Plasmodium parasites multiply within host erythrocytes, which contain high levels of iron, and parasite egress from these cells results in iron release and host anemia. Although Plasmodium requires host iron for replication, how host iron homeostasis and responses to these fluxes affect Plasmodium infection are incompletely understood. We determined that Lipocalin 2 (Lcn2), a host protein that sequesters iron, is abundantly secreted during human (P. vivax) and mouse (P. yoeliiNL) blood-stage malaria infections and is essential to control P. yoeliiNL parasitemia, anemia, and host survival. During infection, Lcn2 bolsters both host macrophage function and granulocyte recruitment and limits reticulocytosis, or the expansion of immature erythrocytes, which are the preferred target cell of P. yoeliiNL. Additionally, a chronic iron imbalance due to Lcn2 deficiency results in impaired adaptive immune responses against Plasmodium parasites. Thus, Lcn2 exerts antiparasitic effects by maintaining iron homeostasis and promoting innate and adaptive immune responses.

BACKGROUND

Although a number of experimental studies have suggested the role of lipocalin-2 (LCN2) and matrix metalloproteinase-9 (MMP-9) in breast cancer progression, limited numbers of epidemiological studies have examined the relationship between the levels of lipocalin-2 and MMP-9 and breast cancer survival.

METHODS

Preoperative serum levels of lipocalin-2 and MMP-9 were measured in 303 breast cancer patients and 74 healthy controls recruited between 2004 and 2007. We examined the association between lipocalin-2 and MMP-9 levels and disease-free survival (DFS) using Cox proportional hazard regression model.

RESULTS

The serum levels of lipocalin-2 and MMP-9 were not significantly different between patients and controls (P>> 0.05). Elevated lipocalin-2 and MMP-9 levels were associated with reduced DFS of breast cancer ( Ptrend = 0.029 and Ptrend = 0.063, respectively). When lipocalin-2 and MMP-9 levels were categorized based on the combined risk score, patients with higher levels of both lipocalin-2 and MMP-9 exhibited poor DFS compared to patients with lower levels (Ptrend = 0.004). Furthermore, these effects were profound in patients with BMI less than 25 kg/m2 (adjusted hazard ratio (aHR), 3.17; 95% confidence intervals (CI), 1.66-6.06, Ptrend < 0.001) or lymph-node negative breast cancer (aHR, 5.36; 95% CI, 2.18-13.2, Ptrend < 0.001).

CONCLUSIONS

Our study suggests that the elevated levels of lipocalin-2 and MMP-9 are associated with reduced breast cancer survival, particularly in patients with lower BMI and lymph-node negative breast cancers.

OBJECTIVE

Glaucoma is a progressive eye disease that leads to blindness due to loss of retinal ganglion cells (RGCs). There are difficulties in using primary cultures of purified RGC to study this pathophysiology. RGC-5, a transformed not RGC line, expresses several markers characteristic of the RGCs. The aim of this study was to generate a genome-wide gene expression of RGC-5 following serum deprivation and to identify candidate genes that may be involved in the signal transduction pathways.

METHODS

Apoptosis in the transformed rat RGC-5 was induced by serum deprivation for 0, 8, 24, 48, and 96 h. Briefly, 400 ng of RNA from each sample was reverse transcribed and labeled with Cy3 dye. Fragmented fluorescent cRNA was mixed with hybridization buffer and incubated at 60 degrees C for 16 h. Labeled cRNA was hybridized to Rat Genome Oligonucleotide Arrays. These arrays contain 22,775 transcripts with one oligonucleotide per transcript (60-mer). Gene expression from scanned images was quantified and analyzed using ArrayVision software. Reproducibility among triplicate arrays was determined by ANOVA statistical analysis. Significant differences in gene expression between apoptotic and nonapoptotic cells were determined based on p-values.

RESULTS

Of the 22,775 transcripts present on the arrays (Agilent rat genome, 60-mer), 713 (8 h), 1,967 (24 h), 1,011 (48 h), and 1,161 (96 h) were differentially expressed relative to the 0 h time point (p-values <0.05). Twenty-three transcripts were common to 8, 24, 48, and 96 h and 130 transcripts were common to the 24, 48, and 96 h time points. The two most highly upregulated genes were Fdft1 and Lgals3 (8 h), C3 and Fcgrt (24 h), C and Lcn2 (48 h), and Mgp and C3 (96 h). A subset of the differentially expressed genes identified in microarray data (Ftl1, C3, C1s, Neu1, Polr2g, Acadm, Nupr1, Gch, Dia1, DNase1, Tgfb2, and Cyr61) were validated using quantitative real time polymerase chain reaction (QRT-PCR). Here we show that complement factor H (CFH), the major inhibitor of the alternative complement pathway is downregulated in serum-deprived RGC-5. CFH protein was detected within RGC-5 cells as well as the rat retina with the aid of immunocytochemistry and confocal microscopy.

