After influenza infection, C57BL/6J mice develop increased slow-wave sleep (SWS) during the dark phase of the day-night cycle, whereas BALB/cByJ mice develop decreased SWS during the light phase. A previous analysis of CXB recombinant inbred mice revealed a quantitative trait locus (QTL) designated Srilp (sleep response to influenza, light phase) that was related to expression of the BALB/cByJ sleep phenotype. Srilp was localized to the 10- to 12-cM region of mouse Chr 6 between D6Mit74 and D6Mit188. Temt (thioether S-methyltransferase), which is located at region B3 of Chr 6, is a potential candidate gene for Srilp. We evaluated the expression of Temt and other Srilp candidate genes in hypothalamus and basal forebrain of uninfected and influenza-infected C57BL/6J and BALB/cByJ mice. We report here that Temt expression varies significantly with respect to mouse strain, health status, brain region, and day-night phase. C57BL/6J mice show day-night variation in Temt expression in hypothalamus, but BALB/cByJ mice do not. Temt expression in basal forebrain is much higher in C57BL/6J mice than in BALB/cByJ mice. During influenza infection, both C57BL/6J and BALB/cByJ mice show reduced Temt mRNA in basal forebrain at 30 h postinoculation, but expression remains much lower in the BALB/cByJ strain. In contrast, prostaglandin-D-synthase (Ptgds) and lipocalin 2 (Lcn2) mRNA increase in basal forebrain of both strains after influenza infection. Administration of the TEMT inhibitor sinefungin reduces sleep in uninfected BALB/cByJ mice and attenuates influenza-induced sleep enhancement in C57BL/6J mice. These data suggest that strain- and infection-related alterations in sleep may be influenced by Temt expression and perhaps by subsequent effects on prostaglandin metabolism.

Lipocalin-2 (Lcn2) is a small glycoprotein involved in a number of biological processes such as inflammation and antibacterial response. In our study, Lcn2 is expressed in the subluminal stromal cells at implantation site on day 5 of pregnancy. The expression of Lcn2 in stromal cells is under the control of progesterone through Akt-c-Myc signaling pathway. Data from Lcn2 knockdown and recombinant protein treatments indicate that Lcn2 promotes mPGES-1 expression in stromal cells. The expression of Lcn2 and mPGES-1 is strongly stimulated by lipopolysaccharide (LPS), indicating that Lcn2 mediates LPS-induced inflammation. These findings shed light on the role of Lcn2 during decidualization.

