OBJECTIVE

A series of mechanisms of immune response, inflammation and apoptosis have been demonstrated to contribute to the appearance and evolution of Crohn's disease (CD) through the overexpression of several cytokines and chemokines in a susceptible host. The aim of this study was to identify the differences in gene expression profiles analyzing a panel of candidate genes in the mucosa from patients with active CD (CD-A), patients in remission (CD-R), and normal controls.

METHODS

Nine individuals were enrolled in the study: six CD patients (three with active lesions, three with mucosal healing) and three controls without inflammatory bowel disease (IBD) seen on endoscopy. All the individuals underwent mucosal biopsy during colonoscopy. Gene expression levels of 84 genes previously associated with CD were evaluated by polymerase chain reaction (PCR) array.

RESULTS

Ten genes out of 84 were found significantly differentially expressed in CD-A (CCL11, CCL25, DEFA5, GCG, IL17A, LCN2, REG1A, STAT3, MUC1, CCR1) and eight genes in CD-R (CASP1, IL23A, STAT1, STAT3, TNF, CCR1, CCL5, and HSP90B1) when compared to controls. A quantitative gene expression analysis revealed that CCR1 gene was more expressed in CD-A than in CD-R.

CONCLUSIONS

Our data suggest that CCR1 gene may be a putative marker of molecular activity of Crohn's disease. Following these preliminary data, a confirmation in larger cohort studies could represent a useful method in order to identify new therapeutic targets.

Recent evidence demonstrated that Lipocalin 2 (LCN2) is garnering interest from a wide spectrum as biomarker. Here, we present an in silico characterization of LCN2 belonging to prominent organisms focusing for their physicochemical and structural differences. We found significant variations in physicochemical properties between organisms and low sequence similarity based on their amino acid properties alone. However, we identified three main structurally distinct motif regions that are conserved among all variants. Further investigation was carried out to understand the functional insights of LCN2. We selected LCN2 sequence from Gallus gallus as an input query to identify unique scoring-framework based on computational tools and confidence scores of various putative associations. Among all ten proteins associated with LCN2; highest confidence of prediction were seen for the associations between LCN2 and metalloproteinase 9 (MMP9) and lipoprotein receptor-related protein 2 (LRP2) which play vital roles in tumor growth and iron transportation, respectively. We attempted to determine binding affinities of LCN2 with MMP9 and LRP2 through molecular modeling and docking. Selected docked models were examined for complex stability by GROMACS. Alteration of binding affinity between LCN2 with MMP9 and LRP2 proteins that we found in this study may provide new direction for future therapeutic targets.

Lipocalin 2 (Lcn2) is an important innate immunity component against bacterial pathogens. In this study, we report that Lcn2 is induced by Brucella (B.) abortus infection and significantly contributes to the restriction of intracellular survival of Brucella in macrophages. We found that Lcn2 prevented iron uptake by B. abortus through two distinct mechanisms. First, Lcn2 is secreted to capture bacterial siderophore(s) and abrogate iron import by Brucella. Second, Lcn2 decreases the intracellular iron levels during Brucella infection, which probably deprives the invading Brucella of the iron source needed for growth. Suppression of Lcn2 signalling resulted in a marked induction of anti-inflammatory cytokine, interleukin 10, which was shown to play a major role in Lcn2-induced antibrucella immunity. Similarly, interleukin 6 was also found to be increased when Lcn2 signalling is abrogated; however, this induction was thought to be an alternative pathway that rescues the cell from infection when the effective Lnc2 pathway is repressed. Furthermore, Lcn2 deficiency also caused a marked decrease in brucellacidal effectors, such as reactive oxygen species and nitric oxide but not the phagolysosome fusion. Taken together, our results indicate that Lcn2 is required for the efficient restriction of intracellular B. abortus growth that is through limiting iron acquisition and shifting cells to pro-inflammatory brucellacidal activity in murine macrophages.

