Lipocalin 2 (LCN2) is a member of the lipocalin family. Elevated expression of LCN2 has been observed in many human tumors, suggesting it might be a potential biomarker and/or therapeutic target in malignancies. In this study, we aimed to explore LCN2 interacting proteins through bioinformatics, as well as their biological functions. Protein-protein interaction networks (PPIN) were constructed using LCN2 and its interacting proteins as the core node. These PPINs were scale free biological networks in which LCN2 and its interacting proteins could connect or cross-talk with at least one partner protein. Both functional and KEGG pathway enrichment analyses identified the known and potential biological functions of the PPIN, such as cell migration and cancer-related pathways. Expression levels of the PPIN proteins, as well as their expression correlations, in five types of brain tumor, were analyzed and integrated into the PPIN to illustrate a dynamic change. A significant correlation was found between the survival time of glioblastoma patients and the expression level of 10 genes (LCN2, MMP9, MMP2, PDE4DIP, L2HGDH, HNRNPA1, DDX31, LOXL2, FAM60A and RNF25). Taken together, our results suggest that LCN2 and its interacting proteins are mostly differentially expressed and have a distinguishing co-expression pattern. They might promote proliferation and migration via cell migration signaling and cancer-related pathways. LCN2 and its interacting proteins might be potential biomarkers in glioblastoma.

Psoriasis is a chronic and relapsing disorder with considerable negative effects on patients' quality of life. The finer details associated with the molecular mechanism of psoriasis and its pathogenesis remain somewhat elusive. Extensive studies have highlighted the crucial role of microRNAs (miRNAs) in the development of psoriasis. Hence, the current study aimed to investigate the effect of miR-383 on a psoriasis rat model and elucidate the underlying molecular mechanism. The rat psoriasis model was established via imiquimod (IMQ) induction followed by verification of miR-383 and LCN2 expression in the skin tissues of the models. ELISA was conducted to determine the secretion of inflammatory factors. Keratinocyte proliferation and apoptosis was evaluated by MTT assay and flow cytometric analysis. Down-regulation of miR-383 and up-regulation of LCN2 were detected in the psoriasis rat model. Our data indicated that miR-383 targeted LCN2 by binding to its 3'UTR and inhibited JAK/STAT pathway activation. Notably, miR-383 overexpression or LCN2 knockdown attenuated psoriasis-like symptoms, suppressed inflammatory response, reduced the expression of JAK3 and STAT3, ceased keratinocyte proliferation, and promoted the apoptosis. The findings of our study suggest that miR-383 may inhibit LCN2 and inactivate the JAK/STAT pathway, suppressing the progression of psoriasis in a rat model. This study provided novel insights into the pathogenesis of psoriasis and offered potential targets for psoriasis treatment.

Keywords: Apoptosis; Cell proliferation; JAK/STAT; Keratinocyte; Lipocalin-2; Psoriasis; microRNA-383.

Inflammatory Breast Cancer (IBC) is an aggressive form of invasive breast cancer, highly metastatic, representing 2-4% of all breast cancer cases in the United States. Despite its rare nature, IBC is responsible for 7-10% of all breast cancer deaths, with a 5-year survival rate of 40%. Thus, targeted and effective therapies against IBC are needed. Here, we proposed Lipocalin-2 (LCN2)-a secreted glycoprotein aberrantly abundant in different cancers-as a plausible target for IBC. In immunoblotting, we observed higher LCN2 protein levels in IBC cells than non-IBC cells, where the LCN2 levels were almost undetectable. We assessed the biological effects of targeting LCN2 in IBC cells with small interference RNAs (siRNAs) and small molecule inhibitors. siRNA-mediated LCN2 silencing in IBC cells significantly reduced cell proliferation, viability, migration, and invasion. Furthermore, LCN2 silencing promoted apoptosis and arrested the cell cycle progression in the G0/G1 to S phase transition. We used in silico analysis with a library of 25,000 compounds to identify potential LCN2 inhibitors, and four out of sixteen selected compounds significantly decreased cell proliferation, cell viability, and the AKT phosphorylation levels in SUM149 cells. Moreover, ectopically expressing LCN2 MCF7 cells, treated with two potential LCN2 inhibitors (ZINC00784494 and ZINC00640089) showed a significant decrease in cell proliferation. Our findings suggest LCN2 as a promising target for IBC treatment using siRNA and small molecule inhibitors.

Keywords: IBC; LCN2; docking; inflammatory breast cancer; lipocalin-2; siRNA; small molecule inhibitors.

Background: Hidradenitis suppurativa (HS) is a chronic, inflammatory disease of the apocrine gland-rich (AGR) skin region. The initial steps of disease development are not fully understood, despite intense investigations into immune alterations in lesional HS skin.

