m6A RNA modification regulates innate lymphoid cell responses in a lineage-specific manner.

Innate lymphoid cells (ILCs) can quickly switch from a quiescent state to an active state and rapidly produce effector molecules that provide critical early immune protection. How the post-transcriptional machinery processes different stimuli and initiates robust gene expression in ILCs is poorly understood. Here, we show that deletion of the N6-methyladenosine (m6A) writer protein METTL3 has little impact on ILC homeostasis or cytokine-induced ILC1 or ILC3 responses but significantly diminishes ILC2 proliferation, migration and effector cytokine production and results in impaired antihelminth immunity. m6A RNA modification supports an increase in cell size and transcriptional activity in activated ILC2s but not in ILC1s or ILC3s. Among other transcripts, the gene encoding the transcription factor GATA3 is highly m6A methylated in ILC2s. Targeted m6A demethylation destabilizes nascent Gata3 mRNA and abolishes the upregulation of GATA3 and ILC2 activation. Our study suggests a lineage-specific requirement of m6A for ILC2 responses.

DNMT1 can induce primary germ layer differentiation through de novo DNA methylation.

DNA methyltransferases and Ten-Eleven Translocation (TET) proteins regulate the DNA methylation and demethylation cycles during mouse embryonic development. Although DNMT1 mainly plays a role in the maintenance of DNA methylation after DNA replication, it is also reported to possess de novo methyltransferase capacity. However, its physiological significance remains unclear. Here, we demonstrate that full-length DNMT1 (FL) and a mutant lacking the N-terminus necessary for its maintenance activity (602) confer the differentiation potential of mouse Dnmt1, Dnmt3a, and Dnmt3b (Dnmts-TKO) embryonic stem cells (ESCs). Both FL and 602 inhibit the spontaneous differentiation of Dnmts-TKO ESCs in the undifferentiated state. Dnmts-TKO ESCs showed loss of DNA methylation and de-repression of primitive endoderm-related genes, but these defects were partially restored in Dnmts-TKO + FL and Dnmts-TKO + 602 ESCs. Upon differentiation, Dnmts-TKO + FL ESCs show increased 5mC and 5hmC levels across chromosomes, including pericentromeric regions. In contrast, Dnmts-TKO + 602 ESCs didn't accumulate 5mC, and sister chromatids showed 5hmC asynchronously. Furthermore, in comparison with DNMT1_602, DNMT1_FL effectively promoted commitment to the epiblast-like cells and beyond, driving cell-autonomous mesendodermal and germline differentiation through embryoid body-based methods. With precise target selectivity achieved by its N-terminal region, DNMT1 may play a role in gene regulation leading to germline development.

Lobulated tumor contour as a predictor of preoperative tumor invasion of the lung or pericardium in thymoma patients.

PURPOSE: Preoperative investigations to detect invasion to neighboring organs are important for deciding on the most appropriate surgical approach for thymoma. We evaluated preoperative computed tomography (CT) findings in thymoma patients to identify the CT features associated with tumor invasion. METHODS: Clinicopathologic information on 193 patients who underwent surgical resection for thymoma at Chiba University Hospital between 2002 and 2016 was collected retrospectively. The surgical pathology identified invasion of thymoma in 35 patients: in the lung (n = 18), pericardium (n = 11), or both (n = 6). Contact lengths between the tumor contour and lung (CLTL) or pericardium (CLTP) were measured at the maximum section of the tumor on axial CT. Univariate and multivariate analyses were performed to analyze the relationship between pathological invasion of the lung or pericardium and the clinicopathologic features. RESULTS: The mean CLTL and CLTP were significantly longer in patients with invasion of the neighboring organs than in those without invasion. A lobulated tumor contour was identified in 95.6% of the patients with invasion of the neighboring organs. A multivariate analysis revealed that a lobulated tumor contour was significantly associated with both lung and pericardial invasion. CONCLUSIONS: A lobulated tumor contour was significantly associated with lung and/or pericardial invasion in thymoma patients.

A simple nomogram for predicting occult lymph node metastasis of non-small cell lung cancer from preoperative computed tomography findings, including the volume-doubling time.

