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Patients with isolated pulmonary embolism (PE) have a distinct clinical profile from those with deep vein thrombosis (DVT)-associated PE, with more pulmonary conditions and atherosclerosis. These findings suggest a distinct molecular pathophysiology and the potential involvement of alternative pathways in isolated PE. To test this hypothesis, data from 532 individuals from the Genotyping and Molecular Phenotyping of Venous ThromboEmbolism Project, a multicenter prospective cohort study with extensive biobanking, were analyzed. Targeted, high-throughput proteomics, machine learning, and bioinformatic methods were applied to contrast the acute-phase plasma proteomes of isolated PE patients (n = 96) against those of patients with DVT-associated PE (n = 276) or isolated DVT (n = 160). This resulted in the identification of shared molecular processes between PE phenotypes, as well as an isolated PE-specific protein signature. Shared processes included upregulation of inflammation, response to oxidative stress, and the loss of pulmonary surfactant. The isolated PE-specific signature consisted of 5 proteins: interferon-γ, glial cell line-derived neurotrophic growth factor, polypeptide N-acetylgalactosaminyltransferase 3, peptidyl arginine deiminase type-2, and interleukin-15 receptor subunit α. These proteins were orthogonally validated using cis protein quantitative trait loci. External replication in an independent population-based cohort (n = 5778) further validated the proteomic results and showed that they were prognostic for incident primary isolated PE in individuals without history of VTE (median time to event: 2.9 years; interquartile range: 1.6-4.2 years), supporting their possible involvement in the early pathogenesis. This study has identified molecular overlaps and differences between VTE phenotypes. In particular, the results implicate noncanonical pathways more commonly associated with respiratory and atherosclerotic disease in the acute pathophysiology of isolated PE., (© 2021 by The American Society of Hematology.)

The emergence of 2019 novel coronavirus disease (COVID-19) is currently a global concern. In this study, our goal was to explore the changing expression levels of acute-phase reaction proteins (APRPs) in the serum of COVID-19 patients and to elucidate the immunological characteristics of COVID-19. In the study design, we recruited 72 COVID-19 patients, including 22 cases of mild degree, 38 cases of moderate degree and 12 cases of severe degree. We also recruited 20 patients with community-acquired pneumonia (CAP) and 20 normal control subjects as a comparison. Fasting venous blood was taken to detect the content of complement 3 (C3), complement 4 (C4), C-reactive protein (CRP), serum amyloid A (SAA) and prealbumin (PA). When compared the COVID-19 group with the CAP and normal control groups, respectively, the mean value of CRP and SAA in the COVID-19 group (including mild, moderate and severe patients) had increased significantly (P < 0.01), whereas the mean values of C3, C4 and PA decreased (P < 0.01). For the asymptomatic or mild symptomatic patients with COVID-19, the actual aggravation of disease may be more advanced than the clinical appearances. Meanwhile, the statistical analyses indicated that the development of COVID-19 brought about a significant increase in the content of CRP and SAA (P < 0.01), and a decline in the content of C3, C4 and PA (P < 0.01). These findings suggested that the changes in the level of APRPs could be used as indicators to identify the degree and progression of COVID-19, and the significant changes might demonstrate the aggravation of disease. This study provided a new approach to improve the clinical management plan and prognosis of COVID-19.

Aims: Cachexia is a severe consequence of cancer. Although cancer-induced heart atrophy leads to cardiac dysfunction and heart failure (HF), biomarkers for their diagnosis have not been identified. Neutrophil gelatinase-associated lipocalin (NGAL) is an aldosterone-responsive gene increased in HF. We studied NGAL and its association with aldosterone levels in a model of cancer cachexia-induced cardiomyopathy., Methods and Results: Rats were injected with Yoshida 10 8 AH-130 hepatoma cells to induce tumour. Cachectic rats were treated daily, for 16 days, with placebo or with 5 or 50 mg/kg/day of spironolactone. Cardiac function was analysed by echocardiography at baseline and at Day 11. Weight loss and atrophy of lean body and fat mass of cachectic rats were significantly attenuated by spironolactone. Cardiac dysfunction of tumour-bearing rats was improved by spironolactone. Plasma aldosterone was up-regulated from 337 ± 7 pg/mL in sham animals to 591 ± 31 pg/mL in the cachectic rats (P < 0.001 vs. sham). Treatment with 50 or 5 mg/kg/day of spironolactone reduced plasma aldosterone to 396 ± 22 and 391 ± 25 pg/mL (P < 0.01 vs. placebo). Plasma levels of NGAL were also increased in cachectic rats (1.462 ± 0.3603 μg/mL) than in controls (0.0936 ± 6 μg/mL, P < 0.001). Spironolactone treatment (50 mg/kg/day) significantly reduced cardiac mRNA and protein NGAL levels (P < 0.05 and P < 0.001 vs. placebo, respectively). NGAL mRNA and protein levels were overexpressed in cachectic animal hearts treated with placebo, compared with control (P < 0.05 and P < 0.01 vs. sham). Spironolactone treatment at 50 mg/kg/day reduced significantly cardiac NGAL (P < 0.05 and P < 0.001 vs. placebo)., Conclusions: Cancer cachexia induced increased levels of aldosterone and NGAL, contributing to worsening cardiac damage in cancer cachexia-induced cardiomyopathy. Spironolactone treatment may greatly attenuate cardiac dysfunction and lean mass atrophy associated with cancer cachexia., (© 2018 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

Background: We sought to confirm that neutrophil gelatinase-associated lipocalin (NGAL) protects against apoptosis during endotoxemia., Methods: Endotoxemia was induced in rats with lipopolysaccharide (LPS; 3.5 mg/kg) and serum creatinine (SCr), urinary NGAL (uNGAL), renal histopathology confirmed acute kidney injury (AKI). Renal caspase 3 and NGAL were assayed with immunohistochemistry 6 h later. A HK-2 cell model was used in which NGAL and caspase 3 mRNA were evaluated by qRT-PCR within 6 h after LPS (50 μM) treatment, and correlations were studied. NGAL and caspase 3 mRNA expression were measured after delivering NGAL siRNA in HK-2 cells and apoptosis was measured with TUNEL and flow cytometry., Results: SCr and uNGAL were significantly increased after LPS treatment and renal morphology data indicated AKI and renal tubular epithelial cell apoptosis. Caspase 3 and NGAL were predominantly expressed in the tubular epithelial cells and there was a correlation between caspase 3 and NGAL protein (r = 0.663, p = 0.01). In vitro, there was a strong correlation between caspase 3 and NGAL mRNA in LPS-injured HK-2 cells within 24 h (r = 0.448, p < 0.05). Suppressing the NGAL gene in HK-2 cells increased caspase 3 mRNA 4.5-fold and apoptosis increased 1.5-fold after LPS treatment., Conclusions: NGAL is associated with caspase 3 in renal tubular cells with endotoxin-induced kidney injury, and may regulate its expression and inhibit apoptosis.

Aim: To examine how enteral feeding affects the intestinal epigenome and gene expression just after preterm birth., Materials & Methods: Intestinal tissue from preterm pigs, modeling preterm infants, was collected at birth and 5 days after gradual introduction of infant formula or bovine colostrum. The intestinal tissue was analyzed by reduced representation bisulfite sequencing and real-time qPCR., Results: Relative to colostrum, formula increased bacterial epithelial adherence and lipopolysaccharide binding protein (LBP) expression, which was regulated by promoter methylation. Diet-dependent changes in DNA methylation and/or mRNA expression were related to innate immune response, hypoxia, angiogenesis and epithelial-mesenchymal transition pathways (e.g., TTC38, IL8, C3, HIF1A and VEGFR1)., Conclusion: Epigenetic changes may mediate important effects of the first feeding on intestinal development in preterm neonates.

