A nationwide epidemiological study of testicular torsion: Analysis of the Japanese National Database.

Testicular torsion is a severe urological emergency caused by the twisting of the spermatic cord. The nationwide incidence of testicular torsion in Japan has not been previously reported. Accordingly, we aimed to estimate the nationwide incidence of testicular torsion using the National Database of Health Insurance Claims and Specific Health Checkups of Japan (NDB) and examine the orchiectomy rate. This cross-sectional study was based on data from the NDB. We extracted data of patients aged < 21 years with documented testicular torsion and relevant treatment from January 2018 to December 2020. Testicular torsion was identified based on the Japanese standardized disease codes. The national incidence rate between 2018 and 2020 was calculated and assessed according to age and region of origin. Orchiectomy rates were evaluated according to age. The nationwide incidence rates of testicular torsion were 14.46, 15.09, and 15.88 per 100,000 males aged < 21 years in 2018, 2019, and 2020, respectively. The orchiectomy rate was 7.1%. Testicular torsion was most frequently observed during winter. A similar trend was observed nationwide. To the best of our knowledge, this study is the first to report the nationwide incidence of testicular torsion in Japan.

Increased neurotoxicity of high-density lipoprotein secreted from murine reactive astrocytes deficient in a peroxisomal very-long-chain fatty acid transporter Abcd1.

X-linked adrenoleukodystrophy (X-ALD) is a genetic neurodegenerative disorder caused by pathogenic variants in ABCD1, resulting in the accumulation of very-long-chain fatty acids (VLCFAs) in tissues. The etiology of X-ALD is unclear. Activated astrocytes play a pathological role in X-ALD. Recently, reactive astrocytes have been shown to induce neuronal cell death via saturated lipids in high-density lipoprotein (HDL), although how HDL from reactive astrocytes exhibits neurotoxic effects has yet to be determined. In this study, we obtained astrocytes from wild-type and Abcd1-deficient mice. HDL was purified from the culture supernatant of astrocytes, and the effect of HDL on neurons was evaluated in vitro. To our knowledge, this study shows for the first time that HDL obtained from Abcd1-deficient reactive astrocytes induces a significantly higher level of lactate dehydrogenase (LDH) release, a marker of cell damage, from mouse primary cortical neurons as compared to HDL from wild-type reactive astrocytes. Notably, HDL from Abcd1-deficient astrocytes contained significantly high amounts of VLCFA-containing phosphatidylcholine (PC) and LysoPC. Activation of Abcd1-deficient astrocytes led to the production of HDL containing decreased amounts of PC with arachidonic acid in sn-2 acyl moieties and increased amounts of LysoPC, presumably through cytosolic phospholipase A2 α upregulation. These results suggest that compositional changes in PC and LysoPC in HDL, due to Abcd1 deficiency and astrocyte activation, may contribute to neuronal damage. Our findings provide novel insights into central nervous system pathology in X-ALD.

Transmembrane helix 6 of ABCD4 is indispensable for cobalamin transport.

ABCD4, which belongs to the ABC protein subfamily D, plays a role in the transport of cobalamin from lysosomes to the cytosol by cooperating with ATP-binding and ATP-hydrolysis. Pathogenic variants in the ABCD4 gene lead to an inherited metabolic disorder characterized by cobalamin deficiency. However, the structural requirements for cobalamin transport in ABCD4 remain unclear. In this study, six proteoliposomes were prepared, each containing a different chimeric ABCD4 protein, wherein each of the six transmembrane (TM) helices was replaced with the corresponding ABCD1. We analyzed the cobalamin transport activities of the ABCD mutants. In the proteoliposome with chimeric ABCD4 replacing TM helix 6, the cobalamin transport activity disappeared without a reduction in ATPase activity, indicating that TM helix 6 contributes to substrate recognition. Furthermore, the substitution of aspartic acid at position 329 or threonine at position 332 in TM helix 6 with the basic amino acid lysine led to a decrease in cobalamin-transport activity without causing a reduction in ATPase activity. The amino acids in TM helix 6 may be critically involved in substrate recognition; the charged state in the C-terminal half of TM helix 6 of ABCD4 is responsible for cobalamin transport activity.

