas the number of p-ERK immunoreactive neurons remained unchanged. The number of double-labeled neurons, that presumably received convergent primary afferent input from the lingual nerve and the IAN, was significantly increased after IAN injury. These results indicated that convergent primary nociceptive input through neighboring intact nerves may contribute to the c-Fos hyperinducibility in the MDH and the pathogenesis of neuropathic pain following trigeminal nerve injury.

Convergent nociceptive input to spinal dorsal horn neurons after peripheral nerve injury.

The number of c-Fos protein-like immunoreactive (c-Fos-IR) neurons in the spinal dorsal horn evoked by noxious stimulation was previously shown to be increased following peripheral nerve injury, and this increase was proposed to reflect the neuropathic pain state. The aim of this study was to investigate whether anomalous convergent primary afferent input to spinal dorsal horn neurons contributed to nerve injury-induced c-Fos hyperinducibility. Double immunofluorescence labeling for c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK) was performed to detect convergent synaptic input from different branches of the sciatic nerve after injury to the tibial nerve. c-Fos expression and the phosphorylation of ERK were induced by noxious heat stimulation of the hindpaw and also by electrical stimulation (ES) of the injured tibial nerve, respectively. The number of c-Fos-IR neurons was significantly decreased 3 days after the injury. However, the number of c-Fos-IR neurons returned to the control level 14 days after the injury. P-ERK immunoreactive (p-ERK-IR) neurons were induced in the central terminal field of the tibial nerve by ES of the tibial nerve. The topographic distribution pattern and number of such p-ERK-IR neurons remained unchanged after the nerve injury. The time course of changes in the number of double-labeled neurons, that presumably received convergent primary afferent input, showed a pattern similar to that of c-Fos-IR neurons after the injury. These results indicate that convergent primary nociceptive input through neighboring intact nerves may contribute to c-Fos hyperinducibility in the spinal dorsal horn.

Effect of adenosine A1 receptor agonist on the enhanced excitability of spinal dorsal horn neurons after peripheral nerve injury.

Neuronal hyperactivity has been implicated in abnormal pain sensation following peripheral nerve injuries. Previous studies have indicated that the activation of adenosine A1 receptors (A1R) in the central and peripheral nervous systems produces an antinociceptive effect. However, the mechanisms involved in the peripheral effect are still not fully understood. The effects of the local application of the selective A1R agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA) on neuronal hyperactivity were examined in this study using a neuropathic pain model induced by a tibial nerve injury. We utilized Fos protein-like immunoreactivity induced by noxious heat stimulation to examine changes in the number of Fos protein like immunoreactive (Fos-LI) neuron profiles in the spinal dorsal horn, and behavioral analysis for mechanical and thermal sensitivities. The nerve injury induced an exaggerated Fos response to noxious heat stimulation. The number of Fos-LI neuron profiles was significantly decreased and their distribution was restricted to the central terminal field of the spared peroneal nerve 3 days after the injury. The number of Fos-LI neuron profiles returned to control levels and a large number of these profiles were observed in the central terminal field of the injured tibial nerve 14 days after the injury. These enhanced Fos responses were attenuated by the local application of CCPA. The nerve injury also resulted in mechanical allodynia and thermal hyperalgesia. The local application of CCPA inhibited thermal hyperalgesia, but was less effective against mechanical allodynia. These results indicated that activation of peripheral A1R plays a role in the regulation of nerve injury-induced hyperalgesia.

Modulation index predicts the effect of ethosuximide on developmental and epileptic encephalopathy with spike-and-wave activation in sleep.

PURPOSE: In developmental and epileptic encephalopathy with spike-and-wave activation in sleep (DEE-SWAS), the thalamocortical network is suggested to play an important role in the pathophysiology of the progression from focal epilepsy to DEE-SWAS. Ethosuximide (ESM) exerts effects by blocking T-type calcium channels in thalamic neurons. With the thalamocortical network in mind, we studied the prediction of ESM effectiveness in DEE-SWAS treatment using phase-amplitude coupling (PAC) analysis. METHODS: We retrospectively enrolled children with DEE-SWAS who had an electroencephalogram (EEG) recorded between January 2009 and September 2022 and were prescribed ESM at Okayama University Hospital. Only patients whose EEG showed continuous spike-and-wave during sleep were included. We extracted 5-min non-rapid eye movement sleep stage N2 segments from EEG recorded before starting ESM. We calculated the modulation index (MI) as the measure of PAC in pair combination comprising one of two fast oscillation types (gamma, 40-80 Hz; ripples, 80-150 Hz) and one of five slow-wave bands (delta, 0.5-1, 1-2, 2-3, and 3-4 Hz; theta, 4-8 Hz), and compared it between ESM responders and non-responders. RESULTS: We identified 20 children with a diagnosis of DEE-SWAS who took ESM. Fifteen were ESM responders. Regarding gamma oscillations, significant differences were seen only in MI with 0.5-1 Hz slow waves in the frontal pole and occipital regions. Regarding ripples, ESM responders had significantly higher MI in coupling with all slow waves in the frontal pole region, 0.5-1, 3-4, and 4-8 Hz slow waves in the frontal region, 3-4 Hz slow waves in the parietal region, 0.5-1, 2-3, 3-4, and 4-8 Hz slow waves in the occipital region, and 3-4 Hz slow waves in the anterior-temporal region. SIGNIFICANCE: High MI in a wider area of the brain may represent the epileptic network mediated by the thalamus in DEE-SWAS and may be a predictor of ESM effectiveness.