A ternary complex comprising plasmid DNA, lipopolysaccharide-binding peptide (LBP), and deoxycholic acid-conjugated polyethylenimine (PEI-DA) is prepared for combinational therapy of acute lung injury (ALI). The LBP is designed as an anti-inflammatory peptide based on the lipopolysaccharide (LPS)-binding domain of HMGB-1. In vitro cytokine assays show that LBP reduces levels of proinflammatory cytokines by inhibiting LPS. PEI-DA is synthesized as the gene carrier by conjugation of deoxycholic acid to low-molecular weight polyethylenimine (2 kDa, PEI2k). PEI-DA has higher transfection efficiency than high-molecular weight polyethylenimine (25 kDa, PEI25k). The ternary complex of an HO-1 plasmid (pHO-1), PEI-DA, and LBP is prepared as a combinational system to deliver the therapeutic gene and peptide. The transfection efficiency of the ternary complex is higher than that of the pHO-1/PEI-DA binary complex. The ternary complex also reduces TNF-α secretion in LPS-activated Raw264.7 macrophage cells. Administration of the ternary complex into the lungs of an animal ALI model by intratracheal injection induces HO-1 expression and reduces levels of proinflammatory cytokines more efficiently than the pHO-1/PEI-DA binary complex or LBP alone. In addition, the ternary complex reduces inflammation in the lungs. Therefore, the pHO-1/PEI-DA/LBP ternary complex may be an effective treatment for ALI., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Long-term excessive alcohol intake predisposes to infectious diseases. The hepatic acute-phase response is a component of the innate immune system and is part of the first line of defense against invading pathogens, which may be compromised by alcohol. We aimed to investigate whether an induced acute-phase response is impaired in long-term ethanol (EtOH)-fed rats., Methods: For 6 weeks, rats were either fed a Lieber-DeCarli EtOH-containing (36% as calories) liquid diet ad libitum or calorically pair-fed. Then, the rats were injected intraperitoneally with a low dose of lipopolysaccharide (LPS) (0.5 mg/kg) to induce an acute-phase response. Two hours after LPS, we measured the plasma concentrations of an array of inflammatory cytokines. Twenty-four hours after LPS, we measured the hepatic mRNA expression and serum concentrations of prominent rat acute-phase proteins., Results: EtOH-fed rats showed either no liver histopathological changes or varying degrees of steatosis. EtOH feeding decreased the spontaneous liver mRNA expression of the prevailing acute-phase protein alpha-2-macroglobulin (α2M) by 30% (p < 0.01). LPS immediately increased plasma tumor necrosis factor-alpha and interleukin-6 more than 100-fold in both feeding groups (p < 0.001, all) and approximately twice as much in the EtOH-fed rats (p < 0.05 and p = 0.08, respectively). LPS also induced a variable but marked amplification of (α2M), haptoglobin, alpha-1-acid glycoprotein, and lipocalin-2 liver mRNA expression levels and serum concentrations in both feeding groups (p ≤ 0.01 to 0.001). However, the LPS-induced increases in serum (α2M) and haptoglobin were less pronounced in the EtOH-fed rats, averaging approximately 60% of the concentrations in the pair-fed rats (p < 0.01 and p < 0.001, respectively)., Conclusions: Long-term EtOH exposure in rats reduces the spontaneous hepatic mRNA expression of (α2M) and markedly impairs the hepatic acute-phase response to endotoxin, despite higher pro-inflammatory cytokine release. The same phenomenon may contribute to the increased susceptibility to infections observed in humans with long-term excessive alcohol intake., (Copyright © 2017 by the Research Society on Alcoholism.)

Diabetic nephropathy is a serious microvascular complication and one of the main causes of end-stage renal disease. L-Glutamine (LG) is naturally occurring amino acids with antidiabetic and antioxidant potential. The aim of present investigation was to evaluate the potential of LG against streptozotocin (STZ)-induced diabetic nephropathy (DN) in laboratory rats. DN was induced in male Wistar rats (200-220 g) by intraperitoneal administration of STZ (55 mg/kg). Animals were treated orally with either distilled water (10 mg/kg) or LG (250, 500, and 1000 mg/kg) or Sitagliptin (5 mg/kg). Various biochemical, molecular, and histological (hematoxylin-eosin and Masson's trichrome stain) parameters were assessed. Administration of LG (500 and 1000 mg/kg) significantly inhibited (p < .05) STZ-induced alterations in serum and urine biochemistry (urine creatinine, uric acid, albumin, and BUN). It also significantly increased creatinine clearance rate. STZ induced increase in renal oxidonitrosative stress was significantly decreased (p < .05) by LG (500 and 1000 mg/kg) treatment. Upregulated renal KIM-1, NGAL, TGF-β1, and collagen-1 mRNA expression after STZ administration was significantly inhibited (p < .05) by LG (500 and 1000 mg/kg) treatment. Correlation analysis also revealed that antidiabetic potential of LG attenuates STZ-induced elevated renal KIM-1, NGAL, TGF-β1, and collagen-1 mRNA expression. Histopathological alteration induced by STZ in renal tissue was ameliorated by LG treatment. In conclusion, results of present investigation suggest that treatment with LG ameliorated STZ-induced DN via the inhibition of oxidonitrosative stress as well as downregulation of KIM-1, NGAL, TGF-β1, and collagen-1 mRNA expressions.

Introduction: Trauma is one of the causes of peripheral nerve injuries. Free radicals increase after tissue damage. Free radicals are usually scavenged and detoxified by antioxidants. In this study, we assessed the antioxidative role of the NGAL molecule in sciatic nerve repair in rats., Materials and Methods: The sciatic nerves of 40 rats were crushed and the total mRNA of samples from day 1 and 3 and week 1, 3, 5 post injury was extracted. The expression of the NGAL gene was confirmed by RT-PCR. For immunohistochemistry analysis, the samples were fixed in paraformaldehyde and cut in 20 micrometer slices by cryostat., Results: The expression of NGAL significantly upregulated in day 1, 3 and week 1 following the crushing of sciatic nerves in comparison with the intact nerves. Immunohistochemistry results also confirmed the protein expression of this gene., Discussion: The NGAL molecule showed upregulation in the degeneration process after nerve injury, so it may play an important role in nerve repair.

Muscular dystrophy (MD) is associated with mutations in genes that stabilize the myofiber plasma membrane, such as through the dystrophin-glycoprotein complex (DGC). Instability of this complex or defects in membrane repair/integrity leads to calcium influx and myofiber necrosis leading to progressive dystrophic disease. MD pathogenesis is also associated with increased skeletal muscle protease levels and activity that could augment weakening of the sarcolemma through greater degradation of cellular attachment complexes. Here, we observed a compensatory increase in the serine protease inhibitor Serpina3n in mouse models of MD and after acute muscle tissue injury. Serpina3n muscle-specific transgenic mice were generated to model this increase in expression, which reduced the activity of select proteases in dystrophic skeletal muscle and protected muscle from both acute injury with cardiotoxin and from chronic muscle disease in the mdx or Sgcd(-/-) MD genetic backgrounds. The Serpina3n transgene mitigated muscle degeneration and fibrosis, reduced creatine kinase serum levels, restored running capacity on a treadmill and reduced muscle membrane leakiness in vivo that is characteristic of mdx and Sgcd(-/-) mice. Mechanistically, we show that increased Serpina3n promotes greater sarcolemma membrane integrity and stability in dystrophic mouse models in association with increased membrane residence of the integrins, the DGC/utrophin-glycoprotein complex of proteins and annexin A1. Hence, Serpina3n blocks endogenous increases in the activity of select skeletal muscle resident proteases during injury or dystrophic disease, which stabilizes the sarcolemma leading to less myofiber degeneration and increased regeneration. These results suggest the use of select protease inhibitors as a strategy for treating MD., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Protein quality refers to the amounts and ratios of essential amino acids in a food. Two methods most commonly used for determining protein quality are the protein digestibility-corrected amino acid score (PDCAAS) and the digestible indispensible amino acid score (DIAAS)., Objective: To use existing literature to compare different amino acid profiles and PDCAAS and DIAAS scores in individuals with acute inflammation and to assess their relationship with weight gain in children with severe acute malnourished (SAM)., Methods: A series stable isotope studies were previously conducted in children with SAM and acute infection, and these data were reviewed with respect to protein synthesis. Eleven published treatment trials for SAM with different therapeutic foods were analyzed to examine the relationship between protein quality scores with weight gain (g/kg/d). Protein scores were calculated with the PDCAAS and DIAAS amino acid reference patterns. A DIAAS score adjusted for the higher weight gain expected in malnourished children was also used. Bivariate correlation analysis was used to examine this relationship., Results: The protein kinetic data supported the hypothesis that a balance of amino acids that matches the composition of acute-phase proteins maximizes amino acid synthesis. Protein quality scores were highly correlated with the rate of weight gain in recovery from SAM, and the DIAAS scoring system adjusted for the higher expected weight gain had the strongest correlation with the observed weight gain., Conclusion: Protein quality scores must account for physiologic status so that they better match with needs and thus better promote health., (© The Author(s) 2016.)

Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1 mucin-type O-glycans (T-antigen). The developed workflow allows the identification and characterization of the major population of the human blood plasma O-glycoproteome and our results provide new insights, which can help to unravel structure-function relationships. The data were deposited to ProteomeXchange PXD003270., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Hypoxia-induced cardiomyocyte apoptosis contributes significantly to the development of numerous cardiac diseases, such as ischemic heart disease, heart failure, etc. Promoting cell survival by inhibiting apoptosis is one of the available strategies to attenuate cardiac dysfunction caused by cardiomyocyte loss. Previous studies have been demonstrated that miR-138 and lipocalin-2 (Lcn2) play important roles in cardiomyocyte apoptosis and survival. We presently determined whether Lcn2 is a target gene of miR-138 involved in hypoxia-induced cardiomyocyte apoptosis. Firstly, mimics of miR-138 were transfected into HL-1 cells to investigate its effect on cell apoptosis. Using 3-(4,5-dimethyl-thiazol-2-y1) 2,5-diphenyl tetrazolium bromide (MTT) and Annexin V-FITC/PI flow cytometer assays, over-expression of miR-138 significantly enhanced the cell growth and significantly attenuated the cell apoptosis in hypoxic conditions. Dual-luciferase reporter gene and western blot results confirmed Lcn2 was a direct target of miR-138. Then, the recombinant plasmid, pcDNA3.1/Lcn2 was transfected into the HL-1 cells that over-expressed miR-138. We further observed that the over-expression of Lcn2 diminished the protection of miR-138 over-expression from hypoxia-induced cell survival and apoptosis. In conclusion, our study demonstrated that up-regulation of miR-138 inhibits the hypoxia-induced cardiomyocyte apoptosis via down-regulating the pro-apoptotic gene expression of Lcn2., (© 2016 by the Society for Experimental Biology and Medicine.)

Tumor initiating cancer stem-like cells (TICSCs) have recently become the object of intensive study. Human-Lipocalin-2 (hLCN2) acts as a biomarker for cancers. The aim of the present study was to explore new insights regarding the potential role of LCN2 in inducing epithelial to mesenchymal transition (EMT) by transfecting LCN2 into CD133+-A549-TICSCs and its cross-talk with the NF-κB signaling pathway in adenocarcinoma of the lung. Furthermore, EMT was confirmed by transcriptomic analysis, immunoblotting and immunocyto/histochemical analyses. Tumorigenesis and metastasis were confirmed by molecular therapeutics tracer 2DG infrared optical probe in BALB/cSIc-nude mice. It was observed that the CD133+-expressing-LCN2-A549 TICSCs population increased in adenocarcinoma of the lung compared to the normal lung tissue. The expressions of genes involved in stemness, adhesion, motility and drug efflux was higher in these cells than in their non-LCN2 expressing counterparts. The present study revealed that elevated expression of LCN2 significantly induced metastasis via EMT. Overexpression of LCN2 significantly increased stemness and tumor metastasis by modulating NF-κB cellular signaling. BRM270, a novel inhibitor of NF-κB plays a significant role in the EMT reversal. BRM270, a naturaceutical induces cell shrinkage, karyorrhexis and programmed cell death (PCD) which were observed by Hoechst 33342 staining while flow cytometry analysis showed significant (P<0.05) decrease in cell population from G0-G1 phases. Also, 2DG guided in vivo model revealed that BRRM270 significantly (P<0.0003) reduced tumor metastasis and increased percent survival in real-time with complete resection. An elaborate study on the novel concept with respect to linking of naturaceutics as selective and potential anticancer agent that eliminates the elevated LCN2 induced EMT and tumor dissemination through cooperation with the NF-κB signaling as the baseline data for the planning of new therapeutic strategies was conducted for the first time. Our results also illustrate a molecular mechanistic approach for 2DG-guided molecular imaging-based cancer therapy using BRM270 as a novel cancer therapeutic drug to enhance the effect of doxorubicin (Dox)-resistant LCN2 induced metastasis of solid tumors in nude mice.

The expression of genes encoding the acute phase proteins (APP) during the course of Trypanasoma carassii infection in the goldfish was determined using quantitative PCR. Significant changes in the mRNA levels of ceruloplasmin (Cp), C-reactive protein (CRP), transferrin (Tf), hemopexin (Hx) and serum amyloid A (SAA) were observed in the kidney, liver and spleen at various days post infection (dpi). Of the five acute phase protein genes examined, CRP and SAA exhibited the highest expression in the tissues during the acute infection. Cp and Tf were up-regulated throughout the acute course of infection in the liver. During the chronic phase of the infection, APP expression in the liver was similar to that in the non-infected control fish. At 7 dpi, Cp, Tf and Hx were down-regulated in the spleen, and Cp and Tf kidney, but their mRNA levels gradually returned to those of control non-infected fish. In contrast, during the chronic phase of the infection, there was an up-regulation of Cp, Hx and Tf in the spleen, and Tf and SAA in the kidney. The goldfish CRP was cloned and functionally characterized. CRP was differentially expressed in normal goldfish immune cells, with highest expression in monocytes and lowest expression in mature macrophages. A recombinant goldfish CRP (rgfCRP) was generated using prokaryotic expression. rgfCRP enhanced complement-mediated killing of trypanosomes in vitro, and the lysis increased after addition of immune serum. rgfCRP did not affect the production of reactive oxygen and nitrogen intermediates by monocytes and macrophages, respectively., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The dysregulation of endometrial immune response to bacterial lipopolysaccharide (LPS) has been implicated in uterine disease and infertility in the postpartum dairy cow, although the mechanisms are not clear. Here, we investigated whole-transcriptomic gene expression in primary cultures of mixed bovine epithelial and stromal endometrial cells. Cultures were exposed to LPS for 6 h, and cellular response was measured by bovine microarray. Approximately 30% of the 1006 genes altered by LPS were classified as being involved in immune response. Cytokines and chemokines (IL1A, CX3CL1, CXCL2, and CCL5), interferon (IFN)-stimulated genes (RSAD2, MX2, OAS1, ISG15, and BST2), and the acute phase molecule SAA3 were the most up-regulated genes. Ingenuity Pathway Analysis identified up-regulation of many inflammatory cytokines and chemokines, which function to attract immune cells to the endometrium, together with vascular adhesion molecules and matrix metalloproteinases, which can facilitate immune cell migration from the tissue toward the uterine lumen. Increased expression of many IFN-signaling genes, immunoproteasomes, guanylate-binding proteins, and genes involved in the intracellular recognition of pathogens suggests important roles for these molecules in the innate defense against bacterial infections. Our findings confirmed the important role of endometrial cells in uterine innate immunity, whereas the global approach used identified several novel immune response pathways triggered by LPS in the endometrium. Additionally, many genes involved in endometrial response to the conceptus in early pregnancy were also altered by LPS, suggesting one mechanism whereby an ongoing response to infection may interfere with the establishment of pregnancy., (© 2015 by the Society for the Study of Reproduction, Inc.)

Purpose: Neutrophil gelatinase associated lipocalin, also known as lipocalin-2, is a 25 kDa protein now considered the biochemical gold standard for the early diagnosis of acute kidney injury. Recently lipocalin-2 was suggested to have an important role in several human neoplasias. In this study we assess lipocalin-2 expression in 2 renal tumor types and analyze its association with clinicopathological parameters and prognosis., Materials and Methods: The study group included 189 patients who underwent surgery for renal lesions between 2003 and 2013. Of these patients 105 had clear cell renal cell carcinoma and 84 had papillary renal cell carcinoma. The association of lipocalin-2 immunoexpression and clinicopathological characteristics was evaluated. Cox proportional hazard regression was performed to estimate the prognostic significance of potential confounders in predicting overall and disease-free survival., Results: Lipocalin-2 expression in different histotypes of analyzed tumors was highly and significantly associated with papillary renal cell carcinoma (p <0.001). In papillary renal cell carcinoma high lipocalin-2 expression was associated with high Fuhrman grade (p <0.001), tumor size greater than 7 cm (p = 0.007), increased TNM stage (p <0.001) and lymph node metastasis (p = 0.009). On univariable and multivariable Cox survival analyses of papillary renal cell carcinoma, after a median followup of 49.1 months (range 7 to 136) lipocalin-2 expression was associated with reduced overall survival (HR 4.10, 95% CI 1.19-14.14, p = 0.026) and disease-free survival (HR 2.56, 95% CI 1.01-6.48, p = 0.047)., Conclusions: Lipocalin-2 over expression may be a prognostic factor in decreased overall and disease-free survival in papillary renal cell carcinoma. The association of lipocalin-2 over expression and poor prognosis with papillary renal cell carcinoma may have potential diagnostic and therapeutic utility., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Orally administrated iron is suspected to increase susceptibility to enteric infections among children in infection endemic regions. Here we investigated the effect of dietary iron on the pathology and local immune responses in intestinal infection models. Mice were held on iron-deficient, normal iron, or high iron diets and after 2 weeks they were orally challenged with the pathogen Citrobacter rodentium. Microbiome analysis by pyrosequencing revealed profound iron- and infection-induced shifts in microbiota composition. Fecal levels of the innate defensive molecules and markers of inflammation lipocalin-2 and calprotectin were not influenced by dietary iron intervention alone, but were markedly lower in mice on the iron-deficient diet after infection. Next, mice on the iron-deficient diet tended to gain more weight and to have a lower grade of colon pathology. Furthermore, survival of the nematode Caenorhabditis elegans infected with Salmonella enterica serovar Typhimurium was prolonged after iron deprivation. Together, these data show that iron limitation restricts disease pathology upon bacterial infection. However, our data also showed decreased intestinal inflammatory responses of mice fed on high iron diets. Thus additionally, our study indicates that the effects of iron on processes at the intestinal host-pathogen interface may highly depend on host iron status, immune status, and gut microbiota composition., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To examine the significance of the Neutrophil gelatinase-associated lipocalin (NGAL) in diagnosing head and neck squamous cell carcinoma (HNSCC) and predicting regional metastasis. We first used GEO dataset to analyze the NGAL gene expression in HNSCC. Then, we summarized the characteristics of patients retrospectively selected in clinic. Expression of NGAL protein in human HNSCC tumor, lymph node and normal samples were analyzed using immunohistochemistry. Next, we further investigated the NGAL expression in a tissue microassay to analyze the relationship between NGAL protein expression and TNM stage. Finally, we tested the NGAL protein expression in head and neck cancer cell lines. Analysis of GEO dataset concluded that NGAL gene expression in HNSCC was lower than that in normal tissue (P<0.01). There was no statistically significant difference of NGAL gene expression between T-stage and N-stage (P>0.05). NGAL protein expression in tumor was lower than that in normal tissue (P<0.01). There was no statistically significant difference of NGAL protein expression between metastasis group and non-metastasis group (P>0.05). Expression of NGAL protein was not correlated with TNM stage of HNSCC. Aggressive HNSCC cell lines have lower NGAL protein expression. Our data demonstrated that the expression of NGAL protein was correlated with tumorigenesis of HNSCC, but not with regional metastasis. It may serve as a novel biomarker for prognostic evaluation of patients with HNSCC.

