Development of an in vitro assay for screening programmed death receptor-1/programmed cell death ligand 1 monoclonal antibody therapy in dogs.

Immunomodulatory antibody drugs that modulate the function of immune checkpoint molecules, such as programmed death receptor-1 (PD-1) and programmed cell death ligand 1 (PD-L1), have been established as new cancer treatments in human medicine. In recent years, there have also been reports on antibodies that inhibit immune checkpoint molecules in dogs, and clinical trials using such antibodies for canine cancer have been gradually increasing in number. Because inhibitory antibodies restore T-cell function by inhibiting the binding of PD-1 on T cells and its ligand PD-L1, the quality of antibody function has been evaluated using activated T cells or peripheral blood mononuclear cells isolated from healthy dogs; however, the assays and dogs used significantly vary. Therefore, in the present study, we developed a reporter gene assay using reporter cells (Jurkat/NFATluc/cPD1) and effector cells (CTAC/OKT3/cPDL1). Jurkat/NFATluc/cPD1 were generated by introducing both of the NFAT-responsive luciferase gene as a marker of T-cell signaling and canine PD-1, into a human T lymphoid cell line, Jurkat. CTAC/OKT3/cPDL1 were generated by introducing single-chain FV (scFV) of anti-human CD3 antibody (OKT3) and canine PD-L1 into a canine thyroid carcinoma cell line, CTAC. Ligation of PD-1 on Jurkat/NFATluc/cPD1 via binding of PD-L1 on CTAC/OKT3/cPDL1 suppressed NFAT luciferase activity induced by CD3 ligation by scFV of OKT3. The addition of anti-canine PD-1 and PD-L1 antibodies, both of which were previously developed in our laboratory, restored this suppression with high sensitivity, although the anti-human PD-L1 antibody atezolizumab induced a very weak restoration. This assay is an useful method for functionally evaluating the inhibition of canine PD-1 and PD-L1 binding.

Discovery of YS-1 as a cell line of gastric inflammatory cancer-associated fibroblasts.

BACKGROUND: Inflammatory cancer-associated fibroblasts (iCAFs) was first identified by co-culture of pancreatic stellate cells and tumor organoids. The key feature of iCAFs is IL-6high/αSMAlow. We examine this phenomenon in gastric cancer using two cell lines of gastric fibroblasts (HGF and YS-1). METHODS AND RESULTS: HGF or YS-1 were co-cultured with MKN7 (a gastric adenocarcinoma cell line) in Matrigel. IL-6 protein levels in the culture supernatant were measured by ELISA. The increased production of IL-6 was not observed in any of the combinations. Instead, the supernatant of YS-1 exhibited the higher levels of IL-6. YS-1 showed IL-6high/αSMA (ACTA2)low in real-time PCR, mRNA-seq and immunohistochemistry. In mRNA-seq, iCAFs-associated genes and signaling pathways were up-regulated in YS-1. No transition to myofibroblastic phenotype was observed by monolayer culture, or the exposure to sonic hedgehog (SHH) or TGF-ß. YS-1 conditioned medium induced changes of morphology and stem-ness/differentiation in NUGC-3 (a human gastric adenocarcinoma cell line) and UBE6T-15 (a human bone marrow-derived mesenchymal stem cell line). CONCLUSIONS: YS-1 is a stable cell line of gastric iCAFs. This discovery will promote further research on iCAFs for many researchers.

Prototype and evaluation of automatic fingertip-blood-sampling system that uses fingertip blood-vessel images to determine puncture position.

We are developing an automatic fingertip-blood-sampling system to reduce the burden on trained medical personnel. For this system to withdraw a consistent volume of sampled blood for blood tests, we developed a mechanism for our system to select and puncture the vicinity of a large blood vessel from the blood-vessel image of an individual's fingertip. We call this mechanism the fingertip-vessel-puncture mechanism. From the results of an experiment in which the fingertips of 20 individuals (men and women in their 20 s to 60 s) were manually punctured at near and far locations from the blood vessel selected with our mechanism, the following conclusions were obtained. The fingertip-vessel-puncture mechanism tends to increase the volume of sampled blood, thus is effective in sampling more than 650 µL of blood for automatic blood analyzers. It was also found that it is more effective in increasing the volume of sampled blood in the men and those who were younger.