CONCLUSIONS

This study was undertaken to generate a genome-wide gene expression profile of RGC-5 after serum deprivation, and to identify candidate and novel genes that may be involved in the signal transduction pathways leading to apoptosis. RGC-5 serum deprivation revealed up-and downregulation in gene expression profiles. The data gathered from this study was the first report that the genes identified in microarray data and validated by real-time RT-PCR may play an important role in RGC-5 cell death. Among the validated genes, C3 and C1s showed significant upregulation of the complement component pathway. The results further indicate that components of the complement pathway are present in neurons of the rat retina. The data indicated that complement factors are likely involved in the pathway leading to ganglion cell death in the serum-deprivation paradigm, which may be similar to the mechanism of cell death in glaucoma.

Characterization of a newly established serous ovarian cancer cell line, YDOV-139 was performed and ER-60 (PDIA3), which was highly expressed in YDOV-139, was evaluated as novel biomarker for ovarian cancer. The YDOV-139 cell line was established using ascites samples from a 67-year-old Korean woman with recurrent ovarian cancer, and was characterized with respect to various biological and genetic features. Gene expression profiles were analyzed using cDNA microarrays, and proteomic evaluation was performed by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time of flight peptide mass fingerprinting (MALDI-TOF/PMF). Four candidate markers that were strongly up-regulated in YDOV-139 were validated by real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC). The epithelial-like characteristics of YDOV-139 were evident from morphologic studies, and the average population doubling time was 120 h. When transplanted into nude mice, YDOV-139 cells successfully induced tumor masses in all three animals. Chemosensitivity tests showed that gemcitabine had the highest chemosensitivity index against YDOV-139 cells. HLA typing revealed A*24/A*31, B*07/B*35, Cw03*(09)/w*07, and DRB1*01/DRB1*15 alleles. Compared with human ovarian surface epithelial (HOSE) cells, 2,520 genes and 23 protein spots were differentially expressed in YDOV-139. Validation by real-time PCR showed that mRNA expression of LCN2, MDK, SLCO4A1, and ER-60 (PDIA3) were strongly elevated in ovarian cancers. In IHC analysis, ER-60 (PDIA3) was significantly overexpressed in both borderline tumors and invasive ovarian cancers (P<0.001). The molecular characteristics of YDOV-139 may have implications for future ovarian cancer research and ER-60 (PDIA3) should be investigated further as a potential biomarker of ovarian cancer.

OBJECTIVE

To determine the recurring DNA copy number alterations (CNA) in classical Hodgkin lymphoma (HL) by microarray-based comparative genomic hybridization (aCGH) using laser capture microdissected CD30(+) Hodgkin and Reed-Sternberg (HRS) cells.

METHODS

Archived tissues from 27 CD30(+) HL plus control samples were analyzed by DNA microarrays. The HL molecular karyotypes were compared with the genomic profiles of germinal center B cells and treatment outcome (chemotherapy responsive vs. primary refractory disease).

RESULTS

Gains and losses observed in more than 35% of HL samples were localized to 22 and 12 chromosomal regions, respectively. Frequent gains (>65%) were associated with growth and proliferation, NF-κB activation, cell-cycle control, apoptosis, and immune and lymphoid development. Frequent losses (>40%) observed encompassed tumor suppressor genes (SPRY1, NELL1, and ID4, inhibitor of DNA binding 4), transcriptional repressors (TXNIP, thioredoxin interacting protein), SKP2 (S-phase kinase-associated protein 2; ubiquitin ligase component), and an antagonist of NF-κB activation (PPARGC1A). In comparison to the germinal center profiles, the most frequent imbalances in HL were losses in 5p13 (AMACR, GDNF, and SKP2), and gains in 7q36 (SHH, sonic hedgehog homolog) and 9q34 (ABL1, CDK9, LCN2, and PTGES). Gains (>35%) in the HL chemoresponsive patients housed genes known to regulate T-cell trafficking or NF-κB activation (CCL22, CX3CL1, CCL17, DOK4, and IL10), whereas the refractory samples showed frequent loss of 4q27 (interleukin; IL21/IL2) and 17p12, and gain of 19q13.3 (BCL3/RELB).

CONCLUSIONS

We identified nonrandom CNAs in the molecular karyotypes of classical HL. Several recurring genetic lesions correlated with disease outcome. These findings may be useful prognostic markers in the counseling and management of patients and for the development of novel therapeutic approaches in primary refractory HL.

