[Autoimmune hemolytic anemia following the third dose of the COVID-19 vaccine].

The development and exacerbation of autoimmune diseases following coronavirus disease 2019 (COVID-19) vaccination have been reported; however, there are few reports of autoimmune hemolytic anemia (AIHA). A 75-year-old woman was admitted to the emergency department 46 days after receiving her third dose of the mRNA-1273 COVID-19 vaccine because of fatigue and general weakness. Initial laboratory analyses revealed severe hemolytic anemia with positive direct and indirect Coombs test and elevation of serum indirect bilirubin and lactate dehydrogenase. The patient had no underlying disease after a close examination and was diagnosed with warm AIHA, which was thought to be associated with COVID-19 vaccination. The anemia improved daily after the administration of prednisolone. Clinicians should be aware of the possibility of AIHA being caused by COVID-19 vaccination, and monotherapy with prednisolone should be considered in cases of severe anemia.

Utility of cerebrospinal fluid liquid biopsy in distinguishing CNS lymphoma from cerebrospinal infectious/demyelinating diseases.

BACKGROUND: Distinguishing between central nervous system lymphoma (CNSL) and CNS infectious and/or demyelinating diseases, although clinically important, is sometimes difficult even using imaging strategies and conventional cerebrospinal fluid (CSF) analyses. To determine whether detection of genetic mutations enables differentiation between these diseases and the early detection of CNSL, we performed mutational analysis using CSF liquid biopsy technique. METHODS: In this study, we extracted cell-free DNA from the CSF (CSF-cfDNA) of CNSL (N = 10), CNS infectious disease (N = 10), and demyelinating disease (N = 10) patients, and performed quantitative mutational analysis by droplet-digital PCR. Conventional analyses were also performed using peripheral blood and CSF to confirm the characteristics of each disease. RESULTS: Blood hemoglobin and albumin levels were significantly lower in CNSL than CNS infectious and demyelinating diseases, CSF cell counts were significantly higher in infectious diseases than CNSL and demyelinating diseases, and CSF-cfDNA concentrations were significantly higher in infectious diseases than CNSL and demyelinating diseases. Mutation analysis using CSF-cfDNA detected MYD88L265P and CD79Y196 mutations in 60% of CNSLs each, with either mutation detected in 80% of cases. Mutual existence of both mutations was identified in 40% of cases. These mutations were not detected in either infectious or demyelinating diseases, and the sensitivity and specificity of detecting either MYD88/CD79B mutations in CNSL were 80% and 100%, respectively. In the four cases biopsied, the median time from collecting CSF with the detected mutations to definitive diagnosis by conventional methods was 22.5 days (range, 18-93 days). CONCLUSIONS: These results suggest that mutation analysis using CSF-cfDNA might be useful for differentiating CNSL from CNS infectious/demyelinating diseases and for early detection of CNSL, even in cases where brain biopsy is difficult to perform.

Choreito, a Kampo medicine attenuates detrusor overactivity and bladder pain symptoms in rat tranilast-induced interstitial cystitis/bladder pain syndrome-like model.

AIMS: Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory condition of the bladder. However, there are only a few medicines that are of pharmaceutical grade and reliably effective for IC/BPS symptoms. Choreito (CRT) is a pharmaceutical-grade Kampo medicine and has been widely prescribed for patients of lower urinary tract symptoms (LUTS) and BPS in Japan. In this study, we exploratory investigated the effects of CRT on the IC/BPS-like symptoms induced by tranilast. METHODS: The rat IC/BPS-like model was induced by feeding administration with 0.4% tranilast. The rats were divided into the three following treatment groups: normal diet (Normal), tranilast treatment (Control), and the groups of 1% CRT (CRT) treatment for IC/BPS-like model. After 4 weeks, continuous cystmetry, locomotor, and vascular permeability was assessed. Furthermore, the cytokine levels in bladder were analyzed by the Bio-Plex suspension array system and plasma monoamine were measured. RESULTS: Control group exhibited 14.3% decrease of locomotor activity in the dark period, and which were 20.3% increase by 1%CRT treatment. The voiding interval was shorter in control than in other groups. 1%CRT suppressed the shortening of voiding interval. Evans blue leakage of bladder wall observed 44.8% higher in control group than in the normal group. The leakage of 1%CRT group was 33.3% less than in the control group. The cytokine level of IFNγ and VEGF were elevated in the control, and CRT treatment suppressed the elevation of IFNγ in the bladder. Plasma noradrenaline was significantly reduced by CRT treatment compared normal group. CONCLUSION: These results suggest that CRT can be an effective therapeutic agent for the treatment of IC/BPS-like symptoms.