Successful conservative treatment for left ventricular free wall rupture after acute myocardial infarction.

Left ventricular free wall rupture (LVFWR) is a rare but fatal complication of acute myocardial infarction (AMI). An 81-year-old female patient with several cardiovascular risk factors presented to the emergency department with symptoms of developing a chronic stomachache and cold sweat. An echocardiograph showed wall motion abnormalities from the lateral to posterior wall, as well as pericardial effusion containing clots of up to 17 mm in the posterior wall that indicated LVFWR after AMI. Although she was conscious after being brought to the initial care unit, she suddenly lost consciousness and fell into electromechanical dissociation (EMD). Endotracheal intubation was immediately initiated and her pericardial drainage and intra aortic balloon pump (IABP) placement, and hemodynamics recovered. Although she had 100% obstruction in the left circumflex artery (LCX) #12 on coronary angiography (CAG), she was discharged to the Intensive Care Unit (ICU) without percutaneous coronary intervention (PCI). Conservative treatment such as intubation, sedation, pericardiocentesis and strict blood pressure management as well as treatment by IABP long-term support led to the patient being uneventfully discharged after 60 days.

Fundamental Characterization of Antibody Fusion-Single-Chain TNF Recombinant Proteins Directed against Costimulatory TNF Receptors Expressed by T-Lymphocytes.

The costimulatory signal regulated by the members of the tumor necrosis factor receptor (TNFR) superfamily expressed by T cells plays essential roles for T cell responses and has emerged as a promising target for cancer immunotherapy. However, it is unclear how the difference in TNFR costimulation contributes to T cell responses. In this study, to clarify the functional significance of four different TNFRs, OX40, 4-1BB, CD27 and GITR, we prepared corresponding single-chain TNF ligand proteins (scTNFLs) connected to IgG Fc domain with beneficial characteristics, i.e., Fc-scOX40L, Fc-sc4-1BBL, Fc-scCD27L (CD70) and Fc-scGITRL. Without intentional cross-linking, these soluble Fc-scTNFL proteins bound to corresponding TNFRs induced NF-kB signaling and promoted proliferative and cytokine responses in CD4+ and CD8+ T cells with different dose-dependencies in vitro. Mice injected with one of the Fc-scTNFL proteins displayed significantly augmented delayed-type hypersensitivity responses, showing in vivo activity. The results demonstrate that each individual Fc-scTNFL protein provides a critical costimulatory signal and exhibits quantitatively distinct activity toward T cells. Our findings provide important insights into the TNFR costimulation that would be valuable for investigators conducting basic research in cancer immunology and also have implications for T cell-mediated immune regulation by designer TNFL proteins.

The impact that myocarditis for post-acute COVID-19 syndrome may be dermatomyositis-like myocarditis: A case report.

Myocarditis is often reported as a complication of COVID-19 infection or post-vaccination, but there are few reports of "myocarditis for Post-acute COVID-19 syndrome", and many unknowns still remain. Apart from that, an association between COVID-19 infection and dermatomyositis has also been reported. We describe the clinical presentation of acute myocarditis in a patient who had developed COVID-19 syndrome one-month earlier. A healthy 49-year-old man experienced typical COVID-19 symptoms. Thirty-two days later, he was admitted because of fever and severe fatigue, chest pain and bradycardia. Blood tests showed major inflammation. PCR for SARS-CoV-2 on nasopharyngeal swab (ID NOW™) was positive, but diagnosed as a previous infection due to a high CT value. Because of haemodynamic worsening with both an increase in cardiac troponin I and NT-pro BNP levels and reduced wall motion on echocardiography, acute myocarditis was suspected. Myocardial biopsy revealed severe lymphocytic infiltration and interstitial edema between myocardial fibers. These findings led to the diagnosis of fulminant myocarditis. Interestingly, myocardium was also stained with human myxovirus resistance protein 1 (MxA). We consider that there may be an aspect of "dermatomyositis-like myocarditis with SARS-CoV-2" in our case. This is the first case of fulminant myocarditis for Post-acute COVID-19 syndrome in which diagnosis of active myocarditis was proven by pathological examination following myocardial biopsy and strong association with dermatomyositis was suggested pathologically.