Objectives: We aimed to systematically investigate the inflammatory molecules involved in three stages of HS pathogenesis, including healthy AGR, non-lesional HS, and lesional HS skin, with the parallel application of multiple mRNA and protein-based methods.

Methods: Immune cell counts (T cells, dendritic cells, macrophages), Th1/Th17-related molecules (IL-12B, TBX21, IFNG, TNFA, IL17, IL10, IL23A, TGFB1, RORC, CCL20), keratinocyte-related sensors (TLR2,4), mediators (S100A7, S100A8, S100A9, DEFB4B, LCN2, CAMP, CCL2), and pro-inflammatory molecules (IL1B, IL6, TNFA, IL23A) were investigated in the three groups by RNASeq, RT-qPCR, immunohistochemistry, and immunofluorescence.

Results: Epidermal changes were already detectable in non-lesional HS skin; the epidermal occurrence of antimicrobial peptides (AMPs), IL-1β, TNF-α, and IL-23 were highly upregulated compared to healthy AGR skin. In lesional HS epidermis, TNF-α and IL-1β expression remained at high levels while AMPs and IL-23 increased even more compared to non-lesional skin. In the dermis of non-lesional HS skin, signs of inflammation were barely detectable (versus AGR), while in the lesional dermis, the number of inflammatory cells and Th1/Th17-related mediators were significantly elevated.

Conclusions: Our findings that non-lesional HS epidermal keratinocytes produce not only AMPs and IL-1β but also high levels of TNF-α and IL-23 confirm the driver role of keratinocytes in HS pathogenesis and highlight the possible role of keratinocytes in the transformation of non-inflammatory Th17 cells (of healthy AGR skin) into inflammatory cells (of HS) via the production of these mediators. The fact that epidermal TNF-α and IL-23 appear also in non-lesional HS seems to prove these cytokines as excellent therapeutic targets.

Keywords: IL-17; antimicrobial peptides; hidradenitis suppurativa; keratinocyte; non-lesional HS; skin.

People living with HIV (PLWH) continue to develop HIV-associated neurocognitive disorders despite combination anti-retroviral therapy. Lipocalin-2 (LCN2) is an acute phase protein that has been implicated in neurodegeneration and is upregulated in a transgenic mouse model of HIV-associated brain injury. Here we show that LCN2 is significantly upregulated in neocortex of a subset of HIV-infected individuals with brain pathology and correlates with viral load in CSF and pro-viral DNA in neocortex. However, the question if LCN2 contributes to HIV-associated neurotoxicity or is part of a protective host response required further investigation. We found that the knockout of LCN2 in transgenic mice expressing HIVgp120 in the brain (HIVgp120tg) abrogates behavioral impairment, ameliorates neuronal damage, and reduces microglial activation in association with an increase of the neuroprotective CCR5 ligand CCL4. In vitro experiments show that LCN2 neurotoxicity also depends on microglia and p38 MAPK activity. Genetic ablation of CCR5 in LCN2-deficient HIVgp120tg mice restores neuropathology, suggesting that LCN2 overrides neuroprotection mediated by CCR5 and its chemokine ligands. RNA expression of 168 genes involved in neurotransmission reveals that neuronal injury and protection are each associated with genotype- and sex-specific patterns affecting common neural gene networks. In conclusion, our study identifies LCN2 as a novel factor in HIV-associated brain injury involving CCR5, p38 MAPK and microglia. Furthermore, the mechanistic interaction between LCN2 and CCR5 may serve as a diagnostic and therapeutic target in HIV patients at risk of developing brain pathology and neurocognitive impairment.

Keywords: Behavior; CCR5; HIV gp120-transgenic; HIV neuropathology; HIV-associated neurocognitive disorders; Knockout; Lipocalin-2; Neurodegeneration; Sexual dimorphism; p38 MAPK.

Screening programs for colorectal cancer (CRC) often rely on detection of blood in stools, which is unspecific and leads to a large number of colonoscopies of healthy subjects. Painstaking research has led to the identification of a large number of different types of biomarkers, few of which are in general clinical use. Here, we searched for highly accurate combinations of biomarkers by meta-analyses of genome- and proteome-wide data from CRC tumors. We focused on secreted proteins identified by the Human Protein Atlas and used our recently described algorithms to find optimal combinations of proteins. We identified nine proteins, three of which had been previously identified as potential biomarkers for CRC, namely CEACAM5, LCN2 and TRIM28. The remaining proteins were PLOD1, MAD1L1, P4HA1, GNS, C12orf10 and P3H1. We analyzed these proteins in plasma from 80 patients with newly diagnosed CRC and 80 healthy controls. A combination of four of these proteins, TRIM28, PLOD1, CEACAM5 and P4HA1, separated a training set consisting of 90% patients and 90% of the controls with high accuracy, which was verified in a test set consisting of the remaining 10%. Further studies are warranted to test our algorithms and proteins for early CRC diagnosis.