PURPOSE: Latent lymph node metastasis is a clinical concern in the surgical treatment of non-small cell lung cancer (NSCLC). The present study identified a simple tool, including the volume-doubling time (VDT), for evaluating the risk of nodal metastasis. METHODS: We reviewed, retrospectively, 560 patients who underwent radical resection for cN0M0 NSCLC. The whole tumor VDT and solid component VDT (SVDT) for differentiating the histological type and adenocarcinoma subtype were analyzed and a nomogram was constructed using variables selected through a stepwise selection method. The model was assessed through a calibration curve and decision curve analysis (DCA). RESULTS: Lymph node metastases were detected in 89 patients (15.9%). The SVDT tended to be longer in patients with adenocarcinoma (294.5 days, p < 0.0001) than in those with other histological types of NSCLC, but was shorter when the solid/micropapillary component was predominant (127.0 days, p < 0.0001). The selected variables (tumor location, solid component diameter, consolidation tumor ratio, SVDT, and carcinoembryonic antigen) demonstrated significant differences and were used for the nomogram. The calibration curve indicated consistency, and the DCA showed validity across most threshold ranges from 0 to 68%. CONCLUSIONS: The established nomogram is a useful tool for the preoperative prediction of lymph node metastasis, and the SVDT was the most influential factor in the nomogram.

Surgical treatment for chronic pulmonary coccidioidomycosis: a retrospective study from a single institution.

PURPOSE: Coccidioidomycosis, caused by the Coccidioides species, is a well-known disease in the Southwestern United States and North Mexico, with scattered reports in Latin America countries. While this disease is still rare in Japan and other Asian countries, its incidence has been increasing over the last two decades. Coccidioides species are highly infectious and require caution when encountered. This study presents a case series of chronic pulmonary coccidioidomycosis surgically treated at a single institution. METHODS: We conducted a retrospective chart review of six patients who underwent lung resection for pulmonary coccidioidomycosis at Chiba University Hospital between January 2007 and December 2021. RESULTS: All six patients had travelled to the Southwestern United States. Preoperative serology was negative for the anti-Coccidioides antibody in four patients and positive in two. Chest computed tomography revealed a single, well-defined round nodule in all patients. Preoperative biopsy taken from three patients failed to obtain a definitive diagnosis. Histopathological examination of the resected pulmonary nodules revealed granulomas that contained numerous spherules with many endospores, thereby confirming the diagnosis of pulmonary coccidioidomycosis. CONCLUSIONS: Pulmonary coccidioidomycosis should be suspected based on travel history and radiological findings. Meticulous care should be taken during specimen processing to prevent cross infection.

[Robot-assisted Thoracic Surgery for Mediastinal Tumor].

In Japan, robot-assisted thoracic surgery (RATS) was introduced in thoracic surgery in 2001, but it did not become widespread. However, surgery for mediastinal tumors and lobectomy for lung cancer with RATS were covered by insurance in 2018 and are currently becoming popular as a general practice, following video-assisted thoracic surgery(VATS). Forty-six patients with mediastinal tumors were treated by RATS from February 2014 to November 2022 in our institution. Theoretically, the RATS approach is performed from one side in a semi-supine position under CO2 insufflation as with the VATS approach of our institution. In the case of extended thymectomy, a bilateral approach is performed by changing the patient's position. The median surgery time was 88 min, and the median surgery time in unilateral and bilateral approaches were 79 and 208 min, respectively. Blood loss during surgery was quite minimum, and no patients required conversion to VATS or thoracotomy. Regarding adverse events, postoperative bleeding was observed in one patient (2.2%). RATS has been successfully introduced and expanded safely for mediastinal tumors. Considering further expansion of RATS indications while conducting verification by comparison with VATS in the future is necessary.

Cyclic DNA remethylation following active demethylation at euchromatic regions in mouse embryonic stem cells.