Decreased thymic output predicts progression of chronic kidney disease.

BACKGROUND: Chronic kidney disease (CKD) is age-related disease, and decreased renal function is associated with the premature aging of T cells and increased incidence of other age-related diseases. However, the relationship between T cell senescence and CKD progression remains unclear. Here, we investigated the relationship between T cell senescence, as indicated by decreased thymic output and increased proportion of highly differentiated CD28- T cells, and CKD progression. RESULTS: A total of 175 patients with non-dialysis-dependent CKD were enrolled in this study. Thymic output was assessed based on the CD45RA+CD31+CD4+ cell (recent thymic emigrant [RTE]) counts (RTEs) (/mm3) and the proportion of RTE among CD4+ T cells (RTE%). Highly differentiated T cells were assessed based on the proportion of CD28- cells among CD4+ T cells (CD28-/CD4+) and CD28- cells among CD8+ T cells (CD28-/CD8+). The primary outcome was estimated glomerular filtration rate (eGFR) decline of ≥40% or initiation of renal replacement therapy. The association between T cell senescence and renal outcomes was examined using Cox proportional hazards models and restricted cubic splines. The median age was 73 years, 33% were women, and the median eGFR was 26 mL/min/1.73 m2. The median RTEs, RTE%, CD28-/CD4+, and CD28-/CD8+ were 97.5/mm3, 16.2, 5.3, and 49.7%, respectively. After a median follow-up of 1.78 years, renal outcomes were observed in 71 patients. After adjusting for age, sex, eGFR, proteinuria, diabetes, and cytomegalovirus seropositivity, decreased RTEs, which corresponded to decreased thymic output, significantly and monotonically increased the risk of poor renal outcome (p = 0.04), and decreased RTE% and increased highly differentiated CD28-/CD4+ T cells also tended to monotonically increase the risk (p = 0.074 and p = 0.056, respectively), but not CD28-/CD8+ T cells. CONCLUSIONS: Decreased thymic output in CKD patients, as well as increased highly differentiated CD4+ T cells, predicted renal outcomes. Thus, the identification of patients prone to CKD progression using T cell senescence, particularly decreased RTE as a biomarker, may help to prevent progression to end-stage kidney disease.

OX40 Ligand-Mannose-Binding Lectin Fusion Protein Induces Potent OX40 Cosignaling in CD4+ T Cells.

OX40, a member of the tumor necrosis factor (TNF) receptor superfamily, is induced on activated T cells. Membrane-bound OX40 ligand (OX40L) expressed by activated antigen-presenting cells induces OX40 signaling, which promotes T cell immunity. OX40 agonism would be a potential target for immunotherapy, however, it remains unclear how the activity of OX40 can be successfully controlled by a designer OX40L protein. We prepared a soluble OX40L protein possessing a PA-peptide tag and a collagenous trimerization domain from mannose-binding lectin (MBL), and tested whether PA-MBL-OX40L fusion protein worked as an agonist for OX40. We found that the majority of recombinant PA-MBL-OX40L protein purified from culture supernatants displayed a trimer structure and bound to cell surface OX40 or OX40-Fc fusion protein in a dose-dependent manner. Upon stimulation of CD4+ T cells with TCR/CD3 without CD28, PA-MBL-OX40L displayed significantly increased proliferative and cytokine responses when compared with a benchmark agonistic monoclonal antibody for OX40. Both soluble and immobilized forms of PA-MBL-OX40L induced potent OX40 signaling in CD4+ T cells. Mice administered with PA-MBL-OX40L displayed significantly augmented T cell-mediated delayed-type hypersensitivity responses. Our results suggest that activity of OX40L could be engineered to elicit better T cell responses by rational design of its assembly and architecture.

Detection of Characteristic Phosphatidylcholine Containing Very Long Chain Fatty Acids in Cerebrospinal Fluid from Patients with X-Linked Adrenoleukodystrophy.