Following spinal cord injury, astrocytes at the site of injury become reactive and exhibit a neurotoxic (A1) phenotype, which leads to neuronal death. In addition, the glial scar, which is composed of reactive astrocytes, acts as a chemical and physical barrier to subsequent axonal regeneration. Biomaterials, specifically electrospun fibers, induce a migratory phenotype of astrocytes and promote regeneration of axons following acute spinal cord injury in preclinical models. However, no study has examined the potential of electrospun fibers or biomaterials in general to modulate neurotoxic (A1) or neuroprotective (A2) astrocytic phenotypes. To assess astrocyte reactivity in response to aligned poly-l-lactic acid microfibers, naïve spinal cord astrocytes or spinal cord astrocytes primed towards the neurotoxic phenotype (A1) were cultured on fibrous scaffolds. Gene expression analysis of the pan-reactive astrocyte makers (GFAP, Lcn2, SerpinA3), A1 specific markers (H2-D1, SerpinG1), and A2 specific makers (Emp1, S100a10) was done using quantitative polymerase chain reaction (qPCR). Electrospun fibers mildly increased the expression of the pan-reactive and A1-specific markers, showing the ability of fibrous materials to induce a more reactive, A1 phenotype. However, when naïve or activated astrocytes were cultured on fibers in the presence of transforming growth factor β3 (TGFβ3), the expression of A1-specific markers was greatly reduced, which in turn improved neuronal survival in culture.

Keywords: Astrocytes; Macrophage conditioned media; Reactivity; Spinal cord injury; Transforming growth factor β3.

Background: Ginsenosides have antioxidant and anti-inflammatory features. This study aimed to evaluate the biologic effects of ginsenoside Rb2 pretreatment on ventilator-induced lung injury (VILI) in rats.

Methods: Rats were divided into four groups with 12 rats per group: control; low tidal volume (TV), TV of 6 mL/kg, VILI, TV of 20 mL/kg, positive end-expiratory pressure of 5 cm H₂O, and respiratory rate of 60 breaths per minute for 3 h at an inspiratory oxygen fraction of 0.21; and ginsenosides, treated the same as the VILI group but with 20 mg/kg intraperitoneal ginsenoside pretreatment. Morphology was observed with a microscope to confirm the VILI model. Wet-to-dry weight ratios, protein concentrations, and pro-inflammatory cytokines in the bronchoalveolar lavage fluid were measured. RNA sequencing of the lung tissues was conducted to analyze gene expression.

Results: High TV histologically induced VILI with alveolar edema and infiltration of inflammatory cells. Ginsenosides pretreatment significantly reduced the histologic lung injury score compared to the VILI group. Wet-to-dry weight ratios, malondialdehyde, and TNF-α in bronchoalveolar lavage fluid were significantly higher in the VILI group and ginsenoside pretreatment mitigated these effects. In the immunohistochemistry assay, ginsenoside pretreatment attenuated the TNF-α upregulation induced by VILI. We identified 823 genes differentially presented in the VILI group compared to the control group. Of the 823 genes, only 13 genes (Arrdc2, Cygb, Exnef, Lcn2, Mroh7, Nsf, Rexo2, Srp9, Tead3, Ephb6, Mvd, Sytl4, and Ube2l6) recovered to control levels in the ginsenoside group.

Conclusions: Ginsenosides inhibited the inflammatory and oxidative stress response in VILI. Further studies are required on the 13 genes, including LCN2.

Keywords: Ginsenosides; LCN2; NGAL; Tumor necrosis factor-alpha; Ventilator-induced lung injury.

The female climacteric or menopausal process characterised by reduced estrogen, associates with an increased risk of recurrent urinary tract infections (rUTIs) linked to uropathogenic Escherichia coli (UPEC). Clinically, topical vaginal estrogen treatment has a prophylactic effect against such infections. The aim of this study was to investigate, in vitro, the effects of a topical estrogen treatment on vaginal epithelial responses following challenge with E.coli flagellin mimicking an UPEC challenge. Immortalised vaginal epithelial cells (VK2 E6/E7), modelling the vaginal epithelium were treated with either 4 nM 17β-estradiol (E) for seven days, 50 ng/ml E.coli flagellin (F) for 12 h, or 4 nM 17β-estradiol plus 50 ng/ml flagellin (E + F(12 h)). RNA was analysed by microarray gene profiling using the Illumina HumanHT-12 v 4 Expression Beadchip. Following E + F treatments expression of genes encoding host defence molecules including DEFβ4A, DEFB103A, LCN2 as well as those associated with keratinisation eg CNFN and SPRR family genes were significantly enhanced (P < 0.05) compared to either E or F treatments alone. Mutation of estrogen responsive elements (EREs) identified in the DEFβ4 gene promoter abolished the augmented gene expression suggesting estrogen functioned directly through a regulatory mechanism involving ESR1/2. Ingenuity pathway analyses also suggested the pro-inflammatory cytokine IL-17A to regulate the vaginal host defences during infection. Pre-treating VK2 E6/E7 cells with estrogen (4 nM) and challenging with 1L-17A & F (12 h) significantly enhanced DEFβ4, DEF103A and S100A7 expression (P < 0.05). Origins of vaginal IL-17 in vivo remain unclear, but patient biopsies support γδ T cells located within the vaginal epithelium. These data suggest that the vaginal antimicrobial response induced by flagellin activation of Toll-like Receptor 5 cell signalling is augmented following topical estrogen application.

