HMGB1 released by mesothelial cells drives the development of asbestos-induced mesothelioma.

Asbestos is the main cause of malignant mesothelioma. Previous studies have linked asbestos-induced mesothelioma to the release of HMGB1 from the nucleus to the cytoplasm, and from the cytoplasm to the extracellular space. In the cytoplasm, HMGB1 induces autophagy impairing asbestos-induced cell death. Extracellularly, HMGB1 stimulates the secretion of TNFα. Jointly, these two cytokines kick-start a chronic inflammatory process that over time promotes mesothelioma development. Whether the main source of extracellular HMGB1 were the mesothelial cells, the inflammatory cells, or both was unsolved. This information is critical to identify the targets and design preventive/therapeutic strategies to interfere with asbestos-induced mesothelioma. To address this issue, we developed the conditional mesothelial HMGB1-knockout (Hmgb1ΔpMeso) and the conditional myelomonocytic-lineage HMGB1-knockout (Hmgb1ΔMylc) mouse models. We establish here that HMGB1 is mainly produced and released by the mesothelial cells during the early phases of inflammation following asbestos exposure. The release of HMGB1 from mesothelial cells leads to atypical mesothelial hyperplasia, and in some animals, this evolves over the years into mesothelioma. We found that Hmgb1ΔpMeso, whose mesothelial cells cannot produce HMGB1, show a greatly reduced inflammatory response to asbestos, and their mesothelial cells express and secrete significantly reduced levels of TNFα. Moreover, the tissue microenvironment in areas of asbestos deposits displays an increased fraction of M1-polarized macrophages compared to M2 macrophages. Supporting the biological significance of these findings, Hmgb1ΔpMeso mice showed a delayed and reduced incidence of mesothelioma and an increased mesothelioma-specific survival. Altogether, our study provides a biological explanation for HMGB1 as a driver of asbestos-induced mesothelioma.

Distinct and opposite effects of leukemogenic Idh and Tet2 mutations in hematopoietic stem and progenitor cells.

Mutations in IDH1, IDH2, and TET2 are recurrently observed in myeloid neoplasms. IDH1 and IDH2 encode isocitrate dehydrogenase isoforms, which normally catalyze the conversion of isocitrate to α-ketoglutarate (α-KG). Oncogenic IDH1/2 mutations confer neomorphic activity, leading to the production of D-2-hydroxyglutarate (D-2-HG), a potent inhibitor of α-KG-dependent enzymes which include the TET methylcytosine dioxygenases. Given their mutual exclusivity in myeloid neoplasms, IDH1, IDH2, and TET2 mutations may converge on a common oncogenic mechanism. Contrary to this expectation, we observed that they have distinct, and even opposite, effects on hematopoietic stem and progenitor cells in genetically engineered mice. Epigenetic and single-cell transcriptomic analyses revealed that Idh2R172K and Tet2 loss-of-function have divergent consequences on the expression and activity of key hematopoietic and leukemogenic regulators. Notably, chromatin accessibility and transcriptional deregulation in Idh2R172K cells were partially disconnected from DNA methylation alterations. These results highlight unanticipated divergent effects of IDH1/2 and TET2 mutations, providing support for the optimization of genotype-specific therapies.

Identification of an Optimized Receptor-Binding Domain Subunit Vaccine against SARS-CoV-2.

