Pembrolizumab-related Immune Thrombocytopenia in a Patient with Lung Adenocarcinoma Treated by Radiotherapy: Potential Immune-related Adverse Event Elicited by Radiation Therapy

The effect of radiotherapy during immunotherapy on immune-related adverse events (irAEs) is not fully understood. We herein report a 74-year-old woman diagnosed with lung adenocarcinoma with programmed death ligand 1 expression ≥50% and treated with pembrolizumab. She developed fatal immune thrombocytopenia associated with pembrolizumab immediately following radiotherapy. A flow cytometry analysis of peripheral blood detected an increased expression of programmed death-1 (PD-1) and Ki-67 in CD4^＋ and CD8^＋ T cells after radiotherapy, compared with pre-irradiation measurements. This case suggests that radiotherapy may evoke irAEs during treatment with anti-PD-1 antibodies, which physicians should consider when using radiotherapy in patients treated with these drugs.     

Combination therapy using tafamidis and neurohormonal blockers for cardiac amyloidosis and a reduced ejection fraction: a case report

Cardiac amyloidosis usually presents with diastolic dysfunction, but sometimes systolic dysfunction develops, particularly at its advanced stage. However, the therapeutic strategy for patients with cardiac amyloidosis and systolic dysfunction remains unknown. We report a 77-year-old man who was diagnosed with wild-type cardiac amyloidosis and systolic dysfunction with a left ventricular ejection fraction of 27%. Following 6-month medical therapy of tafamidis 80 mg and neurohormonal blockers (carvedilol 5.0 mg, enalapril 2.5 mg, and spironolactone 25 mg), the left ventricular ejection fraction improved to 55%. Tafamidis-incorporated neurohormonal blocker therapy might be a promising strategy to facilitate cardiac reverse remodeling in patients with cardiac amyloidosis and systolic dysfunction.     

CXCL12/CXCR4 activation by cancer‐associated fibroblasts promotes integrin β1 clustering and invasiveness in gastric cancer

Cancer‐associated fibroblasts (CAFs) are reportedly involved in invasion and metastasis in several types of cancer, including gastric cancer (GC), through the stimulation of CXCL12/CXCR4 signaling. However, the mechanisms underlying these tumor‐promoting effects are not well understood, which limits the potential to develop therapeutic targets against CAF‐mediated CXCL12/CXCR4 signaling. CXCL12 expression was analyzed in resected GC tissues from 110 patients by immunohistochemistry (IHC). We established primary cultures of normal fibroblasts (NFs) and CAFs from the GC tissues and examined the functional differences between these primary fibroblasts using co‐culture assays with GC cell lines. We evaluated the efficacy of a CXCR4 antagonist (AMD3100) and a FAK inhibitor (PF‐573,228) on the invasive ability of GC cells. High CXCL12 expression levels were significantly associated with larger tumor size, increased tumor depth, lymphatic invasion and poor prognosis in GC. CXCL12/CXCR4 activation by CAFs mediated integrin β1 clustering at the cell surface and promoted the invasive ability of GC cells. Notably, AMD3100 was more efficient than PF‐573,228 at inhibiting GC cell invasion through the suppression of integrin β1/FAK signaling. These results suggest that CXCL12 derived from CAFs promotes GC cell invasion by enhancing the clustering of integrin β1 in GC cells, resulting in GC progression. Taken together, the inhibition of CXCL12/CXCR4 signaling in GC cells may be a promising therapeutic strategy against GC cell invasion.     

Long-term results of thermoradiotherapy for superficial, subsurface tumors using a 430 MHz microwave heating system

Superficial, subsurface tumors of 93 patients were treated with thermoradiotherapy using a 430 MHz microwave heating system (HTS-100). All patients had malignant tumors, and all were treated with a combination of hyperthermia and radiation. Satisfactory temperature data for each thermal parameter were achieved within a 5 cm depth for each tumor. The overall complete response (CR) rate was 39.8%; in the less than or equal to 3 cm (depth of tumor), the CR rate was 66.7%, in the 3-5 cm group, the CR rate was 42.9%. In the multivariate analysis, duration of local control correlated with tumor responses (CR). There were no instances of prominent late complications that were observed for greater than or equal to 6 months. This study suggests that the HTS-100 may improve tumor response and the duration of local control of superficial and subsurface tumors.     