Background: Post-surgical recurrence of the metastatic colorectal cancer (mCRC) remains a challenge, even with adjuvant therapy. Moreover, patients show variable outcomes. Here, we set to identify gene models based on the perspectives of intrinsic cell activities and extrinsic immune microenvironment to predict the recurrence of mCRC and guide the adjuvant therapy. Methods: An RNA-based gene expression analysis of CRC samples (total = 998, including mCRCs = 344, non-mCRCs = 654) was performed. A metastasis-evaluation model (MEM) for mCRCs was developed using the Cox survival model based on the prognostic differentially expressed genes between mCRCs and non-mCRCs. This model separated the mCRC samples into high- and low-recurrence risk clusters that were tested using machine learning to predict recurrence. Further, an immune prognostic model (IPM) was built using the COX survival model with the prognostic differentially expressed immune-related genes between the two MEM risk clusters. The ability of MEM and IPM to predict prognosis was analyzed and validated. Moreover, the IPM was utilized to evaluate its relationship with the immune microenvironment and response to immuno-/chemotherapy. Finally, the dysregulation cause of IPM three genes was analyzed in bioinformatics. Results: A high post-operative recurrence risk was observed owing to the downregulation of the immune response, which was influenced by MEM genes (BAMBI, F13A1, LCN2) and their related IPM genes (SLIT2, CDKN2A, CLU). The MEM and IPM were developed and validated through mCRC samples to differentiate between low- and high-recurrence risk in a real-world cohort. The functional enrichment analysis suggested pathways related to immune response and immune system diseases as the major functional pathways related to the IPM genes. The IPM high-risk group (IPM-high) showed higher fractions of regulatory T cells (Tregs) and smaller fractions of resting memory CD4+ T cells than the IPM-low group. Moreover, the stroma and immune cells in the IPM-high samples were scant. Further, the IPM-high group showed downregulation of MHC class II molecules. Additionally, the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm and GDSC analysis suggested the IPM-low as a promising responder to anti-CTLA-4 therapy and the common FDA-targeted drugs, while the IPM-high was non-responsive to these treatments. However, treatment using anti-CDKN2A agents, along with the activation of major histocompatibility complex (MHC) class-II response might sensitize this refractory mCRC subgroup. The dysfunction of MEIS1 might be the reason for the dysregulation of IPM genes. Conclusions: The IPM could identify subgroups of mCRC with a distinct risk of recurrence and stratify the patients sensitive to immuno-/chemotherapy. Further, for the first time, our study highlights the importance of MHC class-II molecules in the treatment of mCRCs using immunotherapy.

Keywords: bioinformatics; disease recurrence; immune prognostic model; immunotherapy; metastatic colorectal cancer; real-world cohort.

Skin injury can trigger formation of new lesions in psoriasis (Koebner phenomenon). The mechanisms through which injury exacerbates psoriasis are unclear. During wound repair, epidermal keratinocytes are activated and produce abundant IL-36γ, further promoting the skin inflammation. IL-17A is the cornerstone cytokine in the pathogenesis of psoriasis. We sought to investigate the effects of IL-17A on injury-induced keratinocyte activation and IL-36γ production. Here, we demonstrated that dsRNA released from necrotic keratinocytes induced the expression of IL-36γ. Silencing of TLR3 by siRNA decreased the IL-36γ induction by necrotic keratinocyte supernatant. Co-stimulation with dsRNA and IL-17A synergistically increased the expression of IL-36γ and other proinflammatory mediators (CCL20, CXCL8, DEFB4 and LCN2) in keratinocytes. The synergistic effects were not dependent on TLR3 upregulation, TNF receptor signalling and mRNA stabilization. Co-stimulation with dsRNA and IL-17A resulted in an accumulation of IκBζ. The synergistic upregulation of IL-36γ and proinflammatory mediators were inhibited by IκBζ siRNA. Co-stimulation with IL-17A and poly(I:C) markedly activated the p38 MAPK and NF-κB pathway, compared with poly(I:C). Blockade of p38 MAPK and NF-κB suppressed dsRNA/IL-17A-mediated IκBζ and IL-36γ induction. These findings demonstrated that IL-17A synergistically enhanced the dsRNA-mediated IL-36γ production through a p38 MAPK-, NF-κB-, and IκBζ-dependent mechanism.

