Hen egg only diets support healthy aging in adult mice.

Hen eggs (eggs) are a conventional food, known to contain the nutrients required for the growth of chicken embryos. These eggs are rich in important proteins and fats, with a very low amount of carbohydrate, and include all of the vitamins and minerals needed for the development of mice. We found that mice fed eggs grew to the same weight as mice fed a normal chow diet (ND) and remained healthy until the 20-months. As expected, the serological indicators of fat content were higher in egg-only mice than in ND mice. However, surprisingly the serum glucose levels in the egg-only mice were nearly identical to those in the ND mice. Given the high fat content in eggs, we expected that our egg-only mice would develop fatty liver or other metabolic diseases. However, we observed no pathological changes in the livers of egg-only mice until 20-months with their serological indicators (ALT and AST) and histological features (no fat droplets) remaining normal. However, when we examined the pups nursed by mothers of the egg-only diet group we noted that almost the animals died 2 to 4 weeks after birth. This is likely because these pups presented with reduced enzymes for metabolism in their liver when compared to pups of the ND group. In addition, we also found that the expression of various development proteins were severely lacking in liver of these pups. From these results, our report suggested that eggs could support healthy aging in adult mice, but not in pups.

Functional validation of ATF4 and GADD34 in Neuro2a cells by CRISPR/Cas9-mediated genome editing.

Activating transcription factor 4 (ATF4), which is ubiquitously expressed, plays a crucial role in regulating various stress-responsive genes under pathophysiological conditions. Further, growth arrest and DNA damage-inducible gene 34 (GADD34), a downstream target of ATF4, has been reported to negatively regulate ATF4 expression. To understand the relationship between intrinsic ATF4 and GADD34 under resting and ER stress conditions, we used a novel gene editing approach, CRISPR/Cas9, to integrate antibiotic-resistant genes into the target genes, ATF4 and GADD34. First, we manipulated the ATF4 gene in the mouse neuroblastoma cell line, Neuro2a, and compared the ER stress responses between parental and ATF4-edited Neuro2a cells. Next, we established Neuro2a cells with edited GADD34 and ATF4/GADD34 genes and found that ATF4 acts as a proapoptotic factor, but GADD34 depletion did not attenuate the expression of cleaved caspase-3 induced by tunicamycin treatment. These findings provide new insights into the ATF4 signaling cascades. Additionally, the rapid establishment of cells lacking multiple genes using this CRISPR/Cas9 system will be a powerful tool for exploring various cellular issues under pathophysiological conditions.

Transfusion of CD206+ M2 Macrophages Ameliorates Antibody-Mediated Glomerulonephritis in Mice.

Macrophages are multifunctional immune cells that may either drive or modulate disease pathogenesis, depending on the activated phenotype. In this study, we investigated the protective effects of CD206+ M2 macrophages against nephrotoxic serum nephritis in mice. We found that these immunosuppressive macrophages, derived from bone marrow and stimulated with IL-4/IL-13 [CD206+ M2 bone marrow-derived macrophages (M2BMMs)], protected against renal injury, decreased proteinuria, and diminished the infiltration of CD68+ macrophages, neutrophils, and T cells into glomerular tissue. Comparable therapeutic results were obtained with CD206+ M2 cells derived from induced pluripotent stem cells. Notably, CD206+ M2BMMs, which retained an M2 signature, could elicit a switch of M1 to M2 phenotype in co-cultured macrophages. Moreover, these cells were found to induce the production of regulatory T cells in the spleen and renal draining lymph node. Accordingly, mRNA expression of the T helper 1 cytokines tumor necrosis factor-α, interferon-ß, interferon-γ, and IL-12 was significantly reduced in kidneys from mice treated with CD206+ M2BMMs. Taken together, the data suggest that CD206+ M2 may have therapeutic potential against antibody-mediated glomerular injury and presents its therapeutic value for the treatment of crescentic nephritis in humans.

Clinical outcomes of patients 80 years of age and older with soft tissue sarcoma.

