In vitro throughput screening of anticancer drugs using patient-derived cell lines cultured on vascularized three-dimensional stromal tissues.

The development of high-throughput anticancer drug screening methods using patient-derived cancer cell (PDC) lines that maintain their original characteristics in an in vitro three-dimensional (3D) culture system poses a significant challenge to achieving personalized cancer medicine. Because stromal tissue plays a critical role in the composition and maintenance of the cancer microenvironment, in vitro 3D-culture using reconstructed stromal tissues has attracted considerable attention. Here, a simple and unique in vitro 3D-culture method using heparin and collagen together with fibroblasts and endothelial cells to fabricate vascularized 3D-stromal tissues for in vitro culture of PDCs is reported. Whereas co-treatment with bevacizumab, a monoclonal antibody against vascular endothelial growth factor, and 5-fluorouracil significantly reduced the survival rate of 3D-cultured PDCs to 30%, separate addition of each drug did not induce comparable strong cytotoxicity, suggesting the possibility of evaluating the combined effect of anticancer drugs and angiogenesis inhibitors. Surprisingly, drug evaluation using eight PDC lines with the 3D-culture method resulted in a drug efficacy concordance rate of 75% with clinical outcomes. The model is expected to be applicable to in vitro throughput drug screening for the development of personalized cancer medicine. STATEMENT OF SIGNIFICANCE: To replicate the cancer microenvironment, we constructed a cancer-stromal tissue model in which cancer cells are placed above and inside stromal tissue with vascular network structures derived from vascular endothelial cells in fibroblast tissue using CAViTs method. Using this method, we were able to reproduce the invasion and metastasis processes of cancer cells observed in vivo. Using patient-derived cancer cells, we assessed the possibility of evaluating the combined effect with an angiogenesis inhibitor. Further, primary cancer cells also grew on the stromal tissues with the normal medium. These data suggest that the model may be useful for new in vitro drug screening and personalized cancer medicine.

A macrocyclic kinase inhibitor overcomes triple resistant mutations in EGFR-positive lung cancer.

Brigatinib-based therapy was effective against osimertinib-resistant EGFR C797S mutants and is undergoing clinical studies. However, tumor relapse suggests additional resistance mutations might emerge. Here, we first demonstrated the binding mode of brigatinib to the EGFR-T790M/C797S mutant by crystal structure analysis and predicted brigatinib-resistant mutations through a cell-based assay including N-ethyl-N-nitrosourea (ENU) mutagenesis. We found that clinically reported L718 and G796 compound mutations appeared, consistent with their proximity to the binding site of brigatinib, and brigatinib-resistant quadruple mutants such as EGFR-activating mutation/T790M/C797S/L718M were resistant to all the clinically available EGFR-TKIs. BI-4020, a fourth-generation EGFR inhibitor with a macrocyclic structure, overcomes the quadruple and major EGFR-activating mutants but not the minor mutants, such as L747P or S768I. Molecular dynamics simulation revealed the binding mode and affinity between BI-4020 and EGFR mutants. This study identified potential therapeutic strategies using the new-generation macrocyclic EGFR inhibitor to overcome the emerging ultimate resistance mutants.

Idiopathic hypersomnia with a video recording of a spontaneous sleep attack: A case report.

RATIONALE: Although patients with central disorders of hypersomnolence (CDH) exhibit characteristic symptoms of hypersomnia frequently, it takes 5 to 15 years from the onset for its diagnosis due to the lack of symptom recognition. Here, we present a case of idiopathic hypersomnia (IH), a CDH, wherein early diagnosis was aided by a video footage of a spontaneous sleep attack. PATIENT CONCERNS: A 21-year-old man lost consciousness while driving and experienced an accident. He had complained of excessive daytime sleepiness (EDS) over half a year. During his hospitalization for close monitoring of the loss of consciousness, an in-room surveillance camera captured a 14-minutes long spontaneous sleep attack, during which he experienced general muscle weakness and loss of consciousness without warnings or convulsions leading to a fall from the bed. There were no abnormalities in vital signs. DIAGNOSES: There was no significant cataplexy and less than 2 sleep-onset rapid eye movements (SOREM) in 2 sleep latency tests, with a mean sleep latency of 2.1 and 4.6 minutes. Other sleep deprivation syndromes were excluded from differential diagnosis and finally, a diagnosis of IH was confirmed according to the criteria of the Third Edition of the International Classification of Sleep Disorders. During the course of the disease, attention-deficit/hyperactive disorder (ADHD) and a gaming disorder also diagnosed. INTERVENTIONS: Pharmacological treatment with modafinil was administered for IH and methylphenidate for ADHD. Cognitive behavioral therapy was performed for the gaming disorder. OUTCOMES: The EDS improved, and sleep attacks were no longer observed. The disruption of daily life caused by the gaming disorder was also reduced. LESSONS: Video recordings of sleep attacks are beneficial for identifying the cause of loss of consciousness. Home video recordings may be helpful in the early diagnosis of IH.

