Pneumocephalus and Cerebral Abscess in Granulomatosis With Polyangiitis.

Granulomatosis with polyangiitis (GPA) is a systemic autoimmune disease that causes necrotizing vasculitis of small- to medium-sized blood vessels and necrotizing granulomatous inflammation, primarily of the upper and lower respiratory system. A 33-year-old woman presented with a 16-month history of headaches, nasal obstruction, and anosmia.

Inhibition of GCN2 sensitizes ASNS-low cancer cells to asparaginase by disrupting the amino acid response.

General control nonderepressible 2 (GCN2) plays a major role in the cellular response to amino acid limitation. Although maintenance of amino acid homeostasis is critical for tumor growth, the contribution of GCN2 to cancer cell survival and proliferation is poorly understood. In this study, we generated GCN2 inhibitors and demonstrated that inhibition of GCN2 sensitizes cancer cells with low basal-level expression of asparagine synthetase (ASNS) to the antileukemic agent l-asparaginase (ASNase) in vitro and in vivo. We first tested acute lymphoblastic leukemia (ALL) cells and showed that treatment with GCN2 inhibitors rendered ALL cells sensitive to ASNase by preventing the induction of ASNS, resulting in reduced levels of de novo protein synthesis. Comprehensive gene-expression profiling revealed that combined treatment with ASNase and GCN2 inhibitors induced the stress-activated MAPK pathway, thereby triggering apoptosis. By using cell-panel analyses, we also showed that acute myelogenous leukemia and pancreatic cancer cells were highly sensitive to the combined treatment. Notably, basal ASNS expression at protein levels was significantly correlated with sensitivity to combined treatment. These results provide mechanistic insights into the role of GCN2 in the amino acid response and a rationale for further investigation of GCN2 inhibitors for the treatment of cancer.

Studies of CDK 8/19 inhibitors: Discovery of novel and selective CDK8/19 dual inhibitors and elimination of their CYP3A4 time-dependent inhibition potential.

In this article, synthetic studies around a pyridylacrylamide-based hit compound (1), utilizing structure-based drug design guided by CDK8 docking models, is discussed. Modification of the pendant 4-fluorophenyl group to various heteroaromatic rings was conducted aiming an interaction with the proximal amino acids, and then replacement of the morpholine ring was targeted for decreasing potential of time-dependent CYP3A4 inhibition. These efforts led to the compound 4k, with enhanced CDK8 inhibitory activity and no apparent potential for time-dependent CYP3A4 inhibition (CDK8 IC50: 2.5nM; CYP3A4 TDI: 99% compound remaining). Compound 4k was found to possess a highly selective kinase inhibition profile, and also showed favorable pharmacokinetic profile. Oral administration of 4k (15mg/kg, bid. for 2weeks) suppressed tumor growth (T/C 29%) in an RPMI8226 mouse xenograft model.

Discovery of 6-[5-(4-fluorophenyl)-3-methyl-pyrazol-4-yl]-benzoxazin-3-one derivatives as novel selective nonsteroidal mineralocorticoid receptor antagonists.

In the course of our study on selective nonsteroidal mineralocorticoid receptor (MR) antagonists, a series of novel benzoxazine derivatives possessing an azole ring as the core scaffold was designed for the purpose of attenuating the partial agonistic activity of the previously reported dihydropyrrol-2-one derivatives. Screening of alternative azole rings identified 1,3-dimethyl pyrazole 6a as a lead compound with reduced partial agonistic activity. Subsequent replacement of the 1-methyl group of the pyrazole ring with larger lipophilic side chains or polar side chains targeting Arg817 and Gln776 increased MR binding activity while maintaining the agonistic response at the lower level. Among these compounds, 6-[1-(2,2-difluoro-3-hydroxypropyl)-5-(4-fluorophenyl)-3-methyl-1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (37a) showed highly potent in vitro activity, high selectivity versus other steroid hormone receptors, and good pharmacokinetic profiles. Oral administration of 37a in deoxycorticosterone acetate-salt hypertensive rats showed a significant blood pressure-lowering effect with no signs of antiandrogenic effects.

Spontaneous resolution of inflammatory myopathy involving the masseter muscle following COVID-19 mRNA vaccination.