OBJECTIVES: Although soft tissue sarcoma (STS) is rare, its incidence is increasing among older patients. Few studies have compared the outcomes between conservative and surgical treatments for STS patients aged ≥80 years. We assessed the outcomes of both treatments in this population and the association between older age and surgical outcome. METHODS: We recruited consecutive patients with STS aged ≥80 years treated at our institution between January 2006 and May 2014. We recommended surgical resection for all patients without multiple distant metastases. Overall survival and sarcoma-specific survival were assessed using the Kaplan-Meier method. RESULTS: Of the 39 patients with STS who presented at our institution, 37 were included in this analysis (19 men and 18 women with a median age of 85 [range 80-94] years). Tumors were classified as Stage IB (n = 3), IIA (n = 6), IIB (n = 3) or III (n = 24). Four patients underwent conservative therapy and 33 underwent surgical resection. The most common tumor site was the lower extremity, and the majority of tumors were classified as undifferentiated pleomorphic sarcoma. The follow-up rate was 100%. One-year sarcoma-specific survival rates were 25.0% in the conservative therapy group and 90.9% in the surgical resection group. No associations were found between age ≥85 years and perioperative complications or clinical outcome. CONCLUSIONS: Surgical resection had relatively few complications, given the age group, and improved the prognosis of older patients with STS. Surgical resection of STS with curative intent should be considered in older patients.

Predominant role of msr(D) over mef(A) in macrolide resistance in Streptococcus pyogenes.

In Japan, the number of patients with streptococcal toxic shock syndrome is reported to be increasing. mef(A) gene-positive macrolide-resistant emm1 strains are thought to possibly contribute to the rise in the frequency of STSS. Although analyses of macrolide-resistant mechanisms, including mef(A) resistance, have been performed mainly in Streptococcus pneumoniae, the role of this gene in Streptococcus pyogenes has not been completely investigated. Therefore, to the best of our knowledge, we established the first mef(A)-knockout strain using an emm1-type S. pyogenes strain, and tested its susceptibility to erythromycin, clarithromycin and azithromycin. We found that the antimicrobial susceptibilities were almost identical to those of the parental strain. Hence, we established a knockout strain for another gene, msr(D), that is located immediately downstream of mef(A). The macrolide resistances of the resulting strain significantly decreased, and were further altered when both mef(A) and msr(D) were knocked out. The introduction of the msr(D) gene into a macrolide-sensitive strain conferred more resistance than the introduction of the mef(A) gene. The erythromycin susceptibilities of knockout strains were further dissected using two additional emm4- and emm75-type S. pyogenes strains. We found almost identical results for both strains except for the mef(A) knockout emm4 type, whose susceptibility was altered, although the change was less than that for the msr(D) knockout. These results suggest that both mef(A) and msr(D) are involved in macrolide resistance in S. pyogenes, and that the msr(D) gene plays a more predominant role in macrolide resistance than mef(A).

Clinical outcome of dedifferentiated liposarcoma in the extremities: A retrospective case series of 7 patients.

BACKGROUND: Wide resection is the generally recommended surgical treatment for dedifferentiated liposarcoma (DDLPS) in the extremities. However, it may be appropriate to distinguish the surgical margin of low-grade atypical lipomatous tumor (ALT)/well-differentiated liposarcoma (WDLPS) area from the high-grade dedifferentiated area, because the low- and high-grade areas can be clearly separated, both radiologically and histologically. This study re-evaluated the details of surgical margin of DDLPS in the extremities, and aimed to investigate the optimal surgical margin and the usefulness of adjuvant therapy for DDLPS in the extremities. METHODS: Seven patients diagnosed with DDLPS in the extremities and treated between 1995 and 2013 were analyzed. The use of adjuvant therapy before and after surgery was assessed, and the surgical margins for the ALT/WDLPS and dedifferentiated areas were re-evaluated by using the specimens resected at surgery. Subsequently, the recurrence rates, metastatic rates, and oncological outcomes were examined. RESULTS: Four and three patients had wide (adequate wide margin, n = 3; inadequate wide margin, n = 1) and marginal margins for the dedifferentiated area, respectively, while three and four patients had wide (adequate wide margin, n = 2; inadequate wide margin, n = 1) and marginal margins for the ALT/WDLPS area, respectively. Postoperative radiotherapy was performed in three patients with an inadequate wide margin or a marginal margin for the dedifferentiated area. No patient had local recurrence. Distant metastases occurred in two patients. These patients died of their disease. The other five patients were disease-free. CONCLUSION: The ALT/WDLPS and dedifferentiated areas in the tumor margin may be better to be considered separately in determining the appropriate resection extent for DDLPS in the extremities. Postoperative radiotherapy may provide good local control for cases with a narrow surgical margin.