Lipocalin 2 (Lcn2) has previously been characterized as an adipokine/cytokine playing a role in glucose and lipid homeostasis. In this study, we investigate the role of Lcn2 in adipose tissue remodeling during high-fat diet (HFD)-induced obesity. We find that Lcn2 protein is highly abundant selectively in inguinal adipose tissue. During 16 weeks of HFD feeding, the inguinal fat depot expanded continuously, whereas the expansion of the epididymal fat depot was reduced in both wild-type (WT) and Lcn2(-/-) mice. Interestingly, the depot-specific effect of HFD on fat mass was exacerbated and appeared more pronounced and faster in Lcn2(-/-) mice than in WT mice. In Lcn2(-/-) mice, adipocyte hypertrophy in both inguinal and epididymal adipose tissue was more profoundly induced by age and HFD when compared with WT mice. The expression of peroxisome proliferator-activated receptor-γ protein was significantly down-regulated, whereas the gene expression of extracellular matrix proteins was up-regulated selectively in epididymal adipocytes of Lcn2(-/-) mice. Consistent with these observations, collagen deposition was selectively higher in the epididymal, but not in the inguinal adipose depot of Lcn2(-/-) mice. Administration of the peroxisome proliferator-activated receptor-γ agonist rosiglitazone (Rosi) restored adipogenic gene expression. However, Lcn2 deficiency did not alter the responsiveness of adipose tissue to Rosi effects on the extracellular matrix expression. Rosi treatment led to the further enlargement of adipocytes with improved metabolic activity in Lcn2(-/-) mice, which may be associated with a more pronounced effect of Rosi treatment in reducing TGF-β in Lcn2(-/-) adipose tissue. Consistent with these in vivo observations, Lcn2 deficiency reduces the adipocyte differentiation capacity of stromal-vascular cells isolated from HFD-fed mice in these cells. Herein Rosi treatment was again able to stimulate adipocyte differentiation to a similar extent in WT and Lcn2(-/-) inguinal and epididymal stromal-vascular cells. Thus, combined, our data indicate that Lcn2 has a depot-specific role in HFD-induced adipose tissue remodeling.

Myelofibrosis (MF) is characterized by cytopenias, constitutional symptoms, splenomegaly, and marrow histopathological abnormalities (fibrosis, increased microvessel density, and osteosclerosis). The microenvironmental abnormalities are likely a consequence of the elaboration of a variety of inflammatory cytokines generated by malignant megakaryocytes and monocytes. We observed that levels of a specific inflammatory cytokine, lipocalin-2 (LCN2), were elevated in the plasmas of patients with myeloproliferative neoplasms (MF>> polycythemia vera or essential thrombocythemia) and that LCN2 was elaborated by MF myeloid cells. LCN2 generates increased reactive oxygen species, leading to increased DNA strand breaks and apoptosis of normal, but not MF, CD34(+) cells. Furthermore, incubation of marrow adherent cells or mesenchymal stem cells with LCN2 increased the generation of osteoblasts and fibroblasts, but not adipocytes. LCN2 priming of mesenchymal stem cells resulted in the upregulation of RUNX2 gene as well as other genes that are capable of further affecting osteoblastogenesis, angiogenesis, and the deposition of matrix proteins. These data indicate that LCN2 is an additional MF inflammatory cytokine that likely contributes to the creation of a cascade of events that results in not only a predominance of the MF clone but also a dysfunctional microenvironment.

BACKGROUND

Endometrium in polycystic ovary syndrome (PCOS) presents altered gene expression indicating progesterone resistance and predisposing to reduced endometrial receptivity and endometrial cancer.

OBJECTIVE

We hypothesized that an altered endocrine/metabolic environment in PCOS may result in an endometrial "disease phenotype" affecting the gene expression of different endometrial cell populations, including stem cells and their differentiated progeny.

METHODS

This was a prospective study conducted at an academic medical center.

METHODS

Proliferative-phase endometrium was obtained from 6 overweight/obese PCOS (National Institutes of Health criteria) and 6 overweight/obese controls. Microarray analysis was performed on fluorescence-activated cell sorting-isolated endometrial epithelial cells (eEPs), endothelial cells, stromal fibroblasts (eSFs), and mesenchymal stem cells (eMSCs). Gene expression data were validated using microfluidic quantitative RT-PCR and immunohistochemistry.

RESULTS

The comparison between eEP(PCOS) and eEP(Ctrl) showed dysregulation of inflammatory genes and genes with oncogenic potential (CCL2, IL-6, ORM1, TNAIFP6, SFRP4, SPARC). eSF(PCOS) and eSF(Ctrl) showed up-regulation of inflammatory genes (C4A/B, CCL2, ICAM1, TNFAIP3). Similarly, in eMSC(PCOS) vs eMSC(Ctrl), the most up-regulated genes were related to inflammation and cancer (IL-8, ICAM1, SPRR3, LCN2). Immunohistochemistry scoring showed increased expression of CCL2 in eEP(PCOS) and eSF(PCOS) compared with eEP(Ctrl) and eSF(Ctrl) and IL-6 in eEP(PCOS) compared with eEP(Ctrl).