Proof-of-concept study of the caninized anti-canine programmed death 1 antibody in dogs with advanced non-oral malignant melanoma solid tumors.

BACKGROUND: The anti-programmed death 1 (PD-1) antibody has led to durable clinical responses in a wide variety of human tumors. We have previously developed the caninized anti-canine PD-1 antibody (ca-4F12-E6) and evaluated its therapeutic properties in dogs with advance-staged oral malignant melanoma (OMM), however, their therapeutic effects on other types of canine tumors remain unclear. OBJECTIVE: The present clinical study was carried out to evaluate the safety profile and clinical efficacy of ca-4F12-E6 in dogs with advanced solid tumors except for OMM. METHODS: Thirty-eight dogs with non-OMM solid tumors were enrolled prospectively and treated with ca-4F12-E6 at 3 mg/kg every 2 weeks of each 10-week treatment cycle. Adverse events (AEs) and treatment efficacy were graded based on the criteria established by the Veterinary Cooperative Oncology Group. RESULTS: One dog was withdrawn, and thirty-seven dogs were evaluated for the safety and efficacy of ca-4F12-E6. Treatment-related AEs of any grade occurred in 13 out of 37 cases (35.1%). Two dogs with sterile nodular panniculitis and one with myasthenia gravis and hypothyroidism were suspected of immune-related AEs. In 30 out of 37 dogs that had target tumor lesions, the overall response and clinical benefit rates were 6.9% and 27.6%, respectively. The median progression-free survival and overall survival time were 70 days and 215 days, respectively. CONCLUSIONS: The present study demonstrated that ca-4F12-E6 was well-tolerated in non-OMM dogs, with a small number of cases showing objective responses. This provides evidence supporting large-scale clinical trials of anti-PD-1 antibody therapy in dogs.

Preliminary report on ultrashort perioperative partial-breast irradiation with multicatheter interstitial brachytherapy for early-stage breast cancer.

PURPOSE: Perioperative partial-breast irradiation (PBI) with multicatheter interstitial brachytherapy (MIB) is less invasive and more convenient than postoperative one. This study aimed to compare ultrashort perioperative MIB-PBI (uPBI) and conventional perioperative MIB-PBI (cPBI) performed during the same period of time. METHODS: Inclusion criteria of the study were patients aged ≥ 40 years and those with T0-2 (≤ 3 cm), N0-mi, and negative margins on mammography. The locoregional recurrence (LRR) and toxicity rates were compared between uPBI at a dose of 25.2 Gy in four fractions and cPBI at a dose of 32 Gy in eight fractions. RESULTS: In total, 198 patients (151 with uPBI and 47 with cPBI) were evaluated. At a median follow-up of 20.1 months, one (0.66%) patient in the uPBI group had LRR. The 2-year ipsilateral breast tumor recurrence-free survival rates of the uPBI and cPBI groups were 98.7% and 100%, respectively. The highest toxicity grades were grade 1 in 23 (15.2%) and grade 2 in 2 (1.3%) patients in the uPBI group, and grade 1 in 8 (17.0%) and grade 2 in 1 (2.1%) patient in the cPBI group. None of the patients in the two groups presented with grade 3 and higher toxicities. The toxicity rates between the two groups did not significantly differ. Further, 22 (14.6%) patients in the uPBI group and 8 (17.0%) in the cPBI group, and 3 (2.0%) patients in the uPBI group and 1 (2.1%) in the cPBI had acute and late toxicities, respectively. The timing of toxicity development between the two groups did not significantly differ. CONCLUSIONS: Although this preliminary report included a small sample size and had a short follow-up period, the local control and toxicity rates were similar between the uPBI and cPBI groups. Further research is warranted to investigate the ideal dose schedule of MIB-PBI.

Air kerma reference field with high energy photons using a 15 MeV electron beam from a clinical linear accelerator.

In the medical and nuclear fields, there are environments where exposure to photons with energies above several MeV can result in problems. The National Metrology Institute of Japan has developed a high-energy photon field using a 15 MeV electron beam of a clinical linear accelerator with a copper target and an aluminium filter unit to facilitate dosimeter calibration in terms of air kerma. To determine the air kerma rate, the energy fluence distribution at a reference point was calculated, and both calculations and experiments evaluated the effective energy and spatial dose distribution. Moreover, to validate the air kerma measurement, two commercial cavity chambers were calibrated in a developed photon field. The results obtained exhibited a 4% difference compared with those in a Co-60γ-ray reference field.