X-linked Adrenoleukodystrophy (X-ALD) is a rare genetic neurological disorder caused by a mutation of the ABCD1 gene that encodes a peroxisomal ABC protein ABCD1. ABCD1 has a role in transporting very long chain fatty acid (VLCFA)-CoA into the peroxisome for ß-oxidation. ABCD1 dysfunction leads to reduced VLCFA ß-oxidation and in turn increased VLCFA levels in the plasma and the cells of all tissues; these increased plasma levels have been used to diagnose X-ALD. It has been reported that plasma VLCFA is not correlated with the severity and disease phenotype of X-ALD. Therefore, we cannot predict the disease progression by the plasma VLCFA level. Cerebrospinal fluid (CSF) is constantly produced by brain, and thus levels of lipids containing VLCFA in CSF might be informative in terms of assessing X-ALD pathology. LC-MS/MS-based analysis showed that phosphatidylcholine (PC) containing VLCFA signals, such as PC 40 : 0(24 : 0/16 : 0), PC 42 : 0(26 : 0/16 : 0), PC 44 : 4(24 : 0/20 : 4) and PC 46 : 4(26 : 0/20 : 4) were characteristically detected only in the CSF from patients with X- ALD. In the present study, we analyzed limited number of patient's CSF samples (2 patients with X-ALD) due to the limitations of the availability for CSF samples from this rare disease. However, our finding would offer helpful information for studying the disease progression biomarkers in X-ALD. To our knowledge, this is the first report of analyzing lipids containing VLCFA in CSF from patients with X-ALD.

Preoperative hypomagnesemia as a possible predictive factor for postoperative increase of transvalvular pressure gradient in hemodialysis patients treated with transcatheter aortic valve implantation.

BACKGROUND: Patients undergoing maintenance hemodialysis (HD) with severe aortic stenosis are at a high risk for bioprosthetic valve dysfunction after transcatheter aortic valve implantation (TAVI). Currently, preoperative factors that predict the occurrence of valve dysfunction after TAVI on HD patients remain to be elucidated. The aim of this study is to analyze the association between preoperative clinical factors and valve stenosis after TAVI on HD patients. METHODS: Twenty-four of HD patients who underwent TAVI at our institution between April 2012 and January 2016 were analyzed. The mean aortic transvalvular pressure gradient (MPG) and effective orifice area index (EOAi) were assessed by serial echocardiography. Associations between preoperative clinical factors and time-series changes in MPG were examined using mixed-effects linear regression model for repeated measures. RESULTS: Three patients developed severe structural valve deterioration with calcific valve stenosis requiring reoperation. A multivariate linear mixed-effects model showed that lower serum magnesium (sMg) levels were associated with the increase of MPG after TAVI (beta-coefficient = 0.019, p = 0.03). No correlation was observed with serum calcium, phosphorus, or intact parathyroid hormone. Time-series changes of MPG and EOAi had significant difference between lower and higher sMg group. All 3 of the patients who underwent reoperation showed lower preoperative sMgs. CONCLUSION: Among bone-mineral metabolism markers, preoperative hypomagnesemia was associated with the increase of MPG after TAVI, suggesting that hypomagnesemia could predict post-TAVI valve dysfunction in HD patients. Further studies with larger sample sizes are warranted.

Reduction of Cell Surface T-Cell Receptor by Non-Mitogenic CD3 Antibody to Mitigate Murine Lupus.

T-cells are critically involved in the pathogenesis of systemic lupus erythematosus. Although treatment with the anti-CD3 antibody has been reported to be effective in several autoimmune disease animal models including lupus, the immunosuppressive mechanisms remain obscure because of its pleiotropic in vivo kinetics. In this study, a conventional anti-CD3 (2C11C) and a non-mitogenic anti-CD3 with a manipulated Fc region (2C11S) were compared to elucidate the underlying mechanism of action. The efficacy and safety of 2C11S in vivo were demonstrated by sustained TCR reduction for a longer period as compared to 2C11C and no induction of cytokine release or T-cell depletion. Anti-CD3s were administered to NZB/W F1 (BWF1) mice at different time points for individual periods. The short-term treatment with 2C11S in the early phase of lupus suppressed the autoantibody associated with the reduction of germinal center B-cells. Treatment in the late phase attenuated lupus nephritis without affecting autoantibodies or differentiation of effector T-cells. The effect of reduced TCR in the development of autoimmunity was examined by CD3ζ heterozygous-deficient mice, in which T-cells had reduced TCR intensity but showed normal TCR signaling response. Autoantibody and lupus nephritis were attenuated significantly in CD3ζ heterozygous-deficient lupus-prone mice. Collectively, the reduction of surface TCR by non-mitogenic anti-CD3 could sufficiently suppress the development of lupus.