Prostate cancer (PCa) is one of the most common and deadly cancers in men worldwide. The surrounding tumor microenvironment (TME) is important in tumor progression, as cytokines and soluble mediators including tumor necrosis factor (TNF-α) or lipocalin-2 (LCN2) can influence tumor growth and formation of metastasis. The exact mechanisms on how these pleiotropic factors affect PCa are still unknown. In this study, we showed for the first time that LCN2 mRNA and protein expression are strongly inducible by TNF-α in the highly metastatic human PCa cell line PC-3. In addition, we observed higher levels of secreted LCN2 in cell culture medium of TNF-α-treated PC-3 cells. We found that different signaling pathways such as p38, NF-κB or JNK were activated shortly after TNF-α treatment. Moreover, the mRNA levels of IL-1β and IL-8 were also significantly increased after 24 h stimulation. Mechanistically, the NF-κB pathway and the JNK signaling axis are directly responsible for LCN2 upregulation. This was shown by the fact that pretreatment with the JNK inhibitors SP600125 or JNK-IN-8 strongly downregulated phosphorylation of c-Jun protein and markedly reduced TNF-α-mediated LCN2 upregulation in PC-3 cells. Likewise, the NF-κB inhibitor QNZ was able to repress TNF-α-induced LCN2 expression in PC-3 cells. Taking into consideration that LCN2 has been described as a tumor promoting factor in PCa, our results indicate that JNK regulates LCN2 expression and unmasks the JNK signaling axis as a possible therapeutic target for patients with PCa.

Keywords: Cytokines; JNK; LCN2; NF-κB; Prostate cancer; TNF-α; Tumor microenvironment; c-Jun.

Background: The gut microbiome is altered in patients with inflammatory bowel disease, yet how these alterations contribute to intestinal inflammation is poorly understood. Murine models have demonstrated the importance of the microbiome in colitis since colitis fails to develop in many genetically susceptible animal models when re-derived into germ-free environments. We have previously shown that Wiskott-Aldrich syndrome protein (WASP)-deficient mice (Was^-/-) develop spontaneous colitis, similar to human patients with loss-of-function mutations in WAS. Furthermore, we showed that the development of colitis in Was^-/- mice is Helicobacter dependent. Here, we utilized a reductionist model coupled with multi-omics approaches to study the role of host-microbe interactions in intestinal inflammation.

Results: Was^-/- mice colonized with both altered Schaedler flora (ASF) and Helicobacter developed colitis, while those colonized with either ASF or Helicobacter alone did not. In Was^-/- mice, Helicobacter relative abundance was positively correlated with fecal lipocalin-2 (LCN2), a marker of intestinal inflammation. In contrast, WT mice colonized with ASF and Helicobacter were free of inflammation and strikingly, Helicobacter relative abundance was negatively correlated with LCN2. In Was^-/- colons, bacteria breach the mucus layer, and the mucosal relative abundance of ASF457 Mucispirillum schaedleri was positively correlated with fecal LCN2. Meta-transcriptomic analyses revealed that ASF457 had higher expression of genes predicted to enhance fitness and immunogenicity in Was^-/- compared to WT mice. In contrast, ASF519 Parabacteroides goldsteinii's relative abundance was negatively correlated with LCN2 in Was^-/- mice, and transcriptional analyses showed lower expression of genes predicted to facilitate stress adaptation by ASF519 in Was^-/-compared to WT mice.

Conclusions: These studies indicate that the effect of a microbe on the immune system can be context dependent, with the same bacteria eliciting a tolerogenic response under homeostatic conditions but promoting inflammation in immune-dysregulated hosts. Furthermore, in inflamed environments, some bacteria up-regulate genes that enhance their fitness and immunogenicity, while other bacteria are less able to adapt and decrease in abundance. These findings highlight the importance of studying host-microbe interactions in different contexts and considering how the transcriptional profile and fitness of bacteria may change in different hosts when developing microbiota-based therapeutics. Video abstract.

Keywords: Defined consortium; Gut microbiota; Immune dysregulation; Intestinal inflammation; Pathobiont; Wiskott-Aldrich syndrome.

