Multimodal Imaging of Optic Nerve Head in Retinitis Pigmentosa.

BACKGROUND: A pallor optic nerve head (ONH) is one of the three features of retinitis pigmentosa (RP). This study aimed to assess the ONH prospectively by color tone, presence of hyper-reflective tissue, blood flow, retinal nerve fiber layer (RNFL) thickness, ganglion cell complex (GCC) and investigate the change in these parameters with and without ONH pallor. METHODS: The presence of ONH pallor was assessed by three independent examiners through careful examination using fundus photographs. The presence of a hyper-reflective structure on the ONH was carefully evaluated using a volume scan optical coherence tomography (OCT). RNFL thickness and ellipsoid zone (EZ) width around the macula were also evaluated by OCT. Laser speckle flowgraphy was used to measure the mean blur rate of the entire ONH area, which was subsequently divided into the vessel area (MV) and tissue area (MT). RESULTS: Twenty-eight eyes of 28 patients with RP (55.4 ± 16.23 years of age) were included. The pale ONH was observed in 10 (35%) eyes. Hyper-reflective structures were observed in seven (25%) eyes. No significant correlation was found between the pale ONH and the presence of a hyper-reflective structure (Pearson's chi-squared test, p = .364). The average of the ONH area, MV, and MT was 8.65 ± 3.08 AU, 17.81 ± 7.54 AU, and 6.4 ± 2.66 AU, respectively, which significantly decreased in patients with pallor ONH (all p < .05). The global RNFL thickness was 73.54 ± 18.82 µm. The nasal and superior quadrants and global RNFL thickness in patients with a pale ONH were significantly thinner than in patients without a pale ONH (all p < .05). The global and superior and inferior GCC thickness in patients with a pale ONH were significantly thinner than in patients without a pale ONH(all p < .05).There was no difference in the EZ width between patients with and without a pale ONH (p = .107). CONCLUSION: We conducted multiple assessments of the ONH in RP patients and investigated its clinical significance. Our findings suggest that ONH pallor may indicate a comprehensive change that emerges alongside the progression of retinal degeneration in RP. TRIAL REGISTRATION: This trial was retrospectively registered in the UMIN Clinical Trial Registry (UMIN ID: 000048168).

Corrigendum: Citrate synthase insufficiency leads to specific metabolic adaptations in the heart and skeletal muscles upon low-carbohydrate diet feeding in mice.

[This corrects the article DOI: 10.3389/fnut.2022.925908.].

Citrate Synthase Insufficiency Leads to Specific Metabolic Adaptations in the Heart and Skeletal Muscles Upon Low-Carbohydrate Diet Feeding in Mice.

A decrease in TCA cycle activity may lead to impaired nutrition metabolism and cellular energy shortage. Herein, we aimed to characterize the detailed metabolic changes that compensate for energy shortages in energy-consuming organs (heart and skeletal muscles) in mice with knockout of citrate synthase (CS), an important enzyme in the TCA cycle. CS hetero knockout (CS +/-) mice and wild-type mice were fed a low-carbohydrate ketogenic diet (LCKD) or high-fat, high-carbohydrate diet (HFHCD) to induce metabolic changes. Body weight, blood serum parameters, metabolic gene expression, and adenosine triphosphate (ATP) levels were measured in the heart and skeletal muscles. Glycogen content, anabolic and catabolic biomarkers, and morphological changes were also assessed in the skeletal muscles. After diet feeding, there were no differences observed in the body weight and blood serum parameters between wild-type and CS +/- mice. The cardiac expression of genes related to the utilization of fatty acids, monocarboxylates, and branched amino acids increased in LCKD-fed CS +/- mice. In contrast, no significant differences in gene expression were observed in the muscles of LCKD-fed mice or the heart and muscles of HFHCD-fed mice. ATP levels decreased only in the skeletal muscles of LCKD-fed CS +/- mice. Additionally, the decrease in glycogen content, suppression of p70 S6 kinase, and presence of type I fiber atrophy were observed in the muscles of LCKD-fed CS +/- mice. These results suggest that the energy-consuming organs with CS insufficiency may undergo tissue-specific adaption to compensate for energy shortages when the carbohydrate supply is limited.