Arrhythmogenic Right Ventricular Cardiomyopathy Accompanied by Chronic Myocarditis

Patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) classically present with ventricular arrhythmias and less commonly heart failure. ARVC is an inherited cardiomyopathy and generally based on a variant of desmosomal genes. Recently, the association between myocardial inflammation and ARVC has been a matter of great concern. We encountered a patient with ARVC who had a desmoglein-2 mutation with advanced right ventricular failure accompanying a preserved left ventricular function. Concomitant right ventricular myocarditis was detected four years after the diagnosis of ARVC. ARVC and myocarditis might have a deep pathophysiological association, at least in some cases.     

Effects of workplace measures against COVID-19 on psychological distress of full-time employees: A 12-month prospective study in the COVID-19 pandemic

ObjectiveThis study aimed to investigate the prospective effects of corporate and organizational workplace measures against COVID-19 on reducing employees’ psychological distress during a 12-month follow-up in the COVID-19 pandemic.MethodsData were retrieved from an online longitudinal panel survey of full-time employees in Japan, with the 1^st survey in March 2020, and the 2^nd to 6^th surveys in May, August, November 2020, February and March 2021, respectively. Seven area-specific workplace measures were assessed using a self-report 23-item scale at the 2^nd follow-up. Psychological distress was measured using an 18-item scale of the Brief Job Stress Questionnaire at each survey. Linear regressions and mixed model analysis were conducted of psychological distress at follow-ups on scores of the area-specific workplace measures, adjusting for psychological distress and other covariates at the 1^st survey.ResultsA total of 941 employees responded at baseline; most of them (86.9–90.9%) participated in the follow-up surveys. Linear regression analysis indicated that workplace measures of facilitating employees’ preventive measures (ie, hygiene behaviors) statistically significantly and negatively correlated with psychological distress at the 5^th survey [b=-0.518, standard error (SE) 0.259, P=0.046]. A statistically significant and negative interaction between the scores and time of follow-up was observed in the mixed model analysis (b=-0.096, SE 0.047, P=0.041). No such correlation or interaction was found for any of other subcategorical workplace measures.ConclusionsThe study provides prospective evidence for a protective effect of workplace measures to facilitate employee’s hygiene behaviors on reducing psychological distress of full-time employees in the COVID-19 pandemic. The association seems stronger at a later follow-up.     

Shear stress activates mitochondrial oxidative phosphorylation by reducing plasma membrane cholesterol in vascular endothelial cells

Vascular endothelial cells (ECs) sense and respond to hemodynamic shear stress, which is critical for circulatory homeostasis and the pathophysiology of vascular diseases. The mechanisms of shear stress mechanotransduction, however, remain elusive. We previously demonstrated a direct role of mitochondria in the purinergic signaling of shear stress: shear stress increases mitochondrial adenosine triphosphate (ATP) production, triggering ATP release and Ca²⁺ signaling via EC purinoceptors. Here, we showed that shear stress rapidly decreases cholesterol in the plasma membrane, thereby activating mitochondrial ATP production. Imaging using domain 4 mutant-derived cholesterol biosensors showed that the application of shear stress to cultured ECs markedly decreased cholesterol levels in both the outer and inner plasma membrane bilayers. Flow cytometry showed that the cholesterol levels in the outer bilayer decreased rapidly after the onset of shear stress, reached a minimum (around 60% of the control level) at 10 min, and plateaued thereafter. After the shear stress ceased, the decreased cholesterol levels returned to those seen in the control. A biochemical analysis showed that shear stress caused both the efflux and the internalization of plasma membrane cholesterol. ATP biosensor imaging demonstrated that shear stress significantly increased mitochondrial ATP production. Similarly, the treatment of cells with methyl-β-cyclodextrin (MβCD), a membrane cholesterol-depleting agent, increased mitochondrial ATP production. The addition of cholesterol to cells inhibited the increasing effects of both shear stress and MβCD on mitochondrial ATP production in a dose-dependent manner. These findings indicate that plasma membrane cholesterol dynamics are closely coupled to mitochondrial oxidative phosphorylation in ECs.