Malaria is still a major health problem worldwide. This study aimed to investigate the hepatoprotective role of Indigofera oblongifolia leaf extracts (ILE) against mice hepatic injury induced by Plasmodium chabaudi. Female C57BL/6 mice were treated with 100 mg/kg of ILE after infection with erythrocytes parasitized by P. chabaudi. On day 7 post-infection, the extract improved the histological alteration induced by the parasite. This was evidenced by the decreased histological index induced by ILE. Moreover, ILE was able to increase the hepatic antioxidant capacity and could significantly improve the decrease in erythrocyte count and hemoglobin content in mice blood plasma due to infection. ILE was also able to upregulate the expression of 24 genes related to metabolism and of 3 genes related to the immune response. Furthermore, the extract was able to downregulate the expression of 35 genes related to metabolism and of 82 genes related to immune response. Moreover, the microarray study showed that ILE regulated the change in gene expression induced by the parasite. Among these genes, we quantified the expression of cd209f, cyp7a1, Hsd3b5, Sult2a3, Lcn2, CcI8, Nos2, and saa3-mRNAs. These genes were regulated by ILE. Therefore, our results revealed the protective role of Indigofera oblongifolia against hepatic injury induced by blood stage malaria.

BACKGROUND

Allergy is often accompanied by infections and lower levels of antimicrobial peptides (AMPs). Vitamin D has been shown to increase expression of selected AMPs. In this study we investigated whether antimicrobial peptide levels in nasal secretions of allergic asthma patients are lower than in healthy controls, and whether administration of the active form of vitamin D (1,25(OH)2D3) affects these antimicrobial peptide levels.

METHODS

The levels of antimicrobial peptides in nasal secretions were compared between 19 allergic asthma patients and 23 healthy controls. The effect of seven days daily oral treatment with 2 μg 1,25(OH)2D3 on antimicrobial peptides in nasal secretions was assessed in a placebo-controlled cross-over clinical study.

RESULTS

Levels of neutrophil α-defensins (human neutrophil peptides 1-3; HNP1-3) and lipocalin 2 (LCN2; also known as NGAL) were significantly lower in asthmatics, but no differences in LL-37 and SLPI were detected. Treatment with a short-term 1,25(OH)2D3 caused a small increase in HNP1-3, but not when the asthma and control groups were analyzed separately. LL-37, LCN2 and SLPI did not change after treatment with 1,25(OH)2D3.

CONCLUSIONS

Levels of the antimicrobial peptides HNP1-3 and LCN2 are lower in nasal secretions in asthmatics and are not substantially affected by a short-term treatment with active vitamin D.

Lipocalin-2 (LCN2), a multiple bioactive hormone particularly expressed in adipose tissue, neutrophils, and macrophages, is known to exhibit anti-microbial effect, increase inflammatory cytokine levels, and maintain glucose homeostasis. Serum LCN2 level is positively correlated with the severity of coronary artery disease. However, it still remains unknown whether LCN2 affects atherogenesis. We assessed the effects of LCN2 on the inflammatory response and monocyte adhesion in human umbilical vein endothelial cells (HUVECs), inflammatory phenotype and foam cell formation in THP1 monocyte-derived macrophages, and migration and proliferation of human aortic smooth muscle cells (HASMCs) in vitro and aortic lesions in Apoe^-/- mice in vivo. LCN2 and its receptor, low-density lipoprotein (LDL)-related protein-2, were expressed in THP1 monocytes, macrophages, HASMCs, and HUVECs. LCN2 significantly enhanced THP1 monocyte adhesion to HUVECs accompanied with upregulation of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin associated with nuclear factor-κB (NF-κB) upregulation in HUVECs. LCN2 significantly increased HUVEC proliferation and oxidized LDL-induced foam cell formation in THP1 monocyte-derived macrophages. LCN2 significantly increased the inflammatory M1 phenotype associated with NF-κB upregulation during differentiation of THP1 monocytes into macrophages. In HASMCs, LCN2 significantly promoted the migration and collagen-1 expression without inducing proliferation, which are associated with increased protein expression of phosphoinositide 3-kinase and phosphorylation of Akt, extracellular signal-regulated kinase, c-jun-N-terminal kinase, and NF-κB. Chronic LCN2 infusion into Apoe^-/- mice significantly accelerated the development of aortic atherosclerotic lesions, with increased intraplaque monocyte/macrophage infiltration and pentraxin-3 and collagen-1 expressions. Our results suggested that LCN2 accelerates the development of atherosclerosis. Thus, LCN2 could serve as a novel therapeutic target for atherosclerotic diseases.