Current vaccine efforts to combat SARS-CoV-2 are focused on the whole spike protein administered as mRNA, viral vector, or protein subunit. However, the SARS-CoV-2 receptor-binding domain (RBD) is the immunodominant portion of the spike protein, accounting for 90% of serum neutralizing activity. In this study, we constructed several versions of RBD and together with aluminum hydroxide or DDA (dimethyldioctadecylammonium bromide)/TDB (d-(+)-trehalose 6,6'-dibehenate) adjuvant evaluated immunogenicity in mice. We generated human angiotensin-converting enzyme 2 knock-in mice to evaluate vaccine efficacy in vivo following viral challenge. We found that 1) subdomain (SD)1 was essential for the RBD to elicit maximal immunogenicity; 2) RBDSD1 produced in mammalian HEK cells elicited better immunogenicity than did protein produced in insect or yeast cells; 3) RBDSD1 combined with the CD4 Th1 adjuvant DDA/TDB produced higher neutralizing Ab responses and stronger CD4 T cell responses than did aluminum hydroxide; 4) addition of monomeric human Fc receptor to RBDSD1 (RBDSD1Fc) significantly enhanced immunogenicity and neutralizing Ab titers; 5) the Beta version of RBDSD1Fc provided a broad range of cross-neutralization to multiple antigenic variants of concern, including Omicron; and 6) the Beta version of RBDSD1Fc with DDA/TDB provided complete protection against virus challenge in the knock-in mouse model. Thus, we have identified an optimized RBD-based subunit vaccine suitable for clinical trials.

IDH1 mutation contributes to myeloid dysplasia in mice by disturbing heme biosynthesis and erythropoiesis.

Isocitrate dehydrogenase (IDH) mutations are common genetic alterations in myeloid disorders, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Epigenetic changes, including abnormal histone and DNA methylation, have been implicated in the pathogenic build-up of hematopoietic progenitors, but it is still unclear whether and how IDH mutations themselves affect hematopoiesis. Here, we show that IDH1-mutant mice develop myeloid dysplasia in that these animals exhibit anemia, ineffective erythropoiesis, and increased immature progenitors and erythroblasts. In erythroid cells of these mice, D-2-hydroxyglutarate, an aberrant metabolite produced by the mutant IDH1 enzyme, inhibits oxoglutarate dehydrogenase activity and diminishes succinyl-coenzyme A (CoA) production. This succinyl-CoA deficiency attenuates heme biosynthesis in IDH1-mutant hematopoietic cells, thus blocking erythroid differentiation at the late erythroblast stage and the erythroid commitment of hematopoietic stem cells, while the exogenous succinyl-CoA or 5-ALA rescues erythropoiesis in IDH1-mutant erythroid cells. Heme deficiency also impairs heme oxygenase-1 expression, which reduces levels of important heme catabolites such as biliverdin and bilirubin. These deficits result in accumulation of excessive reactive oxygen species that induce the cell death of IDH1-mutant erythroid cells. Our results clearly show the essential role of IDH1 in normal erythropoiesis and describe how its mutation leads to myeloid disorders. These data thus have important implications for the devising of new treatments for IDH-mutant tumors.

Software variability in service robotics.

Robots artificially replicate human capabilities thanks to their software, the main embodiment of intelligence. However, engineering robotics software has become increasingly challenging. Developers need expertise from different disciplines as well as they are faced with heterogeneous hardware and uncertain operating environments. To this end, the software needs to be variable-to customize robots for different customers, hardware, and operating environments. However, variability adds substantial complexity and needs to be managed-yet, ad hoc practices prevail in the robotics domain, challenging effective software reuse, maintenance, and evolution. To improve the situation, we need to enhance our empirical understanding of variability in robotics. We present a multiple-case study on software variability in the vibrant and challenging domain of service robotics. We investigated drivers, practices, methods, and challenges of variability from industrial companies building service robots. We analyzed the state-of-the-practice and the state-of-the-art-the former via an experience report and eleven interviews with two service robotics companies; the latter via a systematic literature review. We triangulated from these sources, reporting observations with actionable recommendations for researchers, tool providers, and practitioners. We formulated hypotheses trying to explain our observations, and also compared the state-of-the-art from the literature with the-state-of-the-practice we observed in our cases. We learned that the level of abstraction in robotics software needs to be raised for simplifying variability management and software integration, while keeping a sufficient level of customization to boost efficiency and effectiveness in their robots' operation. Planning and realizing variability for specific requirements and implementing robust abstractions permit robotic applications to operate robustly in dynamic environments, which are often only partially known and controllable. With this aim, our companies use a number of mechanisms, some of them based on formalisms used to specify robotic behavior, such as finite-state machines and behavior trees. To foster software reuse, the service robotics domain will greatly benefit from having software components-completely decoupled from hardware-with harmonized and standardized interfaces, and organized in an ecosystem shared among various companies.

