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Interstitial lung disease (ILD) is a serious complication of connective tissue diseases (CTDs). The heterogeneity of ILDs reflects differences in pathogenesis among diseases. This study aimed to clarify the characteristics of CTD-ILDs via a detailed analysis of the bronchoalveolar lavage fluid (BALF) and blood immune cells. BALF and blood samples were collected from 39 Japanese patients with newly diagnosed ILD: five patients with Sjögren’s syndrome (SS), eight patients with dermatomyositis (DM), six patients with rheumatoid arthritis (RA), six patients with systemic sclerosis, four patients with anti-neutrophil cytoplasmic antibody-associated vasculitis, and 10 patients with idiopathic interstitial pneumonia. We performed single-cell RNA sequencing to analyze the gene expression profiles in these patients’ immune cells. In patients with SS, B cells in the BALF were increased and genes associated with the innate and acquired immunity were enriched in both the BALF and blood. In contrast, patients with DM showed an upregulation of genes associated with viral infection in both the BALF and blood. In patients with RA, neutrophils in the BALF tended to increase, and their gene expression patterns changed towards inflammation. These disease-specific characteristics may help us understand the pathogenesis for each disease and discover potential biomarkers.

Abstract Mus minutoides is one of the smallest mammals worldwide; however, the regulatory mechanisms underlying its dwarfism have not been examined. Therefore, we aimed to establish M. minutoides induced pluripotent stem cells (iPSCs) using the PiggyBac transposon system for applications in developmental engineering. The established M. minutoides iPSCs were found to express pluripotency markers and could differentiate into neurons. Based on in vitro differentiation analysis, M. minutoides iPSCs formed embryoid bodies expressing marker genes in all three germ layers. Moreover, according to the in vivo analysis, these cells contributed to the formation of teratoma and development of chimeric mice with Mus musculus. Overall, the M. minutoides iPSCs generated in this study possess properties that are comparable to or closely resemble those of naïve pluripotent stem cells (PSCs). These findings suggest these iPSCs have potential utility in various analytical applications, including methods for blastocyst completion.

Abstract Background Oral care is important in preventing aspiration pneumonia in older adults. However, it is not clear what kind of oral care can reduce the number of bacteria in saliva. The purposes of this study are to clarify whether there is a relationship between plaque amounts and salivary bacterial counts, and how bacteria dispersed into the oral cavity by brushing can be reduced. Methods First, saliva samples were collected from 10 healthy adult volunteers after 30 h of unbrushing and after thorough brushing, and the total bacterial count was determined by real-time PCR. Next, 40 older adults attending an outpatient dental clinic were randomly assigned into two groups: a wiping group (20 patients) and a mouthwashing group (20 patients). Saliva was collected before and after brushing, and after wiping in the wiping group and after mouthwashing in the mouthwashing group, and the total bacterial count was quantified by real-time PCR. Results In a study of volunteers, there was no association between plaque amounts and salivary bacterial counts. In a study of older adult patients, salivary bacterial counts were significantly higher in patients with higher oral hygiene index and fewer remaining teeth. Brushing increased salivary bacterial counts. Wiping did not significantly reduce the number of bacteria, while mouthwash returned the increased number of bacteria after brushing to the pre-brushing level. Conclusions There is no direct relationship between the amount of plaque and the number of bacteria in saliva. Brushing disperses bacteria into the oral cavity, resulting in a marked increase in the number of bacteria in saliva. Wiping does not collect the dispersed bacteria, and it seems essential to rinse the mouth after brushing. Trial registration UMIN000045854.

Senescent cells play a detrimental role in age-associated pathogenesis by producing factors involved in senescence-associated secretory phenotype (SASP). The present study was conducted to examine the possibility that senescent cells are present in aged ovaries and, if so, to determine the tissue region where senescent cells accumulate using a mouse model. Female mice at 2–4 and 8–10 months were used as reproductively young and aged models, respectively; the latter included mice with and without reproductive experience. Cells positive for senescence-associated β-galactosidase (SA-β-Gal) staining, one of the markers of cellular senescence, were detected in the stromal region of aged, but not young, ovaries regardless of reproductive experience. Likewise, the localization of cells expressing CDKN2A (cyclin dependent kinase inhibitor 2A), another senescence marker, in the stromal region of aged ovaries was detected with immunohistochemistry. CDKN2A expression detected by western blotting was significantly higher in the ovaries of aged mice with reproductive experience than in those without the experience. Moreover, cells positive for both γH2AX (a senescence marker) and fluorescent SA-β-Gal staining were present in those isolated from aged ovaries. In addition, the transcript levels of several SASP factors were significantly increased in aged ovaries. These results suggest that senescent cells accumulate in the ovarian stroma and may affect ovarian function in aged mice. Additionally, reproductive experience may promote accumulation.