Improving image quality using the pause function combination to PROPELLER sequence in brain MRI: a phantom study.

While some MRI systems offer a "pause" function, combining it with the PROPELLER method for image quality improvement remains underexplored. This study investigated whether repositioning the head after pausing during PROPELLER imaging enhances image quality. All brain phantom images in this study were obtained using a 3.0 T MRI and acquired using the fast spin-echo T2WI-based PROPELLER with motion correction. By combining the angle of rotational motion of the head phantom and the number of repositioning after a pause, two studies including seven trials were performed. Increasing the rotation angle decreased the image quality; however, pausing the image and repositioning the head phantom to the original angle improved the image quality. A similar result was obtained by repositioning the angle closer to its original angle. Experiments with multiple head movements showed that pausing the scan and repositioning the phantom with each movement improved image quality.

Exploration of urine metabolic biomarkers for new-onset, untreated pediatric epilepsy: A gas and liquid chromatography mass spectrometry-based metabolomics study.

OBJECTIVE: The discovery of objective indicators for recent epileptic seizures will help confirm the diagnosis of epilepsy and evaluate therapeutic effects. Past studies had shortcomings such as the inclusion of patients under treatment and those with various etiologies that could confound the analysis results significantly. We aimed to minimize such confounding effects and to explore the small molecule biomarkers associated with the recent occurrence of epileptic seizures using urine metabolomics. METHODS: This is a multicenter prospective study. Subjects included pediatric patients aged 2 to 12 years old with new-onset, untreated epilepsy, who had had the last seizure within 1 month before urine collection. Controls included healthy children aged 2 to 12 years old. Those with underlying or chronic diseases, acute illnesses, or recent administration of medications or supplements were excluded. Targeted metabolome analysis of spot urine samples was conducted using gas chromatography (GC)- and liquid chromatography (LC)-tandem mass spectrometry (MS/MS). RESULTS: We enrolled 17 patients and 21 controls. Among 172 metabolites measured by GC/MS/MS and 41 metabolites measured by LC/MS/MS, only taurine was consistently reduced in the epilepsy group. This finding was subsequently confirmed by the absolute quantification of amino acids. No other metabolites were consistently altered between the two groups. CONCLUSIONS: Urine metabolome analysis, which covers a larger number of metabolites than conventional biochemistry analyses, found no consistently altered small molecule metabolites except for reduced taurine in epilepsy patients compared to healthy controls. Further studies with larger samples, subjects with different ages, expanded target metabolites, and the investigation of plasma samples are required.

Monitoring C-reactive protein levels to predict favourable clinical outcomes from tocilizumab treatment in patients with rheumatoid arthritis.

OBJECTIVES: The inflammatory cytokine interleukin-6 (IL-6) directly stimulates C-reactive protein (CRP) expression. The present study aimed to examine how clinical treatment outcomes of rheumatoid arthritis (RA) with tocilizumab (TCZ), a humanised monoclonal anti-IL-6 receptor antibody, are related to CRP levels monitored for 52 weeks. METHODS: One hundred and twenty-two RA patients who underwent TCZ treatment between May 2008 and September 2009 were registered in the Tsurumai Biologics Communication Registry. Data were collected at initiation of treatment (baseline) and over 52 weeks for Disease Activity Score 28-ESR (DAS28-ESR), Boolean core measurements, serum CRP levels and matrix metalloproteinase-3 levels. To compare clinical results, patients were divided into three groups based on treatment time required to achieve normal CRP levels. RESULTS: Multivariate analysis using the Cox proportional-hazards regression model found that higher CRP levels at baseline was a significant and independent factor in predicting normal CRP levels over 52 weeks (hazard ratio 0.86 per 1 mg/dL). In contrast, disease duration, concomitant methotrexate use and previous tumour necrosis factor inhibitor failure were not significant factors. Patients with normal CRP levels at 12 weeks of TCZ treatment achieved better clinical outcomes, including remission based on DAS28-ESR criteria, compared to patients with elevated CRP levels at 12 weeks. CONCLUSIONS: Adequate suppression of pathological IL-6 signalling during TCZ treatment improves clinical outcomes and can be monitored with serum CRP levels, a readily available biomarker in clinical practice.

A female patient with adolescent-onset progressive myoclonus epilepsy carrying a truncating MECP2 mutation.