Reactive oxygen species modulate macrophage immunosuppressive phenotype through the up-regulation of PD-L1.

The combination of immune checkpoint blockade with chemotherapy is currently under investigation as a promising strategy for the treatment of triple negative breast cancer (TNBC). Tumor-associated macrophages (TAMs) are the most prominent component of the breast cancer microenvironment because they influence tumor progression and the response to therapies. Here we show that macrophages acquire an immunosuppressive phenotype and increase the expression of programmed death ligand-1 (PD-L1) when treated with reactive oxygen species (ROS) inducers such as the glutathione synthesis inhibitor, buthionine sulphoximine (BSO), and paclitaxel. Mechanistically, these agents cause accumulation of ROS that in turn activate NF-κB signaling to promote PD-L1 transcription and the release of immunosuppressive chemokines. Systemic in vivo administration of paclitaxel promotes PD-L1 accumulation on the surface of TAMS in a mouse model of TNBC, consistent with in vitro results. Combinatorial treatment with paclitaxel and an anti-mouse PD-L1 blocking antibody significantly improved the therapeutic efficacy of paclitaxel by reducing tumor burden and increasing the number of tumor-associated cytotoxic T cells. Our results provide a strong rationale for the use of anti-PD-L1 blockade in the treatment of TNBC patients. Furthermore, interrogation of chemotherapy-induced PD-L1 expression in TAMs is warranted to define appropriate patient selection in the use of PD-L1 blockade.

AhR controls redox homeostasis and shapes the tumor microenvironment in BRCA1-associated breast cancer.

Cancer cells have higher reactive oxygen species (ROS) than normal cells, due to genetic and metabolic alterations. An emerging scenario is that cancer cells increase ROS to activate protumorigenic signaling while activating antioxidant pathways to maintain redox homeostasis. Here we show that, in basal-like and BRCA1-related breast cancer (BC), ROS levels correlate with the expression and activity of the transcription factor aryl hydrocarbon receptor (AhR). Mechanistically, ROS triggers AhR nuclear accumulation and activation to promote the transcription of both antioxidant enzymes and the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG). In a mouse model of BRCA1-related BC, cancer-associated AhR and AREG control tumor growth and production of chemokines to attract monocytes and activate proangiogenic function of macrophages in the tumor microenvironment. Interestingly, the expression of these chemokines as well as infiltration of monocyte-lineage cells (monocyte and macrophages) positively correlated with ROS levels in basal-like BC. These data support the existence of a coordinated link between cancer-intrinsic ROS regulation and the features of tumor microenvironment. Therapeutically, chemical inhibition of AhR activity sensitizes human BC models to Erlotinib, a selective EGFR tyrosine kinase inhibitor, suggesting a promising combinatorial anticancer effect of AhR and EGFR pathway inhibition. Thus, AhR represents an attractive target to inhibit redox homeostasis and modulate the tumor promoting microenvironment of basal-like and BRCA1-associated BC.

Holo-lipocalin-2-derived siderophores increase mitochondrial ROS and impair oxidative phosphorylation in rat cardiomyocytes.

