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.

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.

Immunological aspects of age-related diseases.

The proportion of elderly people rises in the developed countries. The increased susceptibility of the elderly to infectious diseases is caused by immune dysfunction, especially T cell functional decline. Age-related hematopoietic stem cells deviate from lymphoid lineage to myeloid lineage. Thymus shrinks early in life, which is followed by the decline of naïve T cells. T-cell receptor repertoire diversity declines by aging, which is caused by cytomegalovirus-driven T cell clonal expansion. Functional decline of B cell induces antibody affinity declines by aging. Many effector functions including phagocytosis of myeloid cells are down regulated by aging. The studies of aging of myeloid cells have some controversial results. Although M1 macrophages have been shown to be replaced by anti-inflammatory (M2) macrophages by advanced age, many human studies showed that pro-inflammatory cytokines are elevated in older human. To solve this discrepancy here we divide age-related pathological changes into two categories. One is an aging of immune cell itself. Second is involvement of immune cells to age-related pathological changes. Cellular senescence and damaged cells in aged tissue recruit pro-inflammatory M1 macrophages, which produce pro-inflammatory cytokines and proceed to age-related diseases. Underlying biochemical and metabolic studies will open nutritional treatment.

Female GADD34 mice develop age-related inflammation and hepatocellular carcinoma.

AIM: To analyze the impact of sex on GADD34 function, we studied the aging of female GADD34-deficient mice and compared them with male GADD34-deficient mice. METHODS: We used GADD34-deficient mice on a C57BL/6 background. These mice were fed a normal diet throughout their life. Alternatively, they were fed a high-fat diet at 3 months-of-age. Liver tissues taken from mice were analyzed by hematoxylin-eosin staining and immunohistochemical methods. Fresh liver cells were analyzed by flow cytometry. RESULTS: We found that female GADD34-deficient mice did not develop obesity or fatty livers. However, female GADD34-deficient mice had infiltrations of myeloid cells in the liver, followed by liver atrophy. Many female GADD34-deficient mice developed hepatocellular carcinoma, whereas female wild-type (WT) mice did not show hepatocellular carcinoma during aging. Female GADD34-deficient mice and female WT mice developed the same percentages of lymphoma. Although a high-fat diet induced a higher level of steatosis in young male GADD34-deficient mice compared with WT mice, a high-fat diet induced the same level of steatosis in young female GADD34-deficient mice compared with WT mice. However, GADD34-deficient female young mice had a higher level of infiltration of myeloid cells and myofibroblasts than WT mice. CONCLUSIONS: In contrast to male GADD34-deficient mice, female GADD34-deficient mice did not show obesity as they aged. However, similar to the males, they developed inflammation followed by hepatocellular carcinoma. Geriatr Gerontol Int 2017; 17: 2593-2601.

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.

GADD34 Promotes Tumor Growth by Inducing Myeloid-derived Suppressor Cells.

BACKGROUND: Tumor hypoxia induces the expression of growth arrest and DNA damage-inducible protein (GADD34). However, the role of GADD34 in tumor growth remains unclear. MATERIALS AND METHODS: Gadd34 expression was knocked-down through lentivirus-mediated short hairpin RNA (shRNA) in tumor cells, which were subsequently injected subcutaneously into mice. Tumor volumes and myeloid-derived suppressor cells (MDSCs) were monitored. Isolated MDSCs were incubated with tumor supernatant to investigate the impact of GADD34 on cytokine secretion of MDSCs. RESULTS: We observed that reduction of GADD34 expression significantly suppressed tumor, and resulted in decreased accumulation of MDSCs and T-cells, and inhibition of GADD34 reduced secretion of vascular epithelial growth factor α and transforming growth factor ß by MDSCs. CONCLUSION: These findings provide a promising strategy for targeting GADD34 activity in order to inhibit tumor growth.

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.

Vancomycin-sensitive bacteria trigger development of colitis-associated colon cancer by attracting neutrophils.