Acute renal failure resulting from radiocontrast-induced nephrotoxicity (RIN) is suggested to occur via medullary ischemia coupled with the generation of free radicals and oxidative injury to tubular cells. The aim of the present study was to assess the effects of erdosteine on prevention of RIN. Thirty-three Wistar-albino rats were divided into five groups: control (group 1, n = 6), radiocontrast media (group 2, n = 6), erdosteine (group 3, n = 7), erdosteine four doses before radiocontrast application (group 4, n = 7) and erdosteine one dose at the same day with radiocontrast application (group 5, n = 7). RIN was induced by administration of intravenous high osmolar contrast media amidotrizoate (6 mL/kg). Total RNA was extracted from the kidney, and the expression levels of Lipocalin 2 (Lcn2) and secreted phosphoprotein 1 (Spp1) genes were evaluated by real time reverse transcription polymerase chain reaction (real-time RT-PCR). Total antioxidant status (TAS) and total oxidant status (TOS) were measured in kidney homogenates and serum samples. Serum creatinine, BUN (Blood Urea Nitrogen) and cystatin-C levels were measured from serum samples. The kidneys were evaluated histopathologically. The expression levels of Spp1 and Lcn2 genes in group 2 were significantly higher than groups 1, 3, 4, and 5. The expression levels of Spp1 and Lcn2 genes in group 4 were four and two times lower than group 5, respectively. Kidney TOS levels in group 2 were significantly higher than groups 1, 3, 4, and 5. Kidney TAS levels in group 3 were higher than group 2. Kidney oxidative stress index (OSI) levels in group 2 were significantly higher than groups 4 and 5. All rats in contrast media group developed tubular necrosis, proteinaceous casts, medullary congestion although these changes were significantly reduced in groups 4 and 5. This study demonstrated that multiple doses of erdosteine before application may have higher protective effects against RIN.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) given as a cotreatment with estrogen exhibits antiestrogenic properties on the rodent adult uterus, but less is understood regarding hormonal responsiveness of the adult uterus from animals having been exposed to TCDD during critical periods of development. We characterized the inhibitory effects of TCDD (T) exposure at gestational day 15 (GD15), 4 weeks, and 9 weeks of age (TTT) on the adult uterus following hormone treatment. TTT-exposed mice in response to hormone treatment exhibited a blunted weight increase, had fewer uterine glands, displayed morphological anomalies, and had marked decreases in the hormonal regulation of genes involved in fluid transport (Aqp3 and Aqp5), cytoarchitectural (Dsc2 and Sprr2A), and immune (Lcn2 and Ltf) regulation. To determine if the 9-week exposure was responsible for the blunted uterine response, due to the 7- to 11-day half-life of TCDD in mice, a second set of experiments was performed to examine exposure to TCDD given at GD15, GD15 only (cross-fostered at birth), only during lactation (cross-fostered at birth), or at GD15 and 4 weeks of age. Our studies demonstrate that a single developmental TCDD exposure at GD15 is sufficient to elicit a blunted adult uterine response to estradiol and is due in part to fewer gland numbers and the reduced expression of forkhead box A2 (FoxA2), a gene involved in gland development. Together, these results provide insight regarding the critical nature of in utero exposure and the potential impact on ensuing uterine biology and reproductive health later in life.

Conditioned medium of mesenchymal stem cells (MSCs) is now being used for its cytoprotective effects, especially when the cells are equipped with cytoprotective factors to strengthen them against unfavorable microenvironments. Overexpression of Lcn2 in MSCs mimics in vivo kidney injury. Hence, unraveling how Lcn2-engineered MSCs affect kidney cells has been investigated. Cisplatin treated HK-2 or HEK293 kidney cells were co-cultivated with Lcn2 overexpressing MSCs in upper and lower chambers of transwell plates. Proliferation, apoptosis, and expression of growth factors and cytokines were assessed in the kidney cells. Co-cultivation with the MSCs-Lcn2 not only inhibited cisplatin-induced cytotoxicity in the HK-2 and HEK293 cells, but increased proliferation rate, prevented cisplatin-induced apoptosis, and increased expression of growth factors and the amount of antioxidants in the kidney cells. Thus Lcn2-engineered MSCs can ameliorate and repair injured kidney cells in vitro, which strongly suggests there are beneficial effects of the MSCs-Lcn2 in cell therapy of kidney injury.

An increase in tumour formation and metastasis are observed upon plakophilin3 (PKP3) loss. To identify pathways downstream of PKP3 loss that are required for increased tumour formation, a gene expression analysis was performed, which demonstrated that the expression of lipocalin2 (LCN2) was elevated upon PKP3 loss and this is consistent with expression data from human tumour samples suggesting that PKP3 loss correlates with an increase in LCN2 expression. PKP3 loss leads to an increase in invasion, tumour formation and metastasis and these phenotypes were dependent on the increase in LCN2 expression. The increased LCN2 expression was due to an increase in the activation of p38 MAPK in the HCT116 derived PKP3 knockdown clones as LCN2 expression decreased upon inhibition of p38 MAPK. The phosphorylated active form of p38 MAPK is translocated to the nucleus upon PKP3 loss and is dependent on complex formation between p38 MAPK and PKP3. WT PKP3 inhibits LCN2 reporter activity in PKP3 knockdown cells but a PKP3 mutant that fails to form a complex with p38 MAPK cannot suppress LCN2 promoter activity. Further, LCN2 expression is decreased upon loss of p38β, but not p38α, in the PKP3 knockdown cells. These results suggest that PKP3 loss leads to an increase in the nuclear translocation of p38 MAPK and p38β MAPK is required for the increase in LCN2 expression.