TNF-α induces Claudin-1 expression in renal tubules in Alport mice.

Claudin-1 (CL-1) is responsible for the paracellular barrier function of glomerular parietal epithelial cells (PEC) in kidneys, but the role of CL-1 in proximal tubules remains to be elucidated. In this study, to evaluate CL-1 as a potential therapeutic drug target for chronic kidney disease, we investigated change of CL-1 expression in the proximal tubules of diseased kidney and elucidated the factors that induced this change. We established Alport mice as a kidney disease model and investigated the expression of CL-1 in diseased kidney using quantitative PCR and immunohistochemistry (IHC). Compared to wild type mice, Alport mice showed significant increases in plasma creatinine, urea nitrogen and urinary albumin excretion. CL-1 mRNA was increased significantly in the kidney cortex and CL-1 was localized on the adjacent cell surfaces of PECs and proximal tubular epithelial cells. The infiltration of inflammatory cells around proximal tubules and a significant increase in TNF-α mRNA were observed in diseased kidneys. To reveal factors that induce CL-1, we analyzed the induction of CL-1 by albumin or tumor necrosis factor (TNF)-α in human proximal tubular cells (RPTEC/TERT1) using quantitative PCR and Western blotting. TNF-α increased CL-1 expression dose-dependently, though albumin did not affect CL-1 expression in RPTEC/TERT1. In addition, both CL-1 and TNF-α expression were significantly increased in UUO mice, which are commonly used as a model of tubulointerstitial inflammation without albuminuria. These results indicate that CL-1 expression is induced by inflammation, not by albuminuria in diseased proximal tubules. Moreover, we examined the localization of CL-1 in the kidney of IgA nephropathy patients by IHC and found CL-1 expression was also elevated in the proximal tubular cells. Taken together, CL-1 expression is increased in the proximal tubular epithelial cells of diseased kidney. Inflammatory cells around the tubular epithelium may produce TNF-α which in turn induces CL-1 expression.

Isonitrogenous low-carbohydrate diet elicits specific changes in metabolic gene expression in the skeletal muscle of exercise-trained mice.

With the renewed interest in low-carbohydrate diets (LCDs) in the sports field, a few animal studies have investigated their potential. However, most rodent studies have used an LCD containing low protein, which does not recapitulate a human LCD, and the muscle-specific adaptation in response to an LCD remains unclear. Therefore, we investigated the effects of two types of LCDs, both containing the same proportion of protein as a regular diet (isonitrogenous LCD; INLCD), on body composition, exercise performance, and metabolic fuel selection at the genetic level in the skeletal muscles of exercise-trained mice. Three groups of mice (n = 8 in each group), one fed a regular AIN-93G diet served as the control, and the others fed either of the two INLCDs containing 20% protein and 10% carbohydrate (INLCD-10%) or 20% protein and 1% carbohydrate (INLCD-1%) had a regular exercise load (5 times/week) for 12 weeks. Body weight and muscle mass did not decrease in either of the INLCD-fed groups, and the muscle glycogen levels and endurance capacity did not differ among the three groups. Only in the mice fed INLCD-1% did the plasma ketone concentration significantly increase, and gene expression related to glucose utilization significantly declined in the muscles. Both INLCD-1% and INLCD-10% consumption increased gene expression related to lipid utilization. These results suggest that, although INLCD treatment did not affect endurance capacity, it helped maintain muscle mass and glycogen content regardless of the glucose intake restrictions in trained mice. Moreover, an INLCD containing a low carbohydrate content might present an advantage by increasing lipid oxidation without ketosis and suppressing muscle glucose utilization.