Inflammatory bowel disease confers an increased risk of developing colitis-associated colon cancer (CAC). During the active colitis or developing tumor stage, commensal bacteria show dynamic translocation. However, whether alteration of the bacterial composition in the gut causes CAC is still unclear. To clarify the effect of commensal bacteria on CAC development, we employed an azoxymethane (AOM) and dextran sodium sulfate (DSS)-induced murine CAC model treated with or without antibiotics. In addition, we analyzed the effects of antibiotics on infiltration of myeloid cells, colonic inflammatory responses, and colorectal cancer formation. We found that vancomycin treatment dramatically suppressed tumor development. In addition, AOM/DSS treatment greatly induced the infiltration of Gr-1(high)/CD11b(high) neutrophils to the colon, which led to the production of tumor necrosis factor α and inducible nitric oxide synthase. Vancomycin treatment suppressed the infiltration of neutrophils induced by AOM/DSS. Moreover, vancomycin treatment greatly reduced the colon injury and DNA damage caused by AOM/DSS-induced NO radicals. Our results indicate that vancomycin-sensitive bacteria induced colon inflammation and DNA damage by attracting neutrophils into damaged colon tissue, thus promoting tumor formation.

Chondroitin sulfate synthase 1 expression is associated with malignant potential of soft tissue sarcomas with myxoid substance.

The glycosyltransferases chondroitin sulfate synthase 1 (CHSY1) and exostoses-like 3 (EXTL3) specifically function in biosynthesis of the glycans chondroitin sulfate and heparan sulfate, respectively. Although these glycans play important roles in pathogenesis of various tumors, their significance in soft tissue sarcoma remains unknown. Here, we asked whether CHSY1 or EXTL3 expression correlates with malignant potential of soft tissue sarcomas with myxoid substance. To do so, we examined 40 samples representing specific types, including 12 cases of myxoid liposarcoma, 14 of myxofibrosarcoma, 12 of malignant peripheral nerve sheath tumor, and 2 of low-grade fibromyxoid sarcoma. We performed immunohistochemistry with anti-CHSY1 and anti-EXTL3 antibodies and compared enzyme expression levels with tumor histologic grade as assessed by the Fédération Nationale des Centres de Lutte Contre le Cancer classification and with patient 5-year survival rate. CHSY1 and EXTL3 were expressed in 72.5% and 32.5% of all tumors, respectively. Notably, CHSY1 was strongly expressed in myxofibrosarcoma and malignant peripheral nerve sheath tumor compared with other tumors and significantly associated with higher- rather than lower-grade tumors (P < .01). High expression of CHSY1 was also significantly associated with poorer patient outcomes (P = .031) and higher stages assessed by American Joint Committee on Cancer staging system (P = .004). By contrast, EXTL3 expression was not correlated with histologic grade or patient prognosis. We conclude that CHSY1 expression is closely associated with malignant potential of soft tissue sarcomas with myxoid substance.

A recurrent solitary fibrous tumor of the thigh with malignant transformation: A case report.

INTRODUCTION: We describe an unusual case of a uniformly high-grade malignant solitary fibrous tumor (SFT) of the thigh with recurrence after wide resection in a 31-year-old man. PRESENTATION OF CASE: Our current case showed a long-term benign course before the operation, although the subcutaneous tumor was larger than 10cm at presentation. The SFT was diagnosed by needle biopsy, and wide resection was performed. Histological findings showed proliferation of capillaries surrounded by masses of spindle-shaped cells without any cytologic atypia, and the percentage of MIB-1-positive nuclei was 2.1%. However, a rapidly enlarging recurrent tumor was observed 11 months after the operation. A second wide resection for the recurrent tumor was performed. Histologically, the tumor cells uniformly displayed significant cytologic atypia and pleomorphism, and had 40-50 mitoses per 10 high-power fields. The proportion of MIB-1-positive nuclei was 48%. Consequently, the tumor was diagnosed as a SFT with malignant transformation. DISCUSSION: The malignant transformation described in past studies showed high-grade areas within benign, low-grade, or intermediate-grade SFTs. Therefore, in contrast to our case, uniformly high-grade malignant histological findings at recurrence were not described. CONCLUSION: Even if a tumor is non-malignant during the clinical course, as confirmed by tissue biopsy, the possibility of tumor progression to high-grade sarcoma at recurrence should be considered, and the treatment strategy should be determined carefully.