Mechanism of immune aging.

Age-related decline of immune system not only increases susceptibility of infection, but also delays of wound healing. Although the number of innate immune cells such as neutro- phils and macrophages does not decline by aging, the functions of these cells such as phagocy- tosis, migration toward infection sites decline by aging. Production of inflammatory cytokines by microglia, one of tissue macrophages increases by aging, which causes Alzheimer disease. Human thymic epithelium, which harbor lymphocytes in thymus for development, show a continuous involution from the first year, which replaced by adipose tissue. Age-related involu- tion of thymic epithelium results in the decrease of nalve T cells. Chronic cytomegalovirus infection induces clonally-expanded CD8 T cells and perturbations in the peripheral T cell repertoire.

Effects of growth arrest and DNA damage-inducible protein 34 (GADD34) on inflammation-induced colon cancer in mice.

BACKGROUND: Growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a) is a family of GADD proteins that are induced by DNA damage. GADD34 protein has been suggested to regulate inflammation or host defense systems. However, the in vivo function of GADD34 in inflammation is still unclear. Long lasting inflammation, such as that seen in Crohn's disease and ulcerative colitis, is associated with a higher incidence of colorectal cancer (CRC). METHODS: Using a colitis-associated cancer model, we analysed GADD34-deficient (KO) mice to study the effect of GADD34 on colitis and colorectal tumorigenesis. RESULTS: We found a higher incidence of CRC in wild-type (WT) mice than in GADD34KO mice. Moreover, dextran sodium sulfate (DSS)-induced inflammatory responses were downregulated by GADD34 deficiency. The expression of pro-inflammatory mediators such as TNFα, IL-6, and iNOS/NOS2 was higher in the colons of WT mice than GADD34KO mice. IL-6 is known to activate STAT3 signalling in colonic epithelial cells and subsequently induced epithelial proliferation. We found that IL-6-STAT3 signalling and epithelial proliferation were higher in WT mice compared with GADD34KO mice. CONCLUSIONS: These results indicated that GADD34 upregulated pro-inflammatory mediator production leading to a higher tumour burden following azoxymethane (AOM)/DSS treatment.

Growth arrest and DNA damage-inducible protein (GADD34) enhanced liver inflammation and tumorigenesis in a diethylnitrosamine (DEN)-treated murine model.

Growth arrest and DNA damage-inducible protein (GADD34/Ppp1r15a) is induced by various stimuli including DNA damage and ER stress. DNA damage and oncogene activation, accompanied by tumor-specific DNA repair defects and a failure to stall the cell cycle, are early markers of hepatocellular carcinoma (HCC). However, whether GADD34 accounts for regulating HCC tumorigenesis remains elusive. Here, we demonstrated that GADD34 expression was upregulated in the liver of mice after exposure to a carcinogen, diethylnitrosamine (DEN). In both acute and chronic DEN treatment models, GADD34 deficiency not only decreased oncogene expression, but also reduced hepatic damage. Moreover, loss of GADD34 attenuated immune cell infiltration, pro-inflammatory cytokine expression and hepatic compensatory proliferation. Finally, GADD34-deficient mice showed impaired hepatocarcinogenesis. Thus, the process of DEN-induced HCC proceeded as follows. First, DEN treatment induced DNA damage in hepatocytes, resulting in elevated expression of GADD34 in the liver. The increased expression of GADD34 augmented hepatic necrosis followed by elevated expression of interleukin (IL)-1ß and monocyte chemoattractant protein 1. This process promoted immune cell infiltration and Kupffer cell/macrophage activation followed by production of reactive oxygen species and pro-tumorigenic cytokines such as IL-6 and tumor necrosis factor-α. The pro-tumorigenic cytokines stimulated compensatory proliferation of surviving and mutant hepatocytes. Together with oncogene c-Myc expression, these processes led to HCC. Our results suggest therapeutic opportunities for HCC by targeting GADD34-related pathways.

Loss of GADD34 induces early age-dependent deviation to the myeloid lineage.