Lipocalin-2 (Lcn2), a critical component of the innate immune response which binds siderophores and limits bacterial iron acquisition, can elicit spillover adverse proinflammatory effects. Here we show that holo-Lcn2 (Lcn2-siderophore-iron, 1:3:1) increases mitochondrial reactive oxygen species (ROS) generation and attenuates mitochondrial oxidative phosphorylation in adult rat primary cardiomyocytes in a manner blocked by N-acetyl-cysteine or the mitochondria-specific antioxidant SkQ1. We further demonstrate using siderophores 2,3-DHBA (2,3-dihydroxybenzoic acid) and 2,5-DHBA that increased ROS and reduction in oxidative phosphorylation are direct effects of the siderophore component of holo-Lcn2 and not due to apo-Lcn2 alone. Extracellular apo-Lcn2 enhanced the potency of 2,3-DHBA and 2,5-DHBA to increase ROS production and decrease mitochondrial respiratory capacity, whereas intracellular apo-Lcn2 attenuated these effects. These actions of holo-Lcn2 required an intact plasma membrane and were decreased by inhibition of endocytosis. The hearts, but not serum, of Lcn2 knockout (LKO) mice contained lower levels of 2,5-DHBA compared with wild-type hearts. Furthermore, LKO mice were protected from ischemia/reperfusion-induced cardiac mitochondrial dysfunction. Our study identifies the siderophore moiety of holo-Lcn2 as a regulator of cardiomyocyte mitochondrial bioenergetics.

Loss of the adipokine lipocalin-2 impairs satellite cell activation and skeletal muscle regeneration.

Lipocalin-2 (LCN2) is an adipokine previously described for its contribution to numerous processes, including innate immunity and energy metabolism. LCN2 has also been demonstrated to be an extracellular matrix (ECM) regulator through its association with the ECM protease matrix metalloproteinase-9 (MMP-9). With the global rise in obesity and the associated comorbidities related to increasing adiposity, it is imperative to gain an understanding of the cross talk between adipose tissue and other metabolic tissues, such as skeletal muscle. Given the function of LCN2 on the ECM in other tissues and the importance of matrix remodeling in skeletal muscle regeneration, we examined the localization and expression of LCN2 in uninjured and regenerating wild-type skeletal muscle and assessed the impact of LCN2 deletion (LCN2-/-) on skeletal muscle repair following cardiotoxin injury. Though LCN2 was minimally present in uninjured skeletal muscle, its expression was increased significantly at 1 and 2 days postinjury, with expression present in Pax7-positive satellite cells. Although satellite cell content was unchanged, the ability of quiescent satellite cells to become activated was significantly impaired in LCN2-/- skeletal muscles. Skeletal muscle regeneration was also significantly compromised as evidenced by decreased embryonic myosin heavy chain expression and smaller regenerating myofiber areas. Consistent with a role for LCN2 in MMP-9 regulation, regenerating muscle also displayed a significant increase in fibrosis and lower ( P = 0.07) MMP-9 activity in LCN2-/- mice at 2 days postinjury. These data highlight a novel role for LCN2 in muscle regeneration and suggest that changes in adipokine expression can significantly impact skeletal muscle repair.

Lipocalin-2 (NGAL) Attenuates Autophagy to Exacerbate Cardiac Apoptosis Induced by Myocardial Ischemia.

Lipocalin-2 (Lcn2; also termed neutrophil gelatinase-associated lipocalin (NGAL)) levels correlate positively with heart failure (HF) yet mechanisms via which Lcn2 contributes to the pathogenesis of HF remain unclear. In this study, we used coronary artery ligation surgery to induce ischemia in wild-type (wt) mice and this induced a significant increase in myocardial Lcn2. We then compared wt and Lcn2 knockout (KO) mice and observed that wt mice showed greater ischemia-induced caspase-3 activation and DNA damage measured by TUNEL than Lcn2KO mice. Analysis of autophagy by LC3 and p62 Western blotting, LC3 immunohistochemistry and transmission electron microscopy (TEM) indicated that Lcn2 KO mice had a greater ischemia-induced increase in autophagy. Lcn2KO were protected against ischemia-induced cardiac functional abnormalities measured by echocardiography. Upon treating a cardiomyocyte cell line (h9c2) with Lcn2 and examining AMPK and ULK1 phosphorylation, LC3 and p62 by Western blot as well as tandem fluorescent RFP/GFP-LC3 puncta by immunofluorescence, MagicRed assay for lysosomal cathepsin activity and TEM we demonstrated that Lcn2 suppressed autophagic flux. Lcn2 also exacerbated hypoxia-induced cytochromc c release from mitochondria and caspase-3 activation. We generated an autophagy-deficient H9c2 cell model by overexpressing dominant-negative Atg5 and found significantly increased apoptosis after Lcn2 treatment. In summary, our data indicate that Lcn2 can suppress the beneficial cardiac autophagic response to ischemia and that this contributes to enhanced ischemia-induced cell death and cardiac dysfunction. J. Cell. Physiol. 232: 2125-2134, 2017. © 2016 Wiley Periodicals, Inc.