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).

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.

GADD34 Facilitates Cell Death Resulting from Proteasome Inhibition.

BACKGROUND: Proteasome inhibition has been proven to be a promising therapeutic strategy in cancer clinical treatment. Inhibition of proteasome leads to failure of amino acid homeostasis, which causes cell death via activation of various mechanisms. MATERIALS AND METHODS: To investigate the role of GADD34 in cell death following proteasome inhibition, we treated WT MEF and GADD34 KO MEF with MG132 and various analyses were performed, including PI staining, western blot, immunofluorescence and ROS production. RESULTS: Expression of GADD34 dramatically enhanced MG132-induced cell death via protein synthesis. GADD34 decreased phosphorylated eIF2α and increased ROS production and the levels of ubiquinated protein. Importantly, we found that accumulation of autophagy following MG132-treatment facilitated cell death in MEF. CONCLUSION: GADD34 plays a vital role in promoting cell death following proteasome inhibition via enhancing protein synthesis to activate death-associated mechanisms, including ER stress, ROS production and autophagy formation.

Thymic Epithelial Cells Induced from Pluripotent Stem Cells by a Three-Dimensional Spheroid Culture System Regenerates Functional T Cells in Nude Mice.

The thymus is mainly composed of two types of epithelial cells, medullary thymic epithelial cells and cortex thymic epithelial cells (mTECs and cTECs). The tissue structure and mechanism for T cell development are complicated, with generation of the thymus regulated by complex molecular and cellular interactions of the thymic microenvironment during embryogenesis. Since the development of organ regeneration techniques became available, complete in vitro regeneration of the thymus has been attempted. Steric induction systems are thought to be optimal for tissue regeneration, but three-dimensional (3-D) induction of TECs from induced pluripotent stem cells (iPSCs) has not yet been reported. Here, we demonstrate the induction of functional TECs from iPSCs by a 3-D spheroid culture system with recruitment of robust numbers of T cells into the peripheral blood. Purified iPSC-derived TECs showed a sufficient expression level of FoxN1 comparable to TECs, and phenotypic analysis revealed that iPSC-derived TECs were expressing K5. Moreover, transplants of cell aggregations into recipient mice were not rejected and there was normal support of T cell development. Functional analysis revealed that T cells showed immune tolerance to both donor and recipient MHCs and could reject an allogeneic third party's skin graft without tumorigenesis. Taken together, these findings raised the possibility of using iPSC-derived TECs induced by 3-D spheroid culture in future regenerative therapy for patients with immunodeficiency.

GADD34-deficient mice develop obesity, nonalcoholic fatty liver disease, hepatic carcinoma and insulin resistance.

The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing in parallel with the prevalence of obesity. DNA damage-inducible protein 34 (GADD34/Ppp1r15a), originally isolated from UV-inducible transcripts in Chinese hamster ovary (CHO) cells, dephosphorylates several kinases that function in important signaling cascades, including dephosphorylation of eIF2α. We examined the effects of GADD34 on natural life span by using GADD34-deficient mice. Here we observed for the first time that with age GADD34-deficient mice become obese, developing fatty liver followed by liver cirrhosis, hepatocellular carcinoma, and insulin resistance. We found that myofibroblasts and immune cells infiltrated the portal veins of aged GADD34-deficient mouse livers. A high-fat diet (HFD) induced a higher level of steatosis in young GADD34-deficient mice compared with WT mice. Differentiation into fat is dependent on insulin signaling. Insulin signaling in young GADD34-deficient mice was higher than that in WT mice, which explained the higher fat differentiation of mouse embryonic fibroblasts (MEFs) observed in GADD34-deficient mice. Through aging or a HFD, insulin signaling in GADD34-deficient liver converted to be down regulated compared with WT mice. We found that a HFD or palmitate treatment converted insulin signaling by up-regulating TNF-α and JNK.

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.