Vascular endothelial cells (ECs) recognize shear stress, a biomechanical force generated by flowing blood, and transduce it into intracellular biochemical signals, thereby causing responses such as changes in cell morphology, function, and gene expression (1). These EC responses play crucial roles in maintaining the homeostasis of the circulatory system, and their impairments cause various vascular diseases such as hypertension, aneurysm, and atherosclerosis (2–4). To date, numerous studies have elucidated the mechanism of EC mechanotransduction and have revealed a unique feature: shear stress activates multiple signal transduction pathways through a variety of membrane molecules, including ion channels, receptors, and adhesion proteins, almost simultaneously (5). Recently, it has become apparent that the plasma membrane itself plays an important role in EC mechanotransduction. EC plasma membranes rapidly respond to shear stress by altering their physical properties, such as fluidity, viscosity, and lipid order (6, 7). We previously demonstrated that shear stress decreases the lipid order of not only EC plasma membranes but also, artificial lipid bilayer membranes, thereby causing a transition from the liquid-ordered state to the liquid-disordered state, along with an increase in membrane fluidity (8). These changes in membrane physical properties were linked to downstream signaling pathways, such as the activation of mitogen-activated protein kinase (9) and the phosphorylation of vascular endothelial growth factor receptors (VEGFRs) (10). This mechanism, in which changes in membrane physical properties occur initially and are then followed by the activation of membrane molecules, may characterize the EC mechanotransduction described above (11).Ca²⁺ signaling is known to play a critical role in EC mechanotransduction (12). ECs rapidly release intrinsic adenosine triphosphate (ATP) in response to shear stress (13, 14), and this ATP activates purinoceptors, such as ligand-gated channel P2X and G protein-coupled P2Y receptors, located in the plasma membranes; these purinoceptors are responsible for extracellular Ca²⁺ influx and Ca²⁺ release from the endoplasmic reticulum (15–17). The increase in cytoplasmic Ca²⁺ activates a variety of EC functions. In P2X4-knockout mice, ECs exhibited neither Ca²⁺ signaling nor the production of a potent vasodilator, nitric oxide, in response to shear stress, thereby impairing the blood flow-dependent vasodilator response and vascular remodeling and resulting in hypertension (4). Our recent study revealed that mitochondria play an important role in shear stress-induced ATP release and Ca²⁺ signaling in ECs (18). Upon shear stress stimulation, the ECs rapidly augmented their mitochondrial ATP production, triggering ATP release and subsequent Ca²⁺ signaling. However, the means by which shear stress acting on the plasma membrane affects mitochondrial oxidative phosphorylation in ECs remains unknown.Cholesterol plays a dominant role in determining the mechanical properties of plasma membranes by affecting the membrane lipid order, fluidity, bending modulus, thickness, stiffness, and bilayer pressure profile; in this manner, cholesterol modulates the conformation and function of membrane proteins (19). Plasma membrane cholesterol is involved in not only the control of various cell functions, such as signaling, adhesion, motility, and remodeling of the cytoskeleton (20), but also EC responses to biomechanical forces (10, 21–23). For instance, the depletion of EC plasma membrane cholesterol inhibits the shear stress-induced activation of extracellular signal-regulated kinase (22), whereas the addition of cholesterol to EC plasma membranes suppresses the shear stress-induced phosphorylation of VEGFRs (10). However, the roles of plasma membrane cholesterol in EC mechanotransduction have not been fully elucidated, partly because of a lack of information concerning how plasma membrane cholesterol behaves in ECs under shear stress.In the last decade, domain 4 (D4) of perfringolysin O (PFO), a cholesterol-binding toxin, has been widely used as a cholesterol-specific molecular sensor for measuring and imaging cholesterol in cellular membranes (24). When D4 and its mutants labeled with fluorophores are added to the extracellular medium, cholesterol in the outer leaflet of the plasma membrane can be visualized in living cells; when these sensors are introduced into the cells by gene transfer or microinjection, cholesterol in the inner leaflets of the membrane can be visualized. A recent study showed that these D4-based cholesterol sensors did not lyse the cells or cross the plasma membrane, allowing trans-bilayer asymmetries in plasma membrane cholesterol to be identified in various mammalian cells (25).In the present study, we applied controlled levels of shear stress to cultured human pulmonary aortic endothelial cells (HPAECs) and examined changes in the amounts and distribution of plasma membrane cholesterol using fluorescence imaging and flow cytometry with D4 mutant-based cholesterol biosensors. We also used biochemical measurements to analyze changes in the amounts of cholesterol in whole cells and isolated plasma membranes. Furthermore, to determine whether changes in plasma membrane cholesterol affect mitochondrial oxidative phosphorylation, we examined changes in mitochondrial ATP concentrations using real-time imaging with a fluorescence resonance energy transfer (FRET)-based ATP biosensor (26).