DNA methylation is an essential epigenetic mark that regulates normal mammalian embryonic development. DNA methylation profiles are not always static, especially during germline development. In zygotes, DNA is typically highly methylated but, during preimplantation, DNA methylation is erased globally. Then, at the start of post-implantation development in mouse embryos, DNA again becomes dramatically hypermethylated. Chromatin structure regulates the accessibility of DNA-modifying enzymes to target DNA. Beyond that, however, our understanding of the pathway by which chromatin regulation initiates changes in global DNA methylation during mouse embryonic development remains incomplete. To analyse the relationship between global regulation of DNA methylation and chromatin status, we examined 5-methylcytosine (5mC), modified by the DNA methyltransferase DNMT, and the oxidative derivative 5-hydroxymethylation (5hmC), converted from 5mC by TET-family enzymes, by means of immunofluorescence staining of mitotic chromosomes in mouse embryonic stem cells (ESCs). Our comparison of immunostaining patterns for those epigenetic modifications in wild-type, DNMT-deficient, and TET-deficient ESCs allowed us to visualise cell cycle-mediated DNA methylation changes, especially in euchromatic regions. Our findings suggest that DNA methylation patterns in undifferentiated mouse ESCs are stochastically balanced by the opposing effects of two activities: demethylation by TET and subsequent remethylation by DNMT.

[The Basic Research of Antigen-antibody Reaction in Lung Transplantation].

Lung transplantation has become popular in Japan, showing better survival rate than other countries. However, the results are still not satisfactory compared with other solid organ transplantation. One of the reasons for this might be that knowledge on donor-specific antibodies or antibody-related rejection, which has been attracting attention these days, is less than that of kidney or liver transplantation. Our laboratory has continued basic research in this field using rodent lung transplantation model. We have previously shown that type V collagen is associated in chronic rejection as an autoimmune, and that oral administration of type V collagen induces tolerance. The murine chronic rejection model of the minor antigen mismatch was developed, and involvement of the humoral immunity and role of the complement activation were shown. We are now studying the effects of immune checkpoint molecules, which play a central role in the field of cancer therapy, on rejection after lung transplantation. We are also working to verify the effects of anti-complement drugs and molecular targeted drugs in the future treatment on rejection.

Two cases of novel coronavirus infection (COVID-19) with transient viral elevation using semi-quantitative real-time reverse transcription PCR and symptom relapse after completion of 10 days of favipiravir treatment.

The coronavirus disease of 2019 (COVID-19), which began in Wuhan, China, at the end of 2019, is spreading around the world and causing many deaths, mainly from pneumonia. Currently, there are no specific drugs to treat COVID-19, and existing antiviral drugs are being used as an alternative. One of these is favipiravir, a new type of influenza drug. However, its efficacy, dosage, and duration of administration are still under study. In this case study, we administered favipiravir to patients with COVID-19 and determined the viral load of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the COVID-19 pathogen, using semi-quantitative real-time reverse transcription PCR in sputum samples. We report on two patients in whom the viral load increased again after completion of 10 days of favipiravir treatment and a transient relapse of symptoms was observed.

Regulatory T-cell dysfunction induces autoantibodies to bullous pemphigoid antigens in mice and human subjects.

BACKGROUND: Regulatory T (Treg) cells play a crucial role in peripheral immune tolerance in multiple organs, including the skin. Thus far, the effect of peripheral immune tolerance failure on autoantibody-related autoimmune reactions to the skin is unclear. OBJECTIVE: We sought to elucidate the target autoantigens in the skin under the condition of Treg cell dysfunction caused by forkhead box P3 (Foxp3) gene mutations in scurfy mice and patients with immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. METHODS: Sera and skin from scurfy mice and sera from patients with IPEX syndrome were analyzed to detect target autoantigens by using immunofluorescence studies, ELISAs, and immunoblotting. The pathogenicity of scurfy IgG was examined by using a passive transfer experiment. CD4+ T cells from scurfy mice were transferred to immunodeficient mice to examine their pathogenicity. Signal transducer and activator of transcription 6 (Stat6)-/- scurfy mice were analyzed to further clarify the molecular pathway of autoantibody production. Follicular helper T-cell counts are measured in Stat6-/- scurfy mice and scurfy mice. RESULTS: Scurfy mice spontaneously generated IgG autoantibodies to the dermal-epidermal junction, which had been class-switched from IgM within 12 days after birth. The target autoantigens were murine BP230 and type XVII collagen (COL17). The scurfy polyclonal autoantibodies did not induce skin fragility in neonatal mice. Autoantibody production was induced by CD4+ T cells from scurfy mice and was ameliorated by Stat6 gene knockout in association with a decrease of follicular helper T cells. We also identified autoantibodies to COL17 and BP230 in patients with IPEX syndrome and found an association between production of autoantibodies to COL17 and an eczematous skin phenotype. CONCLUSIONS: Dysregulation of Treg cells generates autoantibodies to COL17 and BP230 in vivo.