According to previous reports, most cases of inflammatory myopathy following messenger RNA (mRNA) vaccination can be classified as idiopathic inflammatory myopathy, particularly dermatomyositis, owing to their similar clinical features and courses. However, some patients have different clinical features and courses. We report a rare case of transient inflammatory myopathy involving the masseter muscle following the third dose of coronavirus disease 2019 (COVID-19) mRNA vaccination. An 80-year-old woman presented with a history of fever and fatigue for 3 months soon after receiving the third COVID-19 mRNA vaccination. Her symptoms progressed to jaw pain and inability to open her mouth. She also experienced mild proximal muscle weakness in the lower limbs but no skin manifestations or daily difficulties. Fat-saturated T2-weighted magnetic resonance imaging showed bilateral high-intensity signals for the masseter and quadriceps muscles. The patient experienced spontaneous resolution of fever and improvement of symptoms 5 months after onset. The timing of the onset of symptoms, the lack of detectable autoantibodies, and the atypical presentation of myopathy in the masseter muscles, in addition to the spontaneous mild course of the disease, all indicate the substantial role of mRNA vaccination in this myopathy. Since then, the patient has been followed up for 4 months without any recurrence of symptoms or any additional treatment. It is important to recognise that the course of myopathy after COVID-19 mRNA vaccination could be different from that of typical idiopathic inflammatory myopathies.

Persistence of SARS-CoV-2 neutralizing antibodies and anti-Omicron IgG induced by BNT162b2 mRNA vaccine in patients with autoimmune inflammatory rheumatic disease: an explanatory study in Japan.

Background: Autoimmune inflammatory rheumatic disease (AIRD) patients are at high risk of the coronavirus disease 2019 (COVID-19), but the medium-term effects of immunosuppressants on vaccine efficacy are unknown. We investigated the duration of humoral responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wild-type and Omicron variant in AIRD patients administered with two doses of the BNT162b2 (Pfizer-BioNTech) vaccine. Methods: Serum-neutralizing antibody (NAb) and anti-receptor-binding domain (RBD)/spike antibody levels were measured. Short- and medium-term effects of immunosuppressants were analyzed pre-vaccination (Term 1) and 14-42 days (Term 2) and 100-200 days (Term 3) after the second vaccination. Findings: From Feb 1, 2021, to Feb 28, 2022, 439 AIRD patients and 146 healthy controls were investigated. The seropositivity rate and log10-NAb titers were significantly lower in AIRD patients than in controls at Terms 2 and 3. In rheumatoid arthritis patients, tumor necrosis factor-α inhibitors (TNFis) at Term 3, and older age, glucocorticoids, and abatacept at Terms 2 and 3 were risk factors for reduced responses. Anti-Omicron RBD/spike IgG levels strongly correlated with NAb titers. Interpretation: Glucocorticoids, TNFis, and abatacept treatments negatively affect the longevity of humoral responses to SARS-CoV-2, including Omicron, after two vaccine doses. These findings may inform the timing of additional vaccination for AIRD patients. Funding: Cloud Funding of Peace Winds Japan; Center of Innovation Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Japan Society for the Promotion of Science KAKENHI; Japan Agency for Medical Research and Development; Kansai Economic Federation; Mitsubishi Zaidan; and Research Grant from Japan Agency for Medical Research and Development-Core Research for Evolutional Science and Technology.

Celastrol suppresses humoral immune responses and autoimmunity by targeting the COMMD3/8 complex.

Celastrol, a bioactive molecule extracted from the Tripterygium wilfordii plant, has been shown to exhibit anti-inflammatory properties. However, its mechanism of action has not been fully elucidated. Here, we show that celastrol suppresses humoral immune responses and autoimmunity by disabling a protein complex consisting of copper metabolism MURR1 domain-containing (COMMD) 3 and COMMD8 (COMMD3/8 complex), a signaling adaptor for chemoattractant receptors. Having demonstrated the involvement of the COMMD3/8 complex in a mouse model of rheumatoid arthritis, we identified celastrol as a compound that covalently bound to and dissociated the COMMD3/8 complex. Celastrol inhibited B cell migration, reduced antibody responses, and blocked arthritis progression, recapitulating deficiency of the COMMD3/8 complex. These effects of celastrol were abolished in mice expressing a celastrol-resistant mutant of the COMMD3/8 complex. These findings establish that celastrol exerts immunosuppressive activity by targeting the COMMD3/8 complex. Our study suggests that the COMMD3/8 complex is a potentially druggable target in autoimmune diseases and points to celastrol as a lead pharmacologic candidate in this capacity.