BACKGROUND: MECP2 is a well-known causative gene for Rett syndrome but other phenotypes have also been reported. Here, we report a case of a female patient with adolescent-onset progressive myoclonus epilepsy (PME) carrying a novel truncating mutation in the MECP2 gene. CASE REPORT: The patient was a 29-year-old woman with infantile-onset intellectual disability of unspecified cause. She had demonstrated slow but steady development with moderate intellectual disability until the age of 16, when she started having epileptic seizures. Her epilepsy progressed intractably with multiple seizure types accompanied by myoclonus, tremor, and gradual regression. She is currently apathetic and requires extensive assistance in all aspects of life. After an extensive work-up for underlying diseases for PME turned out negative, whole-exome sequencing revealed a de novo 113-bp deletion and 3-bp insertion in MECP2, a variant of NM_004992.4:c.1099_1211delinsGGG, p.(His367Glyfs*32). CONCLUSIONS: The clinical presentation of this case was inconsistent with Rett syndrome, and the rapid regression in the patient's twenties was considered characteristic. Mutations of MECP2 may result in variable neurodevelopmental phenotypes and may also be considered a causative gene for adolescent-onset PME.

A case of giant cell myocarditis mimicking cardiac sarcoidosis successfully maintained by prednisolone and tacrolimus.

A 45-year-old woman with no medical history underwent pacemaker implantation for a symptomatic complete atrioventricular block. On day 6, she noticed diplopia and then fever, general malaise, and elevation of serum creatinine kinase (CK). She was transferred to our hospital on day 21. Serum CK was elevated to 4543 IU/L, and echocardiography revealed a left ventricular ejection fraction of 43 %. We diagnosed her with giant cell myocarditis (GCM) via an emergent myocardial biopsy that revealed a proliferation of lymphocytes, eosinophils, and giant cells without granulomas. Initial treatment with high doses of intravenous methylprednisolone and immunoglobulin improved her symptoms in a few days, and prednisolone was given as follow-up treatment. CK was normalized in a week and a thinning of the interventricular septum mimicking cardiac sarcoidosis (CS) occurred. On day 38, we added a calcineurin inhibitor, tacrolimus, and maintained her with a combination of prednisolone and tacrolimus at a target dose of 10-15 ng/mL. Six months after the onset, there were no signs of relapse despite the persistent mild elevation of troponin I levels. We present a case of GCM mimicking CS successfully maintained by a combination of two immunosuppressive agents. Learning objective: Recommended treatment for giant cell myocarditis (GCM), a potentially fatal disease, is a combination of three immunosuppressive agents. However, GCM shares many characteristics with cardiac sarcoidosis (CS), which is treated using prednisolone alone in many cases. Recent studies on GCM and CS suggest they are different spectrums of a common entity. Although they can clinically overlap, they have different progressive speeds and severities. We present a case of GCM mimicking CS successfully treated with a combination of two immunosuppressive agents.

Comprehensive study of metabolic changes induced by a ketogenic diet therapy using GC/MS- and LC/MS-based metabolomics.

OBJECTIVE: The ketogenic diet (KD), a high-fat and low-carbohydrate diet, is effective for a subset of patients with drug-resistant epilepsy, although the mechanisms of the KD have not been fully elucidated. The aims of this observational study were to investigate comprehensive short-term metabolic changes induced by the KD and to explore candidate metabolites or pathways for potential new therapeutic targets. METHODS: Subjects included patients with intractable epilepsy who had undergone the KD therapy (the medium-chain triglyceride [MCT] KD or the modified Atkins diet using MCT oil). Plasma and urine samples were obtained before and at 2-4 weeks after initiation of the KD. Targeted metabolome analyses of these samples were performed using gas chromatography-tandem mass spectrometry (GC/MS/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS). RESULTS: Samples from 10 and 11 patients were analysed using GC/MS/MS and LC/MS/MS, respectively. The KD increased ketone bodies, various fatty acids, lipids, and their conjugates. In addition, levels of metabolites located upstream of acetyl-CoA and propionyl-CoA, including catabolites of branched-chain amino acids and structural analogues of γ-aminobutyric acid and lactic acid, were elevated. CONCLUSIONS: The metabolites that were significantly changed after the initiation of the KD and related metabolites may be candidates for further studies for neuronal actions to develop new anti-seizure medications.

Early aggressive intervention with tocilizumab for rheumatoid arthritis increases remission rate defined using a Boolean approach in clinical practice.

The goal of treating rheumatoid arthritis (RA) should be remission, for which a new definition was proposed in 2011. To determine which patients can achieve the new Boolean-based definition of remission in clinical practice, we analyzed factors associated with remission in 123 patients who received tocilizumab for 52 weeks. We found that patients with short disease duration (<4.8 years) had a significantly higher rate of remission (31.7%) than those with longer disease duration, and patient global assessment was the most important factor for achieving remission. Multivariate analysis revealed the following predictors of remission: short disease duration [<4.8 years; odds ratio (OR) 2.5, 95% confidence interval (CI) 1.4-4.7] and lower disease activity [28-joint disease activity score-erythrocyte sedimentation rate (DAS28-ESR) <5.23; OR 2.5, 95% CI 1.2-5.1). In this study, we showed that remission, as newly defined using a Boolean approach, is a realistic goal for patients treated with tocilizumab with short disease duration in real-world clinical practice.