IgM to IgG Class Switching Is a Necessary Step for Pemphigus Phenotype Induction in Desmoglein 3-Specific B Cell Receptor Knock-in Mouse.

Pemphigus vulgaris is an autoimmune blistering disease caused by IgG targeting desmoglein 3 (Dsg3), an adhesion molecule of keratinocytes. Anti-Dsg3 IgG production is prevented in healthy individuals, but it is unclear how Dsg3-specific B cells are regulated. To clarify the immunological condition regulating Dsg3-specific B cells, a pathogenic anti-Dsg3 Ig (AK23) knock-in mouse was generated. AK23 knock-in B cells developed normally without undergoing deletion or acquiring an anergic phenotype in vivo. The knock-in B cells showed Ca2+ influx upon IgM cross-linking and differentiated into AK23-IgG+ B cells after LPS and IL-4 stimulation in vitro that induced a pemphigus phenotype after adoptive transfer into Rag2 -/- mice. However, the knock-in mouse itself produced AK23-IgM but little IgG without blisters in vivo. Dsg3 immunization and skin inflammation caused AK23-IgG production and a pemphigus phenotype in vivo. Furthermore, Fcgr2b deficiency or haploinsufficiency spontaneously induced AK23-IgG production and a pemphigus phenotype with poor survival rates in AK23 knock-in mice. To assess Fcgr2b involvement in Ig class-switch efficiency, postswitch transcripts of B cells were quantified and significantly higher in Fcgr2b -/- and Fcgr2b +/- mice than wild-type mice in a gene dose-dependent manner. Finally, RNA sequencing revealed reduced expression of FCGR2B and FcγRIIB-related genes in patient B cells. These results indicated that Dsg3-specific B cells do not spontaneously perform pathogenic class switching in vivo, and pemphigus phenotype induction was prevented under normal conditions. Attenuated FcγRIIB signaling is also one of the drivers for pathogenic class switching and is consistent with immunological features identified from clinical samples. This study unveiled a characteristic immune state silencing autoreactive B cells in mice.

Peripheral tolerance by Treg via constraining OX40 signal in autoreactive T cells against desmoglein 3, a target antigen in pemphigus.

Antigen-specific peripheral tolerance is crucial to prevent the development of organ-specific autoimmunity. However, its function decoupled from thymic tolerance remains unclear. We used desmoglein 3 (Dsg3), a pemphigus antigen expressed in keratinocytes, to analyze peripheral tolerance under physiological antigen-expression conditions. Dsg3-deficient thymi were transplanted into athymic mice to create a unique condition in which Dsg3 was expressed only in peripheral tissue but not in the thymus. When bone marrow transfer was conducted from high-avidity Dsg3-specific T cell receptor-transgenic mice to thymus-transplanted mice, Dsg3-specific CD4+ T cells developed in the transplanted thymus but subsequently disappeared in the periphery. Additionally, when Dsg3-specific T cells developed in Dsg3-/- mice were adoptively transferred into Dsg3-sufficient recipients, the T cells disappeared in an antigen-specific manner without inducing autoimmune dermatitis. However, Dsg3-specific T cells overcame this disappearance and thus induced autoimmune dermatitis in Treg-ablated recipients but not in Foxp3-mutant recipients with dysfunctional Tregs. The molecules involved in disappearance were sought by screening the transcriptomes of wild-type and Foxp3-mutant Tregs. OX40 of Tregs was suggested to be responsible. Consistently, when OX40 expression of Tregs was constrained, Dsg3-specific T cells did not disappear. Furthermore, Tregs obtained OX40L from dendritic cells in an OX40-dependent manner in vitro and then suppressed OX40L expression in dendritic cells and Birc5 expression in Dsg3-specific T cells in vivo. Lastly, CRISPR/Cas9-mediated knockout of OX40 signaling in Dsg3-specific T cells restored their disappearance in Treg-ablated recipients. Thus, Treg-mediated peripheral deletion of autoreactive T cells operates as an OX40-dependent regulatory mechanism to avoid undesired autoimmunity besides thymic tolerance.

