Different effects of crizotinib treatment in two non-small cell lung cancer patients with SDC4::ROS1 fusion variants.

The possibility of stratifying patients according to differences in ROS proto-oncogene 1 (ROS1) fusion partners has been discussed. This study aimed to clarify the clinicopathological differences between two SDC4::ROS1 positive NSCLC cases who had different responses to crizotinib. Cytology and pathology samples from two NSCLC cases with SDC4::ROS1 who were diagnosed and treated with crizotinib at Nihon University Itabashi Hospital were obtained. Case 1 has been well-controlled with crizotinib for over 5 years, but case 2 was worse and overall survival was 19 months. Sequencing analysis of ROS1 fusion genes was performed by reverse-transcription-PCR and Sanger's sequencing methods. In addition, thyroid transcription factor (TTF)-1, ROS-1, Ki67, and phosphorylated extracellular signal-regulated kinase (pERK)1/2 expression were investigated using immunohistochemistry. Sequencing analysis showed SDC4 exon2::ROS1 exon 32 (exon33 deleted) in case 1, and coexistence of SDC4 exon2::ROS1 exon 34 and SDC4 exon2::ROS1 exon35 in case 2. The Ki67 index was not different, but ROS1 and pERK1/2 expression levels tended to be higher in the tumor cells of case 2 than in case 1. Therapeutic response to crizotinib and patients' prognosis in ROS1 rearranged NSCLC may be related to the activation of ROS1 signaling, depending on ROS1 and pERK1/2 overexpression status, even if the ROS1 fusion partner is the same.

Anti-UBE2T antibody: A novel biomarker of progressive-fibrosing interstitial lung disease.

BACKGROUND: Anti-fibrotic therapy has demonstrated efficacy against progressive-fibrosing interstitial lung disease (PF-ILD); therefore, identifying disease behavior before progression has become a priority. As autoimmunity is implicated in the pathogenesis of various ILDs, this study explored circulating biomarkers that could predict the chronic progressive behavior of ILDs. METHODS: A single-center retrospective cohort study was conducted. Circulating autoantibodies in patients with ILD were screened using microarray analysis to identify candidate biomarkers. An enzyme-linked immunosorbent assay was performed with a larger sample set for the quantification of antibodies. After 2 years of follow-up, ILDs were reclassified as PF or non-PF. The relationship between the participants' autoantibody levels measured at enrolment and final diagnosis of PF-ILD was determined. RESULTS: In total, 61 healthy participants and 66 patients with ILDs were enrolled. Anti-ubiquitin-conjugating enzyme E2T (UBE2T) antibody was detected as a candidate biomarker. Anti-UBE2T antibody levels were elevated in patients with idiopathic pulmonary fibrosis (IPF). After following up on the study participants for 2 years, anti-UBE2T levels measured at enrolment significantly correlated with the new PF-ILD diagnosis. Immunohistochemical staining of normal lung tissues revealed sparsely located UBE2T in the bronchiole epithelium and macrophages, whereas IPF lung tissues showed robust expression in the epithelial lining of honeycomb structures. CONCLUSION: To our knowledge, this is the first report to describe an anti-UBE2T antibody, a new biomarker that is significantly elevated in patients with ILD who present future disease progression.

Anti-synthetase syndrome-associated interstitial lung disease possibly caused by atezolizumab in a patient with lung adenocarcinoma: a case report.

BACKGROUND: Anti-aminoacyl-tRNA synthetase (ARS) antibody-positive patients present with a variety of symptoms, including interstitial lung disease (ILD), which is termed anti-synthetase syndrome (ASS). But it is rare that ASS-ILD is considered an immune-related adverse event after the use of immune checkpoint inhibitors (ICIs). CASE PRESENTATION: A 47-year-old male with advanced lung adenocarcinoma was treated with platinum and ICI combination immunotherapy and was followed up as an outpatient. Nine months after the start of treatment, he developed a fever and cough, and imaging findings showed lung consolidations in the bilateral lower lung fields. The patient was positive for anti- ARS antibodies and was considered to have developed ASS-ILD due to ICIs remitted with steroid therapy. The patient was found to be positive for anti-ARS antibodies before ICI administration, and the antibody titer was elevated compared to that before ICI administration. CONCLUSIONS: The examination of anti-ARS antibodies pior to the administration of ICIs may be useful in predicting the development of ASS-ILD.