Concordant pattern of radiologic, morphologic, and genomic changes during compensatory lung growth.

BACKGROUND: Although compensatory lung growth (CLG) after lung resection has been reported in various mammalian species, it has generally been thought that the lung cannot regenerate in adult humans. We recently developed a method for evaluating lung weight using a radiologic analysis and demonstrated that the lung was heavier than expected in adult humans after pulmonary resection. In this study, we serially evaluated the morphologic, radiologic, and genomic status during CLG in pneumonectomized mice. METHODS: The serial changes in morphology and gene expression of the remnant right lung after left pneumonectomy were examined in adult male mice. The alveolar density was determined by the mean linear intercept, and the weight was estimated using the Hounsfield value and volumetric data from micro-computed tomography. The parameters were obtained on days 3, 7, and 30 after left pneumonectomy or thoracotomy only (sham control). RESULTS: After left pneumonectomy, the right lung became significantly progressively larger in volume and weight on postoperative days 3, 7, and 30 in comparison to the sham controls (P < 0.01). The estimated weight also significantly increased in association with the real volume on postoperative days 3, 7, and 30 (P < 0.01). The cardiac lobe markedly increased in size. During the observation period, the alveolar density was always lower in the pneumonectomized mice than in controls. A microarray analysis revealed that multiple genes related to proliferation (but not specific alveolar development) were initially upregulated until postoperative day 7 and then returned to normal after 1 mo. The morphologic and genomic changes were more evident in the cardiac lobe than in the upper lobe during the observation period. CONCLUSIONS: The morphologic, radiologic, and genomic changes during CLG were related to each other in pneumonectomized mice. The present study revealed an association between the radiologically estimated weight and other parameters, indicating a marked CLG reaction of the cardiac lobe.

Surgery for Secondary Spontaneous Pneumothorax with Chronic Lung Diseases.

PURPOSES: Secondary spontaneous pneumothorax (SSP) is occasionally observed in elderly patients suffering from diffuse lung diseases. The purpose of this study was to analyze the outcomes of surgical treatment of SSP patients with chronic lung diseases. METHODS: In total, 242 patients who underwent surgery for spontaneous pneumothorax at Chiba University Hospital from January 2006 to October 2016 were included in this study. The patients' records were reviewed retrospectively for data on their background, surgical treatment, morbidity, mortality, and recurrence. RESULTS: Of the spontaneous pneumothorax cohort, primary spontaneous pneumothorax (PSP) accounted for 144 patients. Among the 98 patients with SSP, 57 cases were caused by chronic obstructive pulmonary disease (COPD) and 21 were caused by interstitial pneumonia (IP). The postoperative complication rate was 19.3% in the COPD group, 42.9% in the IP group, and 11.1% in the PSP group. The recurrence rate was 5.3% in the COPD group, 28.6% in the IP group, and 21.5% in the PSP group. CONCLUSIONS: The morbidity and recurrence were comparable between PSP and SSP cases with COPD, whereas these values were unfavorable in SSP cases with IP compared with PSP ones. Surgical intervention should be carefully considered in SSP patients with IP.

ILC2s navigate tissue redistribution during infection using stage-specific S1P receptors.