Efficacy of denosumab in the treatment of hypercalcemic renal dysfunction in sarcoidosis: a case report.

A 70-year-old female patient was admitted for close examination and treatment of hypercalcemia (corrected serum calcium levels: 3.04 mmol/L) and renal dysfunction (serum creatinine levels: 254.59 µmol/L). The patient had a history of sarcoidosis, diagnosed based on epithelioid cell granulomas in subcutaneous nodule biopsies, uveitis, and bilateral hilar lymphadenopathy, which had spontaneously remitted 10 years before admission. Because the patient was diagnosed with hypercalcemia associated with recurrent sarcoidosis, prednisone (20 mg/day) was initiated, and its dose was tapered following the decrease in serum calcium and creatinine levels. However, the levels of these parameters increased again when the prednisone dose was reduced to ≤ 4 mg/day. We were concerned about glucocorticoid-induced osteoporosis in the patient but hesitated to use first-line bisphosphonates because of renal dysfunction. Therefore, denosumab was initiated to reduce the risk of hypercalcemia, renal dysfunction, and glucocorticoid-induced osteoporosis. Serum creatinine and corrected serum calcium levels subsequently decreased. The prednisone dose could be reduced following repeated denosumab administration.Thus, denosumab can be a multifaceted, beneficial option for sarcoidosis-induced hypercalcemia, as it alleviates renal dysfunction indirectly by normalizing serum calcium levels, facilitates reduction of the glucocorticoid dose, and ameliorates glucocorticoid-induced osteoporosis.

Proximal Renal Tubular Acidosis Complicated by Severe Hypocalcemia Caused by Malnutrition and Inappropriate Long-term Use of Zoledronate: A Case Report and Review of the Literature.

An 80-year-old man presented with electrolyte abnormalities, particularly hypocalcemia (3.6 mg/dL). He was diagnosed with bone and lymph node metastases from prostate cancer seven years earlier and continuously received goserelin, bicalutamide, and zoledronate. He later developed gradually worsening hypocalcemia, hypokalemia, hypophosphatemia, hypouricemia, renal dysfunction, and weight loss. Urinary potassium and phosphate loss, renal glucosuria, metabolic acidosis, and a low urine pH (5.0) were observed. Given the acquired onset and clinical course, we diagnosed the patient with zoledronate-induced proximal renal tubular acidosis. In the present case, severe hypocalcemia may have been caused by malnutrition and inappropriate long-term use of zoledronate.

Increasing averaging beats improves the test accuracy on Holter-based late potentials in patients with myocardial infarction.

BACKGROUND: The prevalence of Holter-based late potentials (H-LPs) in cases of fatal cardiac events has increased. Although the noise level of H-LP is higher than that of conventional real-time late potential (LP) recording, a procedure to reduce the noise severity in H-LP by increasing the averaging beats has not been investigated. METHODS: We enrolled 104 patients with post-myocardial infarction (MI) and 86 control participants. Among the patients, 30 reported sustained ventricular tachycardia (VT), and the remaining 74 had unrecorded VT. H-LPs were measured twice in all groups to evaluate the efficacy of increasing the averaging beats for H-LPs. Thereafter, the average of LP was calculated at 250 (default setting), 300, 400, 500, 600, 700, and 800 beats. RESULTS: Across all three groups (MI-VT group, MI non-VT group, and control group), the noise levels significantly decreased in consonance with the increase in averaging beats. In the MI-VT group, the H-LP positive rate considerably increased with the increase in the averaging beats from 250 to 800 both at night and daytime. In the MI-VT group, the LP parameters significantly deteriorated, which led to a positive judgment corresponding to the increment of the averaged night and day beats. The H-LP positive rates were unchanged in the MI non-VT and control groups, while the LP parameters remained consistent, despite the increased averaging beats in the MI non-VT and control groups. CONCLUSION: Increasing the calculated averaging beats in H-LPs can improve the sensitivity of predicting fatal cardiac events in patients with MI.

Diurnal Variation in and Optimal Time to Measure Holter-Based Late Potentials to Predict Lethal Arrhythmia after Myocardial Infarction.