A Randomized Trial of Home Blood-Pressure Reduction by Alcohol Guidance During Outpatient Visits: OSAKE Study.

BACKGROUND: To evaluate the effectiveness of the nurse-led alcohol guidance to control home blood pressure (HBP) in the morning among male patients with hypertension during outpatient visits. METHODS: We enrolled 53 male patients with an HBP of ≥135/85 mm Hg with excessive drinking (alcohol ≥210 g/week or ≥60 g/day habitually) among outpatients in a randomized trial. Patients were assigned to a nurse-led alcohol guidance intervention or to the control. The primary outcomes were the mean HBP of 5 consecutive days at 6 months and alcohol consumption. RESULTS: Twenty-eight and 25 patients were randomized to intervention and control groups, respectively (mean age; 62.7 years old and 64.5, respectively). At baseline, the groups were well balanced across most characteristics. At 6 months, the mean HBP was 131/82 mm Hg in the intervention group vs. 145/87 mm Hg in the control group (SBP <0.001, DBP = 0.09). An HBP level of less than 135/85 mm Hg was achieved among 55.6% of the participants in the intervention group vs. 16.7% in the control group (P = 0.004). The alcohol consumption at 6 months was 256 ± 206 g/w vs. 413 ± 260 g/w, respectively (P = 0.020). CONCLUSIONS: We confirmed the effectiveness of the nurse-led alcohol guidance to control the HBP in male patients with hypertension during outpatient visits. PUBLIC TRIALS REGISTRY NUMBER: UMIN000017454 (UMIN Clinical Trials Registry).

Antibody to CD137 Activated by Extracellular Adenosine Triphosphate Is Tumor Selective and Broadly Effective In Vivo without Systemic Immune Activation.

Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. SIGNIFICANCE: Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1.

Final analysis of a phase II study of nivolumab in combination with ipilimumab for unresectable chemotherapy-naive advanced melanoma.

Nivolumab plus ipilimumab combination is currently one of the preferred regimens for advanced melanoma in recently updated clinical practice guidelines. However, the evidence on the efficacy of the combination for acral or mucosal subtypes remains less robust. This is the final analysis of a multicenter, open-label, uncontrolled phase II study that investigated the long-term efficacy and safety in treatment-naive Japanese patients with advanced melanoma, including acral or mucosal subtypes, and subsequent therapy after discontinuation of the investigational agents. Patients received four doses of nivolumab (1 mg/kg i.v.) in combination with ipilimumab (3 mg/kg i.v.) at 3-week intervals, followed by doses of nivolumab (3 mg/kg i.v.) at 2-week intervals. The median follow-up period was 20.8 months (range, 5.2-35.0). The centrally and locally assessed objective response rates were both 43.3% (13/30; 95% confidence interval [CI], 25.5-62.6). Median progression-free survival was not reached (95% CI, 3.02-not reached), and median overall survival was also not reached (95% CI, 19.52-not reached). The 30-month progression-free survival and overall survival rates were 50.3% and 54.2%, respectively. No new safety concerns were detected. After discontinuation of the investigational agents, 83.3% of patients received some form of subsequent therapy including 43.3% of patients who received nivolumab monotherapy and 26.7% of patients who received radiotherapy. Of the four patients who discontinued the investigational agents because of immune-related adverse events, two received subsequent therapy (nivolumab and ipilimumab, respectively) and the other two showed long-term treatment-free survival (659 and 590 days, respectively). Long-term survival with nivolumab plus ipilimumab was observed in Japanese patients with melanoma including acral and mucosal subtypes, which is consistent with the CheckMate 067 study. Many patients continued to receive some form of treatment safely after stopping treatment with nivolumab plus ipilimumab.

Association of Baseline Serum Levels of CXCL5 With the Efficacy of Nivolumab in Advanced Melanoma.