Lipocalin 2 binds to membrane phosphatidylethanolamine to induce lipid raft movement in a PKA-dependent manner and modulates sperm maturation.

Mammalian sperm undergo multiple maturation steps after leaving the testis in order to become competent for fertilization, but the molecular mechanisms underlying this process remain unclear. In terms of identifying factors crucial for these processes in vivo, we found that lipocalin 2 (Lcn2), which is known as an innate immune factor inhibiting bacterial and malarial growth, can modulate sperm maturation. Most sperm that migrated to the oviduct of wild-type females underwent lipid raft reorganization and glycosylphosphatidylinositol-anchored protein shedding, which are signatures of sperm maturation, but few did so in Lcn2 null mice. Furthermore, we found that LCN2 binds to membrane phosphatidylethanolamine to reinforce lipid raft reorganization via a PKA-dependent mechanism and promotes sperm to acquire fertility by facilitating cholesterol efflux. These observations imply that mammals possess a mode for sperm maturation in addition to the albumin-mediated pathway.

Altered mitochondrial and peroxisomal integrity in lipocalin-2-deficient mice with hepatic steatosis.

Lipocalin-2 (LCN2) is a secreted adipokine that transports small hydrophobic molecules such as fatty acids and steroids. LCN2 limits bacterial growth by sequestering iron-containing siderophores and in mammalian liver protects against inflammation, infection, injury and other stressors. Because LCN2 modulates hepatic fat metabolism and homeostasis, we performed a comparative profiling of proteins and lipids of wild type (WT) and Lcn2-deficient mice fed either standard chow or a methionine- and choline-deficient (MCD) diet. Label-free proteomics and 2D-DIGE protein expression profiling revealed differential expression of BRIT1/MCPH1, FABP5, HMGB1, HBB2, and L-FABP, results confirmed by Western blotting. Gene ontology enrichment analysis identified enrichment for genes associated with mitochondrial membrane permeabilization and metabolic processes involving carboxylic acid. Measurements of mitochondrial membrane potential, mitochondrial chelatable iron pool, intracellular lipid peroxidation, and peroxisome numbers in primary hepatocytes confirmed that LCN2 regulates mitochondrial and peroxisomal integrity. Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry imaging identified significant changes to sphingomyelins, triglycerides, and glycerophospholipids in livers of mice fed an MCD diet regardless of LCN2 status. However, two arachidonic acid-containing glycerophospholipids were increased in Lcn2-deficient livers. Thus, LCN2 influences peroxisomal and mitochondrial biology in the liver to maintain triglyceride balance, handle oxidative stress, and control apoptosis.

Absence of intestinal PPARγ aggravates acute infectious colitis in mice through a lipocalin-2-dependent pathway.

To be able to colonize its host, invading Salmonella enterica serovar Typhimurium must disrupt and severely affect host-microbiome homeostasis. Here we report that S. Typhimurium induces acute infectious colitis by inhibiting peroxisome proliferator-activated receptor gamma (PPARγ) expression in intestinal epithelial cells. Interestingly, this PPARγ down-regulation by S. Typhimurium is independent of TLR-4 signaling but triggers a marked elevation of host innate immune response genes, including that encoding the antimicrobial peptide lipocalin-2 (Lcn2). Accumulation of Lcn2 stabilizes the metalloproteinase MMP-9 via extracellular binding, which further aggravates the colitis. Remarkably, when exposed to S. Typhimurium, Lcn2-null mice exhibited a drastic reduction of the colitis and remained protected even at later stages of infection. Our data suggest a mechanism in which S. Typhimurium hijacks the control of host immune response genes such as those encoding PPARγ and Lcn2 to acquire residence in a host, which by evolution has established a symbiotic relation with its microbiome community to prevent pathogen invasion.