Lymphocytes can circulate as well as take residence within tissues. While the mechanisms by which circulating populations are recruited to infection sites have been extensively characterized, the molecular basis for the recirculation of tissue-resident cells is less understood. Here, we show that helminth infection- or IL-25-induced redistribution of intestinal group 2 innate lymphoid cells (ILC2s) requires access to the lymphatic vessel network. Although the secondary lymphoid structure is an essential signal hub for adaptive lymphocyte differentiation and dispatch, it is redundant for ILC2 migration and effector function. Upon IL-25 stimulation, a dramatic change in epigenetic landscape occurs in intestinal ILC2s, leading to the expression of sphingosine-1-phosphate receptors (S1PRs). Among the various S1PRs, we found that S1PR5 is critical for ILC2 exit from intestinal tissue to lymph. By contrast, S1PR1 plays a dominant role in ILC2 egress from mesenteric lymph nodes to blood circulation and then to distal tissues including the lung where the redistributed ILC2s contribute to tissue repair. The requirement of two S1PRs for ILC2 migration is largely due to the dynamic expression of the tissue-retention marker CD69, which mediates S1PR1 internalization. Thus, our study demonstrates a stage-specific requirement of different S1P receptors for ILC2 redistribution during infection. We therefore propose a fundamental paradigm that innate and adaptive lymphocytes utilize a shared vascular network frame and specialized navigation cues for migration.

The effect of chest wall surgery on lung volume: a new evaluation concept.

Background: In the surgical treatment of chest wall tumors requiring large chest wall resection, reconstruction of the chest wall defect is required using various autologous tissues or artificial materials. However, no appropriate method has been reported to evaluate whether each reconstruction is successful or not. Therefore, we performed lung volumetry before and after surgery to evaluate the negative effects of chest wall surgery on lung expansion. Methods: Twenty-three patients with chest wall tumors who underwent surgery were included in this study. Lung volume (LV) before and after surgery was measured using SYNAPSE VINSENT (FUJIFILM, Tokyo, Japan). The rate of change in LV was calculated as the postoperative and preoperative LV of the operative side × preoperative/postoperative LV of the opposite side. The excised chest wall area was calculated as vertical diameter × horizontal diameter of the tissue specimen. Results: Reconstruction methods included rigid reconstruction (a combination of titanium mesh and extended polytetrafluoroethylene sheet) in four patients, non-rigid reconstruction (extended polytetrafluoroethylene sheet only) in 11, no reconstruction in five, and no chest wall resection in three. Changes in LV were generally well preserved, regardless of the resected area. In addition, LVs were well maintained in most patients who underwent chest wall reconstruction. However, in some cases, decreased lung expansion was observed with migration and deflection of the reconstructive material into the thorax due to postoperative lung inflammation and shrinking. Conclusions: Lung volumetry can be used to evaluate the effectiveness of chest wall surgery.

Robot-assisted thoracic surgery versus video-assisted thoracic surgery for mediastinal lesions.

Background: Robot-assisted thoracic surgery (RATS) has become widely used for mediastinal procedures since 2018 when it was included in insurance coverage in Japan. Few studies have compared the surgical outcomes of RATS with the more established video-assisted thoracic surgery (VATS) approach to mediastinal surgery. We aimed to compare the perioperative outcomes of VATS and RATS to examine the advantages of the RATS approach in a single institutional cohort. Methods: A total of 144 patients who underwent VATS and 46 who underwent RATS mediastinal surgery between 2014 and 2022 were enrolled. We compared clinicopathological features such as age, sex, smoking history, respiratory function, surgical field, laterality, surgical procedure, board certification of the surgeon, and histology between the two groups. Perioperative outcomes including operation time, volume of blood lost, number of conversion cases to open surgery, duration of chest drainage, postoperative hospital stay, and postoperative complications were also reviewed. Results: The comparison of patient characteristics between the groups showed significant differences in median age (VATS, 52.5 years; RATS, 67.0 years; P=0.001), combined resection of surrounding tissues of the tumor (VATS, 2.1%; RATS, 10.9%; P=0.02), board certification of the surgeon (VATS, 53.5%; RATS, 100.0%; P<0.001), and histology (RATS group had a higher percentage of thymic epithelial tumors, P=0.01). Regarding perioperative outcomes, the median operation time was 120 min in the VATS group and 88 min in the RATS group, showing a significant difference (P=0.03). There were no significant differences in the volume of blood lost, incidence of conversion to open chest surgery, duration of chest drainage, postoperative length of stay in hospital, and incidence of perioperative complications. In the perioperative outcomes of cases operated on by board-certified surgeons, the median operation time (VATS, 117 min; RATS, 88 min; P=0.02) and median postoperative length of stay in hospital (VATS, 7 days; RATS, 6 days; P=0.001) showed significant differences, while other postoperative outcomes were not significantly different. Conclusions: RATS for mediastinal surgery is as safe as the VATS approach and may result in a shorter operative time and postoperative hospital stay. Further analysis of RATS for mediastinal surgery in a larger cohort is warranted.