The expression of plasma proteins changes dramatically as a result of cytokine induction, particularly interleukin-6, and their levels are used as clinical markers of inflammation. miRNAs are important regulators of gene expression and play significant roles in many inflammatory diseases and processes. The interactions between miRNAs and the genes that they regulate during the acute phase response have not been investigated. We examined the effects of IL-6 stimulation on the transcriptome and miRNome of human and mouse primary hepatocytes and the HepG2 cell line. Using an integrated analysis, we identified differentially expressed miRNAs whose seed sequences are significantly enriched in the 3' untranslated regions of differentially expressed genes, many of which are involved in inflammation-related pathways. Our finding that certain miRNAs may de-repress critical acute phase proteins within acute timeframes has important biological and clinical implications., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Sepsis is a major healthcare problem and a leading cause of death worldwide. There is no dependable diagnosis, and treatment for this condition remains mainly supportive. The etiology of sepsis is related to an overwhelming inflammatory response. In this regard, the antimicrobial protein lipocalin-2 (Lcn2) has been associated with several inflammatory conditions, but its contribution to polymicrobial sepsis is unclear. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP), and Lcn2 mRNA levels and protein expression were measured in liver and lung tissues. We observed that Lcn2 expression was robustly induced in liver and lung of C57BL/6 J (B6) mice, and remained elevated during the stage of innate immune dysfunction observed in sepsis. This response was different in A/J mice, suggesting a contribution of the genetic background, probably due to differences in IL-10 expression between these two mouse strains. Indeed, IL-10 was found to regulate Lcn2 expression in both primary and J774A.1 macrophages. Thus, Lcn2 expression is highly regulated during CLP-induced sepsis, suggesting that this antimicrobial protein could have a role as a potential biomarker for the diagnosis of sepsis., (© The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

Background: Metastasis is the primary cause of prostate cancer (PCa) lethality and poses a huge clinical obstacle. Lipocalin 2 (LCN2), a member of the lipocalin family, is aberrantly expressed in some human cancers and has been implicated in the progression of some tumors. However, the role of LCN2 in the metastatic capacity of prostate cancer (PCa) is poorly understood., Methods: LCN2 expression was examined by RT-qPCR and/or immunoblotting in human prostate tissue specimens and prostate cancer cell lines LNCaP, C4-2, 22RV1, PC3, DU-145, and PC3MM2. LCN2 protein level in human serum samples was determined by ELISA. Lentiviruses-mediated over-expression of LCN2 and knockdown of LCN2 was conducted to evaluate the role of LCN2 in cell migratory and invasive capacities of prostate cancer cells. Cell migration and invasion was examined by transwell chamber assay. Knockdown of SLUG by lentivirus was performed to investigate its role in LCN2-promoted cell migration and invasion in vitro (22RV1 cell line) and metastasis in vivo (tail vein metastasis assay in nude mice). Role of ERK signaling in LCN2-mediated up-regulation of SLUG was assayed by using ERK inhibitor U0126., Results: We confirmed that LCN2 levels were correlated positively with invasive prostate cancer in human tissue and serum samples, and were also consistently associated with the invasive capacity of prostate cancer cell lines. The over-expression of LCN2 in 22RV1 cells (not highly invasive) promoted the epithelial-mesenchymal transition (EMT), increasing cell motility and invasiveness, while the knockdown of LCN2 in PC3 cells (highly invasive) inhibited EMT, decreasing cell motility and invasiveness. Among the multiple EMT transcription factors, LCN2 specifically induces the expression of SLUG, which was shown here to be required for the LCN2-induced increase in the invasive capacity of prostate cancer cells both in vitro and in vivo. Mechanistically, LCN2 promoted SLUG expression via activating ERK signaling pathway., Conclusion: LCN2 plays an important role in promoting cell migration and invasion of prostate cancer by inducing EMT through the ERK/SLUG axis. Therefore, targeted inhibition of LCN2 may represent a therapeutic strategy to prevent the metastasis of prostate cancer., (© 2015 Wiley Periodicals, Inc.)

Purpose: The current study was done to determine the role of lipocalin-2 (LCN2) in the pathogenesis of demyelinating optic neuritis using an experimental autoimmune optic neuritis (EAON) model., Methods: The EAON was induced by subcutaneous immunization with an emulsified mixture of myelin oligodendrocyte glycoprotein (MOG35-55) peptide in mice. The LCN2 expression was examined in the optic nerve after MOG peptide injection. Degree of demyelination, inflammatory infiltration, glial activation, and expression profile of inflammatory mediators in the optic nerve were compared between LCN2 knockout (KO) animals and wild-type littermates by histological analysis and real-time PCR following EAON induction. Plasma levels of LCN2 in patients with optic neuritis were measured by ELISA., Results: The expression of LCN2 was notably increased in the optic nerve after EAON induction. Expression of LCN2 was colocalized with reactive astrocytes. A significant reduction of demyelination, inflammatory infiltration, and gliosis was demonstrated in the optic nerve of LCN2 KO mice. The LCN2 KO mice also showed markedly reduced gene expression associated with the M1-polarized glia phenotype and toll-like receptor signaling in the optic nerve. The LCN2 levels in plasma were significantly higher in optic neuritis patients (71.6 ± 10.6 ng/mL) compared to healthy controls (37.4 ± 9.1 ng/mL, P = 0.0284)., Conclusions: In this study, we demonstrated a significant induction of LCN2 expression in astrocytes of the optic nerve following EAON induction. Our results imply that astrocyte-derived LCN2 may have a pivotal role in the development of demyelinating optic neuritis, and LCN2 can be a therapeutic target to alleviate immune and inflammatory damage in the optic nerve.

Prognosis in patients with lung cancer is poor. Neutrophil gelatinase-associated lipocalin (NGAL) and matrix metalloproteinase-9 (MMP-9) are proteins involved in the invasion and metastases of cancer. The objective of this study is to determine if there is a relationship between tumor expression of NGAL and MMP-9 in lung adenocarcinoma patients with prognosis and overall survival. Retrospective analysis was made of patients with lung adenocarcinoma treated at Medica Sur Hospital between 2005 and 2013. Tumor tissue was analyzed for NGAL and MMP-9 expression by immunohistochemistry. We identified 41 patients. Mean overexpression in tumoral tissue of NGAL was 70 % and 30 % for MMP-9. Univariate analysis revealed that prognostic factors associated with overall survival (OS) were NGAL expression and stage at diagnosis. Median OS for NGAL expression < 70 % was 45.7 months (95 % CI; 15.2-76.2) and for patients with ≥ 70 % 4.6 months (95 % CI; 0.5-18.8; P < 0.0001), and for stage at diagnosis (stages I and II mean not reached), stage III mean OS 15.57 months (95 % CI; 9.8-21.2) and stage IV 9.6 months (95 % CI; 0.8-18.4. P = 0.002). No differences in OS were found for expression of MMP-9. Multivariate analysis revealed significance for OS in NGAL expression (HR 5.01 [95 % CI; 1.68-14.93] P = 0.004) and stage at diagnosis (HR 2.05 [95 % CI 1.30-3.22] P = 0.002). Tumoral tissue expression of NGAL ≥ 70 % confers a worse prognosis compared to those who did not. NGAL is an independent prognostic factor of stage at diagnosis.

