Efficient detection of somatic UBA1 variants and clinical scoring system predicting patients with variants in VEXAS syndrome.

OBJECTIVES: To efficiently detect somatic UBA1 variants and establish a clinical scoring system predicting patients with pathogenic variants in VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. METHODS: Eighty-nine Japanese patients with clinically suspected VEXAS syndrome were recruited [81 males and 8 females; median onset age (IQR) 69.3 years (62.1-77.6)]. Peptide nucleic acid-clamping PCR (PNA-PCR), regular PCR targeting exon 3 clustering UBA1 variants, and subsequent Sanger sequencing were conducted for variant screening. Partitioning digital PCR (pdPCR) or targeted amplicon deep sequencing (TAS) was also performed to evaluate the variant allele frequency (VAF). We developed our clinical scoring system to predict UBA1 variant-positive and ­negative patients and assessed the diagnostic value of our system using receiver operating characteristic (ROC) curve analysis. RESULTS: Forty patients with reported pathogenic UBA1 variants (40/89, 44.9%) were identified, including a case having a variant with VAF of 1.7%, using a highly sensitive method. Our clinical scoring system considering >50 years of age, cutaneous lesions, lung involvement, chondritis, and macrocytic anaemia efficiently predicted patients with UBA1 variants (the area under the curve for the scoring total was 0.908). CONCLUSIONS: Genetic screening with the combination of regular PCR and PNA-PCR detected somatic UBA1 variants with high sensitivity and specificity. Our scoring system could efficiently predict patients with UBA1 variants.

Effects of dedifferentiated fat cells on neurogenic differentiation and cell proliferation in neuroblastoma cells.

PURPOSE: Mesenchymal stem cells (MSCs) can induce differentiation of neuroblastoma (NB) cells. Properties of dedifferentiated fat cells (DFATs) are similar to those of MSCs. Here, we investigated whether DFATs can induce NB cell differentiation and suppress cell proliferation. METHODS: DFATs were obtained from mature adipocytes isolated from adipose tissue from a ceiling culture. NB cells were cultured in a medium with or without DFATs and, subsequently, cultured in a DFAT-conditioned medium (CM) with or without phosphatidylinositol 3-kinase (PI3K) inhibitor. The neurite lengths were measured, and mRNA expression levels of the neurofilament (NF) and tubulin beta III (TUBß3) were assessed using quantitative real-time RT-PCR. Cell viability was assessed using the WST-1 assay. RESULTS: NB cells cultured with DFATs caused elongation of the neurites and upregulated the expression of NF and Tubß3. NB cells cultured in DFAT-CM demonstrated increased cell viability. NB cells cultured with DFAT-CM and PI3K inhibitors suppressed cell viability. NB cells cultured with DFAT-CM and PI3K inhibitor demonstrated increased neurite length and expression, and upregulation of Tubß3. CONCLUSION: The combined use of DFAT-CM and PI3K inhibitors suppresses the proliferation of NB cells and induces their differentiation. Thus, DFAT may offer new insights into therapeutic approaches in neuroblastoma.

Diagnosis and management of genital injuries in girls: 14-year experience.

BACKGROUND: Pediatric patients with genital injuries are often recommended to receive an examination under general anesthesia; however, detailed clinical data of such patients are rarely reported. METHODS: A single-center retrospective review was conducted in 45 girls less than 16 years of age with genital injuries between January 2005 and December 2018. RESULTS: The median patient age was 5.0 years. Forty-two patients were hospitalized, of whom 38 required an examination under general anesthesia and all consequently required surgical repair. The diagnosis obtained after a thorough examination under general anesthesia was inconsistent with the diagnosis obtained at the emergency room in five patients. In 20 patients, the source of bleeding was not clarified at the time of initial examination at the emergency room; four of these patients were later revealed to have vaginal or rectal injuries that had been overlooked during the examination at the emergency room. Injuries occurring in the bathroom were the most frequent and tended to be serious. Multiple injuries were found in 10 patients. The exterior of the labia minora was the most commonly injured site, found in 18 patients. CONCLUSIONS: We analyzed the clinical data of girls with genital injuries in detail, which allowed us to find a detailed classification of injured sites and the characteristics of serious cases, and to re-recognize the importance of a thorough examination under general anesthesia.