CONCLUSIONS

Isolated endometrial cell populations in women with PCOS showed altered gene expression revealing inflammation and prooncogenic changes, independent of body mass index, especially in eEP(PCOS) and eMSC(PCOS), compared with controls. The study reveals an endometrial disease phenotype in women with PCOS with potential negative effects on endometrial function and long-term health.

BACKGROUND

We previously reported the overexpression of lipocalin2 (LCN2), a 25kDa secretory protein involved in iron-transportation, in endometrial carcinoma and its possible contribution to endometrial carcinogenesis. Recently, a specific receptor for LCN2, solute carrier family 22 member 17 (SLC22A17), was identified. The present study was undertaken to investigate the expression of SLC22A17 in endometrial carcinoma.

METHODS

The expression of the SLC22A17 and LCN2 proteins was examined immunohistochemically using 69 cases of endometrial carcinoma and adjacent normal endometrial tissues. Immunoreactivity was evaluated according to the percentage of positive cells and described as a positivity index (PI, full score 100).

RESULTS

The expression of SLC22A17 was negligible in normal endometria, but positive staining for SLC22A17 (PI≧1) was observed in 35 cases of endometrial carcinoma. The PI for SLC22A17 was significantly higher in cases with histological grade 3 (P<0.0005), advanced FIGO stage (P=0.002), deep myometrial invasion (P=0.029), positive lymph-vascular space invasion (P=0.029), positive intraperitoneal cytology (P=0.020) and adnexal metastasis (P=0.029). The expression of SLC22A17 and LCN2 was positively correlated with a significant difference (P=0.002), and the patients who overexpressed both SLC22A17 and LCN2 showed poorer survival than those without the expression of SLC22A17 or LCN2 (P=0.002). Moreover, the overexpression of both SLC22A17 and LCN2 was indicated to be an independent prognostic factor by multivariable analysis.

CONCLUSIONS

These results suggested that SLC22A17, in cooperation with LCN2, to be involved in the acquisition of aggressive behavior among endometrial carcinoma cells.

BACKGROUND

Lipocalin 2 (LCN2) has been related to obesity, insulin resistance and metabolic disturbance. However, its relation with non alcoholic fatty liver disease (NAFLD) has hardly been studied.

METHODS

We examined LCN2 circulating levels and its protein and gene expression in liver from women with severe obesity and NAFLD. We analyzed the liver histology of 59 white severely obese women (BMI ≥40 Kg/m²): 15 subjects presented normal liver histology or non-significant liver disease (NL), 18 simple steatosis (SS) and 26 non alcoholic steatohepatitis (NASH). We determined the anthropometric and metabolic features of the women. LCN2 levels were determined by an ELISA and liver mRNA expression by real time RT-PCR. We also studied LCN2 expression in HepG2 liver cells under various inflammatory stimuli.

RESULTS

Liver LCN2 protein and gene expression were higher in NAFLD than in obese with NL. Liver LCN2 gene expression correlated with SS (r=0.351, p=0.016), and its protein expression correlated with NASH (r=0.705, p=0.003). LCN2 expression was detected in HepG2 cells after the administration of TNFα, IL6, resistin or adiponectin. LCN2 expression was induced by TNFα, IL6 and resistin.

CONCLUSIONS

Liver LCN2 is related to NAFLD in severely obese women. Up-regulation of LCN2 expression is detected in HepG2 cells after exposure to TNFα, IL6 and resistin. These results suggest that LCN2 expression is induced under liver harmful conditions.

Although systematic therapeutic approaches have reduced cancer-associated mortality, metastatic breast cancer can still evade therapy, particularly triple-negative breast cancer, which remains associated with high rates of cancer metastasis and has the worst clinical prognosis. Lipocalin 2 (LCN2) is a secreted glycoprotein that transports small lipophilic ligands. Its abnormal expression serves critical roles in the epithelial-to-mesenchymal transition process, angiogenesis, and cell migration and invasion in breast cancer. Notably, LCN2 functions as an initiator of carcinogenesis and metastasis by involving multiple signaling pathways. The present review aims to summarize research findings on the abnormal expression of LCN2 in breast cancer progression. Furthermore, the review highlights the latest developments of potential LCN2-targeting agents and proposed LCN2-associated molecular mechanisms with regard to breast cancer invasion and metastasis.