Background: Binding lipopolysaccharide (LPS) with high-affinity, lipopolysaccharide-binding protein (LBP) and CD14 lower the threshold stimulatory concentrations of LPS dramatically and enhance the rate of cytokine production markedly. This study aimed to investigate the kinetic expression of LBP/CD14 and its possible relationship with tumor necrosis factor alpha (TNF-α) to better understand the pathophysiology of obstructive jaundice., Materials and Methods: The tissues (liver, spleen, intestine, and lung) of male Sprague-Dawley rats were harvested at pre-bile duct ligation in controls and at specific time points (24, 48, 72, 96, and 120 hr) after bile duct ligation. LBP, CD14, and TNF-α mRNA expression were measured in tissues harvested from controls and at the specific time points., Results: Hepatic LBP mRNA expression increased significantly at five days after bile duct ligation. CD 14 mRNA expression increased significantly after five days of bile duct ligation in liver, lung, spleen, and ileum. TNF-α mRNA expression increased significantly in all four organs (liver, lung, spleen, and ileum) after four days of bile duct ligation., Conclusion: Five days of bile duct ligation upregulated CD 14 mRNA expression in liver, lung, spleen, and ileum and increased TNF-α mRNA expression simultaneously in the liver, lung, spleen, and ileum. In addition, five days of bile duct ligation also upregulated LBP mRNA expression in the liver and increased hepatic TNF-α mRNA expression simultaneously. The kinetic expressions of LBP and CD 14 in obstructive jaundice are intriguing and further evaluation is warranted.

Nanoparticles (NP) are pervasive in many areas of modern life, with little known about their potential toxicities. One commercially important NP is cadmium oxide (CdO), which is used to synthesize other Cd-containing NP, such as quantum dots. Cadmium (Cd) is a well-known nephrotoxicant, but the nephrotoxic potential of CdO NP remains unknown, particularly when exposure occurs during pregnancy. Therefore, pregnant CD-1 mice were used to examine the effects of inhaled CdO NP (230 μg CdO NP/m(3)) on maternal and neonatal renal function by examining urinary creatinine and urinary biomarkers of kidney injury, including kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin (NGAL). Inhalation of CdO NP by dams produced a fivefold increase in urinary Kim-1 with no marked effect on urinary creatinine levels. Kim-1 mRNA expression peaked by gestational day (GD) 10.5, and NGAL expression increased from GD 10.5 to 17.5. In addition, histological analyses revealed proximal tubular pathology at GD 10.5. Neonatal Kim-1 mRNA expression rose between postnatal days (PND) 7 and 14, with mammary glands/milk being the apparent source of Cd for offspring. These studies demonstrate that, similar to what is seen with other Cd forms, Cd associated with inhaled CdO NP results in renal injury to both directly exposed dam and offspring. As commercial uses for nanotechnology continue to expand throughout the world, risks for unintentional exposure in the workplace increase. Given the large number of women in the industrial workforce, care needs to be taken to protect these already vulnerable populations.

Aim of the study is to evaluate the expression of CK19, galectin-3, HBME-1, CD56, claudin-1 and neutrophil gelatinase-associated lipocalin (NGAL) in papillary thyroid carcinoma (PTC), including classic, follicular variant and micro-carcinoma. Peritumoral benign thyroid tissues were used as a control (C). Immunohistochemical staining with the EnVision detection system was performed on 59 formalin-fixed, paraffin-embedded thyroid tissues, including 43 PTC and 16 C. CK19, galectin-3, HBME-1, claudin-1 and NGAL were positive in most PTC, but were negative or showed focal weak staining in C. CD56 was positive in C, but absent in PTC. For a single analyzed tumor marker, the sensitivity of the above six thyroid tumor markers was 100%, 95.3%, 86%, 79.1%, 90.7%, 93%, respectively. The specificity was 56.25%, 100%, 100%, 100%, 100%, 100%, respectively. For the combination of the six tumor markers, the sensitivity, specificity, positive predictive value, negative predictive value and the diagnostic accuracy were all 100%, with co-expression of at least four indices. In the diagnosis of PTC, combined application of the above tumor markers is recommended. It can be diagnosed as PTC clearly when thyroid lesions are positive for at least four of the above tumor markers [CD56⁻], and can be excluded as PTC definitely when at least four tumor markers are negative [CD56⁺].

The innate immune system has been implicated in several neurodegenerative diseases, including HIV-1-associated dementia. In this study, we show that genetic ablation of CCR5 prevents microglial activation and neuronal damage in a transgenic model of HIV-associated brain injury induced by a CXCR4-using viral envelope gp120. The CCR5 knockout (KO) also rescues spatial learning and memory in gp120-transgenic mice. However, the CCR5KO does not abrogate astrocytosis, indicating it can occur independently from neuronal injury and behavioral impairment. To characterize further the neuroprotective effect of CCR5 deficiency we performed a genome-wide gene expression analysis of brains from HIVgp120tg mice expressing or lacking CCR5 and nontransgenic controls. A comparison with a human brain microarray study reveals that brains of HIVgp120tg mice and HIV patients with neurocognitive impairment share numerous differentially regulated genes. Furthermore, brains of CCR5 wild-type and CCR5KO gp120tg mice express markers of an innate immune response. One of the most significantly upregulated factors is the acute phase protein lipocalin-2 (LCN2). Using cerebrocortical cell cultures, we find that LCN2 is neurotoxic in a CCR5-dependent fashion, whereas inhibition of CCR5 alone is not sufficient to abrogate neurotoxicity of a CXCR4-using gp120. However, the combination of pharmacologic CCR5 blockade and LCN2 protects neurons from toxicity of a CXCR4-using gp120, thus recapitulating the finding in CCR5-deficient gp120tg mouse brain. Our study provides evidence for an indirect pathologic role of CCR5 and a novel protective effect of LCN2 in combination with inhibition of CCR5 in HIV-associated brain injury., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer. Vitamin D, a pro-hormone, is getting popular due to its hormone-like functions after converted to its active form, 1α,25(OH)2D3. Here, we show that dietary supplementation with 6 IU/g of vitamin D greatly suppressed ICC initiation and progression without apparent toxicity in a chemically induced rat model. Microarray analysis of rat ICC tissues showed vitamin D supplementation modulated the expressions of several unique genes, including lipocalin 2 (Lcn2), confirmed by RT-qPCR and immunohistochemical (IHC) staining. Further, 53 of 80 human ICC specimens (66%) exhibited high LCN2 expression and LCN2 knockdown in SNU308 cells decreased cell growth and migration, suggesting LCN2 be an oncogene in human ICC. As human ICC SNU1079 cells were treated by 1α,25(OH)2D3, LCN2 expression and cell proliferation were attenuated. The downregulation of LCN2 expression was blunted when vitamin D receptor (VDR) was knocked down, implicating that the in vivo Lcn2 downregulation is a direct consequence of vitamin D supplementation Our results support the prevailing concept that vitamin D status is negatively associated with cancer incidence and mortality and suggest LCN2 may be a potential target against ICC. Further studies of application of vitamin D or its analog against ICC are warranted.

Lipocalin-2 (LCN2) is secreted from adipocytes, and its expression is up-regulated in obese and diabetic mice and humans. LCN2 expression and secretion have been shown to be induced by two proinflammatory cytokines, IFNγ and TNFα, in cultured murine and human adipocytes. In these studies, we demonstrated that IFNγ and TNFα induced LCN2 expression and secretion in vivo. Although we observed a strong induction of LCN2 expression and secretion from white adipose tissue (WAT) depots, the induction of LCN2 varied among different insulin-sensitive tissues. Knockdown experiments also demonstrated that STAT1 is required for IFNγ-induced lipocalin-2 expression in murine adipocytes. Similarly, knockdown of p65 in adipocytes demonstrated the necessity of the NF-κB signaling pathway for TNFα-mediated effects on LCN2. Activation of ERKs by IFNγ and TNFα also affected STAT1 and NF-κB signaling through modulation of serine phosphorylation. ERK activation-induced serine phosphorylation of both STAT1 and p65 mediated the additive effects of IFNγ and TNFα on LCN2 expression. Our results suggest that these same mechanisms occur in humans as we observed STAT1 and NF-κB binding to the human LCN2 promoter in chromatin immunoprecipitation assays performed in human fat cells. These studies substantially increase our knowledge regarding the requirements and mechanisms used by proinflammatory cytokines to induce LCN2 expression.