The role of CD98hc in mouse macrophage functions.

CD98hc is a type II transmembrane protein that covalently links to one of several L-type amino acid transporters. CD98hc was first identified as a lymphocyte activation marker. In this study, we examined the role that CD98hc plays in the functions of macrophages using tissue specific knock-out miceCD98hc (CD98hc(flox/-)LysM-cre mice). When isolated peritoneal macrophages were incubated for 48 h, the macrophages obtained from the knock-out mice showed round-shaped morphologies, while almost all of the cells obtained from the control mice were spindle-shaped. The macrophage functions such as the antigen-presenting, phagocytic, and fusion activities, have been reported to decrease in CD98hc-deficient peritoneal macrophages. In addition, when the CD98hc deficient macrophages were stimulated with either IFN-γ/LPS or IL-4, the production of NO(2) or arginase-I decreased in comparison to that observed in the control macrophages. These findings show that the CD98hc molecules play an important role in the activation and functions of macrophages.

Case report: Acute exacerbation of interstitial pneumonia related to messenger RNA COVID-19 vaccination.

Messenger RNA (mRNA) vaccines that protect against COVID-19 are widely used in many countries owing to their high efficacy and safety profiles. Recently, few severe adverse events, such as anaphylaxis and myocarditis, were reported in healthy individuals. The safety of mRNA COVID-19 vaccines has not been adequately studied in patients with interstitial lung disease. We report 2 cases of acute exacerbation of preexisting interstitial pneumonia associated with mRNA COVID-19 vaccination. In both cases, lung disease was stable before the vaccination. Initial responses to steroid therapy were unfavorable, and intravenous cyclophosphamide was administered in both cases. Both patients were diagnosed with vaccine-related exacerbation of interstitial pneumonia based on laboratory results, radiologic features, and the observed clinical course, which lacked other causative events. We suggest that clinicians should note the possibility of acute exacerbation of pneumonia after mRNA COVID-19 vaccination and carefully monitor patients with interstitial lung disease.

Consecutive BNT162b2 mRNA vaccination induces short-term epigenetic memory in innate immune cells.

Consecutive mRNA vaccinations against SARS-CoV-2 reinforced both innate and adaptive immune responses. However, it remains unclear whether the enhanced innate immune responses are mediated by epigenetic regulation and, if so, whether these effects persist. Using mass cytometry, RNA-Seq, and ATAC-Seq, we show that BNT162b2 mRNA vaccination upregulated antiviral and IFN-stimulated gene expression in monocytes with greater effects after the second vaccination than those after the first vaccination. Transcription factor-binding motif analysis also revealed enriched IFN regulatory factors and PU.1 motifs in accessible chromatin regions. Importantly, although consecutive BNT162b2 mRNA vaccinations boosted innate immune responses and caused epigenetic changes in isolated monocytes, we show that these effects occurred only transiently and disappeared 4 weeks after the second vaccination. Furthermore, single-cell RNA-Seq analysis revealed that a similar gene signature was impaired in the monocytes of unvaccinated patients with COVID-19 with acute respiratory distress syndrome. These results reinforce the importance of the innate immune response in the determination of COVID-19 severity but indicate that, unlike adaptive immunity, innate immunity is not unexpectedly sustained even after consecutive vaccination. This study, which focuses on innate immune memory, may provide novel insights into the vaccine development against infectious diseases.

Deaths associated with pandemic (H1N1) 2009 among children, Japan, 2009-2010.