Harmonic radiation contribution and X-ray transmission at the Small Quantum Systems instrument of European XFEL.

Transmission measurements of the soft X-ray beamline to the Small Quantum Systems (SQS) scientific instrument at the SASE3 undulator of European XFEL are presented. Measurements are reported for a wide range of photon energies (650 eV to 2400 eV), using X-ray gas monitors as well as a bolometric radiometer. The results are in good agreement with simulations for the beam transport and show a transmission of up to 80% over the whole photon energy range. The contribution of second- and third-harmonic radiation of the soft X-ray undulator is determined at selected photon energies by performing transmission measurements using a gas absorber to provide variable attenuation of the incoming photon flux. A comparison of the results with semi-analytic calculations for the generation of free-electron laser pulses in the SASE3 undulator reveals an influence of apertures along the beam transport on the exact harmonic content to be accounted for at the experiment. The second-harmonic content is measured to be in the range of 0.1% to 0.3%, while the third-harmonic contributed a few percent to the SASE3 emission. For experiments at the SQS instrument, these numbers can be reduced through specific selections of the mirror reflection angles.

TIM23 facilitates PINK1 activation by safeguarding against OMA1-mediated degradation in damaged mitochondria.

PINK1 is activated by autophosphorylation and forms a high-molecular-weight complex, thereby initiating the selective removal of damaged mitochondria by autophagy. Other than translocase of the outer mitochondrial membrane complexes, members of PINK1-containing protein complexes remain obscure. By mass spectrometric analysis of PINK1 co-immunoprecipitates, we identify the inner membrane protein TIM23 as a component of the PINK1 complex. TIM23 downregulation decreases PINK1 levels and significantly delays autophosphorylation, indicating that TIM23 promotes PINK1 accumulation in response to depolarization. Moreover, inactivation of the mitochondrial protease OMA1 not only enhances PINK1 accumulation but also represses the reduction in PINK1 levels induced by TIM23 downregulation, suggesting that TIM23 facilitates PINK1 activation by safeguarding against degradation by OMA1. Indeed, deficiencies of pathogenic PINK1 mutants that fail to interact with TIM23 are partially restored by OMA1 inactivation. These findings indicate that TIM23 plays a distinct role in activating mitochondrial autophagy by protecting PINK1.

Impact of de-escalating systemic therapy guided by 21-gene assay on locoregional recurrence after partial-breast irradiation.

PURPOSE: Partial-breast irradiation (PBI) has been performed as alternative to whole-breast irradiation (WBI) in breast-conserving therapy (BCT). Recently, the 21-gene recurrence score (RS) was introduced to determine the adjuvant therapy for estrogen receptor (ER)-positive, and human epidermal growth factor receptor 2 (HER2)-negative diseases. However, the impact of RS-based systemic therapy on locoregional recurrence (LRR) following BCT with PBI remains uninvestigated. METHODS AND MATERIALS: Patients with ER-positive, HER2-negative, and node-negative breast cancer who underwent BCT with PBI were examined during May 2012-March 2022. In addition to immunohistochemistry (IHC), RS was available to decide on adjuvant therapy. RESULTS: In total, 431 patients were evaluated with a median followup of 48.6 months. The 4-year LRR-free survival rates were 97.3% and 96.4% in the IHC and RS cohorts, respectively (p = 0.50). Ki67 of >20% was significantly associated with LRR in the multivariate analysis (HR 4.39, p < 0.05). For patients with Ki67 > 20%, 29 of 71 (40.8%) and 46 of 59 (78.0%) patients received only endocrine therapy in the IHC and RS cohorts, respectively (p < 0.0001). For patients with Ki67 >20% who received only endocrine therapy, the 4-year LRR-free survival rates were 91.8% in the IHC cohort and 94.6% in the RS cohort (p = 0.29) CONCLUSIONS: Although the introduction of RS increased the number of patients receiving endocrine therapy alone for Ki67 >20% of disease by two times, the LRR-free survival after BCT with PBI could be maintained. However, further studies from multiple institutions with longer followup data are required.