Background and Objectives: Holter-based late potentials (LPs) are useful for predicting lethal arrhythmias in organic cardiac diseases. Although Holter-based LPs exhibit diurnal variation, no studies have evaluated the optimal timing of LP measurement over 24 h for predicting lethal arrhythmia that leads to sudden cardiac death. Thus, this study aimed to validate the most effective timing for Holter-based LP testing and to explore factors influencing the diurnal variability in LP parameters. Materials and Methods: We retrospectively analyzed 126 patients with post-myocardial infarction (MI) status and 60 control participants who underwent high-resolution Holter electrocardiography. Among the 126 post-MI patients, 23 developed sustained ventricular tachycardia (VT) (the MI-VT group), while 103 did not (the MI-non-VT group) during the observation period. Holter-based LPs were measured at 0:00, 4:00, 8:00, 12:00, 16:00, and 20:00, and heart rate variability analysis was simultaneously performed to investigate factors influencing the diurnal variability in LP parameters. Results: Holter-based LP parameters showed diurnal variation with significant deterioration at night and improvement during the day. Assessment at the time with the longest duration of low-amplitude signals < 40 µV in the filtered QRS complex terminus (LAS40) gave the highest receiver operating characteristics curve (area under the curve, 0.659) and the highest odds ratio (3.75; 95% confidence interval, 1.45-9.71; p = 0.006) for predicting VT. In the multiple regression analysis, heart rate and noise were significant factors affecting the LP parameters in the MI-VT and control groups. In the non-VT group, the LP parameters were significantly influenced by noise and parasympathetic heart rate variability parameters, such as logpNN50. Conclusions: For Holter-based LP measurements, the test accuracy was higher when the LP was measured at the time of the highest or worst value of LAS40. Changes in autonomic nervous system activity, including heart rate, were factors influencing diurnal variability. Increased parasympathetic activity or bradycardia may exacerbate Holter-based LP parameters.

Reference blood pressure values obtained using the auscultation method for 2-year-old Japanese children: from the Japan Environment and Children's Study.

BACKGROUND: Reference blood pressure (BP) values for Japanese children based on a large number of measurements by auscultation have not yet been established. METHODS: This was a cross-sectional analysis of data from a birth-cohort study. The data from the sub-cohort study conducted for children at the age of 2 years in the Japan Environment and Children's Study from April 2015 to January 2017 were analyzed. BP was measured via auscultation using an aneroid sphygmomanometer. Each participant was measured in triplicate, and the average value of two consecutive measurements with a difference of less than 5 mmHg was recorded. The reference BP values were estimated using the lambda-mu-sigma (LMS) method and compared with those obtained via the polynomial regression model. RESULTS: Data from 3361 participants were analyzed. Although the difference between the estimated BP values by the LMS and the polynomial regression model was small, the LMS model was more valid based on the results of the fit curve of the observed values and regression models for each model. For 2-year-old children with heights in the 50th percentile, the 50th, 90th, 95th, and 99th percentile reference values of systolic BP (mmHg) for boys were 91, 102, 106, and 112, and that for girls were 90, 101, 103, and 109, respectively, and those of diastolic BP for boys were 52, 62, 65, and 71, and that for girls were 52, 62, 65, and 71, respectively. CONCLUSION: The reference BP values for 2-year-old Japanese children were determined based on auscultation and were made available.

Analysis of the ratio of urinary beta-2-microglobulin to total protein concentration in children with isolated tubulointerstitial disease.

BACKGROUND: Proteinuria is broadly classified into glomerular and tubular proteinuria. Urinary beta-2-microglobulin (ß2-MG) is known as a marker for detecting tubulointerstitial diseases. However, tubulointerstitial damage can also lead to an increase in urinary ß2-MG level in some patients with glomerular diseases. This study aimed to determine the ratio of urinary ß2-MG to total protein (TP) concentration in patients with both isolated tubulointerstitial and glomerular disease. METHODS: This multicenter, retrospective study included children with Dent disease or lupus nephritis in five facilities. Their urinary ß2-MG levels were > 1000 µg/L. Urinary ß2-MG and TP concentrations were obtained, and the ratio of urinary ß2-MG to TP concentration (µg/mg) was calculated. The Mann-Whitney U test was performed to compare this ratio between these children. The optimal cutoff value of the ratio for considering the presence of glomerular disease was obtained from the receiver operating characteristic (ROC) curve. RESULTS: We obtained information on 23 children with Dent disease and 14 children with lupus nephritis. The median ratios of urinary ß2-MG to TP concentrations in children with Dent disease and lupus nephritis were 84.85 and 1.59, respectively. The ROC curve yielded the optimal cutoff value of this ratio for distinguishing between these diseases, and the cutoff value was found to be 22.3. CONCLUSION: In children with tubulointerstitial diseases, the urinary ß2-MG concentration may be approximately 8.5% of the TP concentration. The possibility of presenting with glomerular disease should be considered in patients with a ratio of urinary ß2-MG to TP concentration of < 22.3 (µg/mg).