Although some forms of phospholipase A2, the initiator of the arachidonic acid cascade, contribute to carcinogenesis in many organs, the contribution of phospholipase A2 group IVc (Pla2g4c) remains to be clarified and the function of the enzyme in cancer development is unknown. The Hirosaki hairless rat (HHR), a mutant rat strain with autosomal recessive inheritance, derived spontaneously from the Sprague-Dawley rat (SDR). The HHRs showed a lower incidence and much smaller volume of mammary tumours induced by 7,12-dimethylbenz[a]anthracene, and a markedly increased number of TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling)-positive apoptotic cells was detected. Array comparative genomic hybridization and PCR analyses revealed the deletion of 50-kb genomic DNA on 1q21, including Pla2g4c, in HHRs. The Pla2g4c gene was expressed in the ductal carcinoma cells and myoepithelial cells in SDRs, but not in HHRs. The direct involvement of Pla2g4c in the prevention of cell death was demonstrated through the inhibition of its expression in rat mammary tumour RMT-1 cells using siRNA. This treatment also induced expression of lipocalin 2 (Lcn2) and other NF-κB (nuclear factor κB)-related genes. siRNA-induced apoptosis was inhibited by Lcn2 repression or NF-κB inhibitors. This is the first report on Pla2g4c gene-deficient rats and their low susceptibility to mammary carcinogenesis by enhancing NF-κB/Lcn2-induced apoptosis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis, which is a demyelinating and an inflammatory disease of central nervous system. Recent studies have established that some molecules such as Lipocaline2 (LCN2), which expresses during inflammatory conditions, play an important role in EAE pathogenesis and might involve in its treatment process. Recently, it has been proved that MS14, an herbal-marine drug, has anti-inflammatory properties through reduction of TNF-α and IL-1β. Thus, the present study investigated the effects of MS14 on the course of EAE and its relation to LCN2 expression in both protein and gene levels.

EAE was induced in female C57BL/6 mice using Hooke kits. Animals were scored for clinical signs of the disease according to a 10-point EAE scoring system. On 21st and 35th days after immunization, mice (n = 4/group) were deeply anesthetized, and the spinal cords were removed. Inflammatory cell infiltration and LCN2 expression in spinal cord were assessed by hematoxylin and eosin staining, immuno-histochemistry, and real-time PCR methods.

MS14 significantly ameliorated EAE symptoms and decreased lymphocyte infiltration into the spinal cord (P<0.05). Our data also revealed that LCN2 expression was significantly down-regulated in acute and chronic phases of EAE both at protein and gene levels after MS14 treatment (P<0.05).

The results demonstrated that MS14 regulatory effect on EAE is accompanied by LCN2 down-regulation after treatment with the herb; however, more studies are required for clarifying the other involved mechanisms.

Smoking is the major cause of lung cancer and the leading cause of cancer-related death in the world. The most current view about lung cancer is no longer limited to individual genes being mutated by any carcinogenic insults from smoking. Instead, tumorigenesis is a phenotype conferred by many systematic and global alterations, leading to extensive heterogeneity and variation for both the genotypes and phenotypes of individual cancer cells. Thus, strategically it is foremost important to develop a methodology to capture any consistent and global alterations presumably shared by most of the cancerous cells for a given population. This is particularly true that almost all of the data collected from solid cancers (including lung cancers) are usually distant apart over a large span of temporal or even spatial contexts. Here, we report a multiple non-Gaussian graphical model to reconstruct the gene interaction network using two previously published gene expression datasets. Our graphical model aims to selectively detect gross structural changes at the level of gene interaction networks. Our methodology is extensively validated, demonstrating good robustness, as well as the selectivity and specificity expected based on our biological insights. In summary, gene regulatory networks are still relatively stable during presumably the early stage of neoplastic transformation. But drastic structural differences can be found between lung cancer and its normal control, including the gain of functional modules for cellular proliferations such as EGFR and PDGFRA, as well as the lost of the important IL6 module, supporting their roles as potential drug targets. Interestingly, our method can also detect early modular changes, with the ALDH3A1 and its associated interactions being strongly implicated as a potential early marker, whose activations appear to alter LCN2 module as well as its interactions with the important TP53-MDM2 circuitry. Our strategy using the graphical model to reconstruct gene interaction work with biologically-inspired constraints exemplifies the importance and beauty of biology in developing any bio-computational approach.

