The sirtuin SIRT6 regulates lifespan in male mice.

The significant increase in human lifespan during the past century confronts us with great medical challenges. To meet these challenges, the mechanisms that determine healthy ageing must be understood and controlled. Sirtuins are highly conserved deacetylases that have been shown to regulate lifespan in yeast, nematodes and fruitflies. However, the role of sirtuins in regulating worm and fly lifespan has recently become controversial. Moreover, the role of the seven mammalian sirtuins, SIRT1 to SIRT7 (homologues of the yeast sirtuin Sir2), in regulating lifespan is unclear. Here we show that male, but not female, transgenic mice overexpressing Sirt6 (ref. 4) have a significantly longer lifespan than wild-type mice. Gene expression analysis revealed significant differences between male Sirt6-transgenic mice and male wild-type mice: transgenic males displayed lower serum levels of insulin-like growth factor 1 (IGF1), higher levels of IGF-binding protein 1 and altered phosphorylation levels of major components of IGF1 signalling, a key pathway in the regulation of lifespan. This study shows the regulation of mammalian lifespan by a sirtuin family member and has important therapeutic implications for age-related diseases.

Inflammation-Induced Expression and Secretion of MicroRNA 122 Leads to Reduced Blood Levels of Kidney-Derived Erythropoietin and Anemia.

BACKGROUND & AIMS: Anemia is associated commonly with acute and chronic inflammation, but the mechanisms of their interaction are not clear. We investigated whether microRNA 122 (MIR122), which is generated in the liver and is secreted into the blood, is involved in the development of anemia associated with inflammation. METHODS: We characterized the primary transcript of the human liver-specific MIR122 using Northern blot, quantitative real-time polymerase chain reaction, and 3' and 5' rapid amplification of cDNA ends analyses. We studied regulation of MIR122 in human hepatocellular carcinoma cell lines (Huh7 and HepG2) as well as in C57BL/6 and mice with disruption of the tumor necrosis factor (Tnf) gene. Liver tissues were collected and analyzed by bioluminescence imaging or immunofluorescence. Inflammation in mice was induced by lipopolysaccharide (LPS) or by cerulein injections. Mice were given 4 successive injections of LPS, leading to inflammation-induced anemia. Steatohepatitis was induced with a choline-deficient, high-fat diet. Hemolytic anemia was stimulated by phenylhydrazine injection. MIR122 was inhibited in mice by tail-vein injection of an oligonucleotide antagonist of MIR122. MicroRNA and messenger RNA levels were determined by quantitative real-time polymerase chain reaction. RESULTS: The primary transcript of MIR122 spanned 5 kb, comprising 3 exons; the third encodes MIR122. Within the MIR122 promoter region we identified a nuclear factor-κB binding site and showed that RELA (NF-κB p65 subunit), as well as activators of NF-κB (TNF and LPS), increased promoter activity of MIR122. Administration of LPS to mice induced secretion of MIR122 into blood, which required TNF. Secreted MIR122 reached the kidney and reduced expression of erythropoietin (Epo), which we identified as a MIR122 target gene. Injection of mice with an oligonucleotide antagonist of MIR122 increased blood levels of EPO, reticulocytes, and hemoglobin. We found an inverse relationship between blood levels of MIR122 and EPO in mice with acute pancreatitis or steatohepatitis, and also in patients with acute inflammation. CONCLUSION: In mice, we found that LPS-induced inflammation increases blood levels of MIR122, which reduces expression of Epo in the kidney; this is a mechanism of inflammation-induced anemia. Strategies to block MIR122 in patients with inflammation could reduce the development or progression of anemia.

Erythrocyte survival is controlled by microRNA-142.