BCL6 inhibition ameliorates resistance to ruxolitinib in CRLF2-rearranged acute lymphoblastic leukemia.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is an intractable disease and most cases harbor genetic alterations that activate JAK or ABL signaling. The commonest subtype of Ph-like ALL exhibits a CRLF2 gene rearrangement that brings about JAK1/2-STAT5 pathway activation. However, JAK1/2 inhibition alone is insufficient as a treatment, so combinatorial therapies targeting multiple signals are needed. To better understand the mechanisms underlying the insufficient efficacy of JAK inhibition, we explored gene expression changes upon treatment with a JAK1/2 inhibitor (ruxolitinib) and found that elevated BCL6 expression was one such mechanism. Upregulated BCL6 suppressed the expression of TP53 along with its downstream cell cycle inhibitor p21 (CDKN2A) and pro-apoptotic molecules, such as FAS, TNFRSF10B, BID, BAX, BAK, PUMA, and NOXA, conferring cells some degree of resistance to therapy. BCL6 inhibition (with FX1) alone was able to upregulate TP53 and restore the TP53 expression that ruxolitinib had diminished. In addition, ruxolitinib and FX1 concertedly downregulated MYC. As a result, FX1 treatment alone had growth-inhibitory and apoptosis- sensitizing effects, but the combination of ruxolitinib and FX1 more potently inhibited leukemia cell growth, enhanced apoptosis sensitivity, and prolonged the survival of xenografted mice. These findings provide one mechanism for the insufficiency of JAK inhibition for the treatment of CRLF2-rearranged ALL and indicate BCL6 inhibition as a potentially helpful adjunctive therapy combined with JAK inhibition.

Influence of different treatment procedures on the temporomandibular joint after mandibular setback in skeletal class III - A retrospective study.

The aim of this study was to clarify the effects of three different orthognathic surgical procedures on the temporomandibular joint after mandibular setback. Conventional sagittal split ramus osteotomy (SSRO) with segmental fixation (conv-SSRO), intraoral vertical ramus osteotomy (IVRO), or SSRO without fixation followed by the physiological positioning strategy (nonfix-SSRO) was performed for mandibular setback. Temporomandibular joint disorder (TMD) symptoms were clinically assessed, and the condylar head angle was measured. In total, 129 patients participated. Preoperative TMD and treatment procedure were related to postoperative TMD. A menton deviation of 3.43 mm was the cutoff for the risk of postoperative TMD. The incidence rate of postoperative TMD in the conv-SSRO group was higher than that in the IVRO (p = 0.0197) and nonfix-SSRO (p = 0.0001) groups in asymmetric cases. There was no significant postoperative change in the temporomandibular joint space in each group. In symmetric and asymmetric cases, the condylar head was rotated inwards by 5.82 ± 4.75° (p < 0.0001) and 5.44 ± 3.10° (p < 0.0001), respectively, in the conv-SSRO group, and outwards by -7.98 ± 5.05° (p < 0.0001) and -8.32 ± 6.38° (p < 0.0001), respectively, in the IVRO group, but it was almost stable in the nonfix-SSRO group. Within the limitations of the study it seems that nonfix-SSRO should be preferred over conv-SSRO and IVRO whenever appropriate.

Map7D2 and Map7D1 facilitate microtubule stabilization through distinct mechanisms in neuronal cells.