The signals of the transforming growth factor β (TGF-β) superfamily play a critical role in follicular development in mammals. ACVR1B/TGFBR1/ACVR1C receptors mediate the signaling of several TGF-β superfamily ligands in granulosa cells. Although the requirement for ACVR1B/TGFBR1/ACVR1C receptor signaling in follicular development has been confirmed using mutant mouse models, the detailed roles of the signaling in granulosa cell and oocyte development have not been clearly defined. In the present study, we examined the requirement for ACVR1B/TGFBR1/ACVR1C receptor signaling in granulosa cells using an in vitro growth culture of oocyte-granulosa cell complexes (OGCs) and SB431542, a potent inhibitor of the receptor signaling. Although cumulus-oocyte complexes isolated from the control OGCs were able to undergo cumulus expansion, those isolated from OGCs grown with the inhibitor were not competent, even in the presence of in vivo-grown oocytes. The diameter of the oocytes in the SB431542-treated OGCs was comparable with that of the control; however, these oocytes were not competent for complete meiotic maturation or preimplantation development. Therefore, ACVR1B/TGFBR1/ACVR1C receptor signaling is not required for oocytes to increase their volume but is essential for the normal development of cumulus cells and oocyte developmental competence.

Abstract Background Clarifying how tongue pressure in convalescent stroke patients affects oral condition and activities of daily living (ADL) is important for developing oral rehabilitation programs and for rehabilitation care to reacquire ADL. Objective To clarify how tongue pressure is associated with oral status, ADL, and nutritional status in stroke patients. Methods Sixty‐eight patients aged 77.9 ± 10.0 years participated. The Japanese version of the Oral Health Assessment Tool was used to evaluate oral status. Data such as the ADL index functional independence measure (FIM), nutritional intake method, serum albumin, and body mass index were extracted from medical records. To examine factors associated with tongue pressure, multiple regression analysis was performed adjusting for confounding factors. The level of statistical significance was set at p

Anaphylaxis is a severe life-threatening hypersensitivity reaction induced by mast cell degranulation. Among the various mediators of mast cells, little is known about the role of tryptase. Therefore, we aimed to elucidate the role of protease-activating receptor-2 (PAR-2), a receptor activated by tryptase, in murine anaphylactic models using PAR-2-deficient mice and newly generated tryptase-deficient mice. Anaphylaxis was induced by IgE-dependent and IgE-independent mast cell degranulation in mice. PAR-2 deficiency exacerbated the decrease in body temperature and hypotension during anaphylaxis; however, the number of skin mast cells, degree of mast cell degranulation, and systemic and local vascular hyperpermeability were comparable in PAR-2 knockout and wild-type mice. Nitric oxide, which is produced by endothelial nitric oxide synthase (eNOS), is an indispensable vasodilator in anaphylaxis. In the lungs of anaphylactic mice, PAR-2 deficiency promoted eNOS expression and phosphorylation, suggesting a protective effect of PAR-2 against anaphylaxis by downregulating eNOS activation and expression. Based on the hypothesis that the ligand for PAR-2 in anaphylaxis is mast cell tryptase, tryptase-deficient mice were generated using CRISPR-Cas9. In wild-type mice, the PAR-2 antagonist exacerbated the body temperature drop due to anaphylaxis; however, the effect of the PAR-2 antagonist was abolished in tryptase-deficient mice. These results suggest that tryptase is a possible ligand of PAR-2 in anaphylaxis and that the tryptase/PAR-2 pathway attenuates the anaphylactic response in mice.

A woman diagnosed with rheumatoid arthritis and treated with Janus kinase (JAK) inhibitors presented with a gradually enlarging bilateral submandibular lymph nodes swelling that had lasted several weeks. A lymph node biopsy showed epithelioid granulomatous lymphadenitis with caseous necrosis. Mycobacteria grew in acid-fast bacteria culture and were identified as Mycobacterium avium by polymerase chain reaction. The patient was diagnosed with cervical lymphadenitis caused by M. avium. A computed tomography scan showed no evidence of a mass or infection at other sites, including the lungs; therefore, the mass was excised without any antimicrobial treatments. Her neck mass had not recurred at 9 months after the excision. JAK inhibitors have emerged as an important new class of oral therapy for rheumatoid arthritis and other diseases. Physicians should be aware of the relatively rare complications, such as cervical lymphadenitis caused by nontuberculous mycobacteria, when using JAK inhibitors.