Liver is the major source of elevated serum lipocalin-2 levels after bacterial infection or partial hepatectomy: a critical role for IL-6/STAT3.

UNLABELLED: Lipocalin-2 (LCN2) was originally isolated from human neutrophils and termed neutrophil gelatinase-associated lipocalin (NGAL). However, the functions of LCN2 and the cell types that are primarily responsible for LCN2 production remain unclear. To address these issues, hepatocyte-specific Lcn2 knockout (Lcn2(Hep-/-)) mice were generated and subjected to bacterial infection (with Klesbsiella pneumoniae or Escherichia coli) or partial hepatectomy (PHx). Studies of Lcn2(Hep-/-) mice revealed that hepatocytes contributed to 25% of the low basal serum level of LCN2 protein (∼ 62 ng/mL) but were responsible for more than 90% of the highly elevated serum LCN2 protein level (∼ 6,000 ng/mL) postinfection and more than 60% post-PHx (∼ 700 ng/mL). Interestingly, both Lcn2(Hep-/-) and global Lcn2 knockout (Lcn2(-/-)) mice demonstrated comparable increases in susceptibility to infection with K. pneumoniae or E. coli. These mice also had increased enteric bacterial translocation from the gut to the mesenteric lymph nodes and exhibited reduced liver regeneration after PHx. Treatment with interleukin (IL)-6 stimulated hepatocytes to produce LCN2 in vitro and in vivo. Hepatocyte-specific ablation of the IL-6 receptor or Stat3, a major downstream effector of IL-6, markedly abrogated LCN2 elevation in vivo. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed that STAT3 was recruited to the promoter region of the Lcn2 gene upon STAT3 activation by IL-6. CONCLUSION: Hepatocytes are the major cell type responsible for LCN2 production after bacterial infection or PHx, and this response is dependent on IL-6 activation of the STAT3 signaling pathway. Thus, hepatocyte-derived LCN2 plays an important role in inhibiting bacterial infection and promoting liver regeneration.

Lipocalin-2 (LCN2) regulates PLIN5 expression and intracellular lipid droplet formation in the liver.

Lipocalin-2 (LCN2) belongs to the superfamily of lipocalins and plays critical roles in the control of cellular homeostasis during inflammation and in responses to cellular stress or injury. In the liver, LCN2 triggers protective effects following acute or chronic injury, and its expression is a reliable indicator of liver damage. However, little is known about LCN2's functions in the homeostasis and metabolism of hepatic lipids or in the development of steatosis. In this study, we fed wild type (WT) and LCN2-deficient (Lcn2(-/-)) mice a methionine- and choline-deficient (MCD) diet as a nutritional model of non-alcoholic steatohepatitis, and compared intrahepatic lipid accumulation, lipid droplet formation, mitochondrial content, and expression of the Perilipin proteins that regulate cellular lipid metabolism. We found that Lcn2(-/-) mice fed an MCD diet accumulated more lipids in the liver than WT controls, and that the basal expression of the lipid droplet coat protein Perilipin 5 (PLIN5, also known as OXPAT) was significantly reduced in these animals. Similarly, the overexpression of LCN2 and PLIN5 were also found in animals that were fed with a high fat diet. Furthermore, the loss of LCN2 and/or PLIN5 in hepatocytes prevented normal intracellular lipid droplet formation both in vitro and in vivo. Restoration of LCN2 in Lcn2(-/-) primary hepatocytes by either transfection or adenoviral vector infection induced PLIN5 expression and restored proper lipid droplet formation. Our data indicate that LCN2 is a key modulator of hepatic lipid homeostasis that controls the formation of intracellular lipid droplets by regulating PLIN5 expression. LCN2 may therefore represent a novel therapeutic drug target for the treatment of liver diseases associated with elevated fat accumulation and steatosis.

Protective effects of lipocalin-2 (LCN2) in acute liver injury suggest a novel function in liver homeostasis.