Microtubule (MT) dynamics are modulated through the coordinated action of various MT-associated proteins (MAPs). However, the regulatory mechanisms underlying MT dynamics remain unclear. We show that the MAP7 family protein Map7D2 stabilizes MTs to control cell motility and neurite outgrowth. Map7D2 directly bound to MTs through its N-terminal half and stabilized MTs in vitro. Map7D2 localized prominently to the centrosome and partially on MTs in mouse N1-E115 neuronal cells, which expresses two of the four MAP7 family members, Map7D2 and Map7D1. Map7D2 loss decreased the resistance to the MT-destabilizing agent nocodazole without affecting acetylated/detyrosinated stable MTs, suggesting that Map7D2 stabilizes MTs via direct binding. In addition, Map7D2 loss increased the rate of random cell migration and neurite outgrowth, presumably by disturbing the balance between MT stabilization and destabilization. Map7D1 exhibited similar subcellular localization and gene knockdown phenotypes to Map7D2. However, in contrast to Map7D2, Map7D1 was required for the maintenance of acetylated stable MTs. Taken together, our data suggest that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms in cell motility and neurite outgrowth.

Phenotypic Characterization of the Endocannabinoid-Degrading Enzyme Alpha/Beta-Hydrolase Domain 6 Knockout Rat.

Introduction: Alpha/beta-hydrolase domain 6 (ABHD6) is an enzyme that hydrolyzes 2-arachidonoylglycerol, a high-efficiency endogenous cannabinoid. Although the endocannabinoid system has been suggested to be involved in regulation of bladder function, the roles of ABHD6 in the control of micturition remain unknown. To elucidate the physiological and pathological roles of ABHD6 in vivo, we examined phenotypes of ABHD6 knockout rats (Abhd6-/-) generated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins system. Materials and Methods: Age-matched knockout and wild-type (WT) rats of both sexes were used. Results: Expression of ABHD6, assessed by quantitative real-time polymerase chain reaction and Western blot analysis, was clearly diminished in Abhd6-/- rats compared with WT rats. Mutant rats had a normal appearance, and the body weight and food consumption were similar to those of WT rats. The interval between bladder contractions assessed by continuous cystometry was significantly shorter in ABHD6 knockout rats than in WT rats when the bladder was stimulated with acetic acid. Mechanical paw withdrawal thresholds measured by von Frey testing were significantly lowered in the knockout rats than in WT rats. The plasma levels of prostaglandin E2 (PGE2) and the stable metabolite of PGE2 in Abhd6-/- rats were twice as high as that in WT rats. Conclusions: Deletion of the ABHD6 gene in rats causes more frequent urination in the stimulated bladder and hyperalgesia to non-noxious mechanical stimuli along with increased plasma PGE2.

Gene-Activated Matrix with Self-Assembly Anionic Nano-Device Containing Plasmid DNAs for Rat Cranial Bone Augmentation.

We have developed nanoballs, a biocompatible self-assembly nano-vector based on electrostatic interactions that arrange anionic macromolecules to polymeric nanomaterials to create nucleic acid carriers. Nanoballs exhibit low cytotoxicity and high transfection efficiently in vivo. This study investigated whether a gene-activated matrix (GAM) composed of nanoballs containing plasmid (p) DNAs encoding bone morphogenetic protein 4 (pBMP4) could promote bone augmentation with a small amount of DNA compared to that composed of naked pDNAs. We prepared nanoballs (BMP4-nanoballs) constructed with pBMP4 and dendrigraft poly-L-lysine (DGL, a cationic polymer) coated by γ-polyglutamic acid (γ-PGA; an anionic polymer), and determined their biological functions in vitro and in vivo. Next, GAMs were manufactured by mixing nanoballs with 2% atelocollagen and ß-tricalcium phosphate (ß-TCP) granules and lyophilizing them for bone augmentation. The GAMs were then transplanted to rat cranial bone surfaces under the periosteum. From the initial stage, infiltrated macrophages and mesenchymal progenitor cells took up the nanoballs, and their anti-inflammatory and osteoblastic differentiations were promoted over time. Subsequently, bone augmentation was clearly recognized for up to 8 weeks in transplanted GAMs containing BMP4-nanoballs. Notably, only 1 µg of BMP4-nanoballs induced a sufficient volume of new bone, while 1000 µg of naked pDNAs were required to induce the same level of bone augmentation. These data suggest that applying this anionic vector to the appropriate matrices can facilitate GAM-based bone engineering.