Surgical Outcomes of Selective Laminectomy for Patients With Cervical Kyphosis: A Retrospective Study of 379 Cases.

STUDY DESIGN: A retrospective study. OBJECTIVES: This study aimed to investigate the impact of cervical kyphosis on patients with cervical spondylotic myelopathy (CSM) following selective laminectomy (SL) regarding posterior spinal cord shift (PSS), and a number of SLs. METHODS: We evaluated 379 patients with CSM after SL. The patients with kyphosis (group K) were compared with those without kyphosis (group L). Moreover, groups K and L were divided into subgroups KS and KL (SLs ≤ 2) and LS and LL (SLs ≥ 3), respectively, and analyzed. Receiver operating characteristic (ROC) curve analysis was performed to determine the cut-off value of the C2-C7 angle for satisfactory surgical outcomes, which was defined as a Japanese Orthopaedic Association (JOA) recovery rate of ≥50% in group KS. RESULTS: The average PSS (mm) in group K was smaller than that in group L (.8 vs 1.4; P < .01), but the JOA recovery rate was comparable between the 2 groups. Meanwhile, the mean PSS and JOA recovery rate (%) in group KS was lower than those in group KL, respectively (.3 vs 1.0; P < .01, 35.1 vs 52.3; P = .047). Moreover, the average PSS of group KS (.6) was smaller than those of other subgroups ( < .01). In addition, the ROC curve analysis showed that the C2-C7 angle of -14.5° could predict satisfactory surgical outcomes in group KS. CONCLUSION: Selective laminectomy is not contraindicated for patients with kyphosis, but a larger number of SLs may be indicated for the patients with C2-C7 angles of ≤ -14.5°.

DEVELOPMENT OF ABSOLUTE MEASUREMENT SYSTEM FOR 𝑾AIR IN 60CO GAMMA RAYS.

In the ICRU Report 90, the uncertainty of the recommendation for ${{W}}_{{air}}$ was changed from 0.15 to 0.35%. The purpose of this study is to develop an absolute measurement system for ${{W}}_{{air}}$ in electrons with lower uncertainty using 60Co gamma rays. ${{W}}_{{air}}$ was determined by the ratio of the energy deposition to the number of ion pairs created in a given volume of air using a graphite calorimeter and a graphite-wall ionisation chamber. The obtained value for ${{W}}_{{air}}$ was 33.91 eV with a relative standard uncertainty of 0.08 eV (k = 1), and was in good agreement with the recommended value of the ICRU Report 90 (33.97 eV). Additionally, the uncertainty for ${{W}}_{{air}}$ obtained in this investigation is 0.23% and is comparable with that reported by Burns et al., which is the lowest uncertainty in recent studies determining ${{W}}_{{air}}$ In the future, the proposed system can be used to measure ${{W}}_{{air}}$ in high-energy electron beams.

Regulatory implementation of the occupational equivalent dose limit for the lens of the eye and underlying relevant efforts in Japan.

In April 2011, the International Commission on Radiological Protection recommended reducing the occupational equivalent dose limit for the lens. Such a new occupational lens dose limit has thus far been implemented in many countries, and there are extensive discussions toward its regulatory implementation in other countries. In Japan, discussions in the Japan Health Physics Society (JHPS) began in April 2013 and in Radiation Council in July 2017, and the new occupational lens dose limit was implemented into regulation in April 2021. To share our experience, we have published a series of papers summarizing situations in Japan: the first paper based on information available by early 2017, and the second paper by early 2019. This paper (our third paper of this series) aims to review updated information available by mid-2022, such as regarding regulatory implementation of the new occupational lens dose limit, recent discussions by relevant ministries based on the opinion from the council, establishment process of safety and health management systems, the JHPS guidelines on lens dose monitoring and radiation safety, voluntary countermeasures of the licensees, development of lens dose calibration method, and recent studies on exposure of the lens in nuclear workers and biological effect on the lens.

Impact of the Coronavirus Disease 2019 Pandemic on the Clinical Features of Pediatric Respiratory Syncytial Virus Infection in Japan.