The molecular mechanisms underlying salivary gland tumorigenesis remain unclear. In order to identify genetic changes that occur during the development of invasive adenocarcinoma from normal salivary gland, we used the Smgb-Tag transgenic mouse model. This transgene induces the progressive development of dysplasia to invasive adenocarcinoma in the submandibular salivary gland. Gene expression patterns from 20 submandibular glands (two normal, nine dysplasia and nine adenocarcinoma samples) were assessed using a mouse 15 K cDNA array. Unsupervised hierarchical clustering was used to group gene expression based on 157 differentially expressed genes distinguishing between dysplasias and adenocarcinomas. Further analysis identified 25 significantly overexpressed and 28 underexpressed cDNA sequences in adenocarcinoma as compared to dysplasia. Differential expression of five genes (Lcn2, Ptn, Cd24a, Mapk6 and Rnps1) was validated by quantitative real-time RT-PCR in a total of 48 mouse salivary gland tissues (seven histologically normal, 13 dysplasias and 28 adenocarcinomas), including the 20 samples analyzed by cDNA arrays. Immunohistochemical analysis was used to validate the expression of Ptn and Cd24a at the protein level in a subset of 16 mouse salivary glands (four normal, five dysplasia and seven adenocarcinoma samples), as well as in 23 human submandibular gland tumors (16 pleomorphic adenomas, three adenoid cystic carcinomas, one acinic cell carcinoma, one adenocarcinoma NOS, one myoepithelial and one mucoepidermoid carcinoma). We thus demonstrated that the Smgb-Tag transgenic mouse model is a useful tool for the identification of genes that are deregulated in salivary gland adenocarcinomas. Our data suggest that Ptn and Cd24a may be genetic markers associated with salivary gland tumorigenesis and/or progression.

BACKGROUND

Lipocalin-2 (LCN2), a small glycoprotein, has a pivotal role in diverse biological processes such as cellular proliferation and differentiation. We previously reported that LCN2 is implicated in osteoclast formation induced by receptor activator of nuclear factor-kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). In the present study, we used a knockout mouse model to further investigate the role of LCN2 in osteoclast development.

METHODS

Osteoclastogenesis was assessed using primary bone marrow-derived macrophages. RANKL and M-CSF signaling was determined by immunoblotting, cell proliferation by bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA), and apoptosis by cell death detection ELISA. Bone morphometric parameters were determined using a micro-computed tomography system.

RESULTS

Our results showed that LCN2 deficiency increases tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclast formation in vitro, a finding that reflects enhanced proliferation and differentiation of osteoclast lineage cells. LCN2 deficiency promotes M-CSF-induced proliferation of bone marrow macrophages (BMMs), osteoclast precursors, without altering their survival. The accelerated proliferation of LCN2-deficient precursors is associated with enhanced expression and activation of the M-CSF receptor, c-Fms. Furthermore, LCN2 deficiency stimulates the induction of c-Fos and nuclear factor of activated T cells c1 (NFATc1), key transcription factors for osteoclastogenesis, and promotes RANKL-induced inhibitor of kappa B (IκBα) phosphorylation. Interestingly, LCN2 deficiency does not affect basal osteoclast formation in vivo, suggesting that LCN2 might play a role in the enhanced osteoclast development that occurs under some pathological conditions.

CONCLUSIONS

Our study establishes LCN2 as a negative modulator of osteoclast formation, results that are in accordance with our previous findings.

The data herein is related to the research article entitled "Microbiota-inducible innate immune siderophore binding protein Lipocalin 2 is critical for intestinal homeostasis" (Singh et al., 2016) [1]. In the present article, we monitored dextran sodium sulfate (DSS)-induced colitis development upon Lipocalin 2 (Lcn2) neutralization, and examined the survival of Lcn2 deficient (Lcn2KO) mice and their WT littermates upon DSS challenge. To dissect the relative contribution of immune and non-immune cells-derived Lcn2 in mediating protection against gut inflammation, we generated respective bone marrow chimera and evaluated their susceptibility to IL-10 receptor neutralization-induced chronic colitis. Neutralization of Lcn2 in WT mice resulted in exacerbated DSS-induced colitis. Notably, mice lacking Lcn2 exhibited 100% mortality whereas only 20% mortality was observed in WT mice upon DSS challenge. Further, data from bone marrow chimera showed that immune cell-derived Lcn2 is the major contributor in conferring protection against colitis.

