Detailed Molecular Interactions between Respiratory Syncytial Virus Fusion Protein and the TLR4/MD-2 Complex In Silico

Molecular interactions between respiratory syncytial virus (RSV) fusion protein (F protein) and the cellular receptor Toll-like receptor 4 (TLR4) and myeloid differentiation factor-2 (MD-2) protein complex are unknown. Thus, to reveal the detailed molecular interactions between them, in silico analyses were performed using various bioinformatics techniques. The present simulation data showed that the neutralizing antibody (NT-Ab) binding sites in both prefusion and postfusion proteins at sites II and IV were involved in the interactions between them and the TLR4 molecule. Moreover, the binding affinity between postfusion proteins and the TLR4/MD-2 complex was higher than that between prefusion proteins and the TLR4/MD-2 complex. This increased binding affinity due to conformational changes in the F protein may be able to form syncytium in RSV-infected cells. These results may contribute to better understand the infectivity and pathogenicity (syncytium formation) of RSV.     

Potential of a glucagon‐like peptide‐1 receptor/glucose‐dependent insulinotropic polypeptide receptor/glucagon receptor triagonist for the treatment of obesity and type 2 diabetes

Triagonists of GLP‐1R/ GIPR /GCGR, including SAR441255, bind to each receptor and induce specific effects through each receptor signaling pathway, thus result in weight loss and glycemic control in obese T2D animal models.