Interleukin-6 (IL-6) is a cytokine with redundant and pleiotropic activities, and its synthesis is tightly regulated by transcriptional and posttranscriptional mechanisms. When infections and tissue injuries occur, IL-6 synthesis is promptly induced and provides an emergent signal that contributes to host defense through the stimulation of acute-phase responses, immune reactions, and hematopoiesis. After the environmental stress is removed from the host, the production of IL-6 is terminated. However, dysregulated continual synthesis of IL-6 is involved in the development of chronic inflammatory autoimmune diseases. For this reason, tocilizumab, a humanized anti-IL-6 receptor antibody, was developed. Worldwide clinical trials have demonstrated the outstanding efficacy of tocilizumab in rheumatoid arthritis, systemic juvenile idiopathic arthritis, and Castleman's disease; thus, a new era has come for the treatment of these diseases, which were previously considered intractable. Moreover, favorable results from off-label use of tocilizumab strongly suggest that it will be widely applicable for various refractory inflammatory autoimmune diseases. In this context, the mechanism for the continual synthesis of IL-6 needs to be elucidated in order to investigate the pathogenesis of specific diseases and to facilitate the development of more specific therapeutic strategies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: Over-expression of the aldo-keto reductase family 1 member C3 (AKR1C3) has been demonstrated in many human cancers. Lipocalin 2 (LCN2) is reported to inhibit cervical cancer metastasis but little is known regarding its relationship with AKR1C3 in the development and progression of uterine cervical cancer. This study aimed to investigate the involvement of AKR1C3 and its relationship with LCN2 in cervical cancer., Methods: The roles of AKR1C3 and LCN2 were investigated using the lentivirus shRNA system in SiHa and Caski cervical cancer cells. LCN2 and matrix metalloproteinase-2 (MMP-2) promoters were constructed to demonstrate transcriptional regulation by shAKR1C3 and shLCN2, respectively. The influences of metastatic phenotypes were analyzed by wound healing, Boyden chamber, and immunofluorescence assays. The activity of MMP-2 was determined by zymography assay. The impacts of AKR1C3 and LCN2 on patient prognosis were evaluated using tissue microarrays by Cox regression and Kaplan-Meier models., Results: Silencing of the AKR1C3 gene increased the expression of LCN2 and decreased the migratory and invasive abilities and changed the cytoskeleton of cervical cancer cells. When AKR1C3 was over-expressed, it decreased LCN2 promoter activity and LCN2 expression and increased cell migration. The mRNA level and enzyme activity of MMP-2 increased in silenced LCN2 cells. Positive AKR1C3 and negative LCN2 were correlated with higher recurrence and poorer survival of cervical cancer patients., Conclusions: Silencing of AKR1C3 increases LCN2 expression and inhibits metastasis in cervical cancer. Both AKR1C3 and LCN2 serve as molecular targets for cancer therapy to improve the clinical outcome of cervical cancer patients., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The kidney proximal tubule is a key target in many forms of acute kidney injury (AKI). The multiligand receptor megalin is responsible for the normal proximal tubule uptake of filtered molecules, including nephrotoxins, cytokines, and markers of AKI. By mediating the uptake of nephrotoxins, megalin plays an essential role in the development of some types of AKI. However, megalin also mediates the tubular uptake of molecules implicated in the protection against AKI, and changes in megalin expression have been demonstrated in AKI in animal models. Thus, modulation of megalin expression in response to AKI may be an important part of the tubule cell adaption to cellular protection and regeneration and should be further investigated as a potential target of intervention. This review explores current evidence linking megalin expression and function to the development, diagnosis, and progression of AKI as well as renal protection against AKI.

NGAL (neutrophil gelatinase-associated lipocalin) is a novel cancer-related protein involves multiple functions in many cancers and other diseases. We previously overexpressed NGAL to analyze its role in esophageal squamous cell carcinoma (ESCC). In this study, a protein-protein interaction (PPI) was constructed and the shortest paths from NGAL to transcription factors in the network were analyzed. We found 28 shortest paths from NGAL to RELA, most of them obeying the principle of extracellular to cytoplasm, then nucleus. These shortest paths were also prioritized according to their normalized intensity from the microarray by the order of interaction cascades. A systems approach was developed in this study by linking differentially expressed genes with publicly available PPI data, Gene Ontology and subcellular localizaton for the integrated analyses. These shortest paths from NGAL to DEG transcription factors or other transcription factors in the PPI network provide important clues for future experimental identification of new pathways.

Background: Lipocalin-2 is a multifaceted modulator in cancer progression. Its clinical significance is not clear in pancreatic cancer. The purpose of this study was to investigate whether lipocalin-2 is associated with good prognosis by reversing epithelial-to-mesenchymal transition (EMT) in pancreatic cancer., Methods: Lipocalin-2, E-cadherin, or vimentin expression was detected in 60 pancreatic adenocarcinoma specimens. Correlations between lipocalin-2 expression and EMT, the clinicopathologic characteristics, and prognosis were investigated. Whether pancreatic cancer cells' migration and invasion (some characteristics of EMT) were affected by lipocalin-2 was also explored., Results: High lipocalin-2 expression was significantly associated with a good prognosis in pancreatic cancer (p < 0.05). Overexpression of lipocalin-2 correlated with a lower extent of EMT (p < 0.05), increased E-cadherin expression (p < 0.05), decreased vimentin expression (p < 0.05), and reduced cancer cell migration and invasion in pancreatic cancer., Conclusions: Lipocalin-2 may be considered an epithelial inducer, which may reverse EMT and predict a good prognosis in pancreatic cancer.

The in vivo effects of the Fusarium toxin deoxynivalenol (DON) on albumin and fibrinogen synthesis in pigs and metabolic activity of porcine peripheral blood mononuclear cells (PBMCs) were studied alone or in combination with lipopolysaccharides (LPSs) in order to examine proposed synergistic effects of both substances. A total of 36 male castrated pigs (initial weight of 26 kg) were used. Uncontaminated (Control) and naturally DON-contaminated (chronic oral DON, 3.1mg/kg diet) wheat was fed for 37 days. On the day of protein synthesis measurement, pigs recruited from the Control group were treated once intravenously with (iv) DON (100 μg/kg live weight (LW)/h), iv LPS (7.5 μg/kgLW/h) or a combination of both substances, and six pigs from the chronic oral group were treated once with iv LPS. A treatment with DON alone exhibited no alterations of acute phase protein synthesis and metabolic activity of PBMC. There was no evidence that the chosen dosing regimen of DON had influences on the induced sub-acute stage of sepsis, as the LPS challenge, irrespective of DON co-exposure, mediated an acute phase reaction with a typical decrease of albumin synthesis, as well as changes in cytokine concentration and a loss of metabolic activity in PBMC., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: Acute-phase proteins, such as C-reactive protein and albumin, may be related with course and outcome in status epilepticus, as changes of cytokine levels and blood-brain barrier breakdown during status epilepticus have been demonstrated. The aim of this study was to elucidate the association of C-reactive protein and albumin with course and outcome of status epilepticus., Design: Observational cohort study., Setting: This study was performed on the ICU of a university-affiliated tertiary care center., Patients: All consecutive patients with status epilepticus from 2005 to 2009 were selected from a prospectively established electroencephalography database., Intervention: None., Measurements: Albumin was assessed at admission and status epilepticus onset, and C-reactive protein was assessed during the first 3 days of status epilepticus. Outcomes were defined as refractory status epilepticus and death., Main Results: One hundred thirty-five consecutive status epilepticus patients were analyzed. Patients with higher levels of albumin at status epilepticus onset had significant lower odds for the development of refractory status epilepticus and death (with every 1g/L: odds ratio 0.91, 95% confidence interval 0.86-0.96, p = 0.001; odds ratio 0.88, 95% confidence interval 0.82-0.95, p < 0.0001, respectively). These associations remained significant after multiple adjustments for possible confounders and correction for multiple comparisons (with every 1g/L: odds ratio 0.92, 95% confidence interval 0.86-0.97, p = 0.004; odds ratio 0.87, 95% confidence interval 0.80-0.94, p = 0.001, respectively). Increased C-reactive protein levels at status epilepticus onset were associated with higher rates of refractory status epilepticus and death (with every 1mg/L: odds ratio 1.01, 95% confidence interval 1.00-1.02, p = 0.021; odds ratio 1.01, 95% confidence interval 1.00-1.02, p < 0.007, respectively). These associations were inconsistent after adjustment for possible confounders and corrections for multiple comparisons (with every 1mg/L: odds ratio 1.01, 95% confidence interval 1.00-1.02, p = 0.109; odds ratio 1.01, 95% confidence interval 1.00-1.02, p = 0.043)., Conclusions: Albumin levels measured early in status epilepticus are independently associated with refractory epileptic activity and death while C-reactive protein levels were inconsistent. Further studies are needed to assess the potential of acute-phase proteins for inclusion in prediction models allowing to identify patients with poor outcome of status epilepticus.