Lysosomal Protein Lamtor1 Controls Innate Immune Responses via Nuclear Translocation of Transcription Factor EB.

Amino acid metabolism plays important roles in innate immune cells, including macrophages. Recently, we reported that a lysosomal adaptor protein, Lamtor1, which serves as the scaffold for amino acid-activated mechanistic target of rapamycin complex 1 (mTORC1), is critical for the polarization of M2 macrophages. However, little is known about how Lamtor1 affects the inflammatory responses that are triggered by the stimuli for TLRs. In this article, we show that Lamtor1 controls innate immune responses by regulating the phosphorylation and nuclear translocation of transcription factor EB (TFEB), which has been known as the master regulator for lysosome and autophagosome biogenesis. Furthermore, we show that nuclear translocation of TFEB occurs in alveolar macrophages of myeloid-specific Lamtor1 conditional knockout mice and that these mice are hypersensitive to intratracheal administration of LPS and bleomycin. Our observation clarified that the amino acid-sensing pathway consisting of Lamtor1, mTORC1, and TFEB is involved in the regulation of innate immune responses.

Therapeutic potential of mature adipocyte-derived dedifferentiated fat cells for inflammatory bowel disease.

PURPOSE: Our previous studies demonstrated that mature adipocyte-derived dedifferentiated fat (DFAT) cells possess similar multipotency as mesenchymal stem cells. Here, we examined the immunoregulatory potential of DFAT cells in vitro and the therapeutic effect of DFAT cell transplantation in a mouse inflammatory bowel disease (IBD) model. METHODS: The effect of DFAT cell co-culture on T cell proliferation and expression of immunosuppression-related genes in DFAT cells were evaluated. To create IBD, CD4+CD45RBhigh T cells were intraperitoneally injected into SCID mice. One week later, DFAT cells (1 × 105, DFAT group) or saline (Control group) were intraperitoneally injected. Subsequently bodyweight was measured every week and IBD clinical and histological scores were evaluated at 5 weeks after T cell administration. RESULTS: The T cell proliferation was inhibited by co-cultured DFAT cells in a cell density-dependent manner. Gene expression of TRAIL, IDO1, and NOS2 in DFAT cells was upregulated by TNFα stimulation. DFAT group improved IBD-associated weight loss, IBD clinical and histological scores compared to Control group. CONCLUSION: DFAT cells possess immunoregulatory potential and the cell transplantation promoted recovery from colon damage and improved clinical symptoms in the IBD model. DFAT cells could play an important role in the treatment of IBD.

Lamtor1 Is Critically Required for CD4+ T Cell Proliferation and Regulatory T Cell Suppressive Function.

Mechanistic target of rapamycin complex (mTORC)1 integrates intracellular sufficiency of nutrients and regulates various cellular functions. Previous studies using mice with conditional knockout of mTORC1 component proteins (i.e., mTOR, Raptor, and Rheb) gave conflicting results on the roles of mTORC1 in CD4+ T cells. Lamtor1 is the protein that is required for amino acid sensing and activation of mTORC1; however, the roles of Lamtor1 in T cells have not been investigated. In this article, we show that Lamtor1-deficient CD4+ T cells exhibited marked reductions in proliferation, IL-2 production, mTORC1 activity, and expression of purine- and lipid-synthesis genes. Polarization of Th17 cells, but not Th1 and Th2 cells, diminished following the loss of Lamtor1. Accordingly, CD4-Cre-driven Lamtor1-knockout mice exhibited reduced numbers of CD4+ and CD8+ T cells at rest, and they were completely resistant to experimental autoimmune encephalomyelitis. In contrast, genetic ablation of Lamtor1 in Foxp3+ T cells resulted in severe autoimmunity and premature death. Lamtor1-deficient regulatory T cells survived ex vivo as long as wild-type regulatory T cells; however, they exhibited a marked loss of suppressive function and expression of signature molecules, such as CTLA-4. These results indicate that Lamtor1 plays essential roles in CD4+ T cells. Our data suggest that Lamtor1 should be considered a novel therapeutic target in immune systems.