Deletion of the lysyl oxidase-like 1 gene induces impaired elastin fiber synthesis and inefficient urethral closure in rats.

We investigated the bladder and urethral function in a rat model lacking the protein lysyl oxidase-like 1 (Loxl1). Female nulliparous rats of Loxl1-/- or age-matched wild type (WT) rats had leak-point pressure testing, cystometry, histopathological analyses of lower urinary tract, and contractile response of isolated detrusor strips to carbachol and electric field stimulation. The Loxl1-/- rats showed increased looseness and redundancy of the skin, the decreased intercontraction interval and voided volume in cystometry, the lower leak-point pressure, thinner elastic fibers of the mesentery, bladder, urethra and vagina, and smaller contractile response of detrusor strips to carbachol when compared to the WT rats. Thus, the insufficient hydrostatic mechanism of urethra via submucosal impaired elastin synthesis might reduce the resting urethral closure pressure and the diminished cholinergic contractile response of detrusor smooth muscle might be involved in bladder activity in the Loxl1-/- rats.

[ASSOCIATION OF BREATH HYDROGEN CONCENTRATION WITH ORAL INTAKE AND URINARY DISEASES].

(Purpose) Ingestion of hydrogen is said to prevent oxidation in the body, but hydrogen is produced by intestinal bacterial flora and excreted in the exhaled breath. We investigated how breath hydrogen concentrations change with the diurnal cycle and under various conditions, including after consuming food or drink, and in people with urological disease. (Subjects and methods) Participants were healthy volunteers (40 men, 45 women; 30-83 years old) and urological outpatients (40 men with benign prostatic hyperplasia, 30 women with overactive bladder; 60 years or older). Breath hydrogen levels were measured before and after eating and drinking in three volunteers, and its diurnal variation was examined in one. The relationship between breath hydrogen and age or urological disease status was also analyzed by gender. Additional measurements were taken in the person with the highest breath hydrogen concentration and the person with the lowest; in these two people, breath hydrogen was measured at the same time for 10 or more days to determine the fluctuation range. (Results) Breath hydrogen concentration increased temporarily after ingestion of tap water, hydrogen water or food. It also increased with food intake and in cases of flatulence with intestinal gas accumulation, but decreased after defecation. In the person with the highest breath hydrogen, concentrations were 11.2-188.6 ppm, whereas in the person with the lowest, they were 0.4-2.3 ppm. Breath hydrogen increased significantly with age in healthy female volunteers. There was no association between breath hydrogen and benign prostatic hyperplasia, overactive bladder or constipation. (Conclusion) Breath hydrogen concentration increases with eating, drinking and aging, and is not associated with benign prostatic hyperplasia, overactive bladder or constipation. Breath hydrogen concentration varies widely between individuals, which may be due to differences in intestinal flora.

Roles of Pyk2 in signal transduction after gonadotropin-releasing hormone receptor stimulation.

The receptor for gonadotropin-releasing hormone (GnRH) is highly expressed in hypothalamic neurons. It has been reported that GnRH treatment of cultured GnRH neurons (GT1-7 cells) activated proline-rich tyrosine kinase 2 (Pyk2), and Pyk2 was involved in the activation of extracellular signal-regulated protein kinase 1 (ERK1) and ERK2 (ERK1/2). In the present study, we first examined the possibility that GnRH treatment might activate epidermal growth factor receptor (EGFR). We found that activation of EGFR after GnRH treatment for 5 min was much less than after EGF or heparin-binding EGF treatment. Next, we examined whether or not Pyk2 bound to growth factor receptor-binding protein 2 (Grb2). We overexpressed FLAG-fused Pyk2 in GT1-7 cells, and immunoprecipitated Pyk2 using an anti-FLAG antibody. The binding of Pyk2 to Grb2 was detected only after GnRH treatment. In contrast, a site-directed mutant of Pyk2 wherein tyrosine 881 was mutated to phenylalanine did not bind to Grb2. Studies with small interfering RNA and inhibitors indicated that the activation of Grb2/Ras/Raf/MEK was a major pathway to ERK1/2 activation after the short-term treatment of GT1-7 cells with GnRH.

