Native Low-Density Lipoproteins Act in Synergy with Lipopolysaccharide to Alter the Balance of Human Monocyte Subsets and Their Ability to Produce IL-1 Beta, CCR2, and CX3CR1 In Vitro and In Vivo: Implications in Atherogenesis.

Increasing evidence has demonstrated that oxidized low-density lipoproteins (oxLDL) and lipopolysaccharide (LPS) enhance accumulation of interleukin (IL)-1 beta-producing macrophages in atherosclerotic lesions. However, the potential synergistic effect of native LDL (nLDL) and LPS on the inflammatory ability and migration pattern of monocyte subpopulations remains elusive and is examined here. In vitro, whole blood cells from healthy donors (n = 20) were incubated with 100 µg/mL nLDL, 10 ng/mL LPS, or nLDL + LPS for 9 h. Flow cytometry assays revealed that nLDL significantly decreases the classical monocyte (CM) percentage and increases the non-classical monocyte (NCM) subset. While nLDL + LPS significantly increased the number of NCMs expressing IL-1 beta and the C-C chemokine receptor type 2 (CCR2), the amount of NCMs expressing the CX3C chemokine receptor 1 (CX3CR1) decreased. In vivo, patients (n = 85) with serum LDL-cholesterol (LDL-C) >100 mg/dL showed an increase in NCM, IL-1 beta, LPS-binding protein (LBP), and Castelli's atherogenic risk index as compared to controls (n = 65) with optimal LDL-C concentrations (≤100 mg/dL). This work demonstrates for the first time that nLDL acts in synergy with LPS to alter the balance of human monocyte subsets and their ability to produce inflammatory cytokines and chemokine receptors with prominent roles in atherogenesis.

Serum amyloid A is a positive acute phase protein in Russian sturgeon challenged with Aeromonas hydrophila.

The immune system of sturgeons, one of the most ancient and economically valuable fish worldwide, is poorly understood. The lack of molecular tools and data about infection biomarkers hinders the possibility to monitor sturgeon health during farming and detect infection outbreaks. To tackle this issue, we mined publicly available transcriptomic datasets and identified putative positive acute-phase proteins (APPs) of Russian sturgeons that could be induced by a bacterial infection and monitored using non-invasive methods. Teleost literature compelled us to focus on five promising candidates: hepcidin, a warm acclimation associated hemopexin, intelectin, serum amyloid A protein (SAA) and serotransferrin. Among them, SAA was the most upregulated protein at the mRNA level in the liver of sturgeons challenged with heat-inactivated or live Aeromonas hydrophila. To assess whether this upregulation yielded increasing SAA levels in circulation, we developed an in-house ELISA to quantify SAA levels in sturgeon serum. Circulating SAA rose upon bacterial challenge and positively correlated with hepatic saa expression. This is the first time serum SAA has been quantified in an Actinopterygii fish. Since APPs vary across different fish species, our work sheds light on sturgeon acute-phase response, revealing that SAA is a positive APP with potential value as infection biomarker.

Acute-phase proteins during inflammatory reaction by bacterial infection: Fish-model.

Acute-phase protein (APPs) serum levels have been studied in many human diseases, and their components contribute to host defense during the evolution of infectious diseases by acting as part of the innate immune system. Based on the importance of establishing new experimental models, the present investigation evaluated the modulation of APPs following inflammatory stimulus by the inoculation of Aeromonas hydrophila in tilapias. Fish were sampled 6 and 24 hours post-infection. Tilapias presented increase of positive APPs such as ceruloplasmin, haptoglobin, alpha-2-macroglobulin and complement C3, as well as decrease of negative APPs such as albumin and transferrin. The protein response of tilapias during the course of bacterial infection showed correlation with the kinetics of cellular accumulation in the inflamed focus with significant increase of granulocytes, thrombocytes, lymphocytes and macrophages. However, granulocytes were the predominant cells, associated with increment in the reactive oxygen species (ROS) production. Showing responses similar to those observed in humans, the modulation of APPs and the kinetics of cellular accumulation in the exudate demonstrate the feasibility of this alternative experimental model for advances and studies to understand changes in pathophysiological mechanisms of acute inflammatory reaction due to bacterial infection.

Functional diets modulate the acute phase protein response in Oncorhynchus mykiss subjected to chronic stress and challenged with Vibrio anguillarum.