Age-related reference intervals for ambulatory electrocardiographic parameters in healthy individuals.

Background: The advent of novel monitoring technologies has dramatically increased the use of ambulatory electrocardiography (AECG) devices. However, few studies have conducted detailed large-scale investigations on the incidence of arrhythmias over 24 h, especially ectopy, in healthy individuals over a wide age range. Objectives: This study aimed to investigate the incidence of arrhythmias detected using AECG and associated factors, in healthy individuals, over a wide age range. Methods: In this cross-sectional study, we performed AECG on 365 healthy volunteers (median [interquartile range]: 48 [36, 67], 20-89 years, 165 men) under free-running conditions for 24 h. Ultrasonic echocardiography and heart rate variability analysis were performed to explore the factors associated with the incidence of arrhythmias. Results: The 97.5th percentile of single ventricular ectopy (VE) was 149/day, 254/day, and 1,682/day in the 20-39-, 40-59- and 60-89-year age groups, respectively; that of single supraventricular ectopy (SVE) was 131/day, 232/day, and 1,063/day, respectively. Multivariate analysis revealed that aging was the only independent significant factor influencing the frequency of VE (ß = 0.207, P = 0.001). Age (ß = 0.642, P < 0.001), body mass index (BMI) (ß = -0.112, P = 0.009), and the root mean square of successive differences in RR intervals (ß = 0.097, P = 0.035) were factors significantly associated with SVE frequency. Conclusions: Age-specific reference intervals of VE and SVE in a large population of healthy participants over a wide age range were generated. VE and SVE increased with age; SVE was influenced by BMI and the aging-induced decrease in parasympathetic tone activity.

Early predictive factors for progression to kidney failure in infants with severe congenital anomalies of the kidney and urinary tract.

BACKGROUND: Severe congenital anomalies of the kidney and urinary tract (CAKUT) progress to infantile kidney failure with replacement therapy (KFRT). Although prompt and precise prediction of kidney outcomes is important, early predictive factors for its progression remain incompletely defined. METHODS: This retrospective cohort study included patients with CAKUT treated at 12 centers between 2009 and 2020. Patients with a maximum serum creatinine level ≤ 1.0 mg/dL during the first 3 days, patients who died of respiratory failure during the neonatal period, patients who progressed to KFRT within the first 3 days, and patients lacking sufficient data were excluded. RESULTS: Of 2187 patients with CAKUT, 92 were finally analyzed. Twenty-five patients (27%) progressed to KFRT and 24 (26%) had stage 3-5 chronic kidney disease without replacement therapy during the median observation period of 52.0 (interquartile range, 22.0-87.8) months. Among these, 22 (24%) progressed to infantile KFRT. The kidney survival rate during the infantile period was significantly lower in patients with a maximum serum creatinine level during the first 3 days (Cr-day3-max) ≥ 2.5 mg/dL (21.8%) compared with those with a Cr-day3-max < 2.5 mg/dL (95.2%) (log-rank, P < 0.001). Multivariate analysis demonstrated Cr-day3-max (P < 0.001) and oligohydramnios (P = 0.025) were associated with higher risk of infantile KFRT. Eighty-two patients (89%) were alive at the last follow-up. CONCLUSIONS: Neonatal kidney function, including Cr-day3-max, was associated with kidney outcomes in patients with severe CAKUT. Aggressive therapy for severe CAKUT may have good long-term life outcomes through infantile dialysis and kidney transplantation. A higher resolution version of the Graphical abstract is available as Supplementary information.

Urinary extracellular vesicles signature for diagnosis of kidney disease.

Congenital disorders characterized by the quantitative and qualitative reduction in the number of functional nephrons are the primary cause of chronic kidney disease (CKD) in children. We aimed to describe the alteration of urinary extracellular vesicles (uEVs) associated with decreased renal function during childhood. By nanoparticle tracking analysis and quantitative proteomics, we identified differentially expressed proteins in uEVs in bilateral renal hypoplasia, which is characterized by a congenitally reduced number of nephrons. This expression signature of uEVs reflected decreased renal function in CKD patients by congenital anomalies of the kidney and urinary tract or ciliopathy. As a proof-of-concept, we constructed a prototype ELISA system that enabled the isolation of uEVs and quantitation of expression of molecules representing the signature. The system identified decreased renal function even in its early stage. The uEVs signature could pave the way for non-invasive methods that can complement existing testing methods for diagnosing kidney diseases.