Semaphorin 4D inhibits neutrophil activation and is involved in the pathogenesis of neutrophil-mediated autoimmune vasculitis.

OBJECTIVES: Inappropriate activation of neutrophils plays a pathological role in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The aim of this study was to investigate the functions of semaphorin 4D (SEMA4D) in regulation of neutrophil activation, and its involvement in AAV pathogenesis. METHODS: Serum levels of soluble SEMA4D were evaluated by ELISA. Blood cell-surface expression of membrane SEMA4D was evaluated by flow cytometry. To determine the functional interactions between neutrophil membrane SEMA4D and endothelial plexin B2, wild-type and SEMA4D-/- mice neutrophils were cultured with an endothelial cell line (MS1) stained with SYTOX green, and subjected to neutrophil extracellular trap (NET) formation assays. The efficacy of treating human neutrophils with recombinant plexin B2 was assessed by measuring the kinetic oxidative burst and NET formation assays. RESULTS: Serum levels of soluble SEMA4D were elevated in patients with AAV and correlated with disease activity scores. Cell-surface expression of SEMA4D was downregulated in neutrophils from patients with AAV, a consequence of proteolytic cleavage of membrane SEMA4D. Soluble SEMA4D exerted pro-inflammatory effects on endothelial cells. Membranous SEMA4D on neutrophils bound to plexin B2 on endothelial cells, and this interaction decreased NET formation. Recombinant plexin B2 suppressed neutrophil Rac1 activation through SEMA4D's intracellular domain, and inhibited pathogen-induced or ANCA-induced oxidative burst and NET formation. CONCLUSIONS: Neutrophil surface SEMA4D functions as a negative regulator of neutrophil activation. Proteolytic cleavage of SEMA4D as observed in patients with AAV may amplify neutrophil-mediated inflammatory responses. SEMA4D is a promising biomarker and potential therapeutic target for AAV.

mTOR Complex Signaling through the SEMA4A-Plexin B2 Axis Is Required for Optimal Activation and Differentiation of CD8+ T Cells.

Mammalian target of rapamycin (mTOR) plays crucial roles in activation and differentiation of diverse types of immune cells. Although several lines of evidence have demonstrated the importance of mTOR-mediated signals in CD4(+) T cell responses, the involvement of mTOR in CD8(+) T cell responses is not fully understood. In this study, we show that a class IV semaphorin, SEMA4A, regulates CD8(+) T cell activation and differentiation through activation of mTOR complex (mTORC) 1. SEMA4A(-/-) CD8(+) T cells exhibited impairments in production of IFN-γ and TNF-α and induction of the effector molecules granzyme B, perforin, and FAS-L. Upon infection with OVA-expressing Listeria monocytogenes, pathogen-specific effector CD8(+) T cell responses were significantly impaired in SEMA4A(-/-) mice. Furthermore, SEMA4A(-/-) CD8(+) T cells exhibited reduced mTORC1 activity and elevated mTORC2 activity, suggesting that SEMA4A is required for optimal activation of mTORC1 in CD8(+) T cells. IFN-γ production and mTORC1 activity in SEMA4A(-/-) CD8(+) T cells were restored by administration of recombinant Sema4A protein. In addition, we show that plexin B2 is a functional receptor of SEMA4A in CD8(+) T cells. Collectively, these results not only demonstrate the role of SEMA4A in CD8(+) T cells, but also reveal a novel link between a semaphorin and mTOR signaling.

Improvement of health status evaluated by Arthritis Impact Measurement Scale 2 (AIMS-2) and Short Form-36 (SF-36) in patients with rheumatoid arthritis treated with tocilizumab.