Type 2 diabetes, which often accompanies obesity, is one of the major health‐threatening diseases worldwide. Incretin‐based therapies, such as dipeptidyl peptidase‐4 inhibitors and glucagon‐like peptide‐1 receptor (GLP‐1R) agonists, are used for the treatment of type 2 diabetes. The GLP‐1R agonists have been shown to improve glycemic control and reduce bodyweight through its various actions, including glucose‐dependent insulin secretion, suppression of glucagon secretion, and inhibition of gastric emptying and food intake ¹ .Recently, dual agonists of GLP‐1R and glucose‐dependent insulinotropic polypeptide receptor (GIPR), and those of GLP‐1R and glucagon receptor (GCGR) have been developed. One of the dual agonists of GLP‐1R and GIPR, named tirzepatide, has been shown to bind and activate both GLP‐1R and GIPR in vitro and in vivo, its efficacy for the treatment of type 2 diabetes has been proven in a phase III trial, and it has recently been approved by the US Food and Drug Administration ^{2 , 3} . In the phase III trial, once‐weekly injection of trizepatide (5, 10 or 15 mg) for 40 weeks reduced glycated hemoglobin (HbA1c) from baseline by 1.87, 1.89 or 2.07%, respectively, and reduced bodyweight from baseline by 7.0, 7.8 or 9.5 kg, respectively, in type 2 diabetes patients. The most frequent adverse events with trizepatide were gastrointestinal events ³ . In the clinical trial that compared trizepatide (5, 10 or 15 mg) with once‐weekly semaglutide (1 mg) in type 2 diabetes patients, trizepatide showed non‐inferior and superior reductions in HbA1c levels and in bodyweight ⁴ . For both treatments, gastrointestinal events were the most common adverse events ⁴ . The possible additional effects of GIP signaling in type 2 diabetes patients might depend on its augmentation of insulin secretion during hyperglycemic states ⁵ .The dual agonists of GLP‐1R and GCGR specific for mice or monkeys have been developed and were investigated their effects in each species. In obese and diabetic cynomolgus monkeys induced by high‐fat diet feeding, the monkey‐specific dual agonist of GLP‐1R and GCGR reduced total energy intake, decreased bodyweight and improved glucose tolerance ⁶ . However, in another study using obese diabetic monkeys, glycemic control was worse when they were co‐administered with both GLP‐1R and GCGR agonists compared with that treated with an GLP‐1R agonist alone ⁷ . Therefore, the significance of the activation of GCGR signaling has not yet been fully clarified.In 2015, Finan et al. ⁸ created a triagonist that activates GLP‐1R, GIPR and GCGR by selecting amino acids from the three peptide hormones, GLP‐1, GIP and glucagon, and reported that the triagonist reduced bodyweight and improved glucose control in rodent models of obesity and diabetes.More recently, Bossart et al. ⁹ developed a novel GLP‐1R, GIPR and GCGR triagonist, SAR441255. SAR441255 was confirmed to efficiently bind to and stimulate all three receptors in in vitro studies. In a diet‐induced obesity mouse model, subcutaneous injection of SAR441255 (0.3, 1, 3, 10 or 30 μg/kg) showed a dose‐dependent effect on bodyweight (+9.7%, +6.9%, +5.8%, −4.8% and −14.1%, respectively) compared with injection of vehicle (+11.5%). Non‐fasted blood glucose levels were significantly lower in mice treated with SAR441255 at doses of ≥1 μg/kg when compared with vehicle‐treated controls. In obese diabetic cynomolgus monkeys, treatment with SAR441255 (11 μg/kg) or a monkey‐specific GLP‐1R/GCGR dual agonist (4 μg/kg) ⁶ significantly reduced the bodyweight by −12.6% or −8.1%, respectively, and decreased the HbA1c levels by −1.37% or −1.85%, respectively. Fasting plasma glucose and alanine aminotransferase levels were significantly reduced with SAR441255, but not with the dual agonist (Figure 1).Open in a separate windowFigure 1Possible mechanisms of glucagon‐like peptide‐1 receptor (GLP‐1R)/glucose dependent insulinotropic polypeptide receptor (GIPR)/glucagon receptor (GCGR) triagonist in weight loss and glycemic control in obese type 2 diabetes animal models. Triagonists of GLP‐1R/GIPR/GCGR, including SAR441255, bind to each receptor and induce specific effects through each receptor signaling pathway, thus resulting in weight loss and glycemic control in obese type 2 diabetes animal models. Potential effects of each receptor signaling that contribute to weight loss and/or better glycemic control are listed below each receptor.To further understand engagement of the different receptors in vivo, receptor occupancy of SAR441255 at the GLP‐1 and the GCG receptors was studied with the use of positron emission tomography imaging after radiotracer administration in lean cynomolgus monkeys. A subcutaneous injection of 11 μg/kg SAR411255 together with radiotracers specific for GLP‐1R and GCGR showed the significant signals of both receptors in the liver and pancreas of monkeys, suggesting the binding of SAR411255 to both receptors existing in these organs. Cardiovascular safety of SAR411255 was further confirmed in lean cynomolgus monkeys.Finally, a phase I study with SAR411255 in lean to overweight healthy participants was carried out to assess the safety, tolerability, pharmacokinetics and pharmacodynamics. After subcutaneous administration, SAR441255 concentration reached the median maximum serum concentration by 3.0–3.5 h, and was eliminated with a mean elimination terminal half‐life of 3.5–6.1 h. After administration of single doses (80 and 150 mg) of SAR441255 in the fasting state, maximal reduction in blood glucose levels were observed at 1 h post‐dose. At this time point, three of six participants who received the 80 mg dose and all six participants who received the 150 mg dose showed hypoglycemia (<70 mg/dL) without any clinical signs or symptoms. No further low blood glucose values were observed in either dose group. Blood glucose levels subsequently returned to baseline levels within 1–2 h. In contrast, fasting insulin and C‐peptide levels were relatively stable, and showed no correlation with the glucose levels. Therefore, the mechanisms of this early phase hypoglycemia have not yet been clarified. After the mixed‐meal test 3 h after the SAR441255 administration (80 and 150 mg), a dose‐dependent reduction in postprandial plasma glucose was observed. Because insulin and C‐peptide levels were also reduced during mixed‐meal test, postprandial plasma glucose reduction was suggested to be due to inhibition of gastric emptying, as observed with GLP‐1R engagement.Gastrointestinal disorders (nausea, vomiting, dry mouth and mouth ulceration) were the most frequent treatment‐emergent adverse events after treatment with SAR441255. All events were mild in severity, and occurred between 3 and 4 h after SAR441255 administration.To clarify the effects of SAR441255 on GIPR and GCGR in humans, specific biomarkers for each receptor were measured. As expected, a biomarker for GIPR activation, C‐telopeptide of cross‐linked type I collagen, which is a marker of bone turnover, was significantly reduced by >50% in participants who received SAR441255 administration (80 and 150 mg). Likely, plasma amino acids levels, which are sensitive biomarkers of GCGR activation, were reduced after SAR441255 administration in these individuals.As aforementioned, the dual agonists of GLP‐1R and GIPR might have comparable or even more stronger effects in reductions of HbA1c and bodyweight in obese type 2 diabetes patients ⁴ ; the significance of the activation of GCGR signaling should be evaluated to confirm the benefits of the triagonists of GLP‐1R, GIPR and GCGR. In this regard, the effects of glucagon to enhance energy expenditure partly through enhancement of fat and glucose oxidation could cover some of the benefits ⁷ , but further investigations should be necessary. In addition, it has recently been reported that GLP‐1R agonists have clinically significant cardiovascular benefits in type 2 diabetes patients ¹ . Therefore, it should be clarified if the dual or triagonists also have comparable cardiovascular benefits, as observed in GLP‐1R agonists in type 2 diabetes patients.Because triagonists have been shown to have better profiles in weight loss and glycemic control in animal models of obese type 2 diabetes compared with GLP‐1R agonists and dual agonists of GLP‐1R and GIPR or GLP‐1R and GCGR ^{8 , 9} , clinical studies that investigate the efficacy in type 2 diabetes patients should be of great interest.     