Background: Mitigation measures implemented during the coronavirus disease 2019 (COVID-19) pandemic remarkably reduced the incidence of infectious diseases among children. However, a re-emergence of respiratory syncytial virus (RSV) infection was observed in 2021 in Japan. We compared the clinical characteristics of hospitalized patients with RSV infection before and during COVID-19. Methods: We retrospectively enrolled children aged <6 years who were hospitalized with RSV infection in 18 hospitals and compared their clinical characteristics before (January 2019 to April 2020, 1675 patients) and during COVID-19 (September 2020 to December 2021, 1297 patients). Results: The mean age of patients with RSV infection was significantly higher during COVID-19 than before (17.4 vs 13.7 months, P < .001). Compared with before COVID-19, a 2.6-fold increase in RSV cases in the 2-5 years age group was observed from sentinel surveillance during COVID-19, whereas a 1.2-fold increase was noted in the same age group among hospitalized patients. On average for all patients, consolidation shadows obtained on radiography were less frequently observed (26.1 vs 29.6%, P = .04), and reduced respiratory assistance (42.2% vs 48.7%, P < .001) and hospitalization stay (5.7 vs 6.0 days, P < .001) was required in patients with RSV infection during COVID-19. Conclusions: Coronavirus disease 2019 and social activity restriction caused epidemiological changes in pediatric RSV infections, and a majority of patients with RSV infection aged ≥2 years did not develop severe symptoms requiring hospitalization. The RSV symptoms during the COVID-19 outbreak were equivalent to or milder than in the previous seasons.

Acoustic camera system for measuring ultrasound communication in mice.

To investigate biological mechanisms underlying social behaviors and their deficits, social communication via ultrasonic vocalizations (USVs) in mice has received considerable attention as a powerful experimental model. The advances in sound localization technology have facilitated the analysis of vocal interactions between multiple mice. However, existing sound localization systems are built around distributed-microphone arrays, which require a special recording arena and long processing time. Here, we report a novel acoustic camera system, USVCAM, which enables simpler and faster USV localization and assignment. The system comprises recently developed USV segmentation algorithms with a modification for overlapping vocalizations that results in high accuracy. Using USVCAM, we analyzed USV communications in a conventional home cage, and demonstrated novel vocal interactions in female ICR mice under a resident-intruder paradigm. The extended applicability and usability of USVCAM may facilitate future studies investigating typical and atypical vocal communication and social behaviors, as well as the underlying mechanisms.

Long-term survival of dogs with stage 4 oral malignant melanoma treated with anti-canine PD-1 therapeutic antibody: A follow-up case report.

A monoclonal antibody targeting programmed cell death-1 (PD-1) is one of the most promising treatments for human cancers. Clinical studies in humans demonstrated that the anti-PD-1 antibody provides a long-lasting tumour response. Previously, we established an anti-canine PD-1 therapeutic antibody (ca-4F12-E6), and the pilot clinical study demonstrated that the antibody was effective in dogs with oral malignant melanoma (OMM). However, two OMM cases were still undergoing treatment when the pilot study was published. Here, we describe the long-term follow-up of those two cases. Although both cases showed long-term survival with complete response (CR), the tumour response differed; the effect onset was slow in one case and a durable response was observed in the second case even after treatment discontinuation. Secondary malignant tumours occurred during treatment in both cases. This follow-up study revealed that ca-4F12-E6 maintains CR in dogs for more than 1 year. In addition, the pattern of tumour response was unique compared to conventional therapy. These results indicate that new evaluation criteria for tumour response may be necessary for immunotherapy in veterinary medicine. Long-term follow-up is necessary regardless of the short-term treatment responsiveness.

Unraveling the reasons behind lead phthalocyanine acting as a good absorber for near-infrared sensitive devices.

Lead phthalocyanine (PbPc) is well known to be used as a good near-infrared (NIR) light absorber for organic solar cells (OSCs) and photodetectors. The monoclinic and triclinic phases have been understood to absorb the visible and NIR regions, respectively, so far. In the present study, we demonstrated from the absorption spectra and theoretical analysis that the visible band considerably originates from not only the monoclinic but also the amorphous and triclinic phases, and revealed the exciton dynamics in the PbPc film from static/time-resolved photoluminescence (PL), which are first reported. By comparing the external quantum efficiency between PbPc- and ZnPc-based OSCs in relation to their structure, morphology, and optical (absorption and PL) characteristics, we unraveled the reasons behind the PbPc film used as a good absorber for NIR-sensitive devices.