Chemoresistance is a major cause of cancer progression and the mortality of cancer patients. Developing a safe strategy for enhancing chemosensitivity is a challenge for biomedical science. Recent studies have suggested that vitamin D supplementation may decrease the risk of many cancers. However, the role of vitamin D in chemotherapy remains unknown. We found that vitamin D sensitised oral cancer cells to cisplatin and partially reversed cisplatin resistance. Using RNA-seq, we discovered that lipocalin 2 (LCN2) is an important mediator. Cisplatin enhanced the expression of LCN2 by decreasing methylation at the promoter, whereas vitamin D enhanced methylation and thereby inhibited the expression of LCN2. Overexpression of LCN2 increased cell survival and cisplatin resistance both in vitro and in vivo. High LCN2 expression was positively associated with differentiation, lymph node metastasis, and T staging and predicted a poor prognosis in oral squamous cell carcinoma (OSCC) patients. LCN2 was also associated with post-chemotherapy recurrence. Moreover, we found that LCN2 promoted the activation of NF-κB by binding to ribosomal protein S3 (RPS3) and enhanced the interaction between RPS3 and p65. Our study reveals that vitamin D can enhance cisplatin chemotherapy and suggests that vitamin D should be supplied during chemotherapy; however, more follow-up clinical studies are needed.

The tumor microenvironment plays a critical regulatory role in cancer progression, especially in central nervous system metastases. Cancer cells within the cerebrospinal fluid (CSF)-filled leptomeninges face substantial microenvironmental challenges, including inflammation and sparse micronutrients. To investigate the mechanism by which cancer cells in these leptomeningeal metastases (LM) overcome these constraints, we subjected CSF from five patients with LM to single-cell RNA sequencing. We found that cancer cells, but not macrophages, within the CSF express the iron-binding protein lipocalin-2 (LCN2) and its receptor SCL22A17. These macrophages generate inflammatory cytokines that induce cancer cell LCN2 expression but do not generate LCN2 themselves. In mouse models of LM, cancer cell growth is supported by the LCN2/SLC22A17 system and is inhibited by iron chelation therapy. Thus, cancer cells appear to survive in the CSF by outcompeting macrophages for iron.

Background: Astrogliosis has the potential to lead to harmful effects, namely, neuroinflammation, and to interfere with synapse sprouting. Previous studies have suggested that Lipocalin-2 (LCN2) acts as a key target in regulating the reaction of astrocytes. However, the underlying molecular mechanism is not fully elucidated. In the present study, we examined the neuroprotective and anti-inflammatory effects of Ginkgo biloba extract (EGB), a well-known extract with potential immunoregulatory properties in the nervous system. Methods: Triphenyltetrazolium chloride staining, hematoxylin-eosin staining, electron microscopy, and neurological assessments were performed in a microsphere-embolized rat model. Human astrocytes exposed to oxygen glucose deprivation (OGD) were used for in vitro experiments. Inflammatory cytokines, multi-labeling immunofluorescence, and Western blotting were used to investigate the molecular mechanisms underlying the EGB-mediated anti-inflammatory effects in vivo and in vitro. Results: EGB markedly attenuated cerebral infarction and neuronal apoptosis, reduced the inflammatory cytokine level, and alleviated neurological deficiencies in cerebral ischemic rats. After surgery, EGB significantly inhibited astrocyte activation, reduced the phosphorylation of STAT3 and JAK2 and decreased LCN2 expression. In vitro, EGB blocked OGD-induced STAT3 activation and the generation of pro-inflammatory cytokines in human astrocytes, and these effects were significantly enhanced by LCN2 overexpression. EGB downregulated these effects enhanced by LCN2 overexpression. Conclusion: EGB is demonstrated to mediate neuroinflammation, which protects against ischemic brain injury by inhibiting astrogliosis and suppresses neuroinflammation via the LCN2-JAK2/STAT3 pathway, providing insight into a promising therapeutic strategy for ischemic stroke.

CCAAT/enhancer binding protein-ε (C/EBP-ε) is considered a master transcription factor regulating terminal neutrophil maturation. It is essential for expression of secondary granule proteins, but it also regulates proliferation, cell cycle, and maturation during granulopoiesis. Cebpe(-/-) mice have incomplete granulocytic differentiation and increased sensitivity toward bacterial infections. The amount of C/EBP-ε messenger RNA (mRNA) increases with maturation from myeloblasts with peak level in myelocytes (MC)/metamyelocytes (MM), when the cells stop proliferating followed by a decline in more mature cells. In contrast, C/EBP-ε protein is virtually detectable only in the MC/MM population, indicating that expression in more immature cells could be inhibited by microRNAs (miRNAs). We found that miRNA-130a (miR-130a) regulates C/EBP-ε protein expression in both murine and human granulocytic precursors. Overexpression of miR-130a in a murine cell line downregulated C/EBP-ε protein and lactoferrin (Ltf), cathelicidin antimicrobial protein (Camp), and lipocalin-2 (Lcn2) mRNA expression giving rise to cells with a more immature phenotype, as seen in the Cebpe(-/-) mouse. Introduction of a C/EBP-ε mRNA without target site for miR-130a restored both C/EBP-ε production, expression of Camp and Lcn2, and resulted in the cells having a more mature phenotype. We conclude that miR-130a is important for the regulation of the timed expression of C/EBP-ε during granulopoiesis.