BACKGROUND

The aim of this study was to investigate the differential plasma levels of lipocalin 2 (LCN2) and its complex with MMP-9 (where MMP is matrix metalloproteinase) before and after antibiotic treatment in hospitalized adult patients with community-acquired pneumonia (CAP).

METHODS

Plasma LCN2 and LCN2/MMP-9 complex levels were measured in 61 adult patients with CAP and 60 healthy controls using commercial enzyme-linked immunosorbent assay (ELISA).

RESULTS

A decrease in the number of white blood cells (WBCs) and neutrophils and decreases in the levels of C-reactive protein (CRP), LCN2, and LCN2/MMP-9 complex were observed after antibiotic treatment. The plasma level of LCN2, but not that of CRP, was correlated with the severity of CAP based on the Pneumonia Severity Index (PSI; r = 0.333, P = 0.009), confusion, urea, respiratory rate and blood pressure (CURB)-65 (r = 0.288, P = 0.024), and Acute Physiology And Chronic Health Evaluation II (APACHE II) scores (r = 0.328, P = 0.010). LCN2 levels were also significantly correlated with LCN2/MMP-9 levels and the numbers of WBCs or neutrophils.

CONCLUSIONS

Plasma levels of LCN2 and the LCN2/MMP-9 complex can act as adjuvant diagnostic biomarkers for CAP. Plasma LCN2 might play a further role in the clinical assessment of the severity of CAP, which could potentially guide the development of future treatment strategies.

Background: Lipocalin-2 (LCN2) is a novel adipokine with potential roles in obesity, insulin resistance, and inflammation. This study aims to assess the concentrations of LCN2 and vascular endothelial growth factor (VEGF) expressed in the vitreous humors of patients with proliferative diabetic retinopathy (PDR).

Methods: The concentrations of LCN2 and VEGF were measured from the vitreous of 67 patients undergoing vitrectomy (20 controls and 47 PDR) via enzyme-linked immunosorbent assay (ELISA). Patients with non-ocular pathology that could elevate the LCN2 level in the vitreous were excluded. PDR activity and a history of panretinal photocoagulation were used for further grouping analysis.

Results: The vitreous concentration of LCN2 was statistically significantly higher in the PDR group compared to the control group (63,522 (30,009) pg/ml versus 1663 (1191) pg/ml, respectively; P < 0.001). VEGF level was also significantly higher in the PDR group than in the control group (1038 (1326) pg/ml versus 9 pg/ml, respectively; P < 0.001). The mean vitreous LCN2 and VEGF levels in active PDR patients were significantly higher than that of the inactive PDR patients. The mean LCN2 concentration in vitreous humor was significantly lower in the 28 PDR patients with a history of complete PRP (37,304 (16,651) pg/mL) in comparison with 19 PDR patients without preperformed panretinal photocoagulation or with preperformed incomplete panretinal photocoagulation (79,796 (24,391) pg/mL). A significant correlation between the vitreous LCN2 level and VEGF level was found in patients with PDR (R = 0.34; P = 0.019).

Conclusions: This report shows a significant increase of LCN2 in the vitreous fluid of patients with PDR and present a significant correlation between LCN2 and VEGF, suggesting LCN2 might be involved in the pathogenesis of PDR.

Keywords: Diabetic retinopathy; Lipocalin-2; Vascular endothelial growth factor.

The adipokine lipocalin 2 (LCN2) is linked to insulin resistance. Its expression in human adipose tissue (AT) can be regulated in a sex-specific manner by a synthetic glucocorticoid, dexamethasone, suggesting an underlying role of sex steroids. We show that 17-β-estradiol (E2) dose-dependently increased LCN2 gene expression in subcutaneous AT from postmenopausal women. This was also seen in the presence of estrogen receptor (ER) α antagonist alone but not with ERβ antagonist, suggesting that E2 effects on LCN2 are mediated via ERβ pathway. Dexamethasone alone or E2+dexamethasone had no significant effect on LCN2. However, E2+dexamethasone increased LCN2 expression with ERα-blockade. Dexamethasone reduced ERα but increased ERβ expression. Dexamethasone can regulate LCN2 expression via inhibition of ERα and stimulation of ERβ and may contribute to the development of glucocorticoid-induced insulin resistance in human AT. In conclusion, ERβ and ERα pathways have opposite effects on LCN2 expression and they interact with glucocorticoid action.