Venous ulcers are related to dysfunctions in extracellular matrix. Both matrix metalloproteinases (MMP) and neutrophil gelatinase-associated lipocalin (NGAL) could play a role in the healing process in patients with chronic venous ulcers. We evaluated the role of MMP-9 and NGAL in the healing process in venous ulceration. We performed an open-label, parallel groups, single clinical center study. Patients with chronic venous leg ulcers represented the test group (Group I), whereas patients without chronic ulcers represented the control group (Group II). In Group I plasma and wound fluid samples were collected at the time of admission, at the time of the surgery, and at the follow-up, while ulcer tissues were taken at the time of the surgery. In Group II, plasma and wound fluid were collected at admission and at the time of the surgery, whereas skin tissues were collected at the time of the surgery. Enzyme-linked immunosorbent assay test was used to evaluate the levels of MMP-9 and NGAL in plasma and wound fluid, whereas Western blot analysis was performed to estimate the expression of MMP-9 and NGAL in tissues. Enzyme-linked immunosorbent assay tests revealed significantly higher levels of MMP-9 and NGAL in both plasma and wound fluid of patients with ulcers compared to patients without ulcers (p < 0.01). Moreover, Western blot analysis documented an increased expression of MMP-9 and NGAL in biopsy tissue of patients with ulcers compared to patients without ulcers (p < 0.01). In conclusion MMP-9 and NGAL may correlate with the clinical course of venous ulcers., (© 2013 by the Wound Healing Society.)

Evidence that lipocalin 2 (LCN2) is oncogenic has grown in recent years and comes from both animal models and expression analysis from a variety of human cancers. In the intestine, LCN2 is overexpressed in colitis patients and its overexpression is a negative prognostic indicator in colorectal cancer. Functionally, LCN2 has a number of different activities that may contribute to its oncogenic potential, including increasing matrix metalloproteinase activity, control of iron availability and stimulating inflammation. In this report, we examined APCmin intestinal tumorigenesis in an LCN2-deficient background. We found that the loss of LCN2 increased tumor multiplicity specifically in the duodenum, suggesting a potential tumor-suppressive activity. Concurrently, however, LCN2 increased the average small intestinal tumor size particularly in the distal small intestine. We found that this increase was correlated to tumor iron(II) content, suggesting that an iron-scavenging role is important for LCN2 oncogenic activity in the intestine.

Mounting evidence suggests that immune disturbances in early life may be implicated in the etiology of non-affective psychoses. Our aim was to assess the levels of neonatal acute phase proteins (APPs), central to innate immune function as well as central nervous system development, in neonatal dried blood spots and their association with later risk of non-affective psychoses. This case-control study included 196 individuals with a verified register-based diagnosis of non-affective psychosis and 502 controls matched on age, sex and hospital of birth. Concentrations of nine different APPs were measured in eluates from dried blood spots using a bead-based multiplex assay. Odds ratios (OR) for non-affective psychoses were calculated for log(2)-transformed (continuous) as well as tertiles of APP concentrations. In continuous analysis, higher concentrations of two APPs, tissue plasminogen activator (tPA; OR: 0.90, 95% confidence interval (CI): 0.85-0.96) and serum amyloid P (SAP; OR: 0.88, 95% CI: 0.78-0.99) were protective in terms of risk of non-affective psychosis. These relationships were not affected by the addition of covariates relevant to maternal health, pregnancy and delivery to the model. Tertile analysis confirmed a protective relationship for higher levels of tPA and SAP, as well as for procalcitonin (highest tertile OR: 0.54, 95% CI:0.32-0.91). Our results suggest that persons who develop non-affective psychoses have lower levels of certain APPs at the time of birth. These differences may render individuals more susceptible to infectious diseases or cause deficiencies in pathways critical for neurodevelopment.

Matrix metalloproteinases (MMPs) are involved in the remodeling of extracellular matrix in various tissues. Their functioning could be related to the formation of complexes, containing MMP9, MMP2, tissue inhibitor of metalloproteinases type 1 (TIMP1), and neutrophil gelatinase-associated lipocalin (NGAL). Such complexes have not been investigated in either myocardial or skeletal muscles. We examined 20 male pigs with heart failure (HF), and 5 sham-operated animals. There were no differences in the mRNA expression of MMP9, MMP2, TIMP1, and NGAL between diseased and healthy animals, in either left ventricle (LV) myocardium or skeletal muscles. In LV from both diseased and healthy animals, in nonreducing and nondenaturing conditions, we demonstrated the presence of high molecular weight (HMW) complexes (130, 170, and 220 kDa) containing MMP9, TIMP1, and NGAL (also MMP2 in 220 kDa complex) without proteolytic activity, and a proteolytically active 115 kDa MMP9 form together with 72 and 68 kDa bands (proMMP2 and MMP2). Proteolytically active bands were also spontaneously released from HMW complexes. In skeletal muscles from both diseased and healthy animals, in nonreducing and nondenaturing conditions, we found no HMW complexes, and proteolytic activity was associated with the presence of 72 and 68 kDa bands (proMMP2 and MMP2).

Background: Elevations of intraabdominal pressure during laparoscopic procedures may lead to oliguria or anuria in mammals. Despite this, previous research has not been able to confirm an associated kidney injury. This study aimed to investigate the occurrence of an early kidney lesion secondary to surgical pneumoperitoneum in a rat model using the expression of neutrophil gelatinase-associated lipocalin (N-GAL) as a biomarker for early kidney injury., Methods: In this study, 20 male Sprague-Dawley rats under general anesthesia and mechanically ventilated were allocated to one of five experimental time-dependent groups: group 1 (1-h control), group 2 (1-h pneumoperitoneum), group 3 (2-h control), group 4 (2-h pneumoperitoneum), and group 5 (positive kidney injury group induced by intravenous administration of cisplatin 7.5 mg/kg). To evaluate the renal expression of N-GAL 24 h after the procedure, all the rats underwent a 2-h urine output evaluation as well as laparotomy and bilateral nephrectomy performed sequentially to investigate the presence of renal injury using immunofluorescence qualification and western blotting., Results: Urine output was reduced and N-GAL expression was increased in the animals from the cisplatin group. The animals undergoing 1- or 2-h pneumoperitoneum displayed urine output and N-GAL expression similar to that of the animals from the matching control groups., Conclusions: Under the experimental conditions of this study, the animals with normal preoperative renal function did not show any type of acute kidney injury associated with the presence of a stabilized surgical pneumoperitoneum.

Kidney transplantation from a large donor to a small recipient, as in pediatric transplantation, is associated with an increased risk of thrombosis and DGF. We established a porcine model for renal transplantation from an adult donor to a small or size-matched recipient with a high risk of DGF and studied GFR, RPP using MRI, and markers of kidney injury within 10 h after transplantation. After induction of BD, kidneys were removed from ∼63-kg donors and kept in cold storage for ∼22 h until transplanted into small (∼15 kg, n = 8) or size-matched (n = 8) recipients. A reduction in GFR was observed in small recipients within 60 min after reperfusion. Interestingly, this was associated with a significant reduction in medullary RPP, while there was no significant change in the size-matched recipients. No difference was observed in urinary NGAL excretion between the groups. A significant higher level of HO-1 mRNA was observed in small recipients than in donors and size-matched recipients indicating cortical injury. Improvement in early graft perfusion may be a goal to improve short- and long-term GFR and avoid graft thrombosis in pediatric recipients., (© 2012 John Wiley & Sons A/S.)