Influences of adenosine 2A receptor (A2AR) activity on the cardiac transcriptome and genesis of endotoxemic myocarditis are unclear. We applied transcriptomic profiling (39 K Affymetrix arrays) to identify A2AR-sensitive molecules, revealed by receptor knockout (KO), in healthy and endotoxemic hearts. Baseline cardiac function was unaltered and only 37 A2AR-sensitive genes modified by A2AR KO (≥1.2-fold change, <5 % FDR); the five most induced are Mtr, Ppbp, Chac1, Ctsk and Cnpy2 and the five most repressed are Hp, Yipf4, Acta1, Cidec and Map3k2. Few canonical paths were impacted, with altered Gnb1, Prkar2b, Pde3b and Map3k2 (among others) implicating modified G protein/cAMP/PKA and cGMP/NOS signalling. Lipopolysaccharide (LPS; 20 mg/kg) challenge for 24 h modified >4100 transcripts in wild-type (WT) myocardium (≥1.5-fold change, FDR < 1 %); the most induced are Lcn2 (+590); Saa3 (+516); Serpina3n (+122); Cxcl9 (+101) and Cxcl1 (+89) and the most repressed are Car3 (-38); Adipoq (-17); Atgrl1/Aplnr (-14); H19 (-11) and Itga8 (-8). Canonical responses centred on inflammation, immunity, cell death and remodelling, with pronounced amplification of toll-like receptor (TLR) and underlying JAK-STAT, NFκB and MAPK pathways, and a 'cardio-depressant' profile encompassing suppressed ß-adrenergic, PKA and Ca2+ signalling, electromechanical and mitochondrial function (and major shifts in transcripts impacting function/injury including Lcn2, S100a8/S100a9, Icam1/Vcam and Nox2 induction, and Adipoq, Igf1 and Aplnr repression). Endotoxemic responses were selectively modified by A2AR KO, supporting inflammatory suppression via A2AR sensitive shifts in regulators of NFκB and JAK-STAT signalling (IκBζ, IκBα, STAT1, CDKN1a and RRAS2) without impacting the cardio-depressant gene profile. Data indicate A2ARs exert minor effects in un-stressed myocardium and selectively suppress NFκB and JAK-STAT signalling and cardiac injury without influencing cardiac depression in endotoxemia.

Fenvalerate (FEN), a pyrethroid insecticide used worldwide, has been shown to produce a potentially adverse effect on male reproduction. However the mechanisms are not completely understood. Thus this study aimed to (1) determine whether cellular apoptosis was involved in FEN-induced testicular damage in rats, and (2) identify the potential mechanism involved in FEN-induced apoptosis in testes. Data demonstrated that FEN markedly decreased serum testosterone levels, increased the inner diameter of seminiferous tubules, decreased the layers of spermatogenic cells, disturbed spermatogenesis and increased the number of apoptotic cells. Further, bioinformatic analysis of gene microarray in rat testis tissue showed that FEN significantly altered the expressions of genes (Krt8, Mal, Cd24, Lcn2, Phlda1, Arg2) related to apoptotic related processes. The expression pattern of these 6 genes was upregulated in FEN-treated rat testicular tissue. qRT-PCR analysis demonstrated that Phlda1, a well-documented pro-apoptotic factor, was significantly elevated by FEN. The expression of PHLDA1 testicular protein was also elevated following FEN exposure. In conclusion, our results suggest that FEN exposure induced deleterious effects on rat testes associated with Phlda1-mediated apoptosis which may act as a molecular mechanism underlying FEN induced rat testicular damage.

Objectives: Central nervous system (CNS) infections are common causes of morbidity and mortality worldwide. We aimed to discover protein biomarkers that could rapidly and accurately identify the likely cause of the infections, essential for clinical management and improving outcome.

Methods: We applied liquid chromatography tandem mass-spectrometry on 45 cerebrospinal fluid (CSF) samples from a cohort of adults with/without CNS infections to discover potential diagnostic biomarkers. We then validated the diagnostic performance of a selected biomarker candidate in an independent cohort of 364 consecutively treated adults with CNS infections admitted to a referral hospital in Vietnam.

Results: In the discovery cohort, we identified lipocalin 2 (LCN2) as a potential biomarker of bacterial meningitis (BM) other than tuberculous meningitis. The analysis of the validation cohort showed that LCN2 could discriminate BM from other CNS infections (including tuberculous meningitis, cryptococcal meningitis and viral/antibody-mediated encephalitis), with the sensitivity: 0.88 (95% confident interval (CI): 0.77-0.94), the specificity: 0.91 (95%CI: 0.88-0.94) and the diagnostic odd ratio: 73.8 (95%CI: 31.8-171.4)). LCN2 outperformed other CSF markers (leukocytes, glucose, protein and lactate) commonly used in routine care worldwide. The combination of LCN2, CSF leukocytes, glucose, protein and lactate resulted in the highest diagnostic performance for BM (area under receiver-operating-characteristic-curve 0.96; 95%CI: 0.93-0.99).

Conclusions: Our results suggest that LCN2 is a sensitive and specific biomarker for discriminating BM from a broad spectrum of other CNS infections. A prospective study is needed to assess the diagnostic utility of LCN2 in the diagnosis and management of CNS infections.