To clarify the cause of deaths associated with pandemic (H1N1) 2009 among children in Japan, we retrospectively studied 41 patients <20 years of age who had died of pandemic (H1N1) 2009 through March 31, 2010. Data were collected through interviews with attending physicians and chart reviews. Median age of patients was 59 months; one third had a preexisting condition. Cause of death was categorized as unexpected cardiopulmonary arrest for 15 patients, encephalopathy for 15, and respiratory failure for 6. Preexisting respiratory or neurologic disorders were more frequent in patients with respiratory failure and less frequent in patients with unexpected cardiopulmonary arrest. The leading causes of death among children with pandemic (H1N1) 2009 in Japan were encephalopathy and unexpected cardiopulmonary arrest. Deaths associated with respiratory failure were infrequent and occurred primarily among children with preexisting conditions. Vaccine use and public education are necessary for reducing influenza-associated illness and death.

Malignant glomus tumor of the palm: a case report.

The author herein reports on a glomus tumor of the palm. A 71-year-old man consulted our hospital because of a tumor on the left palm. The tumor was deeply seated, and MRI and CT showed a deep cystic tumor adjacent to the bone. An excision of the tumor was therefore performed. Grossly, the tumor was red and partly cystic. The tumor was well defined from the surrounding tissues, and measured 25 × 24 × 22 mm. Microscopically, the tumor consisted of epithelioid perivascular cells (glomus cells) located around the blood vessels. Cystic changes and hyalinization areas were scattered. The tumor cells had moderately hyperchromatic nuclei. Nuclear pleomorphism was noticed, nucleoli were absent and apparent mitotic figures were not recognized. There were no areas of necrosis. Immunohistochemically, the glomus cells were positive for vimentin and α-smooth muscle actin. They were negative for cytokeratins, epithelial membrane antigen, CD34, CD31, factor VIII-related antigen, S100 protein, p53 protein, desmin and melanosome. The Ki-67 labeling was 5%. The tumor was diagnosed as a malignant glomus tumor because of its deep location and size > 2 cm , according to the criteria of one group. The tumor recurred 12 months later, and a further excision was performed. No metastases were found. Now, the patient is being strictly followed up.

Pyrrole-imidazole hairpin polyamides with high affinity at 5'-CGCG-3' DNA sequence; influence of cytosine methylation on binding.

To investigate the binding of 5'-CpG-3' sequences by small molecules, two pyrrole (Py)-imidazole (Im) hairpin polyamides, PyImPyIm-gamma-PyImPyIm-beta-Dp (1) and PyIm-beta-Im-gamma-PyIm-beta-Im-beta-Dp (2), which recognize the sequence 5'-CGCG-3', were synthesized. The binding affinities of the 5'-CGCG-3' sequence to the Py-Im hairpin polyamides were measured by surface plasmon resonance (SPR) analysis. SPR data revealed that dissociation equilibrium constants (K(d)) of polyamides 1 and 2 were 1.1 (+/- 0.3) x 10(-6) M and 1.7 (+/- 0.4) x 10(-8) M, respectively. Polyamide 2 possesses great binding affinity for this sequence, 65-fold higher than polyamide 1. Moreover, when all cytosines in 5'-CpGpCpG-3' were replaced with 5-methylcytosines ((m)Cs), the K(d) value of polyamide 2 increased to 5.8 (+/- 0.7) x 10(-9) (M), which indicated about 3-fold higher binding than the unmethylated 5'-CGCG-3' sequence. These results suggest that polyamide 2 would be suitable to target CpG-rich sequences in the genome.

Mutated D4-guanine diphosphate-dissociation inhibitor is found in human leukemic cells and promotes leukemic cell invasion.