Ivabradine as an Adjuvant Agent for Severe Heart Failure Occurring in the Early Phase after Allogeneic Hematopoietic Cell Transplantation

Cardiotoxicity is a critical complication of allogeneic hematopoietic cell transplantation (allo-HCT). In particular, management of severe cardiotoxicity occurring in the early phases of allo-HCT is challenging. We encountered a case of severe cardiotoxicity resulting from AHF six days after allo-HCT, which resisted catecholamines and diuretics. The patient was treated with anthracycline-containing regimens and underwent myeloablative conditioning, including high-dose cyclophosphamide. As invasive circulatory assisting devices were contraindicated because of his immunocompromised status and bleeding tendency, we successfully treated the patient with ivabradine-containing medications. Ivabradine may therefore be considered an alternative drug for the treatment of severe cardiotoxicity induced by cytotoxic agents.     

Denoising using deep-learning-based reconstruction for whole-heart coronary MRA with sub-millimeter isotropic resolution at 3 T: a volunteer study

PURPOSEThe aim of this study was to assess the usefulness of denoising deep-learning-based reconstruction (dDLR) to improve image quality and vessel delineation in noncontrast 3-T whole-heart coronary magnetic resonance angiography (WHCMRA) with sub-millimeter isotropic resolution (Sub-mm) compared with a standard resolution without dDLR (Standard).METHODSFor 10 healthy volunteers, we acquired the WHCMRA with Sub-mm with and without dDLR and Standard to quantify signal- (SNR) and contrast-to-noise ratio (CNR) and vessel edge signal response (VESR) in all the 3 image types. Two independent readers subjectively graded vessel sharpness and signal homogeneity of 8 coronary segments in each patient. We used Kruskal–Wallis test with Bonferroni correction to compare SNR, CNR, VESR, and the subjective evaluation scores among the 3 image types and weighted kappa test to evaluate inter-reader agreement on the scores.RESULTSSNR was significantly higher with Sub-mm with dDLR (P < .001) and Standard (P = .005) than with Sub-mm without dDLR and was comparable between Sub-mm with dDLR and Standard (P = .511). CNR was significantly higher with Sub-mm with dDLR (P < .001) and Standard (P = .005) than with Sub-mm without dDLR and was comparable between Sub-mm with dDLR and Standard (P = .560). VESR was significantly greater with Sub-mm with (P = .001) and without dDLR (P = .017) than with Standard and was comparable between Sub-mm with and without dDLR (P = 1.000). In the proximal, middle, distal, and all the coronary segments, the subjective vessel sharpness was significantly better with Sub-mm with dDLR than Sub-mm without dDLR and Standard (P < .001, for all) and was comparable between Sub-mm without dDLR and Standard (P > .05); the subjective signal homogeneity was significantly improved from Sub-mm without dDLR to Standard to Sub-mm with dDLR (P < .001). The inter-reader agreement was excellent (kappa = 0.84).CONCLUSIONApplication of dDLR is useful for improving image quality and vessel delineation in the WHCMRA with Sub-mm compared with Standard.