Visceral leishmaniasis (VL) is an infectious disease caused by parasitic protozoa of the genus Leishmania and manifests clinical symptoms such as splenomegaly, hepatomegaly, anemia, and fever. It has previously been shown that B-cell-activating factor (BAFF) is involved in splenomegaly during VL. Although BAFF is known to be expressed by a variety of cells, the mechanism of elevated BAFF expression in VL is not clear. In this study, we aimed to identify BAFF-producing cells in the spleens of mice infected with Leishmania donovani. Splenocytes of L. donovani-infected mice showed elevated BAFF expression compared to that of naive mice. In the infected spleen, the number of both CD11b+ and F4/80+ cells increased, and the major BAFF-producing cells were CD11b+ cells, which did not serve as host cells of Leishmania. Immunohistochemical/immunofluorescent staining of spleens of infected mice revealed that the increased CD11b+ cells were primarily MRP14+ mononuclear cells. Together, these results suggest the increased BAFF expression in the spleen of L. donovani-infected mice involves a recruitment of inflammatory macrophages distinct from host macrophages for the parasites.

Abstract Forkhead box L2 (FOXL2) plays a critical role in the development and function of mammalian ovaries. In fact, the causative effects of FOXL2 misregulations have been identified in many ovarian diseases, such as primary ovarian insufficiency and granulosa cell tumor; however, the mechanism by which FOXL2 expression is regulated is not well studied. Here, we showed that FOXL2 expression in ovarian mural granulosa cells (MGCs) requires stimulation by both oocyte-derived signals and estrogen in mice. In the absence of oocytes or estrogen, expression of FOXL2 and its transcriptional targets, Cyp19a1 and Fst mRNA, in MGCs were significantly decreased. Moreover, expression levels of Sox9 mRNA, but not SOX9 protein, were significantly increased in the FOXL2-reduced MGCs. FOXL2 expression in MGCs was maintained with either oocytes or recombinant proteins of oocyte-derived paracrine factors, BMP15 and GDF9, together with estrogen, and this oocyte effect was abrogated with an ALK5 inhibitor, SB431542. In addition, the FOXL2 level was significantly decreased in MGCs isolated from Bmp15 −/− /Gdf9 +/− mice. Therefore, oocyte, probably with estrogen, plays a critical role in the regulation of FOXL2 expression in mural granulosa cells in mice.

B cell activating factor (BAFF) plays an important role in antibody production through differentiation and maturation of B cells mainly in secondary lymphoid organs. On the other hand, the role of BAFF in the bone marrow, the primary lymphoid organ of B cell development, has not been well elucidated. Here, effects of BAFF in bone marrow B cell development were examined by using BAFF-deficient mice. When mRNA expression levels of B cell differentiation markers including Cd19, Bcl2, Igμ, Il7r and Cxcr5 were compared between bone marrow of wild-type and BAFF-KO mice, a lower level of Cxcr5 expression was found in the KO mice. Additionally, protein expression of CXCR5 on IgM+ cells in the bone marrow was decreased by BAFF deficiency. In vitro studies also confirmed the effect of BAFF on CXCR5 by IgM+ cells; culturing bone marrow cells from BAFF-KO mice with BAFF in vitro increased the proportion of CXCR5+ cells in IgM+ cells compared with non-treated bone marrow cells. In addition, BAFF synergized with TNF-α and IL-6 to increase the expression of CXCR5+ on IgM+ cells. The BAFF-mediated up-regulation of CXCR5 expression was reproduced by using CD19+ cells purified from BAFF-KO bone marrow cells, suggesting that BAFF directly affects B-lineage cells in bone marrow to promote CXCR5 expression. Together, this study suggests that BAFF has an important role in B cell differentiation in bone marrow by directly inducing CXCR5 expression which affect their migration to secondary lymphoid organs.

Summary: Systemic inflammation is established as part of late-stage severe lung disease, but molecular, functional, and phenotypic changes in peripheral immune cells in early disease stages remain ill defined. Chronic obstructive pulmonary disease (COPD) is a major respiratory disease characterized by small-airway inflammation, emphysema, and severe breathing difficulties. Using single-cell analyses we demonstrate that blood neutrophils are already increased in early-stage COPD, and changes in molecular and functional neutrophil states correlate with lung function decline. Assessing neutrophils and their bone marrow precursors in a murine cigarette smoke exposure model identified similar molecular changes in blood neutrophils and precursor populations that also occur in the blood and lung. Our study shows that systemic molecular alterations in neutrophils and their precursors are part of early-stage COPD, a finding to be further explored for potential therapeutic targets and biomarkers for early diagnosis and patient stratification.