Unlabelled: Neutrophil gelatinase-associated lipocalin (NGAL) is part of a functionally diverse family of proteins that generally bind small, hydrophobic ligands. Neutrophil gelatinase-associated lipocalin is expressed in a number of human tissues including gastrointestinal, respiratory, and urinary tracts and tends to rise in response to inflammation. For this reason, we hypothesized that levels of NGAL might be expressed at higher levels after blast injury compared with other ballistic injury., Purpose: The purpose of this study is to test the hypothesis that NGAL may be a marker of injury severity in blast injury., Materials: Twenty-three combat casualties (13 blast, 10 gunshot wounds) admitted to the multinational role 3 facility in Helmand province were studied. Serum NGAL was measured using a Biosite Triage point-of-care monitor at 5 time points after injury., Results: Neutrophil gelatinase-associated lipocalin rose in both groups of casualties and was significantly predictive of death or renal failure at intensive care unit admission, 12 and 24 hours after injury., Conclusions: Neutrophil gelatinase-associated lipocalin is not a specific marker of blast injury but is predictive of both renal failure and poor outcome., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Hypoferremia, associated with immune system activation, involves a marked reduction in the levels of circulating iron, coupled with iron sequestration within macrophages. Toll-like receptor (TLR) signaling plays an important role in the development of the hypoferremic response, but how downstream signaling events affect genes involved in iron metabolism is incompletely understood. We investigated the involvement of MyD88-dependent (MyD88) and MyD88-independent (TRIF) TLR signaling in the development of hypoferremia. Using MyD88-deficient and TRIF-deficient mice, we show that MyD88 and TRIF signaling pathways are critical for up-regulation by lipopolysaccharide (LPS) of the iron regulator hepcidin. In addition, MyD88 signaling is required for the induction of lipocalin 2 secretion and iron sequestration in the spleen. Activation of TLR4 and TLR3 signaling through LPS and polyinosinic:polycytidylic acid [poly(I:C)] treatments resulted in rapid down-regulation of HFE protein [encoded by the hemochromatosis gene (Hfe)] and ferroportin [encoded by solute carrier family 40 (iron-regulated transporter), member 1 (Slc40a1)] expression in the spleen, independent of MyD88 or TRIF signaling and proinflammatory cytokine production. However, lack of MyD88 signaling significantly impaired the hypoferremic response triggered by LPS, indicating that ferroportin and HFE protein down-regulation alone are insufficient to maintain hypoferremia. The extent of the hypoferremic response was found to be limited by initial, basal iron levels. Together, these results suggest that targeting specific TLR signaling pathways by affecting the function of adaptor molecules may provide new strategies to counteract iron sequestration within macrophages during inflammation., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Rationale: Bacterial pneumonia is the most common infectious cause of death worldwide and treatment is increasingly hampered by antibiotic resistance. Mesenchymal stem cells (MSCs) have been demonstrated to provide protection against acute inflammatory lung injury; however, their potential therapeutic role in the setting of bacterial pneumonia has not been well studied., Objective: This study focused on testing the therapeutic and mechanistic effects of MSCs in a mouse model of Gram-negative pneumonia., Methods and Results: Syngeneic MSCs from wild-type mice were isolated and administered via the intratracheal route to mice 4 h after the mice were infected with Escherichia coli. 3T3 fibroblasts and phosphate-buffered saline (PBS) were used as controls for all in vivo experiments. Survival, lung injury, bacterial counts and indices of inflammation were measured in each treatment group. Treatment with wild-type MSCs improved 48 h survival (MSC, 55%; 3T3, 8%; PBS, 0%; p<0.05 for MSC vs 3T3 and PBS groups) and lung injury compared with control mice. In addition, wild-type MSCs enhanced bacterial clearance from the alveolar space as early as 4 h after administration, an effect that was not observed with the other treatment groups. The antibacterial effect with MSCs was due, in part, to their upregulation of the antibacterial protein lipocalin 2., Conclusions: Treatment with MSCs enhanced survival and bacterial clearance in a mouse model of Gram-negative pneumonia. The bacterial clearance effect was due, in part, to the upregulation of lipocalin 2 production by MSCs.

Background: Increased expression of lipocalin 2 (LCN2) has been observed in several cancers. The aim of the present study was to investigate LCN2 in endometrial cancer in relation to clinico-pathologic phenotype, angiogenesis, markers of epithelial-mesenchymal transition (EMT), and patient survival., Methods: Immunohistochemical staining was performed using a human LCN2 antibody on a population-based series of endometrial cancer patients collected in Hordaland County (Norway) during 1981-1990 (n = 256). Patients were followed from the time of primary surgery until death or last follow-up in 2007. The median follow-up time for survivors was 17 years. Gene expression data from a prospectively collected endometrial cancer series (n = 76) and a publicly available endometrial cancer series (n = 111) was used for gene correlation studies., Results: Expression of LCN2 protein, found in 49% of the cases, was associated with non-endometrioid histologic type (p = 0.001), nuclear grade 3 (p = 0.001), >50% solid tumor growth (p = 0.001), ER and PR negativity (p = 0.028 and 0.006), and positive EZH2 expression (p < 0.001). LCN2 expression was significantly associated with expression of VEGF-A (p = 0.021), although not with other angiogenesis markers examined (vascular proliferation index, glomeruloid microvascular proliferation, VEGF-C, VEGF-D or bFGF2 expression). Further, LCN2 was not associated with several EMT-related markers (E-cadherin, N-cadherin, P-cadherin, β-catenin), nor with vascular invasion (tumor cells invading lymphatic or blood vessels). Notably, LCN2 was significantly associated with distant tumor recurrences, as well as with the S100A family of metastasis related genes. Patients with tumors showing no LCN2 expression had the best outcome with 81% 5-year survival, compared to 73% for intermediate and 38% for the small subgroup with strong LCN2 staining (p = 0.007). In multivariate analysis, LCN2 expression was an independent prognostic factor in addition to histologic grade and FIGO stage., Conclusion: Increased LCN2 expression is associated with aggressive features and poor prognosis in endometrial cancer.

Tumor invasion is a critical step in the spread of cancer. S2R (sigma-2 receptor)/Pgrmc1 (progesterone receptor membrane component 1) is a cytochrome b(5)-related drug-binding orphan receptor essential for tumor formation and invasion. Secretory proteins drive these processes, so we screened for S2R(Pgrmc1)-dependent secreted proteins using antibody arrays. S2R(Pgrmc1) markedly regulated the expression of NGAL/LCN2 (neutrophil gelatinase-associated lipocalin/lipocalin 2), a secreted glycoprotein that binds to MMP-9 (matrix metalloproteinase 9) and protects it from degradation. S2R(Pgrmc1) knock-down blocked NGAL/LCN2 expression at the protein and RNA levels and decreased MMP9 activity. NGAL expression was required for MMP-9 activity and tumor formation. S2R(Pgrmc1) associates with EGFR and increases EGFR levels at the plasma membrane, and the EGFR inhibitors erlotinib and AG1478, as well as Akt and ERK inhibitors, suppressed the NGAL/LCN2 RNA and protein levels. NGAL is transcriptionally regulated by NFκB, and S2R(Pgrmc1) knock-down decreased the NFκB subunit p65/RelA acetylation, phosphorylation, and activation. In S2R(Pgrmc1) knock-down cells, p65 acetylation was reversed by inhibitors of histone deacetylase 1, and the inhibitors partially restored NGAL levels. Our results are consistent with a model in which S2R(Pgrmc1) increases NGAL/LCN2 levels by activating NFκB via EGFR.

Neutrophil gelatinase-associated lipocalin (NGAL) is a protein which participates in iron trafficking and which is involved in cancerogenesis and cancer progression. Since its over-expression has been documented in thyroid malignancies in comparison to thyroid normal gland, in the present study, we aimed to determine whether the evaluation of NGAL immunoexpression may be of help in the differential diagnosis of follicular-patterned thyroid lesions. Our additional aim was to test the possible interference of endogenous biotin on the immunohistochemical findings. Thus, all the immunohistochemical procedures, carried out with labeled streptavidin biotin method, were doubly performed, with or without the preliminary inhibition of endogenous biotin. No NGAL staining was found in the normal thyroid gland nor in the nodular colloid goiters or in Hashimoto's thyroiditis. NGAL expression appeared to be significantly more frequent in the malignant tumors in comparison to benign ones (P < 0.000001). Even more, NGAL expression appeared to be specific (specificity 93%) for carcinoma and represented a sensitive method (sensitivity 84%), with high negative (88%) and positive (94%) predictive values, as well as high diagnostic accuracy (88%), in the identification of follicular-patterned thyroid malignant tumors. The specificity, positive predictive value and diagnostic accuracy lowered when biotin was not preliminary inhibited, due to the presence of false positives among benign Hürthle cell tumors. In conclusion, the immunohistochemical detection of NGAL may be helpful in the differential diagnosis between malignant and benign follicular-patterned lesions of the thyroid. The use of biotin free system or the preliminary biotin inhibition is warranted for the detection of NGAL in thyroid samples, especially when dealing with Hürthle cell tumors.