Keywords: Meningitis; biomarkers; central nervous system infections; lipocalin 2; mass-spectrometry.

Nearly half of cancer patients suffer from cachexia, a metabolic syndrome characterized by progressive atrophy of fat and lean body mass. This state of excess catabolism decreases quality of life, ability to tolerate treatment and eventual survival, yet no effective therapies exist. Although the central nervous system (CNS) orchestrates several manifestations of cachexia, the precise mechanisms of neural dysfunction during cachexia are still being unveiled. Herein, we summarize the cellular and molecular mechanisms of CNS dysfunction during cancer cachexia with a focus on inflammatory, autonomic and neuroendocrine processes and end with a discussion of recently identified CNS mediators of cachexia, including GDF15, LCN2 and INSL3.

Keywords: GDF15; INSL3; LCN2; autonomic nervous system; cachexia; cancer; cytokines; neuroendocrinology; neuroinflammation.

Aims: To investigate determinants of circulating levels of adipocyte-fatty acid binding protein (A-FABP) and lipocalin-2 (LCN2), their relationships with cardiovascular disease (CVD) and microvascular events, and effects of fenofibrate in type 2 diabetes (T2D).

Methods: A-FABP and LCN2 were quantified in baseline plasma from 2000 T2D adults in a Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial sub-study and correlates thereof determined. In a subset (n=200) adipokines were also measured on-trial.

Results: Female sex, older age, higher body mass index (BMI), HbA1c, insulin resistance index, triglycerides, plasma creatinine and homocysteine, shorter diabetes duration, and use of oral hypoglycaemic agents alone were independent determinants of higher A-FABP. Higher BMI, fibrinogen and homocysteine, Caucasian race, and lower fasting glucose, HDL-cholesterol, apolipoprotein A-II and estimated glomerular filtration rate were independent predictors of higher LCN2 levels. Baseline A-FABP and LCN2 levels were associated with multiple new CVD and microvascular events over 5-years, though significance was lost after risk factor adjustment. Fenofibrate increased A-FABP but did not change LCN2 levels.

Conclusions: Baseline plasma A-FABP and LCN2 levels were associated with concurrent CVD risk factors, and on-trial chronic complications, likely mediated via traditional risk factors. Fenofibrate increased A-FABP modestly but did not affect LCN2 levels.

Keywords: Adipocyte-fatty acid binding protein; adipokine; cardiovascular outcome; fenofibrate; lipocalin-2; microvascular disease.

Extracellular Vesicles (EVs) are becoming increasingly recognized as integral signaling vehicles in several types of cancers, including bone malignancies. However, the specific mechanisms by which EVs influence osteosarcoma progression have not been fully determined. We evaluated the effects of EVs derived from the human osteosarcoma cell line MNNG/HOS (MNNG/HOS-EVs) on bone resident cells. We found that MNNG/HOS-EVs are internalized by osteoblasts and osteoclasts in vitro, with potent inhibitory effects on osteoblast metabolic activity, cell density and alkaline phosphatase activity. Consistently, MNNG/HOS-EVs reduced the expression of cell cycle and pro-osteoblastogenic genes, whilst increasing transcriptional expression and protein release of pro-osteoclastogenic/inflammatory cytokines (RankL, Il1b, Il6 and Lcn2), pro-tumoral cytokines (CCL2,5,6,12 and CXCL1,2,5) and the metalloproteinase MMP3. MNNG/HOS-EVs did not induce osteoclast differentiation, while promoting in vitro and in vivo angiogenesis. Intriguingly, EVs derived from another osteosarcoma cell line (U2OS) reduced ALP activity but had no other effect on osteoblast phenotype. MNNG/HOS-EVs were also found to dramatically increase Serpin b2 expression in osteoblasts. To evaluate the significance of this finding, osteoblasts were forced to overexpress Serpin b2, which however did not affect osteoblast differentiation, while Il6 and Lcn2 mRNAs were up regulated. Overall, we shed light on the interactions of osteosarcoma EVs with the cells of the bone microenvironment, identifying key anti-osteoblastogenic, pro-inflammatory and pro-angiogenic factors that could contribute to osteosarcoma expansion.

Keywords: Angiogenesis; Extracellular Vesicles; Osteoblast; Osteoclast; Osteosarcoma.