Abstract Background Allergen immunotherapy (AIT) is the only treatment that has modified the natural history of allergic diseases. However, since its overall effect on the immune system has not been elucidated, AIT is either absolutely or relatively contraindicated in patients with rheumatic autoimmune diseases (RADs). Therefore, there have been no long-term observations of patients with RADs receiving AIT; thus, the effectiveness and safety of AIT in these patients remain unclear. Methods This was a single-center retrospective observational study. RAD patients receiving AIT for allergic rhinitis at our institution were selected. Changes in the activity of RAD patients were investigated for 2 years from baseline, including those who discontinued AIT. The effectiveness of AIT was also investigated using the Japan Allergic Rhinitis Standard Quality of Life Questionnaire. Results Thirteen patients with RADs were enrolled in the study. All patients received sublingual immunotherapy, of which four discontinued AIT owing to adverse events. Among all patients, the symptoms of RADs in three patients worsened during the observation period; however, none of them were causally related to AIT. Most of the adverse events associated with AIT were mild, in which only one patient required drug intervention due to worsening rhinitis symptoms. In the nine patients who were able to continue AIT, their eye and nasal symptom scores showed a significant improvement from 1.67 (1.5–2.0) at baseline to 0.67 (0–1.17) in the 2nd year of treatment (p = 0.0141). Conclusions AIT is a safe and effective treatment modality for patients with allergic rhinitis complicated by RADs.

The cooperative effects of estrogen and oocyte-derived paracrine factors (ODPFs) play critical roles in the normal development of ovarian follicles; however, the mechanism underlying this cooperation has not been well studied. The present study aimed to determine whether ODPFs affect estrogen signaling by regulating the expression of estrogen receptor (ESR) and its coregulators in mouse granulosa cells. Some transcripts encoding ESR coregulators were differentially expressed between cumulus and mural granulosa cells (MGCs). The transcript levels of ESR coregulators, including nuclear receptor corepressor 1 and activator 2, in cumulus cells were significantly suppressed by ODPFs; however, they increased when cumulus cell-oocyte complexes were treated with the transforming growth factor beta receptor I inhibitor, SB431542. Moreover, MGCs exhibited significantly higher ESR2 protein and transcript levels than those in cumulus cells. ODPFs promoted Esr2 expression in cumulus cells but had no effect on that in MGCs. Overall, regulation of the expression of ESR2 and its coregulators in cumulus cells by oocytes seems to be one of the mechanisms underlying estrogen-oocyte cooperation in well-developed antral follicles in mice.

Leishmaniasis is caused by infection with protozoan parasites of the genus Leishmania. In both clinical and experimental visceral leishmaniasis, macrophage multinucleation is observed in parasitized tissues. However, the feature and the mechanism of macrophage multinucleation remained unclear. Here, we report that infection of Leishmania donovani, a causative agent of visceral leishmaniasis, induces multinucleation of bone marrow-derived macrophages (BMDMs) in vitro. When these infection-induced multinucleated macrophages were compared with cytokine-induced multinucleated giant cells, the former had higher phagocytic activity on red blood cells but no apparent changes on phagocytosis of latex beads. BMDMs infected with L. donovani had increased expression of ATP6V0D2, one of the components of V-ATPase, which was also upregulated in the spleen of infected mice. Infection-induced ATP6V0D2 localized in a cytoplasmic compartment, which did not overlap with the mitochondria, endoplasmic reticulum, or lysosomes. When ATP6V0D2 expression was recombinantly induced in BMDMs, the formation of multinucleated macrophages was induced as seen in the infected macrophages. Taken together, L. donovani infection induces multinucleation of macrophages via ATP6V0D2 upregulation leading to a unique metamorphosis of the macrophages toward hemophagocytes.

Despite its high prevalence, the cellular and molecular mechanisms of chronic obstructive pulmonary disease (COPD) are far from being understood. Here, we determine disease-related changes in cellular and molecular compositions within the alveolar space and peripheral blood of a cohort of COPD patients and controls. Myeloid cells were the largest cellular compartment in the alveolar space with invading monocytes and proliferating macrophages elevated in COPD. Modeling cell-to-cell communication, signaling pathway usage, and transcription factor binding predicts TGF-β1 to be a major upstream regulator of transcriptional changes in alveolar macrophages of COPD patients. Functionally, macrophages in COPD showed reduced antigen presentation capacity, accumulation of cholesteryl ester, reduced cellular chemotaxis, and mitochondrial dysfunction, reminiscent of impaired immune activation.