Background: Gene signatures derived from transcriptomic data using machine learning methods have shown promise for biodosimetry testing. These signatures may not be sufficiently robust for large scale testing, as their performance has not been adequately validated on external, independent datasets. The present study develops human and murine signatures with biochemically-inspired machine learning that are strictly validated using k-fold and traditional approaches. Methods: Gene Expression Omnibus (GEO) datasets of exposed human and murine lymphocytes were preprocessed via nearest neighbor imputation and expression of genes implicated in the literature to be responsive to radiation exposure (n=998) were then ranked by Minimum Redundancy Maximum Relevance (mRMR). Optimal signatures were derived by backward, complete, and forward sequential feature selection using Support Vector Machines (SVM), and validated using k-fold or traditional validation on independent datasets. Results: The best human signatures we derived exhibit k-fold validation accuracies of up to 98% ( DDB2, PRKDC, TPP2, PTPRE, and GADD45A) when validated over 209 samples and traditional validation accuracies of up to 92% ( DDB2, CD8A, TALDO1, PCNA, EIF4G2, LCN2, CDKN1A, PRKCH, ENO1, and PPM1D) when validated over 85 samples. Some human signatures are specific enough to differentiate between chemotherapy and radiotherapy. Certain multi-class murine signatures have sufficient granularity in dose estimation to inform eligibility for cytokine therapy (assuming these signatures could be translated to humans). We compiled a list of the most frequently appearing genes in the top 20 human and mouse signatures. More frequently appearing genes among an ensemble of signatures may indicate greater impact of these genes on the performance of individual signatures. Several genes in the signatures we derived are present in previously proposed signatures. Conclusions: Gene signatures for ionizing radiation exposure derived by machine learning have low error rates in externally validated, independent datasets, and exhibit high specificity and granularity for dose estimation.

Gene mutations play critical roles during cancer development and progression, and therefore represent targets for precision medicine. Here we recapitulated the pharmacogenomic data to delineate novel candidates for actionable mutations and therapeutic target drugs. As a proof-of-concept, we demonstrated that the loss-of-function of SULF2 by mutation (N491K) or inhibition enhanced sorafenib sensitivity in liver cancer cells and in vivo mouse models. This effect was mediated by deregulation of EGFR signaling and downstream expression of LCN2. We also report that the liver cancer patients non-responding to sorafenib treatment exhibit higher expression of SULF2 and LCN2. In conclusion, we suggest that SULF2 plays a key role in sorafenib susceptibility and resistance in liver cancer via deregulation of LCN2. Diagnostic or therapeutic targeting of SULF2 (e.g., OKN-007) and/or LCN2 can be a novel precision strategy for sorafenib treatment in cancer patients.

The GLI (GLI1/GLI2) transcription factors have been implicated in the development and progression of prostate cancer although our understanding of how they actually contribute to the biology of these common tumours is limited. We observed that GLI reporter activity was higher in normal (PNT-2) and tumourigenic (DU145 and PC-3) androgen-independent cells compared to androgen-dependent LNCaP prostate cancer cells and, accordingly, GLI mRNA levels were also elevated. Ectopic expression of GLI1 or the constitutively active ΔNGLI2 mutant induced a distinct cobblestone-like morphology in LNCaP cells that, regarding the former, correlated with increased GLI2 as well as expression of the basal/stem-like markers CD44, β1-integrin, ΔNp63 and BMI1, and decreased expression of the luminal marker AR (androgen receptor). LNCaP-GLI1 cells were viable in the presence of the AR inhibitor bicalutamide and gene expression profiling revealed that the transcriptome of LNCaP-GLI1 cells was significantly closer to DU145 and PC-3 cells than to control LNCaP-pBP (empty vector) cells, as well as identifying LCN2/NGAL as a highly induced transcript which is associated with hormone independence in breast and prostate cancer. Functionally, LNCaP-GLI1 cells displayed greater clonal growth and were more invasive than control cells but they did not form colonies in soft agar or prostaspheres in suspension suggesting that they do not possess inherent stem cell properties. Moreover, targeted suppression of GLI1 or GLI2 with siRNA did not reverse the transformed phenotype of LNCaP-GLI1 cells nor did double GLI1/GLI2 knockdowns activate AR expression in DU145 or PC-3 cells. As such, early targeting of the GLI oncoproteins may hinder progression to a hormone independent state but a more detailed understanding of the mechanisms that maintain this phenotype is required to determine if their inhibition will enhance the efficacy of anti-hormonal therapy through the induction of a luminal phenotype and increased dependency upon AR function.