This study aimed to identify effective targets for carcinogenesis of primary myelofibrosis (PMF), as well as to screen ideal lead compounds with potential inhibition effect on Janus kinase 2 to contribute to the medication design and development. Gene expression profiles of GSE26049, GSE53482, GSE61629 were obtained from the Gene Expression Omnibus database. The differentially expressed genes were identified, and functional enrichment analyses such as Gene Ontology, protein-protein interaction network etc., were performed step by step. Subsequently, highly-precise computational techniques were conducted to identify potential inhibitors of JAK2. A series of structural biology methods including virtual screening, ADMET (absorption, distribution, metabolism, excretion, and toxicity) prediction, molecule docking, molecular dynamics simulation etc., were implemented to discover novel natural compounds. Results elucidated that PMF patients had abnormal LCN2, JAK2, MMP8, CAMP, DEFA4, LTF, MPO, HBD, STAT4, EBF1 mRNA expression compared to normal patients. Functional enrichment analysis revealed that these genes were mainly enriched in erythrocyte differentiation, neutrophil degranulation and killing cells of other organisms. Two novel natural compounds, ZINC000013513540 and ZINC000004099068 were found binding to JAK2 with favorable interaction energy together with high binding affinity. They were predicted with non-Ames mutagenicity, low-rodent carcinogenicity, less developmental toxicity potential as well as non-toxicity with liver. Molecular dynamics simulation demonstrated that these two complexes: ZINC000013513540-JAK2 and ZINC000004099068-JAK2 could exist stably under natural circumstances. In conclusion, this study revealed hub genes in the carcinogenesis of PMF. ZINC000013513540 and ZINC000004099068 were promising drugs in dealing with PMF. This study may also accelerate exploration of new drugs.

Keywords: Janus Kinase 2; bioinformatics; discovery studio; inhibitor; virtual screening.

Optic nerve crush (ONC) induces retinal ganglion cell (RGC) death, which causes vision loss in glaucoma. To investigate early events leading to apoptosis of RGCs, we performed gene expression analysis of injured retinas in the period before RGC loss.

The temporal changes of gene profiles at 0, 1, and 4 days after ONC were determined by DNA microarray. To verify the gene expression changes in RGCs, we enriched RGCs by laser-captured microdissection and performed real-time RT-PCR of 14 selected genes. In situ localization study was performed by immunohistochemistry.

At 1 day and 4 days after ONC, 1423 and 2010 retinal genes were changed compared with 0 day, respectively; these genes were mainly related to apoptotic process, immune process, regulation of cell cycle, and ion transport. RT-PCR analysis revealed that expression levels of Activating transcription factor 3 (Atf3), Lipocalin 2 (Lcn2), and tumor necrosis factor receptor superfamily member 12a (Tnfrsf12a) were remarkably changed in RGC-enriched fraction within 4 days postcrush. Immunohistochemical analysis confirmed that all of these genes expressed highly in the ganglion cell layer of crushed retinas.

In response to ONC, the expression of apoptotic genes was stimulated soon after crush. Atf3, Lcn2, and Tnfrsf12a might be key molecules responsible for RGC loss in glaucoma.

Calcitriol, the active form of vitamin D, is known for its anticancer properties including induction of apoptosis as well as the inhibition of angiogenesis and metastasis. Understanding the mechanisms of action for calcitriol will help with the development of novel treatment strategies. Since vitamin D exerts its cellular actions via binding to its receptor and by altering expressions of a set of genes, we aimed to evaluate the effect of calcitriol on transcriptomic profile of breast cancer cells. We previously demonstrated that calcitriol alters endoplasmic reticulum (ER) stress markers, therefore in this study we have focused on ER-stress-related genes to reveal calcitriols action on these genes in particular. We have treated breast cancer cell lines MCF-7 and MDA-MB-231 with previously determined IC50 concentrations of calcitriol and evaluated the transcriptomic alterations via microarray. During analysis, only genes altered by at least 2-fold with a P value < 0.05 were taken into consideration. Our findings revealed an ER-stress-associated transcriptomic profile induced by calcitriol. Induced genes include genes with a pro-survival function (NUPR1, DNAJB9, HMOX1, LCN2, and LAMP3) and with a pro-death function (CHOP (DDIT3), DDIT4, NDGR1, NOXA, and CLGN). These results suggest that calcitriol induces an ER-stress-like response inducing both pro-survival and pro-death transcripts in the process.