Hematopoietic stem cells (HSCs) generate all known hematopoietic lineages and are capable of self-renewal. Upon aging, myeloid-biased HSCs are maintained, whereas lymphoid-biased HSCs are lost. GADD34 protein is expressed in myeloid-lineage cells and has been cloned from them. However, the function of GADD34 in the myeloid lineage has not yet been elucidated. Here, we show that early age-dependent deviation to the myeloid lineage occurs in GADD34-deficient mice. Early increases of GR-1(int)CD11b(+) and GR-1(high)CD11b(+) neutrophils were observed in the spleen, bone marrow (BM) and blood of GADD34-deficient mice. We found that BM Lin(-) c-Kit(+) Sca1(+) and Lin(-) c-Kit(+) Sca1(-)cells expressed GADD34 protein without stimulation and increased GADD34 expression following intravenous injection of Staphylococcus aureus (S.aureus). These cell populations were high in GADD34-deficient BM and were increased by the injection of S. aureus. Because of the increase in granulocyte colony-stimulating factor (G-CSF) induced by S. aureus injection, we examined the signaling pathway from the G-CSF receptor (G-CSFR). We found that phosphorylation of signal transducer and activator of transcription factor 3 was highly increased in GADD34-deficient Lin(-) BM cells by the stimulation of G-CSF. These results indicate that GADD34 binds to Lyn and inhibit G-CSFR signaling. We show here that GADD34 works to inhibit the proliferation and differentiation of HSCs or myeloid precursor cells and maintains homeostatic differentiation of neutrophil-lineage cells to avoid early immunological senescence.

[Developmental change in facial recognition by premature infants during infancy].

OBJECTIVE: Premature infants are thought to be at increased risk for developmental disorders. We evaluated facial recognition by premature infants during early infancy, as this ability has been reported to be impaired commonly in developmentally disabled children. METHODS: In premature infants and full-term infants at the age of 4 months (4 corrected months for premature infants), visual behaviors while performing facial recognition tasks were determined and analyzed using an eye-tracking system (Tobii T60 manufactured by Tobii Technologics, Sweden). RESULTS: Both types of infants had a preference towards normal facial expressions; however, no preference towards the upper face was observed in premature infants. CONCLUSIONS: Our study suggests that facial recognition ability in premature infants may develop differently from that in full-term infants.

The effect of blood transfusion on cerebral hemodynamics in preterm infants.

BACKGROUND: Anemia of prematurity commonly occurs in infants with very low birth weight; blood transfusion is an important treatment. However, there is no clear evidence to support the criteria currently widely used, based on blood hemoglobin (bHb) and hematocrit indices. Previous studies showed that overtransfusion or a low threshold for transfusion could induce complications or neurologic sequelae, respectively. We hypothesized that a cerebral hemodynamic index may provide an appropriate criterion for determining the need for transfusion in anemic preterm infants. STUDY DESIGN AND METHODS: We used near-infrared time-resolved spectroscopy to measure cerebral hemoglobin oxygen saturation (ScO2 ) and cerebral blood volume (CBV) before and after transfusion in 19 infants (24 measurements) with anemia of prematurity. The median gestational age was 27 weeks 0 days, median birth weight was 751 g, and median postconceptual age at transfusion was 30 weeks 4 days. RESULTS: bHb levels before and after transfusion (mean ± SD) were 9.3 ± 1.4 and 13.7 ± 1.3 g/dL, respectively. After transfusion, CBV significantly decreased from 2.63 ± 0.60 to 2.13 ± 0.26 mL/100 g of brain, and ScO2 significantly increased from 72.8 ± 4.3% to 74.7 ± 4.2%. CONCLUSION: After transfusion, CBV changes were significantly greater with low compared to high pretransfusion Hb levels. This reflected the physiologic response to severe anemia in premature infants, which is to increase CBV and decrease ScO2 . Therefore, CBV and ScO2 may be useful markers for determining the need for transfusion in very-low-birth-weight infants.

CD8+CD122+ regulatory T cells (Tregs) and CD4+ Tregs cooperatively prevent and cure CD4+ cell-induced colitis.