Anti-programmed cell death protein 1 (PD1) antibodies are in wide use for the treatment of various cancers. PD1 antibody-based immunotherapy, co-administration of nivolumab and ipilimumab, is one of the optimal immunotherapies, especially in advanced melanoma with high tumor mutation burden. Since this combined therapy leads to a high frequency of serious immune-related adverse events (irAEs) in patients with advanced melanoma, biomarkers are needed to evaluate nivolumab efficacy to avoid serious irAEs caused by ipilimumab. This study analyzed baseline serum levels of CXCL5, CXCL10, and CCL22 in 46 cases of advanced cutaneous melanoma treated with nivolumab. Baseline serum levels of CXCL5 were significantly higher in responders than in non-responders. In contrast, there were no significant differences in baseline serum levels of CXCL10 and CCL22 between responders and non-responders. These results suggest that baseline serum levels of CXCL5 may be useful as a biomarker for identifying patients with advanced cutaneous melanoma most likely to benefit from anti-melanoma immunotherapy.

Problems, goals and solutions reported by cancer patients participating in group problem-solving therapy.

BACKGROUND: The purpose of the present study was to categorize and develop lists of contents of problems, goals, solution plans generated through the brainstorming work in therapy, and selected solutions for execution, that treated in problem-solving therapy (PST) for cancer patients in clinical settings, and to describe their characteristics. Additionally, examining the associations of problem domains with characteristics of participants, was also aimed. METHODS: We conducted content analysis using records of thirty-one cancer patients (M = 62.6 years old; SD = 10.5) who participated in PST program. RESULTS: Problems were categorized into four domains (e.g. psychological and existential problems; physical problems; social relations; social living environment). Participants under treatment at baseline reported psychological and existential problems most often (P < 0.05). Goals were categorized into four domains (e.g. improving mental health; improving physical functions; improving social relations and improving one's social living environment). Solution plans generated through the brainstorming work in therapy were categorized into four domains (e.g. emotion regulation/cognitive adjustment; health behaviors; adjustment of social relationships and adjusting one's social living environment). Selected solutions for execution were categorized into four domains (e.g. emotion regulation/cognitive adjustment; health behaviors; adjustment of social relationships and adjusting one's social living environment). CONCLUSIONS: We found that various problems, goals and solutions were treated in PST of realistic clinical setting. Creating lists based on our study and making use of it for the materials as aids while implementing the PST or being shared with patients and medical staff would be expected.

A Case of Acute Exacerbation of Chronic Adrenal Insufficiency Due to Ipilimumab Treatment for Advanced Melanoma.

BACKGROUND Ipilimumab is a therapeutic human monoclonal antibody that targets the T-cell inhibitory molecule, cytotoxic T-lymphocyte antigen-4 (CTLA-4), and is classified as an immune checkpoint inhibitor that has been shown to improve prognosis in patients with advanced melanoma. However, several immune-related adverse events have been reported to be associated with ipilimumab Treatment. A case of acute exacerbation of chronic adrenal insufficiency is presented that highlights that glucocorticoid dosage for patients undergoing steroid treatment at the time of ipilimumab treatment has yet to be established. CASE REPORT A 50-year-old Japanese woman was diagnosed with malignant melanoma on the sole of her right foot. During her second course of ipilimumab treatment, she developed acute adrenal insufficiency caused by isolated adrenocorticotropic hormone (ACTH) deficiency, which required treatment with oral hydrocortisone. However, the symptoms of her adrenal insufficiency worsened, and she commenced treatment with 12 courses of nivolumab, a therapeutic human monoclonal antibody that blocks programmed cell death protein 1 (PD-1) on the surface of T-cells. She did not require corticosteroid support during nivolumab treatment. CONCLUSIONS This case report highlights the risk of exacerbating adrenal insufficiency during treatment with ipilimumab. The differences in clinical outcome in this patient between ipilimumab and nivolumab treatment might be explained by the different mechanisms between ipilimumab and nivolumab on immune function.