The shedding of cell surface receptors may bring synergistic outcomes through the loss of receptor-mediated cell signaling and competitive binding of the shed soluble receptor to its ligand. Thus, soluble receptors have both biological importance and diagnostic importance as biomarkers in immunological disorders. Signal regulatory protein α (SIRPα), one of the receptors responsible for the ‘don’t-eat-me’ signal, is expressed by myeloid cells where its expression and function are in part regulated by proteolytic cleavage. However, reports on soluble SIRPα as a biomarker are limited. We previously reported that mice with experimental visceral leishmaniasis (VL) manifest anemia and enhanced hemophagocytosis in the spleen accompanied with decreased SIRPα expression. Here, we report increased serum levels of soluble SIRPα in mice infected with Leishmania donovani, a causative agent of VL. Increased soluble SIRPα was also detected in a culture supernatant of macrophages infected with L. donovani in vitro, suggesting the parasite infection promotes ectodomain shedding of SIRPα on macrophages. The release of soluble SIRPα was partially inhibited by an ADAM proteinase inhibitor in both LPS stimulation and L. donovani infection, suggesting a shared mechanism for cleavage of SIRPα in both cases. In addition to the ectodomain shedding of SIRPα, both LPS stimulation and L. donovani infection induced the loss of the cytoplasmic region of SIRPα. Although the effects of these proteolytic processes or changes in SIRPα still remain unclear, these proteolytic regulations on SIRPα during L. donovani infection may explain hemophagocytosis and anemia induced by infection, and serum soluble SIRPα may serve as a biomarker for hemophagocytosis and anemia in VL and the other inflammatory disorders.

In recent years, magnesium hydroxide has been widely studied due to its bioactivity and biocompatibility. The bactericidal effects of magnesium hydroxide nanoparticles on oral bacteria have also been reported. Therefore, in this study, we investigated the biological effects of magnesium hydroxide nanoparticles on inflammatory responses induced by periodontopathic bacteria. Macrophage-like cells, namely J774.1 cells, were treated with LPS derived from Aggregatibacter actinomycetemcomitans and two different sizes of magnesium hydroxide nanoparticles (NM80/NM300) to evaluate their effects on the inflammatory response. Statistical analysis was performed using an unresponsive Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test. NM80 and NM300 inhibited the expression and secretion of IL-1β induced by LPS. Furthermore, IL-1β inhibition by NM80 was dependent on the downregulation of PI3K/Akt-mediated NF-κB activation and the phosphorylation of MAPK molecules such as JNK, ERK1/2, and p38 MAPK. By contrast, only the deactivation of the ERK1/2-mediated signaling cascade is involved in IL-1β suppression by NM300. Although the molecular mechanism involved varied with size, these results suggest that magnesium hydroxide nanoparticles have an anti-inflammatory effect against the etiologic factors of periodontopathic bacteria. These properties of magnesium hydroxide nanoparticles can be applied to dental materials.

Background Immune cells play a major role in the pathogenesis of COPD. Changes in the distribution and cellular functions of major immune cells, such as alveolar macrophages (AMs) and neutrophils are well known; however, their transcriptional reprogramming and contribution to the pathophysiology of COPD are still not fully understood. Method To determine changes in transcriptional reprogramming and lipid metabolism in the major immune cell type within bronchoalveolar lavage fluid, we analysed whole transcriptomes and lipidomes of sorted CD45+Lin−HLA-DR+CD66b−Autofluorescencehi AMs from controls and COPD patients. Results We observed global transcriptional reprogramming featuring a spectrum of activation states, including pro- and anti-inflammatory signatures. We further detected significant changes between COPD patients and controls in genes involved in lipid metabolism, such as fatty acid biosynthesis in GOLD2 patients. Based on these findings, assessment of a total of 202 lipid species in sorted AMs revealed changes of cholesteryl esters, monoacylglycerols and phospholipids in a disease grade-dependent manner. Conclusions Transcriptome and lipidome profiling of COPD AMs revealed GOLD grade-dependent changes, such as in cholesterol metabolism and interferon-α and γ responses.