Lipocalin-2 is expressed under pernicious conditions such as intoxication, infection, inflammation and other forms of cellular stress. Experimental liver injury induces rapid and sustained LCN2 production by injured hepatocytes. However, the precise biological function of LCN2 in liver is still unknown. In this study, LCN2(-/-) mice were exposed to short term application of CCl4, lipopolysaccharide and Concanavalin A, or subjected to bile duct ligation. Subsequent injuries were assessed by liver function analysis, qRT-PCR for chemokine and cytokine expression, liver tissue Western blot, histology and TUNEL assay. Serum LCN2 levels from patients suffering from liver disease were assessed and evaluated. Acute CCl4 intoxication showed increased liver damage in LCN2(-/-) mice indicated by higher levels of aminotransferases, and increased expression of inflammatory cytokines and chemokines such as IL-1ß, IL-6, TNF-α and MCP-1/CCL2, resulting in sustained activation of STAT1, STAT3 and JNK pathways. Hepatocytes of LCN2(-/-) mice showed lipid droplet accumulation and increased apoptosis. Hepatocyte apoptosis was confirmed in the Concanavalin A and lipopolysaccharide models. In chronic models (4weeks bile duct ligation or 8weeks CCl4 application), LCN2(-/-) mice showed slightly increased fibrosis compared to controls. Interestingly, serum LCN2 levels in diseased human livers were significantly higher compared to controls, but no differences were observed between cirrhotic and non-cirrhotic patients. Upregulation of LCN2 is a reliable indicator of liver damage and has significant hepato-protective effect in acute liver injury. LCN2 levels provide no correlation to the degree of liver fibrosis but show significant positive correlation to inflammation instead.

Development and characterization of a new inbred transgenic rat strain expressing DsRed monomeric fluorescent protein.

The inbred rat is a suitable model for studying human disease and because of its larger size is more amenable to complex surgical manipulation than the mouse. While the rodent fulfills many of the criteria for transplantation research, an important requirement is the ability to mark and track donors cells and assess organ viability. However, tracking ability is limited by the availability of transgenic (Tg) rats that express suitable luminescent or fluorescent proteins. Red fluorescent protein cloned from Discosoma coral (DsRed) has several advantages over other fluorescent proteins, including in vivo detection in the whole animal and ex vivo visualization in organs as there is no interference with autofluorescence. We generated and characterized a novel inbred Tg Lewis rat strain expressing DsRed monomeric (DsRed mono) fluorescent protein under the control of a ubiquitously expressed ROSA26 promoter. DsRed mono Tg rats ubiquitously expressed the marker gene as detected by RT-PCR but the protein was expressed at varying levels in different organs. Conventional skin grafting experiments showed acceptance of DsRed monomeric Tg rat skin on wild-type rats for more than 30 days. Cardiac transplantation of DsRed monomeric Tg rat hearts into wild-type recipients further showed graft acceptance and long-term organ viability (>6 months). The DsRed monomeric Tg rat provides marked cells and/or organs that can be followed for long periods without immune rejection and therefore is a suitable model to investigate cell tracking and organ transplantation.

Neutrophil gelatinase-associated lipocalin is instrumental in the pathogenesis of antibody-mediated nephritis in mice.