The acute phase response to pathogens alters the production of proinflammatory cytokines that, in turn, activate the synthesis of acute phase proteins. These proteins neutralize, prevent, and indicate tissue damage, thereby influencing the specific immune response and allowing the organism to regain homeostasis. Functional diets based in pre- and probiotics are used in aquaculture to improve fish health and resistance to diseases, but there is an information gap on the mechanisms involved in these effects and if these diets are efficient when fish are raised under high stocking densities. This study aimed an evaluation of the acute phase response in Oncorhynchus mykiss fed functional diets supplemented with pre- and probiotics (i.e. mannan-oligosaccharides and Saccharomyces cerevisiae, respectively) and challenged by either Vibrio anguillarum or chronic stress via maintenance under high stocking densities. For this, the relative expression of acute phase response related genes in liver, and of inflammatory response related genes in head kidney was evaluated by RT-qPCR. The supplemented diets differentially modulated the acute phase protein response to the assessed challenge conditions, specifically evidencing an overexpression of the genes HAPT, SAA, LECT2, and IL-1ß under chronic stress and of HAPT, IL-1ß, IL8, and LECT2 at 24 h post-challenge with V. anguillarum. The observed early-stage regulation of acute phase proteins and of the immune response by the probiotic S. cerevisiae and by prebiotic mannan-oligosaccharides suggests that both supplements have high immunostimulatory potentials for fish farmed under high stocking densities.

IGF-1 modulates gene expression of proteins involved in inflammation, cytoskeleton, and liver architecture.

Even though the liver synthesizes most of circulating IGF-1, it lacks its receptor under physiological conditions. However, according to previous studies, a damaged liver expresses the receptor. For this reason, herein, we examine hepatic histology and expression of genes encoding proteins of the cytoskeleton, extracellular matrix, and cell-cell molecules and inflammation-related proteins. A partial IGF-1 deficiency murine model was used to investigate IGF-1's effects on liver by comparing wild-type controls, heterozygous igf1+/-, and heterozygous mice treated with IGF-1 for 10 days. Histology, microarray for mRNA gene expression, RT-qPCR, and lipid peroxidation were assessed. Microarray analyses revealed significant underexpression of igf1 in heterozygous mice compared to control mice, restoring normal liver expression after treatment, which then normalized its circulating levels. IGF-1 receptor mRNA was overexpressed in Hz mice liver, while treated mice displayed a similar expression to that of the controls. Heterozygous mice showed overexpression of several genes encoding proteins related to inflammatory and acute-phase proteins and underexpression or overexpression of genes which coded for extracellular matrix, cytoskeleton, and cell junction components. Histology revealed an altered hepatic architecture. In addition, liver oxidative damage was found increased in the heterozygous group. The mere IGF-1 partial deficiency is associated with relevant alterations of the hepatic architecture and expression of genes involved in cytoskeleton, hepatocyte polarity, cell junctions, and extracellular matrix proteins. Moreover, it induces hepatic expression of the IGF-1 receptor and elevated acute-phase and inflammation mediators, which all resulted in liver oxidative damage.

Synergistic effect of apoptosis and necroptosis inhibitors in cisplatin-induced nephrotoxicity.

Necroptosis is a nonapoptotic cell death pathway. We aim to study the effect of necrostatin-1 (a specific necroptosis inhibitor) in cisplatin-induced injury. We analyzed the effect of the combined use of inhibitors of apoptosis (z-vad) and necroptosis (necrostatin-1) in acute kidney injury by cisplatin in human proximal tubule cells. Our results showed moderate effectiveness in cytoprotection after treatment with z-vad. But the concomitant use of inhibitors (z-vad and necrostatin-1) presented synergistic and additive protection. The present study analyzed the caspase-3 activity and we observed a significant decrease in the group treated with z-vad and cisplatin. However we did not observe changes in the group treated with both inhibitors (z-vad and necrostatin-1) and cisplatin. Thus, demonstrating that necroptosis is a caspase-independent mechanism. We also analyzed the effect of necrostatin-1 in vivo model. C57BL/6 mice were treated with cisplatin and/or inhibitors. The concomitant use of inhibitors (z-vad and necrostatin-1) recovered renal function and decreased levels of urinary Ngal. Additionally, we analyzed the expression of RIP-1, a specific marker for necroptosis. In animals treated with cisplatin and z-VAD levels of RIP-1 were higher. This result reinforces that necroptosis occurs only in conditions where apoptosis was blocked. However, the use of both inhibitors (z-vad and necrostatin-1) provided additional protection. In conclusion, our study has a significant potential to show in vitro and in vivo protection obtained by necrostatin-1. Therefore, our results suggest that necroptosis may be an important mechanism of cell death after kidney injury.

Neutrophil gelatinase-associated lipocalin (NGAL) and matrix metalloproteinase-9 (MMP-9) prognostic value in lung adenocarcinoma.

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.