The kidney's inherent complexity has made identifying cell-specific pathways challenging, particularly when temporally associating them with the dynamic pathophysiology of acute kidney injury (AKI). Here, we combine renal cell-specific luciferase reporter mice using a chemoselective luciferin to guide the acquisition of cell-specific transcriptional changes in C57BL/6 background mice. Hydrogen peroxide generation, a common mechanism of tissue damage, was tracked using a peroxy-caged-luciferin to identify optimum time points for immunoprecipitation of labeled ribosomes for RNA-sequencing. Together, these tools revealed a profound impact of AKI on mitochondrial pathways in the collecting duct. In fact, targeting the mitochondria with an antioxidant, ameliorated not only hydrogen peroxide generation, but also significantly reduced oxidative stress and the expression of the AKI biomarker, LCN2. This integrative approach of coupling physiological imaging with transcriptomics and drug testing revealed how the collecting duct responds to AKI and opens new venues for cell-specific predictive monitoring and treatment.

BACKGROUND

The incidence of thyroid cancer is fast increasing in both adults and children. The pediatric thyroid cancer had often already progressed to a more advanced stage of the disease at diagnosis. Early detection of pediatric thyroid cancer has been a problem for many years. Lipocalin-2 (Lcn2) has been reported to be over-expressed in cancers of diverse histological origin and it facilitates tumorigenesis by promoting survival, growth, and metastasis.

METHODS

The plasma Lcn2 concentration of 28 Chinese papillary thyroid cancer (PTC) children and 24 healthy controls was measured. Immunostaining for Ki-67 of tumor tissue from PTC children was performed. The expression levels of Lcn2 and NFκB in PTC tissue and peri-carcinoma tissue of PTC children were measured through Western blot.

RESULTS

The plasma concentration of Lcn2 was significantly elevated in pediatric PTC patients compared with healthy controls. Besides, the plasma Lcn2 concentration significantly correlated with clinical characteristics, NFκB level, and Ki-67 positive rate of nucleus in tissue of PTC.

CONCLUSIONS

This is the first study to evaluate the plasma Lcn2 in pediatric PTC patients. It is possible that the plasma Lcn2 may be a new biomarker of pediatric thyroid cancer. Further studies are needed to explore the definite role and mechanism of Lcn2 in thyroid cancer, which will help to explore novel diagnostic or therapeutic strategies.

Unfolded protein response (UPR) is an adaptive mechanism allowing the endoplasmic reticulum (ER) to react to an accumulation of unfolded proteins in its lumen, also known as ER stress. The UPR is interconnected with inflammation through several pathways such as reactive oxygen species (ROS) production resulting from the protein folding or alternatively, activation of nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) via IRE1, or induction of acute phase response (APR). Lipocalin 2 (LCN2) is one of the APR proteins induced under inflammatory conditions and up-regulated during ER stress. Upon incubation of Lcn2^-/- and wild type (wt) primary hepatocytes with tunicamycin (TM) or thapsigargin (TG) we found the Lcn2^-/- hepatocytes to react with strong UPR to the ER stress, as evidenced by significantly increased levels of Grp94, Bip and Chop mRNA and protein compared to the wt. TM and TG-treated hepatocytes activated p65 NF-κB and JNK, the pathways that respond to stress stimuli and playing a central role in inflammation and apoptosis, respectively. ER stress further activated and cleaved full-length CREBH/CREB3L3, the hepatocyte specific transcription factor to induce systemic inflammatory responses. Upregulation of the C/EBP homologous protein (CHOP) was very prominent in Lcn2^-/- hepatocytes and sustained until 48 h, resulting in hepatocyte apoptosis as evidenced by increased cleaved caspase 3. We also explored the UPR of the Lcn2 null mouse livers in acute intoxication and inflammation stages with a single application of lipopolysaccharide (LPS) or carbon tetrachloride (CCl₄). The Lcn2 null mice clearly developed stronger UPR in LPS- and CCl₄-induced ER stress compared to the wt. Our findings indicate that the upregulation of LCN2 during ER stress-induced inflammatory responses protects hepatocytes from being overwhelmed by UPR upon liver injury.