Hematopoietic-specific microRNA-142 is a critical regulator of various blood cell lineages, but its role in erythrocytes is unexplored. Herein, we characterize the impact of microRNA-142 on erythrocyte physiology and molecular cell biology, using a mouse loss-of-function allele. We report that microRNA-142 is required for maintaining the typical erythrocyte biconcave shape and structural resilience, for the normal metabolism of reactive oxygen species, and for overall lifespan. microRNA-142 further controls ACTIN filament homeostasis and membrane skeleton organization. The analyses presented reveal previously unappreciated functions of microRNA-142 and contribute to an emerging view of small RNAs as key players in erythropoiesis. Finally, the work herein demonstrates how a housekeeping network of cytoskeletal regulators can be reshaped by a single micro-RNA denominator in a cell type specific manner.

Parathyroid hormone activates the orphan nuclear receptor Nurr1 to induce FGF23 transcription.

Parathyroid hormone (PTH) increases FGF23 mRNA and protein levels in vivo and in vitro. Here we tested whether the increased FGF23 expression by PTH is mediated by the orphan nuclear receptor Nurr1. PTH increased Nurr1 mRNA levels prior to elevation of FGF23 mRNA levels in UMR-106 rat osteoblast-like cells. Activation of PKA increased both FGF23 and Nurr1 mRNA levels. Modification of Nurr1 expression showed that Nurr1 is essential for the PTH-mediated increase in FGF23 and luciferase reporter gene experiments identified a functional promoter region containing several potential Nurr1 binding sites. Chromatin immunoprecipitation assays confirmed the binding of Nurr1 to these regions in the FGF23 promoter. In vivo, Nurr1 mRNA and protein levels were increased in calvaria from rats with experimental CKD together with high PTH and FGF23 expression. Calcimimetics decrease PTH and FGF23 levels in CKD patients. Importantly, in rats with experimental CKD, the calcimimetic R568 decreased PTH expression, calvaria Nurr1 mRNA and protein levels, and FGF23 mRNA. Immunohistochemistry for Nurr1 showed an increase in the number of Nurr1 expressing osteocytes in the femurs of rats with CKD and this was decreased by R568. Thus, the effect of PTH to increase FGF23 transcription is mediated by Nurr1 in vitro and in vivo. In CKD, calcimimetics decrease PTH, which in turn decreases Nurr1 and consequently FGF23.

Pulmonary epithelioid hemangioendothelioma.

Pulmonary epithelioid hemangioendothelioma (PEH), previously known as "intravascular bronchoalveolar tumor," is a rare vascular malignancy with an unpredictable prognosis. Treatment can vary from observation in asymptomatic patients to surgery in patients with resectable disease or chemotherapy in patients with disseminated disease. This report describes the clinical, radiological and pathological features of three cases of PEH and a review of the current literature.

Association of cell cycle-related gene products and NF-kappaB with clinical parameters in Langerhans cell histiocytosis.

BACKGROUND: Langerhans cell histiocytosis (LCH) is a rare disorder characterized by the accumulation of abnormal Langerhans cells in one or several organs where they cause local tissue damage. The pathophysiology is not well understood. The aim of this study was to examine expression of various gene products that play a role in cell cycle and cell death and to look for an association with the extent of the disease at the time of diagnosis and with "risk bone" involvement. PROCEDURE: Histologic slides from cases with biopsy proven disease were stained immunohistochemically for Bcl-2, caspase-3, Ki-67, p53, and nuclear factor-kappaB (NF-kappaB) and the results were quantitated and compared with the clinical extent of the disease. RESULTS: In patients with multisystem disease and "risk" bone involvement, a higher percentage of Langerhans cells stained with the anti-apoptotic gene product Bcl-2 (P = 0.0004; P = 0.001 respectively) and a lower percentage of these cells stained with the apoptosis marker caspase-3 compared to patients with single system disease (P = 0.0001; P = 0.01 respectively). Proliferation marker Ki-67 was expressed more frequently in multisystem disease compared to single system disease (P = 0.02) but an association with "risk" bone involvement was not found. Expression of p53 and NF-kappaB did not discriminate between clinical subgroups. CONCLUSIONS: The findings suggest that cell proliferation and suppression of apoptosis may be mechanisms of cell survival in the more aggressive forms of LCH (multisystem, risk bone involvement).