We identified CD8(+)CD122(+) regulatory T cells (Tregs) and demonstrated their importance in the maintenance of immune homeostasis and in the recovery from experimental autoimmune encephalomyelitis. In this paper, we show that CD8(+)CD122(+) Tregs effectively prevent and cure colitis in a mouse model. In our experiments, colitis was induced in lymphocyte-deficient RAG-2(-/-) mice by transferring CD4(+)CD45RB(high) cells that were excluded with CD4(+) Tregs. Cotransfer of CD8(+)CD122(+) cells clearly suppressed the development of colitis, and this suppressive effect was similar to that of CD4(+)CD45RB(low) cells that were mostly CD4(+) Tregs. CD8(+)CD122(+) cells obtained from IL-10(-/-) mice were unable to suppress colitis, indicating that IL-10 is an important effect-transmitting factor in the suppression of colitis. CD8(+)CD122(+) cells showed a suppressive effect when they were transferred 4 wk after CD4(+)CD45RB(high) cells, indicating the therapeutic potential of CD8(+)CD122(+) cells. A mixture of CD8(+)CD122(+) cells and CD4(+)CD45RB(low) cells was far more effective than single Tregs, indicating the synergistic effect of these Tregs. These overall findings demonstrate the potential role of CD8(+) Tregs, and possibly together with CD4(+) Tregs, in the medical care of inflammatory bowel disease patients.

Hour-specific nomogram for transcutaneous bilirubin in Japanese neonates.

BACKGROUND: The measurement of transcutaneous bilirubin (TcB) is very important to screen for hyperbilirubinemia in newborns. Until now, however, there has been no hour-specific, percentile-based TcB nomogram during the early neonatal period in Japanese neonates. The aim of this study was to develop a TcB nomogram within 72 h of birth in Japanese neonates. METHODS: A total of 3152 TcB measurements for 181 healthy Japanese neonates (gestational age ≥36 weeks, birthweight ≥2300 g) were obtained within 72 h of birth. All measurements were performed with a Konica Minolta jaundice meter, the JM-103. A nomogram curve was plotted to show the trend of TcB level over time. RESULTS: The nomogram curves rose almost linearly for all percentiles until 72 h after birth. CONCLUSION: An hour-specific, percentile-based TcB nomogram during the first 72 h after birth in Japanese neonates was successfully developed. Because Japanese neonates have higher and later peak bilirubin, an original hour-specific 97.5th percentile-based TcB nomogram may be needed to identify early-onset jaundice and manage neonatal hyperbilirubinemia.

Heterozygosity with respect to Zfp148 causes complete loss of fetal germ cells during mouse embryogenesis.

Zfp148 belongs to a large family of C2H2-type zinc-finger transcription factors. Zfp148 is expressed in fetal germ cells in 13.5-d-old (E13.5) mouse embryos. Germ-line transmission of mutations were not observed in chimeric Zfp148(+/-) mice, and some of these mice completely lacked spermatogonia. The number of primordial germ cells in Zfp148(+/-) tetraploid embryos was normal until E11.5, but declined from E11.5 to E13.5 and continued to decline until few germ cells were present at E18.5. This phenotype was not rescued by wild-type Sertoli or stromal cells, and is therefore a cell-autonomous phenotype. These results indicate that two functional alleles of Zfp148 are required for the normal development of fetal germ cells. Recent studies have shown that Zfp148 activates p53, which has an important role in cell-cycle regulation. Primordial germ cells stop proliferating at approximately E13.5, which correlates with induction of phosphorylation of p53 and its translocation to the nucleus. Phosphorylation of p53 is impaired in Zfp148(+/-) embryonic stem cells and in fetal germ cells from chimeric Zfp148(+/-) embryos. Thus, Zfp148 may be required for regulating p53 in the development of germ cells.

Preoperative radiographic and histopathologic evaluation of central chondrosarcoma.