OBJECTIVE: Rho GTPase may be involved in human cancer invasion via the augmentation of cell motility and adhesion. We report on two point mutations of the D4-guanine diphosphate (GDP)-dissociation inhibitor (GDI) gene, one of the Rho-GDIs, which were found in a human leukemic cell line, Reh, and the mutated D4-GDI functions as an accelerator of leukemic cell invasion. MATERIAL AND METHODS: We investigated the altered activity of GDP dissociation by mutated (mt) D4-GDI and the functions of this mt and wild-type (wt) D4-GDI in invasion. The mice inoculated with wt or mt D4-GDI vector-transfected Raji cells were observed and examined pathologically. Adhesiveness and cell motility of wt or mt D4-GDI vector-transfected Raji cells were examined. Finally, it was examined whether Rho activation was changed by mutation of D4-GDI under the condition of Rho-GDI knockdown. RESULTS: Two point mutations of the D4-GDI gene were found in Reh cells. The region of mutations is conserved among members of the Rho-GDI family at the amino acid level. D4-GDI with two mutations (V68L and V69A) functioned in a dominant negative manner in the inhibition of GDP dissociation from Rho. Severe combined immune-deficient mice inoculated with Raji cells developed hemiparalysis. The Raji cells were present in bone marrow and peripheral blood, and hepatic invasion was observed in 20% of the mice. Mice inoculated with wt D4-GDI vector-transfected Raji cells (wt D4) showed later paralysis and none developed hepatic invasion. Mice inoculated with mt D4-GDI-transfected Raji cells (mt D4) showed a 5-day reduction in the time to paraplegia and death. In addition, hepatic invasion was evident in 80% of mice transplanted with mt D4 cells. There were no differences in growth rates and amounts of guanine triphosphate (GTP)-bound Rho, cdc42, or Rac among all clones, however, GTP-bound Rho in mt D4 clone with short hairpin RNA (shRNA) vector for Rho-GDI knockdown was increased compared with wt D4 clone with shRNA vector for Rho-GDI knockdown. The mt D4 cells showed an augmentation of adhesiveness and cell motility. On the other hand, wt D4 cells showed a decreased ability of cell motility. CONCLUSION: These results suggest the mutated D4-GDI functions as a dominant negative molecule against the wt D4-GDI and accelerates invasion via regulation of cytoskeletal machinery.

The COMMD3/8 complex determines GRK6 specificity for chemoattractant receptors.

Lymphocyte migration is mediated by G protein-coupled receptors (GPCRs) that respond to chemoattractive molecules. After their activation, GPCRs are phosphorylated by different GPCR kinases (GRKs), which produces distinct functional outcomes through ß-arrestins. However, the molecular machinery that targets individual GRKs to activated GPCRs remains elusive. Here, we identified a protein complex consisting of copper metabolism MURR1 domain-containing (COMMD) 3 and COMMD8 (COMMD3/8 complex) as an adaptor that selectively recruits a specific GRK to chemoattractant receptors and promotes lymphocyte chemotaxis. COMMD8, whose stability depended on COMMD3, was recruited to multiple chemoattractant receptors. Deficiency of COMMD8 or COMMD3 impaired B cell migration and humoral immune responses. Using CXC-chemokine receptor 4 (CXCR4) as a model, we demonstrated that the COMMD3/8 complex selectively recruited GRK6 and induced GRK6-mediated phosphorylation of the receptor and activation of ß-arrestin-mediated signaling. Thus, the COMMD3/8 complex is a specificity determinant of GRK targeting to GPCRs and represents a point of regulation for immune responses.

Identification of Novel, Potent, and Orally Available GCN2 Inhibitors with Type I Half Binding Mode.

General control nonderepressible 2 (GCN2) is a master regulator kinase of amino acid homeostasis and important for cancer survival in the tumor microenvironment under amino acid depletion. We initiated studies aiming at the discovery of novel GCN2 inhibitors as first-in-class antitumor agents and conducted modification of the substructure of sulfonamide derivatives with expected type I half binding on GCN2. Our synthetic strategy mainly corresponding to the αC-helix allosteric pocket of GCN2 led to significant enhancement in potency and a good pharmacokinetic profile in mice. In addition, compound 6d, which showed slow dissociation in binding on GCN2, demonstrated antiproliferative activity in combination with the asparagine-depleting agent asparaginase in an acute lymphoblastic leukemia (ALL) cell line, and it also displayed suppression of GCN2 pathway activation with asparaginase treatment in the ALL cell line and mouse xenograft model.

Perylene-conjugated pyrrole polyamide as a sequence-specific fluorescent probe.

Perylene-conjugated pyrrole (Py)-polyamide 2 was designed and synthesized using the Fmoc solid-phase synthesis and a subsequent Sonogashira coupling reaction with 3-bromoperylene. Interestingly, conjugate 2 did not luminesce in water at 313 nm irradiation but was turned on in the presence of target double-stranded (ds) DNA, and showed strong emission with increasing DNA concentration, in particularly, by the binding to the target telomere sequences through heterodimer formation with partner 3. Importantly, the excitation spectrum of 2 clearly indicates that the Py and Imidazole (Im) moieties in the polyamide effectively sensitize the perylene moiety to give rise to fluorescence emission. Energy transfer would occur from the Py moiety to the perylene. Thus, screening of perylene-conjugates will allow us to develop a novel "molecular light switch" with sequence-specificity.