Iodine-containing ointment-induced hypothyroidism in a patient with anorexia nervosa and sacral decubitus ulcer: a case report with literature review.

We present a case of a 58-year-old woman with anorexia nervosa and a sacral decubitus ulcer who developed hypothyroidism because of an iodine-containing ointment. Considering the absence of autoimmune thyroid diseases, the development of hypothyroidism after the use of an iodine-containing ointment, and the recovery of thyroid function after the discontinuation of the ointment, we presumed that her hypothyroidism was induced by the iodine-containing ointment. Although the hypothyroidism improved after discontinuing the iodine-containing ointment, she developed aspiration pneumonia and required long-term hospitalization. Many patients with autoimmune thyroid diseases develop hypothyroidism after excessive iodine intake. However, anorexia nervosa may have exacerbated the iodine-induced hypothyroidism in our patient. To the best of our knowledge, no previous study has reported a case of hypothyroidism caused by iodine-containing ointment in a patient with anorexia nervosa. Hence, physicians must pay careful attention to a patient's background factors to ensure the early diagnosis of hypothyroidism due to iodine-containing ointments.

Subacute Thyroiditis after COVID-19: A Literature Review.

Subacute thyroiditis (SAT), potentially caused by severe acute respiratory syndrome coronavirus 2 infection, has been reported as a complication of COVID-19 since 2020. The clinical characteristics and outcomes of SAT after COVID-19 remain incompletely defined. Therefore, we aimed to collect and survey case reports of SAT after COVID-19. We performed a systematic search of PubMed/MEDLINE, Web of Science, and Google Scholar. The keywords and MeSH terms used for the searches were "subacute thyroiditis" and "COVID-19." A total of 38 patients from 26 case reports, case series, and letters on SAT associated with COVID-19 were included and analyzed. The most frequent SAT symptom was neck pain (27 cases), followed by fever (22 cases). Of the 25 cases with information on the duration between onset of COVID-19 symptoms and onset of SAT symptoms, the shortest was simultaneous occurrence, and the longest was 4 months. In most cases, patients developed SAT at several days or weeks after the onset of COVID-19. All patients with SAT recovered with no severe complications or sequelae. Clinicians should be aware of the possibility of SAT development in patients with neck pain and fever following COVID-19. Further research is necessary to determine the relationship between SAT and COVID-19.

Fibroblast growth factor receptor 3 overexpression mediates ALK inhibitor resistance in ALK-rearranged non-small cell lung cancer.

The rearrangement of anaplastic lymphoma kinase (ALK) occurs in 3%-5% of patients with non-small cell lung cancer (NSCLC) and confers sensitivity to ALK-tyrosine kinase inhibitors (TKIs). For the treatment of patients with ALK-rearranged NSCLC, various additional ALK-TKIs have been developed. Ceritinib is a second-generation ALK-TKI and has shown great efficacy in the treatment of patients with both newly diagnosed and crizotinib (a first-generation ALK-TKI)-refractory ALK-rearranged NSCLC. However, tumors can also develop ceritinib resistance. This may result from secondary ALK mutations, but other mechanisms responsible for this have not been fully elucidated. In this study, we explored the mechanisms of ceritinib resistance by establishing ceritinib-resistant, echinoderm microtubule-associated protein-like 4 (EML4)-ALK-positive H3122 cells and ceritinib-resistant patient-derived cells. We identified a mechanism of ceritinib resistance induced by bypass signals that is mediated by the overexpression and activation of fibroblast growth factor receptor 3 (FGFR3). FGFR3 knockdown by small hairpin RNA or treatment with FGFR inhibitors was found to resensitize the resistant cells to ceritinib in vitro and in vivo. FGFR ligands from either human serum or fetal bovine serum were able to activate FGFR3 and induce ceritinib resistance. A detailed analysis of ceritinib-resistant patient-derived specimens confirmed that tyrosine-protein kinase Met (cMET) amplification induces ceritinib resistance. Amplified cMET counteractivated EGFR and/or Her3 and induced ceritinib resistance. These results reveal multiple ceritinib resistance mechanisms and suggest that ceritinib resistance might be overcome by identifying precise resistance mechanisms.