Exportin 6, which functions specifically in the nuclear export of actin family proteins, has been reported to be absent in immature Xenopus oocytes, which have a huge nucleus containing a large amount of actin. In mammalian oocytes, however, the presence and the function of exportin 6 remain uninvestigated. In this study, we assessed the expression and effects of exportin 6 on meiotic resumption in porcine oocytes after cloning porcine exportin 6 cDNA and carrying out overexpression and expression inhibition by mRNA and antisense RNA injection, respectively. We found for the first time that exportin 6 was expressed in mammalian full-grown germinal-vesicle-stage oocytes and was involved in the nuclear export of actin. In contrast, exportin 6 was absent from the growing oocytes, which are meiotically incompetent and maintain the germinal-vesicle structure in the long term; the regulatory mechanism appeared to be active degradation. We examined the effects of exportin 6 on meiotic resumption of porcine oocytes and noted that its expression did not affect the onset time but increased the rate of germinal vesicle breakdown at 24 h via regulation of the nuclear actin level, which directly influences the physical strength of the germinal-vesicle membrane. Our results suggest that exportin 6 affects the nuclear transport of actin and meiotic resumption in mammalian oocytes.

Myeloid-related protein 14 (MRP14) belongs to the S100 calcium-binding protein family and is expressed in neutrophils and inflammatory macrophages. Increase in the number of MRP14+ cells or serum level of MRP14 is associated with various diseases such as autoimmune diseases and infectious diseases, suggesting the involvement of the molecule in pathogenesis of those diseases. In this study, to examine the pathological involvement of MRP14 during cutaneous and visceral leishmaniasis, wild-type (WT) and MRP14 knockout (MRP14KO) mice were infected with Leishmania major and L. donovani. Increase in the number of MRP14+ cells at the infection sites in wild-type mice was commonly found in the skin during L. major infection as well as the spleen and liver during L. donovani infection. In contrast, the influence of MRP14 to the pathology seemed different between the two infections. MRP14 depletion exacerbated the lesion development and ulcer formation in L. major infection. On the other hand, the depletion improved anemia and splenomegaly but not hepatomegaly at 24 weeks of L. donovani infection. These results suggest that, distinct from its protective role in CL, MRP14 is involved in exacerbation of some symptoms during VL.

Extracellular vesicles such as exosomes contain several types of transcripts, including mRNAs and micro RNAs (miRNAs), and have emerged as important mediators of cell-to-cell communication. Exosome-like vesicles were identified in the ovarian follicles of several mammalian species. Although the miRNA contents have been extensively characterized, the detailed investigation of their mRNA profiles is lacking. Here, we characterize the mRNA profiles of exosome-like vesicles in ovarian follicles in a pig model. The mRNA contents of the exosome-like vesicles isolated from porcine follicular fluid were analyzed and compared with those from mural granulosa cells (MGCs) using the Illumina HiSeq platform. Bioinformatics studies suggested that the exosomal mRNAs are enriched in those encoding proteins involved in metabolic, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) -protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) pathways. While the mRNA profile of the exosome-like vesicles resembled that of MGCs, the vesicles contained mRNAs barely detectable in MGCs. Thus, while the majority of the vesicles are likely to be secreted from MGCs, some may originate from other cell types, including theca cells and oocytes, as well as the cells of non-ovarian organs/tissues. Therefore, the mRNA profiles unveiled several novel characteristics of the exosome-like vesicles in ovarian follicles.

Hepatic dysfunction is one of the clinical features in severe malaria. However, the mechanism of hepatic injury during malaria is still unknown. Myeloid-related protein (MRP) 14 is abundantly expressed by myeloid cells and involved in various inflammatory diseases. We previously reported that serum MRP14 is elevated in mice infected with Plasmodium berghei ANKA. In order to verify whether extracellular MRP14 is involved in the pathology of hepatic injury during rodent malaria, we intravenously administrated recombinant MRP14 (rMRP14) to mice infected with P. berghei ANKA. The administration of rMRP14 did not affect parasite number or hematocrit. On the other hand, the hepatic injury was exacerbated in rMRP14-treated mice, and their serum concentration of hepatic enzymes increased significantly more than PBS-treated controls. Immunohistochemical analysis of the liver showed that more MRP14+ macrophages accumulated in rMRP14-treated mice than PBS-treated controls after infection. The administration of rMRP14 also promotes the up-regulation of pro-inflammatory molecules in the liver, such as iNOS, IL-1β, IL-12, and TNF-α. Even in the absence of Plasmodium infection, administration of rMRP14 could induce the accumulation of MRP14+ macrophages and up-regulation of the pro-inflammatory molecules in the liver of naïve mice. The results indicate that MRP14 promotes the accumulation of MRP14+ cells and the up-regulation of pro-inflammatory molecules and NO, which amplify inflammatory cascade leading to hepatic injury. In conclusion, MRP14 is a one of key molecules for liver inflammation during rodent malaria.