Fabry disease is a lysosomal storage disease caused by a deficiency of alpha-galactosidase A, which results in aberrant glycosphingolipid metabolism and accumulation of globotriaosylceramide (Gb3). Since a correlation between the level of Gb3 and clinical manifestations of Fabry disease has not been observed, we investigated potential diagnostic biomarkers. Hepatic and renal gene expression of male alpha-galactosidase A-deficient mice (Fabry mice) was compared with that of wild-type mice. Microarray analyses were performed using samples taken before and after intravenous infusion of alpha-galactosidase A. The identified genes were validated using quantitative real-time PCR and Western blot assay. Expression of hepatic Serum Amyloid A1 (Saa1), S100 Calcium-binding protein A8 and A9 (S100a8 and a9), and Lipocalin 2 (Lcn2) and renal Neuropeptide Y (Npy), Thrombospondin 2 and 4 (Tsp-2 and -4) was significantly upregulated in Fabry mice compared with wild-type mice and normalized by enzyme replacement therapy. Plasma concentrations of Lcn2 and Npy were also greater in Fabry mice and reduced to wild-type levels after enzyme replacement therapy, although the plasma concentrations of these proteins show heterogeneity. Upregulation of Saa1, S100a8, S100a9 and Lcn2 may modulate inflammation and Lcn2, Npy and Tsp may be associated with vascular and renal involvement in Fabry disease. Furthermore, these genes are promising targets for developing biomarkers for monitoring disease progression and therapeutic efficacy in patients with Fabry disease.

Psoriasis is one of the most common immune‑mediated inflammatory diseases of the skin. The identification of the pivotal molecular mechanisms responsible for the disease pathogenesis may lead to the development of novel therapeutic options. The present study aimed to identify pivotal differentially expressed genes (DEGs) and methylated DEGs in psoriasis. The raw data from gene microarrays were obtained from the Gene Expression Omnibus database. The data were processed using packages in Bioconductor. In total, 352 upregulated and 137 downregulated DEGs were identified. The upregulated DEGs were primarily enriched in the 'innate immune defense' response and the 'cell cycle'. The downregulated DEGs were primarily enriched in 'cell adhesion' and 'tight junction pathways'. A total of 95 methylated DEGs were identified, which were significantly enriched in the 'interleukin (IL)‑17 signaling pathway' and the 'response to interferon'. Based on a comprehensive evaluation of all algorithms in cytoHubba, the key epigenetic‑associated hub genes (S100A9, SELL, FCGR3B, MMP9, S100A7, IL7R, IRF7, CCR7, IFI44, CXCL1 and LCN2) were screened out. In order to further validate these genes, the present study constructed a model of imiquimod (IMQ)‑induced psoriasiform dermatitis using mice. The levels of these hub genes were increased in the IMQ group. The knockdown of methylation‑regulating enzyme ten‑eleven translocation (TET) 2 expression in mice attenuated the expression levels of S100A9, SELL, IL7R, MMP9, CXCL1 and LCN2. Furthermore, the hydroxymethylated level of S100A9 was highly expressed in the IMQ group and was significantly decreased by TET2 deficiency in mice. On the whole, using an integrative system bioinformatics approach, the present study identified a series of characteristic enrichment pathways and key genes that may serve as potential biomarkers in psoriasis.