Vanilla scent reduces frequency of urination in urethane-anesthetized rats.

OBJECTIVES: The scent of vanilla has a relaxing effect and is used to treat sleep disorders. Sleep disorders can both cause and be caused by nocturia. Therefore, we examined whether vanilla inhalation would reduce the frequency of urination in rats under light urethane anesthesia. METHODS: Twenty-four rats were anesthetized with 0.6 g/kg urethane subcutaneously (half the usual dose) to induce a sleep-like state. In 12 rats, continuous cystometry was performed via a transurethral catheter before, during and after inhalation of vanilla (n = 7) or the citrus fruit shiikuwasa (n = 5) for 60 minutes. The remaining 12 rats did not undergo cystometry but underwent vanilla inhalation treatment for 60 minutes (n = 6), or no inhalation treatment (n = 6); blood was then collected from these two groups and serum monoamine levels were compared. RESULTS: Intervals between bladder contractions were significantly longer after vanilla inhalation than before. However, baseline bladder pressure, maximum bladder contraction pressure, and residual volume remained unchanged. During shiikuwasa inhalation, the body movement of each rat increased but cystometric parameters did not change. Serum concentrations of adrenaline, noradrenaline and dopamine, but not serotonin, were significantly lower in rats that had inhaled vanilla than in those that had not. CONCLUSIONS: Vanilla scent decreased serum catecholamine levels and urination frequency in rats under light urethane anesthesia. These results suggest that the scent of vanilla may reduce nocturia.

Assessment of safety and efficacy of antimicrobial photodynamic therapy for peri-implant disease.

BACKGROUND: There is no reliable treatment procedure for peri-implant disease, despite the rise in its incidence. This study sought to evaluate the short-term safety and efficacy of antimicrobial photodynamic therapy (a-PDT) on peri-implantitis by assessing the volume of pus discharge after a-PDT. METHODS: Patients with pus discharge from a peri-implant pocket were recruited from December 1st, 2019 to April 30th, 2020. The enrolled implants were randomly assigned to one of two groups, the irrigation and a-PDT groups. Their peri-implant pocket was irrigated with normal saline in the irrigation group, and a saline irrigation and subsequent a-PDT with toluidine blue (TB) was performed in the a-PDT group. The safety and efficacy of a-PDT were assessed 7 days after treatment. RESULTS: Twenty-five implants in 21 patients (irrigation group; 13 implants, a-PDT group; 12 implants) were registered. No complication was observed after a-PDT. Pus discharge was decreased in 7 of 12 implants (58.3 %) in the a-PDT group, and in 2 of 13 implants (15.4 %) in the irrigation group. According to Fisher's exact test, a-PDT resulted in a statistically significant decrease in pus discharge compared to irrigation alone (p = 0.0414). CONCLUSIONS: a-PDT was confirmed to be a safe treatment for peri-implantitis, and the short-term efficacy of a-PDT with TB on peri-implantitis was clarified. Nevertheless, its efficacy remains restricted, and a new combination therapy of a-PDT and decontamination procedures is expected to be developed in future.

Staurosporine and venetoclax induce the caspase-dependent proteolysis of MEF2D-fusion proteins and apoptosis in MEF2D-fusion (+) ALL cells.

MEF2D-fusion (M-fusion) genes are newly discovered recurrent gene abnormalities that are detected in approximately 5 % of acute lymphoblastic leukemia (ALL) cases. Their introduction to cells has been reported to transform cell lines or increase the colony formation of bone marrow cells, suggesting their survival-supporting ability, which prompted us to examine M-fusion-targeting drugs. To identify compounds that reduce the protein expression level of MEF2D, we developed a high-throughput screening system using 293T cells stably expressing a fusion protein of MEF2D and luciferase, in which the protein expression level of MEF2D was easily measured by a luciferase assay. We screened 3766 compounds with known pharmaceutical activities using this system and selected staurosporine as a potential inducer of the proteolysis of MEF2D. Staurosporine induced the proteolysis of M-fusion proteins in M-fusion (+) ALL cell lines. Proteolysis was inhibited by caspase inhibitors, not proteasome inhibitors, suggesting caspase dependency. Consistent with this result, the growth inhibitory effects of staurosporine were stronger in M-fusion (+) ALL cell lines than in negative cell lines, and caspase inhibitors blocked apoptosis induced by staurosporine. We identified the cleavage site of MEF2D-HNRNPUL1 by caspases and confirmed that its caspase cleavage-resistant mutant was resistant to staurosporine-induced proteolysis. Based on these results, we investigated another Food and Drug Administration-approved caspase activator, venetoclax, and found that it exerted similar effects to staurosporine, namely, the proteolysis of M-fusion proteins and strong growth inhibitory effects in M-fusion (+) ALL cell lines. The present study provides novel insights into drug screening strategies and the clinical indications of venetoclax.