OBJECTIVE: The mechanism by which anti-DNA antibodies mediate lupus nephritis has yet to be conclusively determined. Previously, we found that treatment of mesangial cells with anti-DNA antibodies induced high expression of neutrophil gelatinase-associated lipocalin (NGAL), an iron-binding protein up-regulated in response to kidney injury. We undertook this study to determine whether NGAL is instrumental in the pathogenesis of nephritis, is induced as part of repair, or is irrelevant to damage/repair pathways. METHODS: To investigate the role of NGAL in antibody-mediated nephritis, we induced nephrotoxic nephritis by passive antibody transfer to 129/SyJ and C57BL/6 mice. To determine if NGAL up-regulation is instrumental, we compared the severity of renal damage in NGAL wild-type mice and NGAL-knockout mice following induction of nephrotoxic nephritis. RESULTS: We found that kidney NGAL expression, as well as urine NGAL levels, were significantly increased in mice with nephrotoxic nephritis as compared to control-injected mice. Tight correlations were observed between NGAL expression, renal histopathology, and urine NGAL excretion. NGAL-knockout mice had attenuated proteinuria and improved renal histopathology compared to wild-type mice. Similarly, following nephritis induction, NGAL injection significantly exacerbated nephritis and decreased survival. NGAL induced apoptosis via caspase 3 activation and up-regulated inflammatory gene expression in kidney cells in vitro and when injected in vivo. CONCLUSION: We conclude that kidney binding of pathogenic antibodies stimulates local expression of NGAL, which plays a crucial role in the pathogenesis of nephritis via promotion of inflammation and apoptosis. NGAL blockade may be a novel therapeutic approach for the treatment of nephritis mediated by pathogenic antibodies, including anti-glomerular basement membrane disease and lupus nephritis.

Disruption of the Lcn2 gene in mice suppresses primary mammary tumor formation but does not decrease lung metastasis.

Based largely on studies in xenograft models, lipocalin-2 (Lcn2) has been implicated in the progression of multiple types of human tumors, including breast cancer. Here we examine the role of Lcn2 in mammary tumorigenesis and lung metastasis using an in vivo molecular genetics approach. We crossed a well-characterized transgenic mouse model of breast cancer, the MMTV-PyMT (mouse mammary tumor virus-polyoma middle T antigen) mouse, with two independent gene-targeted Lcn2(-/-) mouse strains of the 129/Ola or C57BL/6 genetic background. The onset and progression of mammary tumor development and lung metastasis in the female progeny of these crosses were monitored over a 20-week period. Female Lcn2(-/-)MMTV-PyMT mice of the 129/Ola background (Lcn2(-/-)PyMT(129)) showed delayed onset of mammary tumors, and both Lcn2(-/-)PyMT(129) mice and Lcn2(-/-)MMTV-PyMT mice of the C57BL/6 background (Lcn2(-/-)PyMT(B6)) exhibited significant decreases in multiplicity and tumor burden (approximately 2- to 3-fold), as measured by total tumor weight and volume. At the molecular level, mammary tumors derived from Lcn2(-/-)PyMT(B6) females showed reduced matrix metalloproteinase-9 (MMP-9) activity and a lack of high molecular weight MMP activity. However, although increased MMP-9 activity has been linked to tumor progression, neither Lcn2(-/-)PyMT(B6) nor Lcn2(-/-)PyMT(129) female mice showed a reduction in lung metastases compared to Lcn2(+/+)PyMT controls. Our results demonstrate, using an in vivo animal model approach, that Lcn2 is a potent inducer of mammary tumor growth but not a significant promoter of lung metastasis.

Identification of BERP (brain-expressed RING finger protein) as a p53 target gene that modulates seizure susceptibility through interacting with GABA(A) receptors.

p53 is a central player in responses to cellular stresses and a major tumor suppressor. The identification of unique molecules within the p53 signaling network can reveal functions of this important transcription factor. Here, we show that brain-expressed RING finger protein (BERP) is a gene whose expression is up-regulated in a p53-dependent manner in human cells and in mice. We generated BERP-deficient mice by gene targeting and demonstrated that they exhibit increased resistance to pentylenetetrazol-induced seizures. Electrophysiological and biochemical studies of cultured cortical neurons of BERP-deficient mice showed a decrease in the amplitude of GABA(A) receptor (GABA(A)R)-mediated miniature inhibitory postsynaptic currents as well as reduced surface protein expression of GABA(A)Rs containing the gamma2-subunit. However, BERP deficiency did not decrease GABA(A)Rgamma2 mRNA levels, raising the possibility that BERP may act at a posttranscriptional level to regulate the intracellular trafficking of GABA(A)Rs. Our results indicate that BERP is a unique p53-regulated gene and suggest a role for p53 within the central nervous system.