Main points

• A denoising method with deep-learning-based reconstruction (dDLR) uses a deep convolution neural network to reduce image noise in magnetic resonance images without additional scan time.
• dDLR can be applied to improve signal- (SNR) and contrast-noise ratio in noncontrast 3-T whole-heart coronary magnetic resonance angiography (WHCMRA) using a spoiled gradient-echo sequence, which offers lower SNR than a steady-state free precession sequence commonly applied at 1.5 T.
• Combined application of dDLR and sub-millimeter isotropic resolution is useful for improving vessel sharpness, signal homogeneity, and delineation of the coronary arteries in the WHCMRA.

Three-dimensional (3D) whole-heart coronary magnetic resonance angiography (WHCMRA) is a noninvasive imaging method for assessing coronary artery stenosis and is advantageous over coronary computed tomography angiography (CCTA) because it does not require radiation exposure or contrast media administration and is only slightly susceptible to calcium-related artifacts.^1-3 Because WHCMRA is commonly limited in delineation of distal coronary segments and quantification of vessel lumen stenosis due to its insufficient spatial resolution and anisotropy, some investigators have used various techniques to acquire WHCMRA with sub-millimeter isotropic resolution (Sub-mm) whose image quality is not necessarily satisfactory at 1.5 T, within an acceptable acquisition time.^4-6 While a steady-state free precession (SSFP) sequence has been commonly applied in noncontrast WHCMRA at 1.5 T, increased B1 field inhomogeneity, frequency offset from tissue susceptibility variation, and specific absorption rate limit consistency of SSFP at 3 T. As such, the spoiled gradient-echo sequence, which decreases the signal-to-noise ratio (SNR) of the coronary arteries, has often been used at 3 T.^7-10A 3-T clinical MR scanner with a maximum gradient magnetic field of 100 mT/m (the slew rate: 200 mT/m/ms) has been recently introduced and can offer thinner slice images at the same bandwidth (i.e., the same sampling interval/time). With this scanner, a denoising method with deep-learning-based reconstruction (dDLR) has been newly developed using a convolution neural network (CNN) to improve SNR in high-resolution MR images without additional scan time.¹¹ Currently, this dDLR algorithm (Advanced intelligent Clear-IQ Engine [AiCE], Canon Medical Systems Corporation) is clinically available only from the single vendor. While some smoothing filters frequently applied in clinical settings are designed to reduce high-frequency noise at the cost of image blurring, we hypothesized that dDLR should only reduce image noise without any negative influence on the delineation of the coronary vessels in WHCMRA. Thus, we performed volunteer studies to assess usefulness of dDLR to improve image quality and coronary vessel delineation in noncontrast WHCMRA with Sub-mm compared with a standard resolution without dDLR (Standard) using this 3-T scanner.     

Biodistribution of Insulin Following Massive Insulin Subcutaneous Injection

A man in his 30s injected insulin several times into his abdomen and was found dead several hours later. Micropathological findings showed alveolar injury with hemorrhaging and cerebral parietal lobe nerve cell edema. Biochemical examinations showed that the blood insulin level was high, significantly so at the insulin injection sites. The blood glucose and C-peptide levels were low. The insulin level in the kidneys was low. In forensic medicine, a postmortem diagnosis of insulin subcutaneous injection is often difficult. When insulin injection is suspected, particularly high insulin levels can be expected at the insulin injection site, rather than in the blood.     