Detection of triplet repeat sequences in the double-stranded DNA using pyrene-functionalized pyrrole-imidazole polyamides with rigid linkers.

Methods for sequence-specific detection in double-stranded DNA (dsDNA) are becoming increasingly useful and important as diagnostic and imaging tools. Recently, we designed and synthesized pyrrole (Py)-imidazole (Im) polyamides possessing two pyrene moieties, 1, which showed an increased excimer emission in the presence of (CAG)(12)-containing oligodeoxynucleotides (ODN) 1 and 2. In this study, we synthesized bis-pyrenyl Py-Im polyamides with rigid linkers 2, 3, and 4 to improve their fluorescence properties. Among the conjugates, 2 showed a marked increase in excimer emission, which was dependent on the concentration of the target ODN and the number of CAG repeats in the dsDNA. Unlike conjugate 1, which has flexible linkers, the excimer emission intensity of 2 was retained at over 85%, even after 4h. Py-Im polyamides have the potential to be important diagnostic molecules for detecting genetic differences between individuals.

Requirement of beta-alanine components in sequence-specific DNA alkylation by pyrrole-imidazole conjugates with seven-base pair recognition.

To investigate the effect of incorporation of beta-alanine in alkylating N-methylpyrrole (Py)-N-methylimidazole (Im) polyamide, seco-CBI conjugates 2-8 were synthesized by an Fmoc solid-phase method and subsequent coupling with an alkylating moiety. DNA-alkylating activities of conjugates 2-8 were evaluated by high-resolution denaturing gel electrophoresis with 202-base pair (bp) DNA fragments. Alkylation by conjugates 2 and 3, which have antiparallel pairings of beta-alanine (beta) opposite beta (beta/beta) and Py/beta, occurred mainly at the adenine (A) of the matching sequences, 5'-AGCTCCA-3' (site 1) and 5'-AGCACCA-3' (site 3). However, conjugate 4, with beta/Py, did not show any DNA-alkylating activities. Similarly, conjugate 5, which possessed a Py/Py pair, weakly alkylated the matching sites at micromolar concentrations. Conjugates 6 and 7, which possessed beta/beta and Py/beta pairs, respectively, alkylated at the A of the matching sequences, 5'-ACTACCA-3' (site 2) and 5'-ACAACCA-3' (site 4). In contrast, conjugated 8, with a Py/Py pair, showed lower activity and less alkylated DNA at sites 2 and 4 with mismatched alkylation at site 1 at a higher concentration than that of 6 and 7. These results demonstrate that incorporation of beta-alanine is required for the sequence-specific alkylation by seco-CBI Py-Im conjugates with a seven-base pair sequence.

Discovery of 3,5-Diphenyl-4-methyl-1,3-oxazolidin-2-ones as Novel, Potent, and Orally Available Δ-5 Desaturase (D5D) Inhibitors.

The discovery and optimization of Δ-5 desaturase (D5D) inhibitors are described. Investigation of the 1,3-oxazolidin-2-one scaffold was inspired by a pharmacophore model constructed from the common features of several hit compounds, resulting in the identification of 3,5-diphenyl-1,3-oxazolidin-2-one 5h as a novel lead showing potent in vitro activity. Subsequent optimization focused on the modification of two metabolic sites, which provided (4S,5S)-5i, a derivative with improved metabolic stability. Moreover, adding a substituent into the upper phenyl moiety further enhanced the intrinsic activity, which led to the discovery of 5-[(4S,5S)-5-(4fluorophenyl)-4-methyl-2-oxo-1,3-oxazolidin-3-yl]benzene-1,3-dicarbonitrile (4S,5S)-5n, endowed with excellent D5D binding affinity, cellular activity, and high oral bioavailability in a mouse. It exhibited robust in vivo hepatic arachidonic acid/dihomo-γ-linolenic acid ratio reduction (a target engagement marker) in an atherosclerosis mouse model. Finally, an asymmetric synthetic procedure for this compound was established.