Atlantoaxial stenosis after muscle-preserving selective laminectomy.

There are few reports of degenerative atlantoaxial stenosis and new stenosis after cervical decompression. We experienced four cases of atlantoaxial stenosis after muscle-preserving selective laminectomy. We compared these four cases with no stenosis cases after long-term follow-up of selective laminectomy, as well as healthy subjects. A total of 1205 patients who underwent muscle-preserving selective laminectomy due to cervical disorders were included in this study. Postoperative atlantoaxial stenosis, which needed decompression, appeared in 4 cases, and 30 patients did not have radiological stenosis for more than 10 years after surgery. Twenty healthy volunteers were also used as controls. The radiographic parameters measured were C2-C7 angle, C2-C7 sagittal vertical axis (SVA), C2 slope, C7 slope, C2-C5 angle, C5-C7 angle, C1-C2 angle, and atlantodental interval (ADI). We measured the anterior-posterior (AP) diameters of the spinal cord (SC) and dural tube (Dura) at C1/C2 with sagittal MRI. In the cases of atlantoaxial stenosis, the AP of SC and Dura at C1/C2 were smaller preoperatively, and the residual space for SC (SAC) was also smaller. The preoperative ADI was significantly higher in patients with atlantoaxial stenosis, suggesting preoperative instability at C1/C2. Analysis of the ROC curve showed that patients with a preoperative SAC of less than 3.6 mm and an ADI of more than 1.35 mm were more likely to develop postoperative atlantoaxial stenosis. When we perform a muscle-preserving selective laminectomy, decompression of C1/C2 is suggested when the SAC at C1/C2 is less than 3.6 mm and the ADI is more than 1.35 mm.

Effect of skin colors due to hemoglobin or melanin modulation on facial expression recognition.

Facial color is thought to contribute to the perception of emotional expression. Previous studies on facial recognition have shown that reddish and bluish faces enhance the perceptions of anger and sadness, respectively. However, the effect of facial color on the recognition of facial expressions in the range of natural skin colors remains unexplored. In this study, we examined the effect of natural skin color changes on the perception of facial expressions of anger, sadness, and happiness. We used five skin color conditions for each facial expression: standard, and hemoglobin or melanin decrease/increase (H-, H+, M-, and M + ). In the experiment, we used images of different facial expressions ranging from neutral to maximum intensity. Observers judged whether a face stimulus was perceived as "angry" ("happy" or "sad") or "not angry" ("not happy" or "not sad"). The results showed that H+, M+, H-, and M- enhanced the perception of anger and happiness, respectively. However, the change in the perception of sadness due to these modulations was small. These results suggest that the range of natural facial colors by hemoglobin and melanin modulation affects facial expression recognition. We further consider the possibility that these results could be partially explained by the lightness of the stimulus.

Therapeutic effects of anti-amyloid ß antibody after intravenous injection and efficient nose-to-brain delivery in Alzheimer's disease mouse model.

Antibody drugs that target amyloid ß (Aß) are considered possible treatments for Alzheimer's disease; however, most have been dropped from clinical trials. We hypothesized that administration route for antiAß antibody (AntiAß) might affect its therapeutic potential and thus compared delivery of antibodies to the brain and their effect on cognitive dysfunction and amyloid disposition via intravenous (i.v.) and intranasal routes with and without the cell-penetrating peptide, L-penetratin. We demonstrated that intranasal administration with L-penetratin more efficiently delivered human immunoglobulin G (IgG), a model molecule for AntiAß, to the brain compared with i.v. injection. We found that multiple intranasal treatments with Alexa 594-labeled AntiAß (A594-AntiAß) with L-penetratin significantly improved learning by mice with aged amyloid precursor protein (APP) knock-in (App KI mice). Further, intranasal administration of A594-AntiAß increased the amount of soluble Aß (1-42) in the brain, suggesting suppression of Aß aggregation in insoluble form and involvement of activated microglia in Aß clearance. Thus, administration route may be critical for efficient delivery of AntiAß to the brain, and the nose-to-brain delivery with L-penetratin can maximize its therapeutic efficacy.