Foreign-body granulomas within intramyocardial arteries in a transcoronary safety assessment in pigs.

Foreign-body granulomas within intramyocardial arteries were detected in three domestic pigs (Sus scrofa domestica) in a routine transcoronary safety assessment study. The foreign bodies stained light grayish-blue by hematoxylin and eosin (H&E) and were strongly birefringent by polarized light microscopy. By their morphological features, they were identified as cotton fibers. Embolization of foreign-body material into the myocardial arteries following catheter-based procedures is an unusual event, and its occurrence may lead to granulomatous foreign-body reaction and misinterpretation of histological results.

Cutaneous toxoplasmosis after bone marrow transplantation with molecular confirmation.

Toxoplasmosis is a rare and often fatal complication of hematopoietic stem cell transplantation (HSCT). The diagnosis of toxoplasmosis is usually made at autopsy because of the variety of systemic manifestations and the difficulty of diagnosis by serologic methods in the severely immunocompromised patient. Cutaneous toxoplasmosis in this setting is extremely rare and is difficult to diagnose with certainty because of the morphologic similarity of Toxoplasma gondii to other organisms, such as Leishmania and Histoplasma species. We report a patient who developed systemic toxoplasmosis, manifested as encephalitis and cutaneous lesions, after HSCT. Findings of a skin biopsy led to a tentative histologic diagnosis of toxoplasmosis, confirmed by polymerase chain reaction (PCR) examination of the skin biopsy and cerebrospinal fluid. This is, to our knowledge, the first report of cutaneous toxoplasmosis diagnosed by skin biopsy confirmed by PCR and sequencing. This disease may be more common than is generally appreciated in severely immunocompromised patients. PCR is a valuable adjunct to diagnosis.

Matrix metalloproteinase 12 promotes tumor propagation in the lung.

OBJECTIVE: Past studies are inconsistent with regard to the role of matrix metalloproteinase 12 in lung tumorigenesis. This is due, in part, to differential tumorigenesis based on tumor-derived versus immune-derived matrix metalloproteinase 12 expression. Our study aims to thoroughly dissect the role of matrix metalloproteinase 12 in lung tumorigenesis. METHODS: We tested matrix metalloproteinase 12 expression and the association with prognosis using a tissue array and a published non-small cell lung cancer gene expression database. In addition, we characterized the contribution of matrix metalloproteinase 12 to tumor propagation in the lung using a series of in vitro and in vivo studies. RESULTS: Tumor cells of a diverse set of human lung cancers stained positive for matrix metalloproteinase 12, and high matrix metalloproteinase 12 mRNA levels in the tumor were associated with reduced survival. The lung microenvironment stimulated endogenous production of matrix metalloproteinase 12 in lung cancer cells (human 460 lung cancer cell line, Lewis lung carcinoma). In vitro, matrix metalloproteinase 12 knockout Lewis lung carcinoma and Lewis lung carcinoma cells had the same proliferation rate, but Lewis lung carcinoma showed increased invasiveness. In vivo, deficiency of matrix metalloproteinase 12 in Lewis lung carcinoma cells, but not in the host, reduced tumor growth and invasiveness. CONCLUSIONS: We suggest that tumor cell-derived matrix metalloproteinase 12 promotes tumor propagation in the lung and that in the context of pulmonary malignancies matrix metalloproteinase 12 should further be tested as a potential novel therapeutic target.

Severe Epstein-Barr virus-associated hemophagocytic syndrome in six adult patients.