OBJECTIVE: To evaluate the improvement of health status in patients with rheumatoid arthritis (RA) treated with tocilizumab. METHODS: Thirty-nine patients were treated with 8 mg/kg tocilizumab every 4 weeks for 24 weeks. Disease activity was assessed by Clinical Disease Activity Index (CDAI) and Simplified Disease Activity Index (SDAI). Improvement of health status was assessed by Arthritis Impact Measurement Scale 2 (AIMS-2) and Short Form-36 (SF-36). RESULTS: Tocilizumab improved CDAI and SDAI significantly at week 4 compared with at baseline. In the components of AIMS-2, "physical score", "symptom" and "affect" improved significantly at week 4 compared with at baseline, while "social interaction" did not improve significantly during 24 weeks of tocilizumab therapy. Similarly in SF-36, "bodily pain", "general health", "vitality" and "mental health" improved significantly at week 4. The most correlative component of AIMS-2 with CDAI was "symptom", while "social interaction" did not correlate with CDAI during tocilizumab treatment. CONCLUSION: The time-course diversity in improvement of health status should be considered to provide proper healthcare when treated with tocilizumab.

Successful Pregnancy Outcome in a Patient with Juvenile Idiopathic Arthritis in Adulthood, Amyloid A Amyloidosis, and Chronic Kidney Disease Using Tocilizumab with Strict Blood Pressure Control.

Pregnancies with chronic kidney disease (CKD) and high disease activity in rheumatic diseases are high-risk events with adverse outcomes for both the mother and fetus. We herein report a 35-year-old woman with juvenile idiopathic arthritis (JIA), amyloid A (AA) amyloidosis related to JIA, and CKD stage G4A2 who wished to have children. She achieved a successful pregnancy, even in the presence of these multiple risk factors, using tocilizumab to control the disease activity of JIA and AA amyloidosis, along with antihypertensive drugs to control her blood pressure before and during pregnancy.

Successful laparoscopic-assisted partial splenectomy and splenopexy with umbilical approach to wandering spleen with an enlarged cyst in a pediatric patient.

A 10-year-old girl with a wandering spleen with an enlarged cyst was successfully treated by laparoscopic-assisted partial splenectomy and splenopexy. The patient visited our hospital with a complaint of a lower abdominal mass. Abdominal computed tomography showed malposition of the spleen and the presence of a 10 cm diameter splenic cyst (SC) in the lower pole. In surgery, the navel was opened with an inverted Y-shaped incision. The SC was punctured and aspirated the contents of the cyst, the migrating spleen was pulled out of navel and the partial splenectomy was done. The residual spleen was laparoscopically fixed by creating an extraperitoneal pocket. Pathologically, the cyst was covered with a vitrified fibrotic capsule and was diagnosed as a pseudocyst. We considered it a traumatic cyst. The postoperative course was uneventful. This minimally invasive laparoscopic procedure was feasible and effective for treating wandering spleen with a large SC in a pediatric patient.

A case report of a patient with rheumatoid arthritis complicated with Mycobacterium avium during tocilizumab treatment.

A female patient with rheumatoid arthritis (RA) suffered from Mycobacterium avium (M. avium) infection during tocilizumab treatment. Tocilizumab was discontinued and she was treated with a recommended chemotherapy, resulting in improvement of M. avium. Tocilizumab retreatment did not aggravate M. avium infection, and radiographic abnormalities improved over 1 year after cessation of the recommended therapy. Tocilizumab may be one candidate for intractable RA patients with M. avium if any biologic is required.

Relationship between pedestrian detection specifications of parking sensor and potential safety benefits.

Measures to protect vulnerable road users during low-speed maneuvers are required. For example, systems that use cameras to display the vehicle's rearview are popular. However, some vehicles are difficult to equip with a rear view camera system. To avoid a crash when driving in reverse, it is also effective to identify the presence of pedestrians via an audible warning using a device (e.g., clearance sonar). It may be cheaper to install than a rearview camera system. Installation cost is also important for the spread of equipment that reduces a crash. It is necessary to clarify the minimum specifications that balance cost and reduce crashes. Device specifications (e.g., detection distance and response delay) may affect the crash reduction rate. There should be a detection distance required for the sonar to have the same crash reduction effect as the rear view camera system. Thus, in this study, we conducted experiments and obtained data about how a vehicle moves and driver reactions to audible warnings when driving in reverse. Based on the acquired data, a numerical simulation was performed to determine whether a driver could avoid a crash under various circumstances. As a result, it was shown that the clearance sonar must have a detection distance of 0.8 m or more to expect the same effect as a rearview camera system. In other words, to expect sonar to have the same performance as a rearview camera, a detection distance of at least 0.8 m should be set as a specification.