Chronic obstructive pulmonary disease (COPD) is a diverse respiratory disease characterised by bronchiolitis, small airway obstruction, and emphysema. Innate immune cells play a pivotal role in the disease’s progression, and in particular, lung macrophages exploit their prevalence and strategic localisation to orchestrate immune responses. To date, alveolar and interstitial resident macrophages as well as blood monocytes have been described in the lungs of patients with COPD contributing to disease pathology by changes in their functional repertoire. In this review, we summarise recent evidence from human studies and work with animal models of COPD with regard to altered functions of each of these myeloid cell populations. We primarily focus on the dysregulated capacity of alveolar macrophages to secrete proinflammatory mediators and proteases, induce oxidative stress, engulf microbes and apoptotic cells, and express surface and intracellular markers in patients with COPD. In addition, we discuss the differences in the responses between alveolar macrophages and interstitial macrophages/monocytes in the disease and propose how the field should advance to better understand the implications of lung macrophage functions in COPD.

A 26-year-old man highly suspected of having antiglomerular basement membrane (GBM) disease was treated with corticosteroid pulse therapy 9 days after initial infection-like symptoms with high procalcitonin value. The patient required hemodialysis the next day of the treatment due to oliguria. In addition to corticosteroid therapy, plasmapheresis was introduced and the patient could discontinue hemodialysis 43 days after the treatment. Kidney biopsy after initiation of hemodialysis confirmed anti-GBM disease with 86.3% crescent formation. Physician should keep in mind that active anti-GBM disease shows even high procalcitonin value in the absence of infection. To pursue recovery of renal function, the challenge of the immediate and persistent treatment with high-dose corticosteroids plus plasmapheresis for highly suspected anti-GBM disease is vitally important despite the presence of reported predictors for dialysis-dependence including oliguria and requiring hemodialysis at presentation.

Non-specific lipid transfer protein (LTP) in barley grain reacted with the IgE in sera drawn from food allergy patients. A sandwich-type of enzyme-linked immunosorbent assay (ELISA) was developed with mouse monoclonal antibodies raised against LTP purified with barley flour. This ELISA showed a practical working range of 0.3–3 ng/mL and no cross-reactivity with wheat, adlay and rye. Using this ELISA, LTP was determined in several types of barley-foods, including fermented foods such as malt vinegar, barley-malt miso and beer. LTP content in beer of the same kind was approximately constant, even if manufacturing factory and production days were different. Not only as a factor of foam formation and stability but also as an allergen, controlling and monitoring of LTP in beer should be considered. Taken together, our LTP-detecting ELISA can be proposed as an appropriate system for the quality control of beer.

Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in many critical cellular processes, including proliferation, migration, and angiogenesis, through interaction with a family of five G protein-coupled receptors (S1P1-5). Some reports have implicated S1P as an important inflammatory mediator of the pathogenesis of airway inflammation, but the role of S1P3 in the pathogenesis of lung diseases is not completely understood. We used S1P3-deficient (knockout (KO)) mice to clarify the role of S1P3 receptor signaling in the pathogenesis of pulmonary inflammation and fibrosis using a bleomycin-induced model of lung injury. On the seventh day after bleomycin administration, S1P3 KO mice exhibited significantly less body weight loss and pulmonary inflammation than wild-type (WT) mice. On the 28th day, there was less pulmonary fibrosis in S1P3 KO mice than in WT mice. S1P3 KO mice demonstrated a 56% reduction in total cell count in bronchoalveolar lavage fluid (BALF) collected on the seventh day compared with WT mice; however, the differential white blood cell profiles were similar. BALF analysis on the seventh day showed that connective tissue growth factor (CTGF) levels were significantly decreased in S1P3 KO mice compared with WT mice, although no differences were observed in monocyte chemotactic protein-1 (MCP-1) or transforming growth factor β1 (TGF-β1) levels. Finally, S1P levels in BALF collected on the 7th day after treatment were not significantly different between WT and S1P3 KO mice. Our results indicate that S1P3 receptor signaling plays an important role in pulmonary inflammation and fibrosis and that this signaling occurs via CTGF expression. This suggests that this pathway might be a therapeutic target for pulmonary fibrosis.