Retrospective Analysis of the Incidence of Drug-Induced Interstitial Lung Disease by Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors and Survival in Patients Aged 75 Years or Older with Lung Cancer.

Introduction: To date, the appropriate epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) for patients aged ≥75 years with advanced EGFR mutation-positive, nonsmall cell lung cancer remain unknown. Methods: This study included a total of 89 patients aged ≥75 years who were diagnosed with EGFR mutation-positive, nonsmall cell lung cancer and treated with EGFR-TKIs at the Tokyo Metropolitan Geriatric Hospital and Nihon University ITABASHI Hospital from 2009 to 2020. The patients were classified into five groups based on their treatment: gefitinib (n = 23), erlotinib (n = 4), afatinib (n = 3), first-line osimertinib (n = 23), and TKI to TKI (n = 36). The efficacy and safety of each EGFR-TKI were analyzed. Results: No significant differences in the overall survival and progression-free survival were observed among the groups. However, a significantly higher incidence of drug-induced interstitial lung disease (ILD) was detected with osimertinib than with the first-generation EGFR-TKIs (p = 0.008). Conclusions: In older patients with EGFR mutation-positive lung cancer, the incidence of drug-induced ILD was significantly increased during osimertinib treatment. This outcome should be noted when treating older patients with osimertinib who may not always want to live longer but want to live better.

Comprehensive genetic analysis of histological components of combined small cell carcinoma.

BACKGROUND: Combined small-cell lung cancer (cSCLC) is a rare type of small-cell lung cancer (SCLC) that includes both SCLC and non-small-cell lung cancer (NSCLC). The molecular biological mechanisms underlying the heterogeneity of histological types in combined or metachronously transformed SCLC (mtSCLC) remain unclear. This study aimed to investigate the relationship between genetic alterations and each histological component heterogeneously detected in cSCLC and mtSCLC. METHODS: This study included four cSCLC cases and one mtSCLC case. Formalin-fixed and paraffin-embedded sections of each histological component of these tumors were subjected to next-generation sequencing (NGS) and quantitative reverse transcription-polymerase chain reaction to investigate the genetic mutations and expression levels of neuroendocrine cell-specific transcription factors (achaete-scute homolog-1 [ASCL1], brain-2 [BRN2] also known as POU domain class 3 transcription factor 2, nuclear factor 1 B [NF1B], insulinoma-associated protein 1 [INSM1], and thyroid transcription factor-1 [TTF-1]). RESULTS: NGS analysis revealed that SCLC and NSCLC components share the same somatic mutations detected most frequently in TP53, and also in RB1 and EGFR. Gene expression analysis showed ASCL1 expression was significantly lower in the NSCLC component than in the SCLC component. CONCLUSION: We conclude that the morphological evolution of heterogeneous histological components in cSCLC may be associated with differences in ASCL1 expression levels, but not in acquired somatic gene mutations.

Successful treatment of refractory brain metastases from ALK-positive lung cancer with lorlatinib.

A 44-year-old woman with ALK-positive advanced adenocarcinoma of the lung was treated with crizotinib, and the lung lesions disappeared. The patient was treated with alectinib and chemotherapy, but brain metastases worsened; therefore, we performed an ALK resistance gene mutation test using plasma samples. Since no ALK resistance gene mutations were detected, we speculated that ALK inhibitors failed to achieve therapeutic effects due to poor transport to the central nervous system. Therefore, we switched to lorlatinib, and found a reduction in brain metastases. In ALK-positive advanced lung cancer, plasma-based resistance gene testing may be useful for treatment decisions.

Amyloid conformation-dependent disaggregation in a reconstituted yeast prion system.

Disaggregation of amyloid fibrils is a fundamental biological process required for amyloid propagation. However, due to the lack of experimental systems, the molecular mechanism of how amyloid is disaggregated by cellular factors remains poorly understood. Here, we established a robust in vitro reconstituted system of yeast prion propagation and found that heat-shock protein 104 (Hsp104), Ssa1 and Sis1 chaperones are essential for efficient disaggregation of Sup35 amyloid. Real-time imaging of single-molecule fluorescence coupled with the reconstitution system revealed that amyloid disaggregation is achieved by ordered, timely binding of the chaperones to amyloid. Remarkably, we uncovered two distinct prion strain conformation-dependent modes of disaggregation, fragmentation and dissolution. We characterized distinct chaperone dynamics in each mode and found that transient, repeated binding of Hsp104 to the same site of amyloid results in fragmentation. These findings provide a physical foundation for otherwise puzzling in vivo observations and for therapeutic development for amyloid-associated neurodegenerative diseases.