Rheumatoid arthritis (RA) is a debilitating, chronic, inflammatory, autoimmune disease associated with cachexia. The substitutive therapy of gut hormone ghrelin has been pointed at as a potential countermeasure for the management of metabolic and inflammatory complications in RA. The recent discovery of liver-expressed antimicrobial peptide 2 (LEAP2) as an endogenous inverse agonist/antagonist of the ghrelin receptor makes feasible the development of a more rational pharmacological approach. This work aimed to assess the serum LEAP2 levels, in a cohort of RA patients, in comparison with healthy individuals and determine its correlation with inflammatory parameters. LEAP2 levels were determined by a commercial ELISA kit, plasma C-reactive protein (CRP) levels were evaluated using immunoturbidimetry, and serum levels of inflammatory mediators, namely IL-6, IL-8, IL-1β, MIP1α, MCP1, and LCN2, were measured by XMap multiplex assay. LEAP2 serum levels were significantly increased in RA patients (n = 101) compared with control subjects (n = 26). Furthermore, the LEAP2 levels significantly correlated with CRP and inflammatory cytokines, but not with BMI. These data reveal LEAP2 as a new potential RA biomarker and indicated the pharmacological control of LEAP2 levels as a novel approach for the treatment of diseases with alterations on the ghrelin levels, such as rheumatoid cachexia.

Investigations of gene expression in allergic rhinitis (AR) typically rely on invasive nasal biopsies (site of inflammation) or blood samples (systemic immunity) to obtain sufficient genetic material for analysis. New methodologies to circumvent the need for invasive sample collection offer promise to further the understanding of local immune mechanisms relevant in AR.

A within-subject design was employed to compare immune gene expression profiles obtained from nasal washing/brushing and whole blood samples collected during peak pollen season. Twelve adults (age: 46.3 ± 12.3 years) with more than a 2-year history of AR and a confirmed grass pollen allergy participated in the study. Gene expression analysis was performed using a panel of 760 immune genes with the NanoString nCounter platform on nasal lavage/brushing cell lysates and compared to RNA extracted from blood.

A total of 355 genes were significantly differentially expressed between sample types (9.87 to -9.71 log2 fold change). The top 3 genes significantly upregulated in nasal lysate samples were Mucin 1 (MUC1), Tight Junction Protein 1 (TJP1), and Lipocalin-2 (LCN2). The top 3 genes significantly upregulated in blood samples were cluster of differentiation 3e (CD3E), FYN Proto-Oncogene Src Family Tyrosine Kinase (FYN) and cluster of differentiation 3d (CD3D).

Overall, the blood and nasal lavage samples showed vastly distinct gene expression profiles and functional gene pathways which reflect their anatomical and functional origins. Evaluating immune gene expression of the nasal mucosa in addition to blood samples may be beneficial in understanding AR pathophysiology and response to allergen challenge.

Acute liver failure (ALF) is characterized by loss of liver function in response to sustained augmentation of the acute-phase response (APR) in the liver, which can progress even to death. Although the inflammatory interleukin-6 (IL-6)-axis is a crucial factor that drives the hepatic APR by releasing diverse acute-phase proteins (APPs), therapeutic strategies to block the IL-6-STAT3-mediated APR are not well developed. Here, we show that the nuclear receptor retinoic acid-related orphan receptor α (RORα) limits APR-mediated liver injury by inhibiting the hepatic IL-6-STAT3 signaling pathway. Administration of JC1-40, an RORα activator, diminished diethylnitrosamine-induced acute liver injury and repressed transcriptional expression of APPs such as CXCL1 and LCN2 in mice. IL-6-mediated activation of STAT3 was repressed after RORα activation by either adenoviral infusion of RORα or JC1-40 treatment in primary hepatocytes. Activation of RORα decreased transcriptional expression of IL-6 receptor α, an upstream activator of STAT3, both in vitro and in vivo. This may be one mechanism underlying the RORα-mediated inhibition of STAT3. Taken together, our results suggest that RORα is a regulator of the hepatic IL-6-STAT3 signaling pathway and may be a new therapeutic target for treating APR-associated inflammatory ALF.

Lipocalin 2 (Lcn2) has been identified in mammals, however, the in vivo function of fish Lcn2 is essentially unknown. Triploid crucian carp (3n = 150) of red crucian carp (female, 2n = 100) and allotetraploid (male, 4n = 200) shows better resistance to pathogenic infections. To elucidate the antimicrobial mechanism of triploid crucian carp, we examined the function of a novel Lcn2 from triploid crucian carp (3nLcn2). 3nLcn2 is 183 residues in length and contains a conserved lipocalin domain. Quantitative real time reverse transcription PCR (qRT-PCR) analysis showed that 3nLcn2 expression occurred in multiple tissues and was upregulated by bacterial infection in a time-dependent manner. We found that purified recombinant 3nLcn2 (r3nLcn2) exerted bactericidal activity to Aeromonas hydrophila and Escherichia coli. qRT-PCR detected increased expression of pro-inflammatory cytokines and tight junctions in fish with 3nLcn2 overexpression. Fish administered with 3nLcn2 exhibited enhanced intestinal barrier and resistance against bacterial infection. These results provide the first evidence that 3nLcn2 is a functional lipocalin with antimicrobial activity and plays a positive role in the immune defense during bacterial infection.