Increased expression of EGR1 and KLF4 by polysulfide via activation of the ERK1/2 and ERK5 pathways in cultured intestinal epithelial cells.

Sodium trisulfide (Na2S3) releases hydrogen polysulfide (H2Sn) and is useful for the investigation of the effects of H2Sn on the cell functions. In the present study, we first examined the effects of Na2S3 on the gene expression of IEC-6 cells, a rat intestinal epithelial cell line. Microarray analysis and reverse transcription-polymerase chain reaction analysis revealed that Na2S3 increased the gene expression of early growth response 1 (EGR1) and Kruppel-like transcription factor 4 (KLF4). It was interesting that U0126, an inhibitor of the activation of extracellular signal-regulated kinase 1 (ERK1), ERK2, and ERK5, inhibited the Na2S3-induced gene expression of EGR1 and KLF4. Na2S3 activated ERK1 and ERK2 (ERK1/2) within 15 min. In addition to ERK1/2, Na2S3 activated ERK5. We noticed that the electrophoretic mobility of ERK5 was decreased after Na2S3 treatment. Phos-tag analysis and in vitro dephosphorylation of the cell extracts indicated that the gel-shift of ERK5 was due to its phosphorylation. The gel-shift of ERK5 was inhibited completely by both U0126 and ERK5-IN-1, a specific inhibitor of ERK5. From these results, we concluded that the gel-shift of ERK5 was induced through autophosphorylation by activated ERK5 after Na2S3 treatment. The present study suggested that H2Sn affected various functions of intestinal epithelial cells through the activation of the ERK1/2 and ERK5 pathways.

Truncated RUNX1 Generated by the Fusion of RUNX1 to Antisense GRIK2 via a Cryptic Chromosome Translocation Enhances Sensitivity to Granulocyte Colony-Stimulating Factor.

Fusions of the Runt-related transcription factor 1 (RUNX1) with different partner genes have been associated with various hematological disorders. Interestingly, the C-terminally truncated form of RUNX1 and RUNX1 fusion proteins are similarly considered important contributors to leukemogenesis. Here, we describe a 59-year-old male patient who was initially diagnosed with acute myeloid leukemia, inv(16)(p13;q22)/CBFB-MYH11 (FAB classification M4Eo). He achieved complete remission and negative CBFB-MYH11 status with daunorubicin/cytarabine combination chemotherapy but relapsed 3 years later. Cytogenetic analysis of relapsed leukemia cells revealed CBFB-MYH11 negativity and complex chromosomal abnormalities without inv(16)(p13;q22). RNA-seq identified the glutamate receptor, ionotropic, kinase 2 (GRIK2) gene on 6q16 as a novel fusion partner for RUNX1 in this case. Specifically, the fusion of RUNX1 to the GRIK2 antisense strand (RUNX1-GRIK2as) generated multiple missplicing transcripts. Because extremely low levels of wild-type GRIK2 were detected in leukemia cells, RUNX1-GRIK2as was thought to drive the pathogenesis associated with the RUNX1-GRIK2 fusion. The truncated RUNX1 generated from RUNX1-GRIK2as induced the expression of the granulocyte colony-stimulating factor (G-CSF) receptor on 32D myeloid leukemia cells and enhanced proliferation in response to G-CSF. In summary, the RUNX1-GRIK2as fusion emphasizes the importance of aberrantly truncated RUNX1 in leukemogenesis.