Genomic analysis between idiopathic pulmonary fibrosis and associated lung cancer using laser-assisted microdissection: A case report.

Lung cancer (LC) is the most fatal complication of idiopathic pulmonary fibrosis (IPF). However, the molecular pathogenesis of the development of LC from IPF is still unclear. Here, we report a case of IPF-associated LC for which we investigated the genetic alterations between IPF and LC. We extracted formalin-fixed paraffin-embedded DNA from each part of the surgical lung tissue using a laser-assisted microdissection technique. The mutations in each part were detected by next-generation sequencing (NGS) using 72 lung cancer-related mutation panels. Five mutations were found in IPF and four in LC. Almost all somatic mutations did not overlap between the IPF and LC regions. These findings suggest that IPF-associated LC may not be a result of the accumulation of somatic mutations in the regenerated epithelium of the honeycomb lung in the IPF region.

Murine neonatal ketogenesis preserves mitochondrial energetics by preventing protein hyperacetylation.

Ketone bodies are generated in the liver and allow for the maintenance of systemic caloric and energy homeostasis during fasting and caloric restriction. It has previously been demonstrated that neonatal ketogenesis is activated independently of starvation. However, the role of ketogenesis during the perinatal period remains unclear. Here, we show that neonatal ketogenesis plays a protective role in mitochondrial function. We generated a mouse model of insufficient ketogenesis by disrupting the rate-limiting hydroxymethylglutaryl-CoA synthase 2 enzyme gene (Hmgcs2). Hmgcs2 knockout (KO) neonates develop microvesicular steatosis within a few days of birth. Electron microscopic analysis and metabolite profiling indicate a restricted energy production capacity and accumulation of acetyl-CoA in Hmgcs2 KO mice. Furthermore, acetylome analysis of Hmgcs2 KO cells revealed enhanced acetylation of mitochondrial proteins. These findings suggest that neonatal ketogenesis protects the energy-producing capacity of mitochondria by preventing the hyperacetylation of mitochondrial proteins.

Cryopreservation of mouse resources.

The cryopreservation of sperm and embryos is useful to efficiently archive valuable resources of genetically engineered mice. Till date, more than 60,000 strains of genetically engineered mice have been archived in mouse banks worldwide. Researchers can request for the archived mouse strains for their research projects. The research infrastructure of mouse banks improves the availability of mouse resources, the productivity of research projects, and the reproducibility of animal experiments. Our research team manages the mouse bank at the Center for Animal Resources and Development in Kumamoto University and continuously develops new techniques in mouse reproductive technology to efficiently improve the system of mouse banking. In this review, we introduce the activities of mouse banks and the latest techniques used in mouse reproductive technology.

DsRNA induction of microRNA-155 disrupt tight junction barrier by modulating claudins.

BACKGROUND: The impaired barrier function of the airway epithelium due to RNA virus infection is closely related to the development and exacerbation of allergic airway inflammation. OBJECTIVE: In this study, we investigated the roles of microRNAs on the mechanisms of double-stranded RNA (dsRNA)-induced epithelial barrier dysfunction. METHODS: 16HBE14o- human bronchial epithelial cells were grown to confluence on Transwell inserts and exposed to poly-I:C. We studied epithelial barrier function by measuring transepithelial electrical resistance and paracellular flux of fluorescent markers and structure of tight junctions by immunofluorescence microscopy. RESULTS: Poly-I:C treated 16HBE14o- cells increased paracellular permeability. Knockdown of Toll-like receptor 3 and TRIF abrogated these effects. The expression of microRNA-155 (miR-155) was increased by poly-I:C in dose-dependent manner. Transfection of mir155 mimics into 16HBE14o- cells increased permeability and inhibited tight junction formation. Transfection of miR-155 inhibitor suppressed poly-I:C-induced barrier disruption. Poly-I:C treatment significantly decreased the expression of claudin members-claudin-1, -3, -4, -5, -9, -11, -16, -18 and -19. Transfection of miR-155 mimics showed similar changing expression pattern of claudin members with those of poly-I:C treatment. CONCLUSION: These results suggest that RNA virus infection can impair the epithelial barrier disruption mechanism by down-regulation of claudin members through the induction of miR-155.