How to avoid unnecessary surgical treatment for neonatal small left colon syndrome.

We report a case of neonatal small left colon syndrome (NSLCS) that underwent surgery. A female infant was born at 38 weeks of gestation. The mother had gestational diabetes requiring insulin therapy. The baby was admitted for respiratory distress. Abdominal distension was observed, and the gastric residue increased. Contrast enema revealed a small caliber of the left colon up to the splenic flexure. At 14 days, the full-thickness biopsy of the sigmoid and transverse colons was performed. Pathological diagnosis showed that the sigmoid colon had few ganglion cells, therefore the transverse colostomy was performed. At 6 months of age, a rectal biopsy was performed to confirm the diagnosis of Hirschsprung's disease; the intestinal plexus and ganglion cells were normal. The surgery was changed from a pull-through to a stoma closure. The postoperative diagnosis was NSLCS, and the course up to 3 years was good without defecation or growth problems.

[A case with liver resection of metastasis from rectal cancer after bevacizumab treatment].

The advancement of systemic chemotherapy for colorectal carcinoma has improved a clinical response rate and expanded a possibility of resection, which we thought we could not have been operable at the initial visit. It also improved a prognosis of patients. We report here a case with liver resection of metastasis from rectal cancer followed by bevacizumab treatment.

[Two cases of venous thrombosis confirmed during the bevacizumab combination chemotherapy for colorectal cancer].

We report two cases of venous thrombosis confirmed during the bevacizumab combination chemotherapy for colorectal cancer. Case 1 was a 59-year-old man. We performed an operation for cancer of the rectum. At 2 years after the operation, he received mFOLFOX6 + bevacizumab therapy for a recurrence in the pelvis and lungs metastasis. After the 14th courses, He had a right shoulder pain and contrast enhanced computed tomography revealed deep vein thrombosis to the right arms. Case 2 was a 65-year-old man. We performed an operation for cancer of the rectum. At 6 months after the operation, he received mFOLFOX6 + bevacizumab therapy for lung metastases. After the 6th courses, contrast enhanced computed tomography revealed deep venous and pulmonary thrombosis for both sides, pulmonary thrombosis.

[Resection of local recurrent gastric cancer following total gastrectomy].

We report a case of patient who underwent resection for local recurrent gastric cancer at the anastomotic site curatively. The patient was a 72 years old male with a history of undergoing total gastrectomy for gastric cancer located at the gastric cardia in February 2005. The histological findings of the resected tumor showed a Type 3 advanced gastric cancer invaded into subserosa in the cardia of the stomach with positive lymphatic and venous invasion and lymph node metastasis. The histological diagnosis was moderately differentiated tubular adenocarcinoma. Both the proximal and distal margins were negative for cancer. Endoscopy, 4 years after the first operation, showed a recurrent tumor at the site of esophago-jejunal anastomosis. A resection of the tumor was carried out curatively through the left thoraco-abdominal approach in June 2009. We recommend a resection of anastomotic recurrence especially if it occurs from the first operation in the long interval.

[A case of synchronous double cancer of stomach and lung responding to neoadjuvant chemotherapy].

A 66-year-old male was admitted to our hospital because of dyspnea in 2007. Cancerous pleural effusion and gastric cancer was diagnosed, and the chemotherapy consisted of S-1 + DOC was started for Stage IV gastric cancer. In 2009, lung cancer was found. The chemotherapy was changed to CDDP + CPT-11. This chemotherapy was effective for both lung and gastric cancers. Operation was performed for both tumors in 2010, and the pathological diagnosis revealed that gastric cancer was pStage I, Cur A, and the lung cancer was pStage IA, R0. Pathologic histology inspection of both tumors was judged to be effective for the chemotherapy prior to resection.