Interlayer Modification of a Layered Silicate RUB-18 with 4-Phosphonophenylsilane and Its Surface Acidic Functions.

The interlayer silylation of a layered silicate H-RUB-18 (Si4O7(OH)2) using a new aromatic silylating reagent containing a phosphonic acid group (4-phosphonophenylsilane: PPS) was demonstrated (H-PPS-RUB-18). The phosphonic acid groups were attached to the silicate layers through the reaction of H-RUB-18 with (4-diethoxyphosphorylphenyl)-triethoxysilane (p-PPS-E), and the ester moieties were subsequently hydrolyzed with hydrochloric acid. H-PPS-RUB-18 is a solid acid, as indicated by the intercalation of various alkylamines and the catalytic acetalization of ketones. A systematic increase in interlayer spacing leading to surface acidic properties was obtained through intercalation with a series of alkylamines. In addition, H-PPS-RUB-18 was exfoliated, resulting in single-layer nanosheets with ca. 2.0 nm thickness. The catalytic acetalization of ketones was related to the interlayer spacing of the modified RUB-18.

TNF Receptor-Associated Factor 5 Limits IL-27 Receptor Signaling in CD4+ T Lymphocytes.

TNF receptor-associated factor 5 (TRAF5) restrains early signaling activity of the IL-6 receptor in naive CD4+ T cells by interacting with the shared gp130 chain, although TRAF5 was initially discovered as a cytoplasmic adaptor protein to activate signaling mediated by TNF receptor family molecules. This leads to the question of whether TRAF5 limits signaling via the receptor for IL-27, which is composed of gp130 and WSX-1. The aim of this study is to clarify the role of TRAF5 in IL-27 receptor signaling and to understand the differential role of TRAF5 on cytokine receptor signaling. We found that Traf5 -/- CD4+ T cells displayed significantly higher levels of phosphorylated STAT1 and STAT-regulated genes Socs3 and Tbx21, as early as 1 h after IL-27 exposure when compared with Traf5 +/+ CD4+ T cells. Upon IL-27 and TCR signals, the Traf5 deficiency significantly increased the induction of IL-10 and promoted the proliferation of CD4+ T cells. Traf5 -/- mice injected with IL-27 displayed significantly enhanced delayed-type hypersensitivity responses, demonstrating that TRAF5 works as a negative regulator for IL-27 receptor signaling. In contrast, IL-2 and proliferation mediated by glucocorticoid-induced TNF receptor-related protein (GITR) and TCR signals were significantly decreased in Traf5 -/- CD4+ T cells, confirming that TRAF5 works as a positive regulator for cosignaling via GITR. Collectively, our results demonstrate that TRAF5 reciprocally controls signals mediated by the IL-27 receptor and GITR in CD4+ T cells and suggest that the regulatory activity of TRAF5 in gp130 is distinct from that in TNF receptor family molecules in a T cell.

Clinical course involving thrombocytosis and thrombocytopenia in a patient with bladder cancer treated with gemcitabine and cisplatin.

Gemcitabine induce thrombocytopenia and thrombocytosis as toxicity. In this report, we show detailed time-course for platelet fluctuation. Our case emphasis attention to monitor see-saw-like toxicity on platelet count.

The lysosomal protein ABCD4 can transport vitamin B12 across liposomal membranes in vitro.