Zinc finger nuclease (ZFN) is a useful tool for endogenous site-directed genome modification. The development of an easier, less expensive and repeatedly usable construction method for various sequences of ZFNs should contribute to the further widespread use of this technology. Here, we establish a novel construction method for ZFNs. Zinc finger (ZF) fragments were synthesized by PCR using short primers coding DNA recognition helices of the ZF domain. DNA-binding domains composed of 4 to 6 ZFs were synthesized by overlap extension PCR of these PCR products, and the DNA-binding domains were joined with a nuclease vector by TA cloning. The short primers coding unique DNA recognition helices can be used repeatedly for other ZFN constructions. By using this novel OLTA (OverLap extension PCR and TA-cloning) method, arbitrary ZFN vectors were synthesized within 3 days, from the designing to the sequencing of the vector. Four different ZFN sets synthesized by OLTA showed nuclease activities at endogenous target loci. Genetically modified mice were successfully generated using ZFN vectors constructed by OLTA. This method, which enables the construction of intended ZFNs repeatedly and inexpensively in a short period of time, should contribute to the advancement of ZFN technology.

Despite its high prevalence, the cellular and molecular mechanisms of chronic obstructive pulmonary disease (COPD) are far from being understood. Here, we determine disease-related changes in cellular and molecular compositions within the alveolar space and peripheral blood of a cohort of COPD patients and controls. Myeloid cells were the largest cellular compartment in the alveolar space with invading monocytes and proliferating macrophages elevated in COPD. Modeling cell-to-cell communication, signaling pathway usage, and transcription factor binding predicts TGF-b1 to be a major upstream regulator of transcriptional changes in alveolar macrophages of COPD patients. Functionally, macrophages in COPD showed reduced antigen presentation capacity, accumulation of cholesteryl ester, reduced cellular chemotaxis, and mitochondrial dysfunction, reminiscent of impaired immune activation. [ABSTRACT FROM AUTHOR]

Despite its high prevalence, the cellular and molecular mechanisms of chronic obstructive pulmonary disease (COPD) are far from being understood. Here, we determine disease-related changes in cellular and molecular compositions within the alveolar space and peripheral blood of a cohort of COPD patients and controls. Myeloid cells were the largest cellular compartment in the alveolar space with invading monocytes and proliferating macrophages elevated in COPD. Modeling cell-to-cell communication, signaling pathway usage, and transcription factor binding predicts TGF-β1 to be a major upstream regulator of transcriptional changes in alveolar macrophages of COPD patients. Functionally, macrophages in COPD showed reduced antigen presentation capacity, accumulation of cholesteryl ester, reduced cellular chemotaxis, and mitochondrial dysfunction, reminiscent of impaired immune activation. [ABSTRACT FROM AUTHOR]

Both oocytes and extracellular vesicles (EV) have emerged as critical regulators of mammalian follicular development; however, the possible interaction between the oocyte-derived paracrine factor (ODPF) and EV signals has never been examined. Therefore, to explore the possibility of an interaction between oocyte and EV signals, the effects of ODPFs on the biogenesis of EVs as well as the expression levels of transcripts related to EV biogenesis in mural granulosa cells (MGCs) were examined using mice. The results showed that, while oocyte coculture has some effects on the expression levels of transcripts related to EV biogenesis, the number of EV particles present in the conditioned medium were not significantly different between ODPFtreated and non-treated MGCs. Therefore, oocytes have no effects on the EV biogenesis by MGCs, at least with respect to the numbers of EV particles. [ABSTRACT FROM AUTHOR]

PKC-potentiated phosphorylation-dependent inhibitory protein of protein phosphatase 1 (CPI-17), an endogenous myosin phosphatase inhibitory protein, is considered a key molecule for Ca2+ sensitization of the contractile apparatus. Here, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 to generate CPI-17-deficient [knockout (KO)] and threonine 38 (T38)-phospho-resistant mice [threonine mutant into alanine (TA)], and then effects of CPI-17 on vascular contractility in vitro and mean blood pressure (MBP) in vivo were investigated. In isolated thoracic aorta, phorbol 12, 13-dibutyrate induced a sustained contraction of wild-type (WT) mice, whereas no contraction showed from TA or KO mice. A high concentration of KCl solution-induced contraction was not different between transgenic and WT mice. In contrast, phenylephrine (PE)-induced contractions in both mutant strains were significantly smaller than those of WT mice in association with a low level of myosin phosphorylation, suggesting that at least part of PE-induced contraction is regulated by phosphorylation of CPI-17 at T38. Finally, the physiologic role of CPI-17 in the regulation of blood pressure was investigated using radio telemetry. MBP was decreased significantly in both transgenic mice, even with a compensatory increase in heart rate. In summary, we generated KO and constitutively phospho-resistant mouse models of CPI-17 for the first time. p-CPI-17 at T38, possibly by PKC, could be important to maintain vascular contractility and blood pressure in vivo. [ABSTRACT FROM AUTHOR]