BACKGROUND: Distinguishing grade 1 chondrosarcoma from grade 2 chondrosarcoma is critical both for planning the surgical procedure and for predicting the outcome. We aimed to review the preoperative radiographic and histologic findings, and to evaluate the reliability of preoperative grading. METHODS: We retrospectively reviewed the medical records of 17 patients diagnosed with central chondrosarcoma at our institution between 1996 and 2011. In these cases, we compared the preoperative and postoperative histologic grades, and evaluated the reliability of the preoperative histologic grading. We also assessed the preoperative radiographic findings obtained using plain radiography, computed tomography (CT), and magnetic resonance imaging (MRI). RESULTS: Preoperative histologic grade was 1 in 12 patients, 2 in 4 patients, and 3 in 1 patient. However, 6 of the 12 cases classified as grade 1 before surgery were re-classified as grade 2 postoperatively. In the radiographic evaluation, grade 1 was suspected by the presence of a ring-and-arc pattern of calcification on plain radiography and CT and entrapped fat and ring-and-arc enhancement on MRI. Grades 2 and 3 were suspected by the absence of calcification and the presence of cortical penetration and endosteal scalloping on plain radiography and CT, as well as soft-tissue mass formation on MRI. CONCLUSION: Although the combination of radiographic interpretation and histologic findings may improve the accuracy of preoperative grading in chondrosarcoma, the establishment of a standard evaluation system with the histologic and radiographic findings and/or the development of new biologic markers are necessary for preoperative discrimination of low-grade chondrosarcoma from high-grade chondrosarcoma.

Gadd34 induces autophagy through the suppression of the mTOR pathway during starvation.

Several types of cellular stress induce expression of growth arrest and DNA damage protein 34 (Gadd34). Autophagy occurs under both basal conditions and conditions of stress, such as starvation. Gadd34 and autophagy are both induced under starvation conditions. In this study we found that starvation induced the expression of Gadd34, reduced mTOR activity, and induced autophagy in wild type mice, but not Gadd34 KO mice. Gadd34 bound to and dephosphorylated pTSC2 at Thr1462. Dephosphorylation of TSC2 during the starvation time period leads to the suppression of mTOR, which is a potent inhibitor of autophagy. We concluded that starvation-induced Gadd34 suppresses mTOR and, thereby, induces autophagy.

Differentiation of induced pluripotent stem cells to thymic epithelial cells by phenotype.

Thymic epithelial cells (TECs) are present in both cortical and medullary thymic areas, and have crucial roles in functional T-cell development. In this study, we studied the differentiation of induced pluripotent stem cells (iPSCs) to TEC. When iPSC were cultured for 4 days in collagen IV-coated dishes in the presence of both activin A and lithium chloride (LiCl), the cells differentiated to definitive endoderm through mesendoderm. Further treatment with Fgf8 followed by Fgf7, Fgf10 and BMP4 differentiated iPSC to thymic epithelial progenitor cells (TEPCs) by phenotype. Gene expression of Hoxa3, Pax1 and Pax9 was observed and cell surface proteins EpCAM1 and MTS24 were detected at day 14 of iPSC differentiation. TEPCs differentiated to medullary TECs (mTECs) by phenotype following the addition of receptor activator nuclear factor B ligand with LiCl. Thus, we successfully induced efficient differentiation from mouse iPSC to TEPCs and mTEC by phenotype using chemically defined conditions.

Long-lasting effects of inescapable-predator stress on brain tryptophan metabolism and the behavior of juvenile mice.

The kynurenine (KYN) pathway, which is initiated by indoleamine 2,3-dioxygenase, is the main tryptophan (TRP) metabolic pathway. It shares TRP with the serotonin (5-HT) pathway. We investigated the influence of inescapable-predator (rat) stress on behavior and brain TRP metabolism in mice. Male ICR mice (4W) were exposed to 20-min inescapable-predator stress. Behavior on an elevated plus-maze, and TRP, KYN, and 5-HT levels in the prefrontal cortex, hippocampus, amygdala, and dorsal raphe nuclei were measured 1 and 4 weeks after stress exposure. Predator stress increased the number of open-arm entries (NOA) 4 weeks after stress exposure without altering the number of closed-arm entries (NCA). Thus, the open/closed-arm entry ratio (NOA/NCA) increased after stress exposure. Predator stress increased KYN levels in the prefrontal cortex (until 4 weeks after stress exposure) and dorsal raphe nuclei (for 1 week after stress exposure), decreased 5-HT levels in all brain regions (until 4 weeks after stress exposure). Thus, predator stress increased the KYN/5-HT ratio in all regions, in particular in the prefrontal cortex and hippocampus until 4 weeks after stress exposure. Predator stress shifted the balance between the KYN and 5-HT pathways to the KYN pathway, and induced behavioral disinhibition.