Cetuximab delivery and antitumor effects are enhanced by mild hyperthermia in a xenograft mouse model of pancreatic cancer

Even with current promising antitumor antibodies, their antitumor effects on stroma‐rich solid cancers have been insufficient. We used mild hyperthermia with the intent of improving drug delivery by breaking the stromal barrier. Here, we provide preclinical evidence of cetuximab + mild hyperthermia therapy. We used four in vivo pancreatic cancer xenograft mouse models with different stroma amounts (scarce, MIAPaCa‐2; moderate, BxPC‐3; and abundant, Capan‐1 and Ope‐xeno). Cetuximab (1 mg/kg) was given systemically, and the mouse leg tumors were concurrently heated using a water bath method for 30 min at three different temperatures, 25°C (control), 37°C (intra‐abdominal organ level), or 41°C (mild hyperthermia) (n = 4, each group). The evaluated variables were the antitumor effects, represented by tumor volume, and in vivo cetuximab accumulation, indirectly quantified by the immunohistochemical fluorescence intensity value/cell using antibodies against human IgG Fc. At 25°C, the antitumor effects were sufficient, with a cetuximab accumulation value (florescence intensity/cell) of 1632, in the MIAPaCa‐2 model, moderate (1063) in the BxPC‐3 model, and negative in the Capan‐1 and Ope‐xeno models (760, 461). By applying 37°C or 41°C heat, antitumor effects were enhanced shown in decreased tumor volumes. These enhanced effects were accompanied by boosted cetuximab accumulation, which increased by 2.8‐fold (2980, 3015) in the BxPC‐3 model, 2.5‐ or 4.8‐fold (1881, 3615) in the Capan‐1 model, and 3.2‐ or 4.2‐fold (1469, 1922) in the Ope‐xeno model, respectively. Cetuximab was effective in treating even stroma‐rich and k‐ras mutant pancreatic cancer mouse models when the drug delivery was improved by combination with mild hyperthermia.     

Properties of Corneas Reconstructed with Cultured Human Corneal Endothelial Cells and Human Corneal Stroma

Purpose

To examine the properties of corneas tissue-engineered with cultured human corneal endothelial cells (HCEC) and human corneal stroma.

Methods

Primary HCEC cultures were established from endothelial cell layer explants and propagated on culture dishes coated with bovine corneal endothelial extracellular matrix. A cell suspension of HCEC at the fifth passage was transferred onto human corneal stroma deprived of endothelial cells, and the corneas were gently centrifuged to enhance cell attachment. The cell density of the tissue-engineered corneas was examined after staining with alizarin red and trypan blue. The tissue-engineered corneas were histologically examined by light and electron microscopy. The pump function of the tissue-engineered corneas was measured using an Ussing chamber.

Results

The mean endothelial cell density of four tissue-engineered corneas was 2380 ± 264 cells/mm² (mean ± SD). HCEC on the tissue-engineered corneas had a morphology similar to HCEC in vivo. The pump function parameters of the tissue-engineered corneas were 55%–75% of those of normal corneas.

Conclusions

HCEC on the tissue-engineered corneas have morphology and cellular density similar to HCEC in vivo, whereas the pump function of the tissue-engineered corneas was lower than in normal corneas. Jpn J Ophthalmol 2005;49:448–452 © Japanese Ophthalmological Society 2005     

Rhinovirus Infection and Virus-Induced Asthma

While the aetiology of asthma is unclear, the onset and/or exacerbation of asthma may be associated with respiratory infections. Virus-induced asthma is also known as virus-associated/triggered asthma, and the reported main causative agent is rhinovirus (RV). Understanding the relationship between viral infections and asthma may overcome the gaps in deferential immunity between viral infections and allergies. Moreover, understanding the complicated cytokine networks involved in RV infection may be necessary. Therefore, the complexity of RV-induced asthma is not only owing to the response of airway and immune cells against viral infection, but also to allergic immune responses caused by the wide variety of cytokines produced by these cells. To better understand RV-induced asthma, it is necessary to elucidate the nature RV infections and the corresponding host defence mechanisms. In this review, we attempt to organise the complexity of RV-induced asthma to make it easily understandable for readers.     