Anti-CD20 Antibody and Calcineurin Inhibitor Combination Therapy Effectively Suppresses Antibody-Mediated Rejection in Murine Orthotopic Lung Transplantation.

Antibody-mediated rejection (AMR) is a risk factor for chronic lung allograft dysfunction, which impedes long-term survival after lung transplantation. There are no reports evaluating the efficacy of the single use of anti-CD20 antibodies (aCD20s) in addition to calcineurin inhibitors in preventing AMR. Thus, this study aimed to evaluate the efficacy of aCD20 treatment in a murine orthotopic lung transplantation model. Murine left lung transplantation was performed using a major alloantigen strain mismatch model (BALBc (H-2d) → C57BL/6 (BL/6) (H-2b)). There were four groups: isograft (BL/6→BL/6) (Iso control), no-medication (Allo control), cyclosporine A (CyA) treated, and CyA plus murine aCD20 (CyA+aCD20) treated groups. Severe neutrophil capillaritis, arteritis, and positive lung C4d staining were observed in the allograft model and CyA-only-treated groups. These findings were significantly improved in the CyA+aCD20 group compared with those in the Allo control and CyA groups. The B cell population in the spleen, lymph node, and graft lung as well as the levels of serum donor-specific IgM and interferon γ were significantly lower in the CyA+aCD20 group than in the CyA group. Calcineurin inhibitor-mediated immunosuppression combined with aCD20 therapy effectively suppressed AMR in lung transplantation by reducing donor-specific antibodies and complement activation.

Long 3'UTRs predispose neurons to inflammation by promoting immunostimulatory double-stranded RNA formation.

Loss of RNA homeostasis underlies numerous neurodegenerative and neuroinflammatory diseases. However, the molecular mechanisms that trigger neuroinflammation are poorly understood. Viral double-stranded RNA (dsRNA) triggers innate immune responses when sensed by host pattern recognition receptors (PRRs) present in all cell types. Here, we report that human neurons intrinsically carry exceptionally high levels of immunostimulatory dsRNAs and identify long 3'UTRs as giving rise to neuronal dsRNA structures. We found that the neuron-enriched ELAVL family of genes (ELAVL2, ELAVL3, and ELAVL4) can increase (i) 3'UTR length, (ii) dsRNA load, and (iii) activation of dsRNA-sensing PRRs such as MDA5, PKR, and TLR3. In wild-type neurons, neuronal dsRNAs signaled through PRRs to induce tonic production of the antiviral type I interferon. Depleting ELAVL2 in WT neurons led to global shortening of 3'UTR length, reduced immunostimulatory dsRNA levels, and rendered WT neurons susceptible to herpes simplex virus and Zika virus infection. Neurons deficient in ADAR1, a dsRNA-editing enzyme mutated in the neuroinflammatory disorder Aicardi-Goutières syndrome, exhibited intolerably high levels of dsRNA that triggered PRR-mediated toxic inflammation and neuronal death. Depleting ELAVL2 in ADAR1 knockout neurons led to prolonged neuron survival by reducing immunostimulatory dsRNA levels. In summary, neurons are specialized cells where PRRs constantly sense "self" dsRNAs to preemptively induce protective antiviral immunity, but maintaining RNA homeostasis is paramount to prevent pathological neuroinflammation.