The kidneys are one of the main dose-limiting organs in peptide receptor radionuclide therapy and due to large inter-individual variations in renal toxicity, biomarkers are urgently needed in order to optimize therapy and reduce renal tissue damage. The aim of this study was to investigate the transcriptional, functional, and morphological effects on renal tissue after 177Lu-octreotate administration in normal mice, and to identify biomarkers for radiation induced renal toxicity.

METHODS

C57BL/6N mice were i.v. injected with 0, 30, 60, 90, 120, or 150 MBq 177Lu-octreotate (0, 16, 29, 40, 48, and 54 Gy to the kidneys). At 4, 8, and 12 months after administration, radiation-induced effects were evaluated in relation to (a) global transcriptional variations in kidney tissues, (b) morphological changes in the kidneys, (c) changes in white and red blood cell count as well as blood levels of urea, and (d) changes in renal function using 99mTc-DTPA/99mTc-DMSA scintigraphy.

RESULTS

In general, the highest number of differentially regulated transcripts was observed at 12 months after administration. The Cdkn1a, C3, Dbp, Lcn2, and Per2 genes displayed a distinct dose-dependent regulation, with increased expression level with increasing absorbed dose. Ifng, Tnf, and Il1B were identified as primary up-stream regulators of the recurrently regulated transcripts. Furthermore, previously proposed biomarkers for kidney injury and radiation damage were also observed. The functional investigation revealed reduced excretion of 99mTc-DTPA after 150 MBq, an increased uptake of 99mTc-DMSA at all dose levels compared with the controls, and markedly increased urea level in blood after 150 MBq at 12 months.

CONCLUSIONS

Distinct dose-response relationships were found for several of the regulated transcripts. The Cdkn1a, Dbp, Lcn2, and Per2 genes are proposed as biomarkers for 177Lu-octreotate exposure of kidney. Correlations to functional and morphological effects further confirm applicability of these genes as markers of radiation damage in kidney tissue.

Kidney surface cooling was used during implantation to assess the effect of warm ischemia elimination on allograft function, histological changes and immune-related gene expression. 23 recipients were randomly assigned to a group operated on with kidney surface cooling during implantation (ice bag technique, IBT group), and the other 23 recipients receiving the contralateral kidney from the same donor were operated on with a standard technique. Three consecutive kidney core biopsies were obtained during the transplantation procedure: after organ recovery, after cold ischemia and after reperfusion. Gene expression levels were determined using low-density arrays (Format 32, TaqMan). The IBT group showed a significantly lower rate of detrimental events (delayed graft function and/or acute rejection, p = 0.015) as well as higher glomerular filtration rate on day 14 (p = 0.026). A greater decrease of MMP9 and LCN2 gene expression was seen in the IBT group during total ischemia (p = 0.003 and p = 0.018). Elimination of second warm ischemia reduced the number of detrimental events after kidney transplantation, and thus had influence on the short-term but not long-term allograft function. Surface cooling of the kidney during vascular anastomosis may reduce some detrimental effects of immune activation resulting from both brain death and ischemia-reperfusion injury.

Neutrophil gelatinase-associated lipocalin is an extracellular protein produced mostly in kidney. Recently, it has become a promising biomarker of renal damage in vivo. On the other hand, the validation of NGAL as a biomarker for nephrotoxicity estimation in vitro has not been characterized in detail yet. Since the HK-2 cells are frequently used human kidney cell line, we aimed to characterize the production of NGAL in these cells and to evaluate NGAL as a possible marker of cell impairment. We used heavy metals (mercury, cadmium), peroxide, drugs (acetaminophen, gentamicin) and cisplatin to mimic nephrotoxicity. HK-2 cells were incubated with selected compounds for 1-24h and cell viability was measured together with extracellular NGAL production. We proved that HK-2 cells possess a capacity to produce NGAL in amount of 2pg/ml/h. We found a change in cell viability after 24h incubation with all tested toxic compounds. The largest decrease of the viability was detected in mercury, acetaminophen, cisplatin and gentamicin. Unexpectedly, we found also a significant decrease in NGAL production in HK-2 cells treated with these toxins for 24h: to 11±5%, 54±5%, 57±6% and 76±9% respectively, compared with controls (=100%). Our results were followed with qPCR analysis when we found no significant increase in LCN2 gene expression after 24h incubation. We conclude that extracellular NGAL production negatively correlates with HK-2 cell impairment.