Vitamin B12 (cobalamin) is an essential micronutrient for human health, and mutation and dysregulation of cobalamin metabolism are associated with serious diseases, such as methylmalonic aciduria and homocystinuria. Mutations in ABCD4 or LMBRD1, which encode the ABC transporter ABCD4 and lysosomal membrane protein LMBD1, respectively, lead to errors in cobalamin metabolism, with the phenotype of a failure to release cobalamin from lysosomes. However, the mechanism of transport of cobalamin across the lysosomal membrane remains unknown. We previously demonstrated that LMBD1 is required for the translocation of ABCD4 from the endoplasmic reticulum to lysosomes. This suggests that ABCD4 performs an important function in lysosomal membrane cobalamin transport. In this study, we expressed human ABCD4 and LMBD1 in methylotrophic yeast and purified them. We prepared ABCD4 and/or LMBD1 containing liposomes loaded with cobalamin and then quantified the release of cobalamin from the liposomes by reverse-phase HPLC. We observed that ABCD4 was able to transport cobalamin from the inside to the outside of liposomes dependent on its ATPase activity and that LMBD1 exhibited no cobalamin transport activity. These results suggest that ABCD4 may be capable of transporting cobalamin from the lysosomal lumen to the cytosol. Furthermore, we examined a series of ABCD4 missense mutations to understand how these alterations impair cobalamin transport. Our findings give insight into the molecular mechanism of cobalamin transport by which ABCD4 involves and its importance in cobalamin deficiency.

Generation of an immortalized astrocytic cell line from Abcd1-deficient H-2KbtsA58 mice to facilitate the study of the role of astrocytes in X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is an inherited metabolic disease characterized by inflammatory demyelination, and activated astrocytes as well as microglia are thought to be involved in its pathogenesis. Conditionally immortalized astrocytic cell clones were prepared from wild-type or Abcd1-deficient H-2KbtsA58 transgenic mice to study the involvement of astrocytes in the pathogenesis of X-ALD. The established astrocyte clones expressed astrocyte-specific molecules such as Vimentin, S100ß, Aldh1L1 and Glast. The conditionally immortalized astrocytes proliferated vigorously and exhibited a compact cell body under a permissive condition at 33 °C in the presence of IFN-γ, whereas they became quiescent and exhibited substantial cell enlargement under a non-permissive condition at 37 °C in the absence of IFN-γ. An Abcd1-deficient astrocyte clone exhibited a decrease in the ß-oxidation of very long chain fatty acid (VLCFA) and an increase in cellular levels of VLCFA, typical features of Abcd1-deficiency. Upon stimulation with LPS, the Abcd1-deficient astrocyte clone expressed higher levels of pro-inflammatory genes, such as Il6, Nos2, Ccl2 and Cxcl10, compared to wild-type (WT) astrocytes. Furthermore, the Abcd1-deficient astrocytes produced higher amounts of chondroitin sulfate, a marker of reactive astrocytes. These results suggest that dysfunction of Abcd1 renders astrocytes highly responsive to innate immune stimuli. Conditionally immortalized cell clones which preserve astrocyte properties are a useful tool for analyzing the cellular and molecular pathology of ALD.

Acyl-CoA thioesterase activity of peroxisomal ABC protein ABCD1 is required for the transport of very long-chain acyl-CoA into peroxisomes.

The ABCD1 protein, one of the four ATP-binding cassette (ABC) proteins in subfamily D, is located on the peroxisomal membrane and is involved in the transport of very long chain fatty acid (VLCFA)-CoA into peroxisomes. Its mutation causes X-linked adrenoleukodystophy (X-ALD): an inborn error of peroxisomal ß-oxidation of VLCFA. Whether ABCD1 transports VLCFA-CoA as a CoA ester or free fatty acid is controversial. Recently, Comatose (CTS), a plant homologue of human ABCD1, has been shown to possess acyl-CoA thioesterase (ACOT) activity, and it is suggested that this activity is required for transport of acyl-CoA into peroxisomes. However, the precise transport mechanism is unknown. Here, we expressed human His-tagged ABCD1 in methylotrophic yeast, and characterized its ACOT activity and transport mechanism. The expressed ABCD1 possessed both ATPase and ACOT activities. The ACOT activity of ABCD1 was inhibited by p-chloromercuribenzoic acid (pCMB), a cysteine-reactive compound. Furthermore, we performed a transport assay with ABCD1-containing liposomes using 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled acyl-CoA as the substrate. The results showed that the fatty acid produced from VLCFA-CoA by ABCD1 is transported into liposomes and that ACOT activity is essential during this transport process. We propose a detailed mechanism of VLCFA-CoA transport by ABCD1.