Serum Level of Soluble CD163 May Be a Predictive Marker of the Effectiveness of Nivolumab in Patients With Advanced Cutaneous Melanoma.

Antibodies against programmed cell death protein 1, such as nivolumab and pembrolizumab, are widely used for treating various cancers, including advanced melanoma. Nivolumab significantly prolongs survival in patients with metastatic melanoma, and sequential administration with lipilimumab may improve outcomes when switched at the appropriate time. Biomarkers are therefore needed to evaluate nivolumab efficacy soon after first administration. This study analyzed serum levels of soluble cluster of differentiation 163 (sCD163) in 59 cases of advanced cutaneous melanoma and 16 cases of advanced mucosal melanoma treated using nivolumab. Serum levels of sCD163 were significantly increased after 6 weeks in responders compared to non-responders after initial administration of nivolumab for cutaneous melanoma. In contrast, no significant difference between responders and non-responders was seen among patients with non-cutaneous melanoma. These results suggest that sCD163 may be useful as a biomarker for selecting patients with advanced cutaneous melanoma most likely to benefit from anti-melanoma immunotherapy.

Efficacy and safety of nivolumab in combination with ipilimumab in Japanese patients with advanced melanoma: An open-label, single-arm, multicentre phase II study.

AIM: The aim of the study was to evaluate the efficacy and safety of nivolumab combined with ipilimumab in treatment-naïve Japanese patients with advanced melanoma. METHODS: In this multicentre, single-arm study, treatment-naïve Japanese patients with unresectable stage III/IV or recurrent melanoma received nivolumab (1 mg/kg) plus ipilimumab (3 mg/kg) every 3 weeks for four doses, followed by biweekly doses of nivolumab (3 mg/kg). The primary end-point was centrally assessed objective response rate (ORR). Secondary end-points included overall survival (OS), progression-free survival (PFS), disease control rate and safety. RESULTS: The subtypes of the thirty patients enrolled were: 12, mucosal; eight, non-acral cutaneous; seven, acral; two, uveal and one, unknown primary melanoma. The ORR was 43.3% (95% confidence interval [CI]: 25.5, 62.6) with central and local assessment. The centrally and locally assessed disease control rate (95% CI) were 73.3% (54.1, 87.7) and 86.7% (69.3, 96.2), respectively. At the median follow-up period of 14.1 months (range 5.2-27.7), median OS and centrally assessed PFS were not reached. OS (95% CI) at 6, 12, 18 and 24 months was 93.3% (75.9, 98.3), 83.3% (64.5, 92.7), 72.9% (50.0, 86.5) and 65.6% (40.4, 82.2), respectively. Treatment-related adverse events (AEs) occurred in all patients. Grade III-IV and serious AEs occurred, mostly during the combination phase, in 23 (76.7%) and 20 (66.7%) patients, respectively. No treatment-related deaths occurred. CONCLUSIONS: This study confirmed the efficacy and safety of nivolumab plus ipilimumab in treatment-naïve Japanese patients with advanced melanoma including rare subtypes. Incidence rates for grade III-IV AEs were high but manageable with appropriate medical attention and treatment. TRIAL REGISTRATION: JapicCTI-152869.

Activation of central/effector memory T cells and T-helper 1 polarization in malignant melanoma patients treated with anti-programmed death-1 antibody.