Among Alzheimer's disease (AD) brain hallmarks, the presence of reactive astrocytes was demonstrated to correlate with neuronal loss and cognitive deficits. Evidence indeed supports the role of reactive astrocytes as mediators of changes in neurons, including synapses. However, the complexity and the outcomes of astrocyte reactivity are far from being completely elucidated. Another key role in AD pathogenesis is played by alterations in brain cholesterol metabolism. Oxysterols (cholesterol oxidation products) are crucial for brain cholesterol homeostasis, and we previously demonstrated that changes in the brain levels of various oxysterols correlate with AD progression. Moreover, oxysterols have been shown to contribute to various pathological mechanisms involved in AD pathogenesis. In order to deepen the role of oxysterols in AD, we investigated whether they could contribute to astrocyte reactivity, and consequently impact on neuronal health. Results showed that oxysterols present in mild or severe AD brains induce a clear morphological change in mouse primary astrocytes, accompanied by the upregulation of some reactive astrocyte markers, including lipocalin-2 (Lcn2). Moreover, astrocyte conditioned media analysis revealed a significant increase in the release of Lcn2, cytokines, and chemokines in response to oxysterols. A significant reduction of postsynaptic density protein 95 (PSD95) and a concurrent increase in cleaved caspase-3 protein levels have been demonstrated in neurons co-cultured with oxysterol-treated astrocytes, pointing out that mediators released by astrocytes have an impact on neurons. Among these mediators, Lcn2 has been demonstrated to play a major role on synapses, affecting neurite morphology and decreasing dendritic spine density. These data demonstrated that oxysterols present in the AD brain promote astrocyte reactivity, determining the release of several mediators that affect neuronal health and synapses. Lcn2 has been shown to exert a key role in mediating the synaptotoxic effect of oxysterol-treated astrocytes.

Keywords: Alzheimer's disease; Astrocyte reactivity; Astrocytes; Lipocalin-2; Oxysterols; Synaptotoxicity.

OBJECTIVE

Despite the benefits of recombinant t-PA (rt-PA) for stroke patients some of them suffer from adverse hemorrhagic transformations (HTs) following treatment. Our objective is to study the transcriptomics of HTs patients.

METHODS

We studied by microarrays 11 blood samples from patients with stroke that had received rt-PA of whom six of them had suffered a HT. For replication step RNA was collected from 14 new subjects (seven with HT, seven without) and then analyzed by real-time PCR. Four proteins were measured by ELISA in 72 new subjects to analyze their role as potential protein biomarkers.

RESULTS

The microarray analysis revealed that 14 genes were altered among the HT patients. The replication study confirmed these results for six genes. Two of them (BCL2 and OLFM4) are associated with apoptosis, whereas the other four (LTF, LCN2 [also known as NGAL], CEACAM8 and CRISP3) are involved in the regulation of neutrophil processes.

CONCLUSIONS

Our data revealed that genes related to apoptosis and neutrophil regulation pathways could be associated with HTs after rt-PA.

To characterize the changes in global gene expression in the distal colon of constipated SD rats in response to the laxative effects of aqueous extracts of Liriope platyphylla (AEtLP), including isoflavone, saponin, oligosaccharide, succinic acid and hydroxyproline, the total RNA extracted from the distal colon of AEtLP-treated constipation rats was hybridized to oligonucleotide microarrays. The AEtLP treated rats showed an increase in the number of stools, mucosa thickness, flat luminal surface thickness, mucin secretion, and crypt number. Overall, compared to the controls, 581 genes were up-regulated and 216 genes were down-regulated by the constipation induced by loperamide in the constipated rats. After the AEtLP treatment, 67 genes were up-regulated and 421 genes were down-regulated. Among the transcripts up-regulated by constipation, 89 were significantly down-regulated and 22 were recovered to the normal levels by the AEtLP treatment. The major genes in the down-regulated categories included Slc9a5, klk10, Fgf15, and Alpi, whereas the major genes in the recovered categories were Cyp2b2, Ace, G6pc, and Setbp1. On the other hand, after the AEtLP treatment, ten of these genes down-regulated by constipation were up-regulated significantly and five were recovered to the normal levels. The major genes in the up-regulated categories included Serpina3n, Lcn2 and Slc5a8, whereas the major genes in the recovered categories were Tmem45a, Rerg and Rgc32. These results indicate that several gene functional groups and individual genes as constipation biomarkers respond to an AEtLP treatment in constipated model rats.