BACKGROUND: EBV associated hemophagocytic syndrome (HPS) is an aggressive and potentially life-threatening condition. So far, most EBV associated HPS has been characterized mainly in infants and children in Asian countries. RESULTS: Here, we report six cases of EBV associated HPS occurring in previously healthy adults in a non-endemic area within a short period of 3 years. All patients presented with fever, hepatosplenomegaly and pancytopenia as well as disturbed liver function tests and coagulopathy. Half were diagnosed as having lymphoma. While EBV-specific serological assays were non-diagnostic in four of the six patients, the presence of EBV DNA in plasma allowed the diagnosis of EBV associated HPS in all patients. CONCLUSION: EBV associated HPS may be more prevalent in non-Japanese adults than was previously considered. Screening for hemophagocytic syndrome, in adults as well as in children, should include real-time PCR for EBV.

Expression of the RNA-binding protein VICKZ in normal hematopoietic tissues and neoplasms.

BACKGROUND AND OBJECTIVES: VICKZ family members are RNA-binding regulatory proteins expressed during embryogenesis but not usually found in normal adult tissue. The presence of VICKZ in normal germinal centers (GC) prompted us to characterize the expression pattern of this protein in lymphoid and hematopoietic tissues. DESIGN AND METHODS: We generated a pan-VICKZ antibody that recognized all three isoforms of VICKZ protein and screened 889 patients' samples by immunohistologic methods. We also analyzed the expression of VICKZ in normal hematopoiesis tissue by staining samples of tonsils, lymph nodes RESULTS: VICKZ protein expression was documented for the first time in normal human GC and in follicular (126/165), mediastinal large B-cell (9/10), Burkitt (2/2), diffuse large B-cell (DLBCL, 155/200), lymphocyte-predominant Hodgkin's (12/13), classical Hodgkin's (101/108), and anaplastic large cell (6/8) lymphomas and in lymphoid and myeloid leukemias. Since DLBCL may derive from GC or non-GC B cells we performed hierarchical cluster analysis for VICKZ, HGAL, BCL6, CD10, MUM1/IRF4 and BCL2 which showed that VICKZ is expressed in both subtypes. In addition, VICKZ mRNA isoforms were differentially expressed in lymphoma subtypes and over 40% of DLBCL expressed hVICKZ2, an isoform not usually present in normal GC B cells. INTERPRETATION AND CONCLUSIONS: We show that in normal lymphoid tissues VICKZ is expressed in GC lymphocytes but in lymphoid neoplasms its expression is not limited to GC-derived lymphoma subtypes. However, VICKZ exhibits differential expression in lymphoma subtypes and thus may be a marker of potential value in the diagnosis and study of hematopoietic neoplasia. The aberrant expression of its isoforms in DLBCL raises the possibility that these isoforms may be associated with different functions and suggests that further study of their role in normal and neoplastic lymphoid cells is warranted.

Ethnic variation in medical and lifestyle risk factors for B cell non-Hodgkin lymphoma: A case-control study among Israelis and Palestinians.