The expression levels of a number of genes associated with inflammation and immune function change with advancing age. Melatonin modulates gene expression levels of several of these genes. Therefore the declining levels of melatonin associated with age may play a role in the physiological effects of aging. We used oligonucleotide microarrays to measure age-related changes in mRNA expression in the murine CNS, and to study the effect of prolonged administration of dietary melatonin upon these changes. CB6F1 male mice were fed 40 ppm melatonin for 2.1 months prior to sacrifice at age 26.5 months, and compared with both age-matched controls and young, 4.5-month-old untreated controls. Total RNA was extracted from whole brain (excluding cerebellum and brain stem) and individual samples were hybridized to Affymetrix Mouse 430-2.0 arrays. The expression of a substantial number of genes was modulated by melatonin treatment and changes in selected genes were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). A subset of these genes did not change with age. Conversely, some genes modulated by age were also modulated by melatonin treatment. In general, melatonin treatment drove the expression levels of these genes closer to the expression levels detected in the younger animals. Notably, the abundance of lipocalin 2 (Lcn2) mRNA increased with age and was decreased in old animals treated with melatonin. Lcn2 is a member of the acute phase response family of proteins and its mRNA levels in the brain increase in response to inflammation. Many of the genes with expression reduced by melatonin are involved in inflammation and the immune system. This suggests that melatonin treatment may influence the inflammatory responses of old animals, driving them to resemble more closely those occurring in young animals.

Despite many advantages of mesenchymal stem cells (MSCs) that make them suitable for cell therapy purposes, their therapeutic application has been limited due to their susceptibility to several stresses (e.g., nutrient-poor environment, oxidative stress, and hypoxic and masses of cytotoxic factors) to which they are exposed during their preparation and following transplantation. Hence, reinforcing MSCs against these stresses is a challenge for both basic and clinician scientists. Recently, much attention has been directed toward equipping MSCs with cytoprotective factors to strengthen them against unfavorable microenvironments. Here, we engineered MSCs with lipocalin 2 (Lcn2), a cytoprotective factor that is naturally induced following exposure of cells to stresses imposed by the microenvironment. Lcn2 overexpression not only did not interfere with the multidifferentiation capacity of the MSCs but also granted many protective properties to them. Lcn2 potentiated MSCs to withstand oxidative, hypoxia, and serum deprivation (SD) conditions via antagonizing their induced cytotoxicity and apoptosis. Adhesion rate of MSCs to coated culture plates was also enhanced by Lcn2 overexpression. In addition, Lcn2 induced antioxidants and upregulated some growth factors in MSCs. Our findings suggested a new strategy for prevention of graft cell death in MSC-based cell therapy.

OBJECTIVE

Interleukin-23 (IL-23) has emerged as a new therapeutic target for the treatment of inflammatory bowel disease (IBD). As biomarkers of disease state and treatment efficacy are becoming increasingly important in drug development, we sought to identify efficacy biomarkers for anti-IL-23 therapy in Crohn's disease (CD).

METHODS

Candidate IL-23 biomarkers, downstream of IL-23 signaling, were identified using shotgun proteomic analysis of feces and colon lavages obtained from a short-term mouse IBD model (anti-CD40 Rag2(-/-)) treated preventively with monoclonal antibodies (mAbs) to the IL-23 receptor (IL-23R). The biomarkers were then measured in an IBD T-cell transfer model treated therapeutically with a mAb to IL-23 (p19), confirming their association with IBD. To assess the clinical relevance of these markers, we assessed their concentrations in clinical serum, colon tissue, and feces from CD patients.

RESULTS

We identified 57 proteins up or downregulated in diseased animals that returned to control values when the mice were treated with mAbs to IL-23R. Among those, S100A8, S100A9, regenerating protein 3β (REG), REG3γ, lipocalin 2 (LCN2), deleted in malignant tumor 1 (DMBT1), and macrophage migration inhibitory factor (MIF) mRNA levels correlated with disease score and dose titration of mAbs to IL-23R or IL-23(p19). All biomarkers, except DMBT1, were also downregulated after therapeutic administration of mAbs to IL-23(p19) in a T-cell transfer IBD mouse model. In sera from CD patients, we confirmed a significant upregulation of S100A8/A9 (43%), MIF (138%), pancreatitis-associated protein (PAP, human homolog of REG3β/γ; 49%), LCN2 (520%), and CCL20 (1280%), compared with control samples, as well as a significant upregulation of S100A8/A9 (887%), PAP (401%), and LCN2 (783%) in human feces from CD patients compared with normal controls.