BACKGROUND

Streptococcus pneumoniae is the leading cause of bacterial pneumonia worldwide. Previous reports showed that IL-20 cytokines (IL-19, IL-20 and IL-24) are induced and have an immuno-regulatory function during cutaneous infection. In the current study, our aim was to demonstrate the implication of IL-20 cytokines and their receptors and their role during experimental pneumococcal infection.

METHODS

C57BL/6 mice were infected with S. pneumoniae by intranasal route. The bacterial burden, the immune response and the cytokine production were evaluated after treatment with an anti-IL-20 receptor-b (IL-20Rb) neutralizing antibody (anti-IL-20Rb).

RESULTS

Of interest, expression of IL-20 cytokines mRNA and protein were transiently increased in the lung tissue during infection. Blocking of the IL-20Rb decreased the bacterial burden both in the bronchoalveolar lavage and the lung whereas there was no significant drop in the blood. This treatment also reduced the pulmonary damages (as shown by the alveolar wall thickening), the recruitment of neutrophils and dendritic cells, and the levels of the pro-inflammatory cytokines IL-1β and IL-6 in the lung. Administration of the anti-IL-20Rb antibody enhanced the synthesis of the antibacterial peptide LCN2. However, this effect is transient and did not affect the survival of the infected mice.

CONCLUSIONS

Collectively, this study highlights the implication of IL-20 related cytokines during lung infection by S. pneumoniae and might have therapeutic applications in bacterial pneumonia.

UNASSIGNED

This work was supported by CNRS, INSERM, INSERM-transfert, the University of Lille and the Fondation du Souffle (Paris, France).

Prostate cancer is one of the leading causes of cancer mortality in men worldwide. An unusual but unique environment for studying tumor cell processes is provided by microgravity, either in space or simulated by ground-based devices like a random positioning machine (RPM). In this study, prostate adenocarcinoma-derived PC-3 cells were cultivated on an RPM for time periods of 3 and 5 days. We investigated the genes associated with the cytoskeleton, focal adhesions, extracellular matrix, growth, survival, angiogenesis, and metastasis. The gene expression of signaling factors of the vascular endothelial growth factor (VEGF), mitogen-activated protein kinase (MAPK), and PI3K/AKT/mTOR (PAM) pathways was investigated using qPCR. We performed immunofluorescence to study the cytoskeleton, histological staining to examine the morphology, and a time-resolved immunofluorometric assay to analyze the cell culture supernatants. When PC-3 cells were exposed to simulated microgravity (s-µg), some cells remained growing as adherent cells (AD), while most cells detached from the cell culture flask bottom and formed multicellular spheroids (MCS). After 3-day RPM exposure, PC-3 cells revealed significant downregulation of the VEGF, SRC1, AKT, MTOR, and COL1A1 gene expression in MCS, whereas FLT1, RAF1, MEK1, ERK1, FAK1, RICTOR, ACTB, TUBB, and TLN1 mRNAs were not significantly changed. ERK2 and TLN1 were elevated in AD, and FLK1, LAMA3, COL4A5, FN1, VCL, CDH1, and NGAL mRNAs were significantly upregulated in AD and MCS after 3 days. After a 5-day culture in s-µg, the PC-3 cells showed significant downregulations of VEGF mRNA in AD and MCS, and FN1, CDH1, and LAMA3 in AD and SCR1 in MCS. In addition, we measured significant upregulations in FLT1, AKT, ERK1, ERK2, LCN2, COL1A1, TUBB, and VCL mRNAs in AD and MCS, and increases in FLK1, FN1, and COL4A5 in MCS as well as LAMB2, CDH1, RAF1, MEK1, SRC1, and MTOR mRNAs in AD. FAK1 and RICTOR were not altered by s-µg. In parallel, the secretion rate of VEGFA and NGAL proteins decreased. Cytoskeletal alterations (F-actin) were visible, as well as a deposition of collagen in the MCS. In conclusion, RPM-exposure of PC-3 cells induced changes in their morphology, cytoskeleton, and extracellular matrix protein synthesis, as well as in their focal adhesion complex and growth behavior. The significant upregulation of genes belonging to the PAM pathway indicated their involvement in the cellular changes occurring in microgravity.