BACKGROUND: Risk factors for B-cell non-Hodgkin lymphoma (B-NHL) have not been assessed among Palestinian Arabs (PA) and Israeli Jews (IJ). METHODS: In a case-control study we investigated self-reported medical and lifestyle exposures, reporting odds ratios (ORs) and 95% confidence intervals [CIs], by ethnicity, for overall B-NHL and subtypes. RESULTS: We recruited 823 cases and 808 healthy controls. Among 307 PA/516 IJ B-NHL cases (mean age at diagnosis = 51 [±17] versus 60 [±15] years, respectively) subtype distributions differed, with diffuse large B-cell lymphoma (DLBCL) being prominent among PA (71%) compared to IJ (41%); follicular lymphoma (FL), was observed in 14% versus 28%, and marginal zone lymphoma, in 2% versus 14%, respectively. Overall B-NHL in both populations was associated with recreational sun exposure OR = 1.43 [CI:1.07-1.91], black hair-dye use OR = 1.70 [CI:1.00-2.87], hospitalization for infection OR = 1.68 [CI:1.34-2.11], and first-degree relative with hematopoietic cancer, OR = 1.69 [CI:1.16-2.48]. An inverse association was noted with alcohol use, OR = 0.46 [CI:0.34-0.62]. Subtype-specific exposures included smoking (FL, OR = 1.46 [CI:1.01-2.11]) and >monthly indoor pesticide use (DLBCL, OR = 2.01 [CI:1.35-3.00]). Associations observed for overall B-NHL in PA only included: gardening OR = 1.93 [CI:1.39-2.70]; history of herpes, mononucleosis, rubella, blood transfusion (OR>2.5, P<0.01 for all); while for IJ risk factors included growing fruits and vegetables, OR = 1.87 [CI:1.11-3.15]; and self-reported autoimmune diseases, OR = 1.99 [CI:1.34-2.95]. CONCLUSIONS: In these geographically proximate populations we found some unique risk factors for B-NHL. Heterogeneity in the observed associations by ethnicity could reflect differences in lifestyle, medical systems, and reporting patterns, while variations by histology infer specific etiologic factors for lymphoma subtypes.

WWOX and p53 Dysregulation Synergize to Drive the Development of Osteosarcoma.

Osteosarcoma is a highly metastatic form of bone cancer in adolescents and young adults that is resistant to existing treatments. Development of an effective therapy has been hindered by very limited understanding of the mechanisms of osteosarcomagenesis. Here, we used genetically engineered mice to investigate the effects of deleting the tumor suppressor Wwox selectively in either osteoblast progenitors or mature osteoblasts. Mice with conditional deletion of Wwox in preosteoblasts (WwoxΔosx1) displayed a severe inhibition of osteogenesis accompanied by p53 upregulation, effects that were not observed in mice lacking Wwox in mature osteoblasts. Deletion of p53 in WwoxΔosx1 mice rescued the osteogenic defect. In addition, the Wwox;p53Δosx1 double knockout mice developed poorly differentiated osteosarcomas that resemble human osteosarcoma in histology, location, metastatic behavior, and gene expression. Strikingly, the development of osteosarcomas in these mice was greatly accelerated compared with mice lacking p53 only. In contrast, combined WWOX and p53 inactivation in mature osteoblasts did not accelerate osteosarcomagenesis compared with p53 inactivation alone. These findings provide evidence that a WWOX-p53 network regulates normal bone formation and that disruption of this network in osteoprogenitors results in accelerated osteosarcoma. The Wwox;p53Δosx1 double knockout establishes a new osteosarcoma model with significant advancement over existing models. Cancer Res; 76(20); 6107-17. ©2016 AACR.

Simultaneous donor marrow cell transplantation with reduced intensity conditioning prevents tracheal allograft obliteration in a bronchiolitis obliterans murine model.

STUDY OBJECTIVES: Prolonged survival of transplanted kidney or liver allografts has been associated with prolonged chimerism resulting from donor-origin leukocytes carried within the allograft parenchyma. The present study, performed in a murine model, examined whether simultaneous administration of donor bone marrow cells after reduced intensity conditioning allows acceptance of heterotopic tracheal allografts and prevents the formation of the airway fibroproliferative lesion, which mimics bronchiolitis obliterans (BO). METHODS: Allogeneic tracheal allografts from C57BL/6 mice were grafted subcutaneously into BALB/c mice (n = 6) [day 0]. Conditioning consisted of total lymphoid irradiation (200 cGy) at day - 1, donor marrow cells (3 x 10(7)) administered IV on day 0, intraperitoneal cyclophosphamide (200 mg/kg) on day 1 to eliminate alloreactive marrow cells, followed by a repeated dose of donor marrow cells on day 2. Control groups consisted of one group (n = 4) that underwent similar conditioning without donor marrow cells, and another group (n = 4) that received syngeneic BALB/c marrow cells. None of these groups were administered maintenance immunosuppression. Grafts were harvested and histopathology findings were evaluated semiquantitatively at day 28, day 55, and day 95. RESULTS: Tracheal allografts from donor marrow cell recipients still maintained a patent airway with intact airway epithelium at 95 days after transplant. However, grafts from control animals not receiving donor marrow cells or mice administered syngeneic marrow cells had lumen obliteration by 28 days after transplant. Chimerism in animals receiving allogeneic bone marrow was confirmed. Graft vs host disease did not develop in animals receiving allogeneic marrow cells. CONCLUSIONS: Further investigation may verify this approach to be applicable for the prevention of posttransplantation BO.