Human anti-programmed death-1 (PD-1) antibody possesses the capability to revitalize host T cells and has been an effective therapy for metastatic malignant melanoma (MM). The precise subsets of T cells predominantly activated by anti-PD-1, however, have not yet been clarified. In this study, peripheral blood mononuclear cells obtained from MM patients scheduled to receive anti-PD-1 (nivolumab) therapy, and healthy subjects (HS), were systematically examined on flow cytometry to identify changes in the proportion of immune cell subsets. Compared with HS, MM patients prior to therapy had an increased proportion of activated CD8+ T cells with effector memory phenotypes (Tem), and PD-1 positive subsets of CD4+ central memory T cells (Tcm) and T-helper (Th)17 cells. After a single course of anti-PD-1 therapy, MM patients had an increase in activated Tem and Tcm subsets of CD4+ and CD8+ T cells, and activated Th1 plus T-helper follicular 1 cells. There was no consistent change in the proportion of Tfh cells, B cells, natural killer cells, or dendritic cells. The observed activated phenotypes were attenuated during the course of therapy, but regulatory T cells belonging to the CD3+CD4+CD45RO+CD25high fraction increased at disease progression. Taken together, anti-PD-1 therapy modulates systemic immune reactions and exerts anti-tumor effects, not only by revitalizing Tem and Tcm of CD4+ and CD8+ T cells, but also via a shift to a Th1 phenotype.

Nivolumab-induced thyroid dysfunction lacking antithyroid antibody is frequently evoked in Japanese patients with malignant melanoma.

BACKGROUND: Nivolumab, an anti-programmed cell death-1 monoclonal antibody, has improved the survival of patients with malignant melanoma. Despite its efficacy, nivolumab inconsistently induces thyroid dysfunction as an immune-related adverse event (irAE). This study aimed to evaluate nivolumab-induced thyroid dysfunction to determine the risks and mechanisms of thyroid irAEs. METHODS: After excluding 10 patients, data of 24 patients with malignant melanoma (aged 17-85 years; 54% female) were retrospectively analyzed. RESULTS: Thyroid irAEs were observed in seven patients (29%). Three patients had hypothyroidism after preceding transient thyrotoxicosis, and the other four patients had hypothyroidism without thyrotoxicosis. Levothyroxine-Na replacement was required in three patients. Antithyroid antibody (ATA) titer was elevated in one of four assessable patients. The average (±SD) time to onset of thyroid irAE was 33.6 (±21.9) weeks. The administration period of nivolumab was longer in patients with thyroid irAEs than in those without thyroid irAEs (P < 0.01). There were no significant differences between patients with and without thyroid irAEs regarding age, sex, tumor stage, response to nivolumab therapy, baseline thyroid function, antithyroid peroxidase antibody (anti-TPO Ab) and antithyroglobulin antibody (anti-Tg Ab). CONCLUSIONS: Thyroid dysfunction was a common irAE of nivolumab in malignant melanoma. Neither anti-TPO Ab nor anti-Tg Ab was associated with the risk for nivolumab-induced thyroid dysfunction. A conventional ATA-independent mechanism might be involved in thyroid irAEs. Further studies are required to clarify the mechanism and identify the predictive factors of thyroid irAEs.

Bufalin induces DNA damage response under hypoxic condition in myeloma cells.

Hypoxia serves a crucial role in the development of drug resistance in various cancer cells. Therefore, many attempts targeting hypoxia are underway to overcome the drug resistance mediated by hypoxia. This strategy is useful for multiple myeloma (MM) cells, as MM cells reside within the bone marrow, where oxygen concentrations are relatively low. A natural compound library was screened to identify compounds exerting cytotoxicity in MM cells under hypoxic conditions. Bufalin exhibited marked cytotoxicity to MM cells under normoxic and hypoxic conditions. No significant toxicity was observed in lymphocytes obtained from healthy donors. Under normoxic conditions, bufalin induced a DNA double strand break (DSB) response, ROS induction and apoptosis within 24 with a rapid response compared with melphalan. Interestingly, the bufalin-induced DSB response was not impaired by low oxygen concentrations while the DSB response by melphalan was reduced. Furthermore, treatment with bufalin abolished HIF-1α expression under hypoxia, suggesting that bufalin exerts cytotoxicity under hypoxia by regulating HIF-1α. These results indicate that bufalin induces apoptosis in MM cells through DSB under hypoxic conditions by inhibiting HIF-1α, suggesting that bufalin could be useful for eradication of drug-resistant MM cells in the hypoxic microenvironment.