Six1 is required for signaling center formation and labial-lingual asymmetry in developing lower incisors.

BACKGROUND: The structure of the mouse incisor is characterized by its asymmetric accumulation of enamel matrix proteins on the labial side. The asymmetric structure originates from the patterning of the epithelial incisor placode through the interaction with dental mesenchymal cells. However, the molecular basis for the asymmetric patterning of the incisor germ is largely unknown. RESULTS: A homeobox transcription factor SIX1 was shown to be produced in the mandibular mesenchyme, and its localization patterns changed dynamically during lower incisor development. Six1-/- mice exhibited smaller lower incisor primordia than wild-type mice. Furthermore, Six1-/- mice showed enamel matrix production on both the lingual and labial sides and disturbed odontoblast maturation. In the earlier stages of development, the formation of signaling centers, the initiation knot and the enamel knot, which are essential for the morphogenesis of tooth germs, were impaired in Six1-/- embryos. Notably, Wnt signaling activity, which shows an anterior-posterior gradient, and the expression patterns of genes involved in incisor formation were altered in the mesenchyme in Six1-/- embryos. CONCLUSION: Our results indicate that Six1 is required for signaling center formation in lower incisor germs and the labial-lingual asymmetry of the lower incisors by regulating the anterior-posterior patterning of the mandibular mesenchyme.

Regenerating islet-derived protein (Reg)3ß plays a crucial role in attenuation of ileitis and colitis in mice.

Regenerating islet-derived protein (Reg)3ß belongs to a member of the Reg family of proteins and has pleiotropic functions, including antimicrobial activity and tissue repair. However, whether Reg3ß plays a protective role in the development of colitis and ileitis has not been fully investigated. We generated transgenic mice expressing a short form of cellular FLICE-inhibitory protein (cFLIPs) that promotes necroptosis, a regulated form of cell death. cFLIPs transgenic (CFLARs Tg) mice develop severe ileitis in utero. Although Reg3ß is undetectable in the small intestine of wild-type embryos, its expression is aberrantly elevated in the small intestine of CFLARs Tg embryos. To test whether elevated Reg3ß attenuates or exacerbates ileitis in CFLARs Tg mice, we generated a Reg3b -/- strain. Reg3b -/- mice grew to adulthood without apparent abnormalities. Deletion of Reg3b in CFLARs Tg mice exacerbated the embryonic lethality of CFLARs Tg mice. Dextran sulfate sodium-induced colitis, characterized by body weight loss and infiltration of neutrophils, was exacerbated in Reg3b -/- compared to wild-type mice. Moreover, the expression of Interleukin 6, an inflammatory cytokine and Chitinase-like 3, a marker for tissue repair macrophages was elevated in the colon of Reg3b -/- mice compared to wild-type mice after DSS treatment. Together, these results suggest that attenuation of colitis and ileitis is a result of Reg3ß's real function.

The Diagnosis of Exogenous Lipoid Pneumonia Caused by the Silent Aspiration of Vegetable Oil Using a Lipidomic Analysis.

We herein report a case of refractory exogenous lipoid pneumonia that was successfully attributed to vegetable oil through a lipidomic analysis of bronchoalveolar lavage fluid (BALF). As a 25-year-old woman diagnosed with lipoid pneumonia experienced repeated exacerbations and improvement, we performed a BALF lipidomic analysis. The major lipid components were oleic acid, linoleic acid, and α-linolenic acid, which are constituents of vegetable oil. She stopped consuming any vegetable oil and has since experienced no instances of lipoid pneumonia relapse. A lipidomic analysis appears to be useful for identifying causative lipids, since patients with lipoid pneumonia are sometimes unaware of aspiration episodes.

Generation of and characterization of anti-IL-11 antibodies using newly established Il11-deficient mice.