Background: Recent clinical and basic research implicated a strong correlation between NAFLD/NASH phenotypes with ectopic manifestations including neuroinflammation and neurodegeneration, but the mediators and critical pathways involved are not well understood. Lipocalin 2 (Lcn2) is one of the important mediators exclusively produced in the liver and circulation during NASH pathology.

Methods: Using murine model of NASH, we studied the role of Lcn2 as a potent mediator of neuroinflammation and neurodegeneration in NASH pathology via the liver-brain axis.

Results: Results showed that high circulatory Lcn2 activated 24p3R (Lipocalin2 receptor) in the brain and induced the release of high mobility group box 1 (HMGB1) preferably from brain cells. Released HMGB1 acted as a preferential ligand to toll-like receptor 4 (TLR4) and induced oxidative stress by activation of NOX-2 signaling involving activated p65 protein of the NF-κB complex. Further, the HMGB1-derived downstream signaling cascade activated NLRP3 inflammasome and release of proinflammatory cytokines IL-6 and IL-1β from brain cells. In addition, to advance our present understanding, in vitro studies were performed in primary brain endothelial cells where results showed high circulatory Lcn2 influenced HMGB1 secretion. Mechanistically, we also showed that elevated Lcn2 level in underlying NASH might be a likely cause for induction of blood-brain barrier dysfunction since the adipokine decreased the expression of tight junction protein Claudin 5 and caused subsequent elevation of pro-inflammatory cytokines IL-6 and IL-1β.

Conclusion: In conclusion, the NASH-induced brain pathology might be because of increased Lcn2-induced release of HMGB1 and accompanying neuroinflammation.

Keywords: Lcn-2; MCD; NASH; NLRP3; NOX-2; Redox signaling; TLR4.

BACKGROUND

Neutrophil gelatinase-associated lipocalin (NGAL or LCN2) is an iron-transporting factor which possesses various activities such as amelioration of kidney injury and host defense against pathogens. Its circulating concentrations are elevated in acute and chronic kidney diseases and show a positive correlation with poor renal outcome and mortality, but its clinical significance in maintenance hemodialysis (HD) patients remains elusive.

METHODS

Serum NGAL levels were determined by enzyme-linked immunosorbent assay in out-patient, Japanese HD subjects. Their correlation to laboratory findings and morbidity (as development of severe infection or serum albumin reduction) was investigated using linear regression analysis and χ2 test.

RESULTS

Pre-dialysis serum NGAL levels in HD patients were elevated by 13-fold compared to healthy subjects (n=8, P<0.001). In a cross-sectional study of 139 cases, serum NGAL concentrations were determined independently by % creatinine generation rate (an indicator of muscle mass, standardized coefficient β=0.40, P<0.001), peripheral blood neutrophil count (β=0.38, P<0.001) and anion gap (which likely reflects dietary protein intake, β=0.16, P<0.05). Iron administration to anemic HD patients caused marked elevation of peripheral blood hemoglobin, serum ferritin and iron-regulatory hormone hepcidin-25 levels, but NGAL levels were not affected. In a prospective study of 87 cases, increase in serum albumin levels a year later was positively associated to baseline NGAL levels by univariate analysis (r=0.36, P<0.01). Furthermore, within a year, patients with the lowest NGAL tertile showed significantly increased risk for marked decline in serum albumin levels (≥0.4 g/dl; odds ratio 5.5, 95% confidence interval 1.5-20.3, P<0.05) and tendency of increased occurrence of severe infection requiring admission (odds ratio 3.1, not significant) compared to the middle and highest tertiles.