CONCLUSIONS

These studies identify multiple protein biomarkers downstream of IL-23 that could be valuable tools to assess the efficacy of this new therapeutic agent.Clinical and Translational Gastroenterology (2012) 3, e10; doi:10.1038/ctg.2012.2; published online 16 February 2012.

BACKGROUND

We examined the alterations in gene expression associated with the development of crescentic glomerulonephritis in murine chronic graft-versus-host disease, a model for human systemic lupus erythematosus.

METHODS

The disease was induced in (C57BL/6 x DBA/2) F(1) hybrids by injection of DBA/2 lymphocytes leading to deposition of auto-antibodies in the glomeruli, and a lupus type of nephritis morphologically. After extensive crescent formation at week 9 of disease, cDNA microarray analysis was performed and highly expressed genes were evaluated as molecular markers by real-time reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, immunohistochemistry and immunoassay of urine proteins.

RESULTS

Six genes, secreted acidic cysteine-rich glycoprotein (Sparc), thymosin beta 10 (Tmsb10), S100 calcium-binding protein A6 (S100a6), annexin A2 (Anxa2), osteopontin (OPN) and lipocalin 2 (Lcn2), were quantified by real-time RT-PCR in laser microdissected glomeruli in a time course manner. Sparc was detected early before the onset of proteinuria and continued to increase throughout the course of the disease. The expression of Tmsb10, S100a6 and Anxa2 coincided with heavy proteinuria. By week 9, OPN and Lcn2 were highly expressed. The expression of proteins encoded by these genes was predominant in the glomerular crescent. The protein levels of Sparc, OPN and Lcn2 in urine were significantly elevated.

CONCLUSIONS

These findings implicate these six genes in the development of glomerular crescents. More importantly, detection of Sparc, OPN and Lcn2 in urine may mean that these molecules could serve as important biomarkers for non-invasive diagnosis of glomerular crescents.

Lipocalin 2 (LCN2) has recently emerged as a novel adipokine involved in different processes including arthritis and chondrocyte inflammatory response. However, little is known about its activity on chondrocyte homeostasis and its regulation by nitric oxide (NO) Hence, we performed a set of experiments aimed to achieve a better understanding of this relationship. Cell vitality was tested in the ATDC5 cell line by the MTT colorimetric assay. Protein expression and gene expression was evaluated by Western blot and real time RT-PCR, respectively. NO production (determined as nitrite accumulation) was assayed by the Griess reaction. First, we demonstrated that LCN2 decreased murine chondrocytes vitality. Next, LCN2 co-stimulation with LPS enhanced NOS2 protein expression by murine chondrocytes. In addition, inhibition of LPS-induced nitric oxide production by aminoguanidine, a selective NOS2 inhibitor, significantly reduced LPS-mediated LCN2 expression. In contrast, treatment of murine chondrocytes with sodium nitroprussiate (SNP), a classic NO donor, scarcely induced LCN2 expression. Intriguingly, SNP addition to LPS-challenged chondrocytes, treated with aminoguanidine, provoked a strong induction of LCN2 expression. Finally, murine ATDC5 cells, co-cultured with LPS pre-challenged macrophages, had higher LCN2 expression in comparison with murine chondrocytes co-cultured with non pre-challenged macrophages. In this work we have described for the first time that NO is able to exert a control on LCN2 expression, suggesting the existence of a feedback loop regulating its expression.

OBJECTIVE

Oleocanthal (OC), a phenolic compound present in extra virgin olive oil (EVOO), has attracted attention since its discovery for its relevant pharmacological properties in different pathogenic processes, including inflammation. Here, we investigated the involvement of OC in LPS-activated osteoarthritis (OA) human primary chondrocytes.

METHODS

Human primary chondrocytes were harvested from articular cartilage samples obtained from OA patients. The effects of OC on the viability of chondrocytes were tested by MTT assay. Protein and mRNA expression of several catabolic and pro-inflammatory factors after OC treatment were measured by RT-qPCR and western blot respectively. Moreover, we analysed the NO production by Griess reaction. Finally, several pathways mediators were analysed by western blot.

RESULTS

We demonstrated that OC did not have any cytotoxic effect. Oleocanthal inhibited NO production and strongly decreased NOS2 and COX-2 protein and mRNA expression in LPS-activated human primary OA chondrocytes. Interestingly, OC also inhibits MMP-13 and ADAMTS-5. In addition, OC downregulates several pro-inflammatory factors, such as IL-6, IL-8, CCL3, LCN2 and TNF-α induced by LPS in human primary OA chondrocytes. Finally, we demonstrated that OC exerts its effects through the MAPK/P38/NF-kB pathways.

CONCLUSIONS

These data show that OC is able to block LPS-mediated inflammatory response and MMP-13 and ADAMTS-5 induction in human primary OA chondrocytes via MAPKs/NF-kB pathways, suggesting that OC may be a promising agent for the treatment of inflammation in cartilage and a potential molecule to prevent disease progression by inhibiting metalloproteases and aggrecanases.