Obstructing tracheal pulmonary Langerhans cell histiocytosis.

Pulmonary Langerhans cell histiocytosis, also known as eosinophilic granuloma, is an uncommon interstitial lung disease. A solitary nodule, usually parenchymal, may rarely be the only manifestation of the disease. We describe a case of Langerhans cell histiocytosis presenting as an obstructing tracheal lesion in a 55-year-old woman. Following complete resection of the lesion via flexible bronchoscopy, full recovery was achieved. This case represents a unique cause for tracheal obstruction, as well as an unreported manifestation of pulmonary Langerhans cell histiocytosis.

CD68 staining correlates with the size of residual mass but not with survival in classical Hodgkin lymphoma.

The prognostic role of CD68 tumor-associated macrophages in classic Hodgkin lymphoma (cHL) remains controversial. We stained diagnostic biopsies and scored for CD68 using the PGM1 antibody among 98 consecutive patients with cHL from our center followed over a median of 45 months for progression-free survival (PFS). Among 79 patients we assessed interim and post-treatment positron emission tomography-computed tomography (PET-CT). Residual mass (RM) size was based on the greatest diameter of the largest mass seen in post-treatment imaging, and percent reduction was calculated by comparing RM size with its greatest pretreatment diameter. We found a significant association between CD68 positivity and absolute size of initial disease mass (p = 0.014) and residual mass at the end of therapy (p = 0.006) but no association was observed with interim PET-CT results or PFS. Our findings suggest that macrophages may influence tumor size by altering the microenvironment. This study does not support a prognostic role of CD68 positivity in predicting survival.

Expression of p53 gene product and cell proliferation marker Ki-67 in Ewing's sarcoma: correlation with clinical outcome.

Overexpression of tumor suppressor gene p53, cell proliferation nuclear antigen Ki-67, and proto-oncogene HER-2/neu are associated with poor prognosis in some tumors. We studied p53, Ki-67, and HER-2/neu immunohistochemical expression in archival biopsies of 37 patients with Ewing's sarcoma (ES). Patients with ES were treated at four Israeli hospitals between 1982 and 2000. Formalin-fixed paraffin-embedded tissue sections were stained by immunohistochemistry for p53, Ki-67, and HER-2/neu. More than 300 cells were counted on each slide, and the percentage of positively stained nuclei was computed. p53 overexpression was defined as nuclear staining of >2.3% of cells, Ki-67 overexpression as nuclear staining of >8.3% malignant cells. HER-2/neu staining was scored semiquantitatively on a scale of 0 to 4+. Twenty-two of 37 patients are alive and well, with mean follow-up time of 38 months. There was overexpression of p53 in 16 patients (43%) and of Ki-67 in 21 patients (57%). The correlation between p53 and Ki-67 overexpressions was 0.61. We found no overexpression of HER-2/neu. Median relapse-free survival (RFS) was statistically significantly shorter for patients with p53 overexpression (25 months) than for patients with negative staining (>92 months). The prognostic value of p53 overexpression was also significant after adjusting for tumor location and age. Median RFS was shorter for patients with positive Ki-67 staining (40 months) than for patients with negative staining (80 months) but did not reach statistical significance. Our study suggests that p53 is a predictor of RFS in patients with ES. More patients must be studied to assess the validity of this observation.