CONCLUSIONS

Low serum NGAL levels appear to be associated with current malnutrition and also its progressive worsening in maintenance HD patients.

Lipocalin-2 (LCN2) is a stress protein, and can be hyper-produced by many kinds of cells after exposure to injury or disease conditions. In this study, we asked whether LCN2 may play a protective role in cerebral endothelium. After focal cerebral ischemia in rats, plasma levels of LCN2 were significantly elevated at 6, 12, and 24 hrs, and persisted until 3 days post-stroke. To assess the vascular mechanisms of LCN2, we used brain endothelial cell cultures to investigate its effects on neutrophil adhesion and endothelial barrier integrity. LCN2 did not affect neutrophil adhesion to endothelial cells either under normal conditions or after TNFα stimulation. TNFα significantly increased endothelial permeability, and LCN2 rescued endothelial permeability. Concomitantly, LCN2 restored the membrane distribution of the tight junction protein ZO-1 and the adherens junction protein VE-cadherin. Our findings suggest that elevated LCN2 in the blood after ischemic stroke might affect endothelial function, in part by reducing damage to endothelial junctional proteins and maintain blood-brain barrier integrity.

Non-small cell lung cancer (NSCLC) represents approximately 85% of all lung cancers, which are the leading cause of cancer-related deaths in the world. Tyrosine kinase inhibitors such as erlotinib represent one therapeutic options presently recommended for tumors produced by activating mutations in the gene coding of epidermal growth factor receptor (EGFR). The aim of this study is the identification of possible biomarkers for tumor sensitivity to erlotinib in the absence of the main EGFR mutations. The erlotinib sensitivity of cells isolated from 41 untreated NSCLC patients was determined and compared with the presence of the more frequent EGFR mutations. Several patients had tumor cells highly sensitive to erlitinib in the absence of the EGFR mutations analyzed. The gene expression profile of 3 erlotinib-sensitive tumors was compared with that of 4 resistant tumors by DNA microarray hybridization. Sixteen genes were expressed at significantly higher levels in the resistant tumors than in the sensitive tumors. The possible correlation between erlotinib sensitivity and the expression of these genes was further analyzed using the data for the NSCLC, breast cancer and colon cancer cell lines of the NCI60 collection. The expression of these genes was correlated with the overall survival of 5 patients treated with erlotinib, according to The Cancer Genome Atlas (TCGA) database. Overlapping groups of 7, 5 and 3 genes, including UGT1A6, TRIB3, MET, MMP7, COL17A1, LCN2 and PTPRZ1, whose expression correlated with erlotinib activity was identified. In particular, low MET expression levels showed the strongest correlation.

Lipocalin-2 (LCN2) is a secreted molecule, expressed in various cell types, that is involved in the progression of numerous diseases and disorders. The biological functions and expression levels of LCN2 in diseases including pancreatic cancer, pancreatitis (acute and chronic), and diabetes mellitus, suggest the potential role of LCN2 as a biomarker and/or therapeutic target. However, findings on the role of LCN2 in pancreatic diseases have been contradictory. In pancreatic cancer and pancreatitis, LCN2 has been identified as a potential biomarker; increased expression levels in various biological specimens correlate with the presence of the disease and may be able to differentiate cancer and chronic pancreatitis from healthy subjects. LCN2 is also known to be an adipokine; it is upregulated in obesity and is a common co-factor in the development of pancreatic diseases. Emerging research suggests LCN2 is elevated in type 2 diabetes mellitus, but the exact role of LCN2 in this disease is not clear. In this review, we summarize research on LCN2 as it relates to pancreatic diseases, highlighting the discrepancies in the literature. By explaining and clarifying the role of LCN2 in these disorders, we aim to promote research in developing novel diagnostic and treatment strategies to reduce the burden of pancreatic diseases.