OBJECTIVE

Recent evidence demonstrated that lipocalin (LCN)2 is induced in many types of human cancer, while the detection of its complex with matrix metalloproteinase (MMP)-9 is correlated with the cancer disease status. We attempted to evaluate plasma expressions of LCN2, MMP-9, and their complex (LCN2/MMP-9) during the diagnostic work-up of patients with oral squamous cell carcinoma (OSCC) and investigated their correlations with disease progression.

METHODS

In total, 195 patients with OSCC and 81 healthy controls were recruited. Expression levels of LCN2, MMP-9, and LCN2/MMP-9 were determined with immunoenzymatic assays.

RESULTS

Patients with OSCC exhibited significantly higher levels of LCN2, MMP-9, and LCN2/MMP-9 compared with healthy controls (LCN2: P < 0.001; MMP-9: P < 0.001; LCN2/MMP-9: P < 0.01). Plasma levels of LCN2, MMP-9, and LCN2/MMP-9 in patients with OSCC were significantly correlated with each other and were associated with more-advanced clinical stages (P < 0.05) and/or a larger tumor size (P < 0.05), but were not associated with positive lymph-node metastasis or distal metastasis.

CONCLUSIONS

Our results suggest that plasma levels of LCN2 and the LCN2/MMP-9 complex may be useful in non-invasively monitoring OSCC progression, while supporting their potential role as biomarkers of oral cancer disease status.

OBJECTIVE

Lipocalin-2 is a pro-inflammatory adipokine up-regulated in obese human subjects and animal models. Its circulating levels are positively correlated with the unfavourable lipid profiles, elevated blood pressure and insulin resistance index. Augmented lipocalin-2 has been found in patients with cardiovascular abnormalities.The present study was designed to investigate the role of lipocalin-2 in regulating endothelial function and vascular reactivity.

METHODS

Wild-type and lipocalin-2 knockout (Lcn2-KO) mice were fed with either a standard chow or a high-fat diet. Blood pressures and endothelium-dependent relaxations/contractions were monitored at 2 week intervals.

RESULTS

Systolic blood pressure was elevated by high-fat diet in wild-type mice but not in Lcn2-KO mice. Endothelial dysfunction, reflected by the impaired endothelium-dependent relaxations to insulin and augmented endothelium-dependent contractions to ACh, was induced by high-fat diet in wild-type mice. In contrast, Lcn2-KO mice were largely protected from the deterioration of endothelial function caused by dietary challenges. The eNOS dimer/monomer ratio, NO bioavailability, basal and insulin-stimulated PKB/eNOS phosphorylation responses were higher in aortae of Lcn2-KO mice. Administration of lipocalin-2 attenuated endothelium-dependent relaxations to insulin and promoted endothelium-dependent contractions to ACh. It induced eNOS uncoupling and elevated COX expression in the arteries. Treatment with sulphaphenazole, a selective inhibitor of cytochrome P450 2C9, improved endothelial function in wild-type mice and blocked the effects of lipocalin-2 on both endothelium-dependent relaxations to insulin and endothelium-dependent contractions to ACh, as well as eNOS uncoupling.

CONCLUSIONS

Lipocalin-2, by modulating cytochrome P450 2C9 activity, is critically involved in diet-induced endothelial dysfunction.

Multidrug and toxin extrusion 1 (MATE1/solute carrier 47A1) mediates cellular transport of a variety of structurally diverse compounds. Paraquat (PQ), which has been characterized in vitro as a MATE1 substrate, is a widely used herbicide and can cause severe toxicity to humans after exposure. However, the contribution of MATE1 to PQ disposition in vivo has not been determined. In the present study, we generated Mate1-deficient (Mate1-/-) mice and performed toxicokinetic analyses of PQ in Mate1-/- and wild-type (Mate1+/+) mice. After a single intravenous administration of PQ (50 mg/kg), Mate1-/- mice exhibited significantly higher plasma PQ concentrations than Mate1+/+ mice. The renal PQ concentration was markedly increased in Mate1-/- mice compared with Mate1+/+ mice. The subsequent nephrotoxicity of PQ were examined in these mice. Three days after intraperitoneal administration of PQ (20 mg/kg), the transcript levels of N-acetyl-β-D-glucosaminidase (Lcn2) and kidney injury molecule-1 (Kim-1) in the kidney were remarkably enhanced in the Mate1-/- mice. This was accompanied by apparent difference in renal histology between Mate1-/- and Mate1+/+ mice. In conclusion, we demonstrated that Mate1 is responsible for renal elimination of PQ in vivo and the deficiency of Mate1 function confers deteriorated kidney injury caused by PQ in mice.