Interleukin (IL)-11 belongs to the members of the IL-6 family of cytokines and is involved in a variety of biological responses, including hematopoiesis, bone development, and carcinogenesis. However, the cellular sources of IL-11 and regulation of IL-11 expression under physiological and pathological conditions are not fully understood. One of the causes to prevent characterization of IL-11 in vivo is due to the lack of reliable antibodies that detect IL-11 by immunohistochemistry. Moreover, although mice lacking Il11ra have been generated and extensively characterized, Il11-deficient mice have not been characterized yet. Here we generated two anti-IL-11 antibodies that blocked biological activities of IL-11 and detected IL-11 by immunohistochemistry, respectively. One clone of anti-IL-11 antibodies blocked IL-11-, but not IL-6-induced cell proliferation and IL-11-induced phosphorylation of STAT3 of an IL-11-dependent cell line. Moreover, we used recently established Il11-deficient mice to test the specificity of anti-IL-11 antibodies for immunohistochemistry. Another clone of anti-IL-11 antibodies stained stromal cells surrounding tumors of the colon of wild-type, but not Il11-deficient mice following treatment with Azoxymethane plus dextran sulfate sodium. Together, these newly developed anti-IL-11 antibodies provide a better understanding of the functions of IL-11 in vivo under various physiological and pathological conditions.

Electroporation-mediated genome editing in vitrified/warmed mouse zygotes created by IVF via ultra-superovulation.

Recently, genome editing in mouse zygotes has become convenient and scalable, in association with various technological developments and improvements such as novel nuclease tools, alternative delivery methods, and contemporary reproductive engineering techniques. We have so far demonstrated the applicability of ultra-superovulation, in vitro fertilization (IVF), and vitrification/warming of zygotes in microinjection-mediated mouse genome editing. Moreover, an electroporation-mediated method has rapidly become established for simple gene knockout and small precise modifications including single amino acid substitutions. Here, we present an updated example of an application coupling the following three latest technologies: 1) CRISPR-Cas9 ribonucleoprotein as the most convenient genome-editing reagent, 2) electroporation as the most effortless delivery method, and 3) cryopreserved oocytes created by IVF via ultra-superovulation as the most animal welfare- and user-friendly strategy. We successfully created gene knockout and knock-in mice carrying insertion/deletion mutations and single amino acid substitutions, respectively, using the streamlined production system of mouse genome editing described above, referred to as the CREATRE (CARD-based Reproductive Engineering-Assisted Technology for RNP Electroporation) system. Owing to its accessibility, robustness, and high efficiency, we believe that our CREATRE protocol will become widely used globally for the production of genome-edited mice.

Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice.

Robust reproductive engineering techniques are required for the efficient and rapid production of genetically modified mice. We have reported the efficient production of genome-edited mice using reproductive engineering techniques, such as ultra-superovulation, in vitro fertilization (IVF) and vitrification/warming of zygotes. We usually use vitrified/warmed fertilized oocytes created by IVF for microinjection because of work efficiency and flexible scheduling. Here, we investigated whether the culture time of zygotes before microinjection influences the efficiency of producing knock-in mice. Knock-in mice were generated using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system and single-stranded oligodeoxynucleotide (ssODN) or PITCh (Precise Integration into Target Chromosome) system, a method of integrating a donor vector assisted by microhomology-mediated end-joining. The cryopreserved fertilized oocytes were warmed, cultured for several hours and microinjected at different timings. Microinjection was performed with Cas9 protein, guide RNA(s), and an ssODN or PITCh donor plasmid for the ssODN knock-in and the PITCh knock-in, respectively. Different production efficiencies of knock-in mice were observed by changing the timing of microinjection. Our study provides useful information for the CRISPR-Cas9-based generation of knock-in mice.

C-Type Lectin Receptor DCAR Recognizes Mycobacterial Phosphatidyl-Inositol Mannosides to Promote a Th1 Response during Infection.

Phosphatidyl-inositol mannosides (PIM) are glycolipids unique to mycobacteria and other related bacteria that stimulate host immune responses and are implicated in mycobacteria pathogenicity. Here, we found that the FcRγ-coupled C-type lectin receptor DCAR (dendritic cell immunoactivating receptor; gene symbol Clec4b1) is a direct receptor for PIM. Mycobacteria activated reporter cells expressing DCAR, and delipidation of mycobacteria abolished this activity. Acylated PIMs purified from mycobacteria were identified as ligands for DCAR. DCAR was predominantly expressed in small peritoneal macrophages and monocyte-derived inflammatory cells in lungs and spleen. These cells produced monocyte chemoattractant protein-1 (MCP-1) upon PIM treatment, and absence of DCAR or FcRγ abrogated MCP-1 production. Upon mycobacterial infection, Clec4b1-deficient mice showed reduced numbers of monocyte-derived inflammatory cells at the infection site, impaired IFNγ production by T cells, and an increased bacterial load. Thus, DCAR is a critical receptor for PIM that functions to promote T cell responses against mycobacteria.