Efficacy and Safety of a Strategy for Reviewing Intravenous Antibiotics for Hospitalized Japanese Patients with Uncomplicated Diverticulitis: A Single-center Observational Study

Objective Treatment for uncomplicated diverticulitis (UD) is not well established. We evaluated the strategy of reviewing intravenous antibiotics for hospitalized Japanese patients with UD. Methods Treatment was based on the physician''s choice until August 2018; the indications for hospitalization and treatment have been standardized since September 2018. In this study, we monitored the use of intravenous antibiotics administered to patients hospitalized for UD and then reviewed the need for them on hospital day 3. We compared patients'' length of antibiotic use, hospital stay, health care cost, and complications via the review strategy from September 2018 to December 2020 and via the previous physicians'' choice strategy from January 2016 to August 2018. Results Two hundred and forty-seven patients were admitted to our hospital because of acute colonic diverticulitis from January 2016 to December 2020. After excluding complicated cases, 106 individuals were enrolled during the period of physician''s choice; 87 were enrolled when treatment review was employed. There were no significant differences in age, sex, inflammation site, or severity during the first hospital visit. The median duration of antibiotic use was significantly reduced from 5 to 4 days (p=0.0075), with no marked increase in rates of transfer to surgery, mortality, or readmission due to recurrence. A more significant proportion of patients completed 3-day antibiotic treatment with the review strategy than with the physician''s choice strategy (6.6% vs. 25.3%, p=0.0004). However, the length of hospital stay and total medical costs did not decrease. Conclusion The strategy of reviewing treatment on day 3 after hospitalization for UD safety reduced the duration of antibiotic use, but the hospital stay and health care costs did not decrease.     

Identifying the Optimal Arm Priming Exercise Intensity to Improve Maximal Leg Sprint Cycling Performance

Priming exercises improve subsequent motor performance; however, their effectiveness may depend on the workload and involved body areas. The present study aimed to estimate the effects of leg and arm priming exercises performed at different intensities on maximal sprint cycling performance. Fourteen competitive male speed-skaters visited a lab eight times, where they underwent a body composition measurement, two V̇O_2max measurements (leg and arm ergometers), and five sprint cycling sessions after different priming exercise conditions. The five priming exercise conditions included 10-minute rest (Control); 10-minute arm ergometer exercise at 20% V̇O_2max (Arm 20%); 10-minute arm ergometer exercise at 70% V̇O_2max (Arm 70%); 1-min maximal arm ergometer exercise at 140% V̇O_2max (Arm 140%); and 10-min leg ergometer exercise at 70% V̇O_2max (Leg 70%). Power outputs of 60-s maximal sprint cycling, blood lactate concentration, heart rate, muscle and skin surface temperature, and rating of perceived exertion were compared between the priming conditions at different measurement points. Our results showed that the Leg 70% was the optimal priming exercise among our experimental conditions. Priming exercise with the Arm 70% also tended to improve subsequent motor performance, while Arm 20% and Arm 140% did not. Mild elevation in blood lactate concentration by arm priming exercise may improve the performance of high-intensity exercise. Key points

• Maximal sprint cycling performance was comparable between the Arm 70% condition and the Leg 70% condition
• A mild elevation in BLC by arm priming exercise may improve the performance of high-intensity exercise
• Low (i.e., Arm 20%) and high (i.e., Arm 140%) workloads did not provide any performance benefits

Key words: Exercise, arm, heart rate, lactic acid