In vivo mechanism study of NGAL in rat renal ischemia-reperfusion injury.

This study aimed to determine the protective effect and mechanism of neutrophil gelatinase-associated lipocalin (NGAL) in rat kidney on ischemia/reperfusion injury (I/R). The rat I/R model was set up by cutting one kidney and clamping the contralateral renal pedicle for 45 min. Male SD rats were randomly divided into sham-operation, I/R and NGAL groups. Hematoxylin-eosin staining was performed to observe the renal pathological changes in the 3 groups; serum creatinine (Scr) and blood urea nitrogen (BUN) determined in blood samples taken from the inferior vena cava 24 h after the reperfusion were measured; TUNEL was used to observe the apoptosis of renal tubular epithelial cells; immunohistochemistry was performed to evaluate the expressions of Bax and activated caspase-3; Western blotting was used to determine the expression changes in apoptotic proteins Fas and Bcl-2. Compared with the I/R group, Scr and BUN of the NGAL group were 63.400 ± 11.908 vs 121.857 ± 17.151 µM and 14.840 ± 2.868 vs 28.557 ± 6.434 mM, respectively. The number of apoptotic tubular epithelial cells was reduced (7.800 ± 1.924 vs 15.400 ± 3.049); the expression of renal tissue Fas mRNA of the NGAL group was decreased (2.34 ± 0.51 vs 6.84 ± 2.34); the expression of the Bax protein was lower (7.440 ± 1.640 vs 15.456 ± 1.955%); the expression of the CC3 protein was decreased (3.171 ± 0.321 vs 7.291 ± 1.059%), while the expression of the Bcl-2 protein increased (6.91 ± 1.64 vs 5.30 ± 1.48), P < 0.05. NGAL had a protective effect towards the renal tubular epithelial cells in I/R, and the effect might have been associated with the reduction in apoptosis and the altered expression of apoptotic proteins, which would thereby reduce tissue damage and protect the kidney.

Clinical significance of neutrophil gelatinase-associated lipocalin (NGAL) in colorectal cancer: a meta-analysis.

Growing evidence has implicated that neutrophil gelatinase-associated lipocalin (NGAL) plays a role in a spectrum of human cancers. Several observational studies from different parts of the world have been devoted to elucidate the clinical relationship between NGAL and colorectal cancer. This meta-analysis aimed to explore the overall accuracy of NGAL detection for the diagnosis and prognosis of colorectal cancer. Based on comprehensive literature screening on Pubmed, Ovid, and CNKI databases, our screening covered all published papers until March 2013. The relevant papers were selected according to some stringent inclusion criteria. Essential data were extracted from the recruited papers and further processed by systematic meta-analysis. The meta-analysis included 5 studies for diagnosis of colorectal cancer. Overall, the pooled sensitivity and specificity of all studies were 73% (95%CI=0.69-0.76) and 89% (95%CI=0.85-0.93). The pooled positive likelihood ratio and negative likelihood ratio were 5.41 (95%CI=3.85- 7.59) and 0.37 (95%CI=0.22-0.62). The pooled diagnostic odds ratios was 18.05 (95%CI=11.77-27.69). The area under the summary receiver operating characteristic curve for the diagnosis of colorectal cancer was 0.87. Meanwhile, 3 studies were included to evaluate the prognostic significance of NGAL overexpression in colorectal cancer patients. The pooled hazard ratio was 2.12 (95%CI=1.35-3.33). High level of NGAL predicted poor disease-free survival. Thus, NGAL is a potential biomarker for the diagnosis and prognosis of colorectal cancer.

Novel genes detected by transcriptional profiling from whole-blood cells in patients with early onset of acute coronary syndrome.

BACKGROUND: Genome-wide expression analysis using microarrays has been used as a research strategy to discovery new biomarkers and candidate genes for a number of diseases. We aim to find new biomarkers for the prediction of acute coronary syndrome (ACS) with a differentially expressed mRNA profiling approach using whole genomic expression analysis in a peripheral blood cell model from patients with early ACS. METHODS AND RESULTS: This study was carried out in two phases. On phase 1 a restricted clinical criteria (ACS-Ph1, n=9 and CG-Ph1, n=6) was used in order to select potential mRNA biomarkers candidates. A subsequent phase 2 study was performed using selected phase 1 markers analyzed by RT-qPCR using a larger and independent casuistic (ACS-Ph2, n=74 and CG-Ph2, n=41). A total of 549 genes were found to be differentially expressed in the first 48 h after the ACS-Ph1. Technical and biological validation further confirmed that ALOX15, AREG, BCL2A1, BCL2L1, CA1, COX7B, ECHDC3, IL18R1, IRS2, KCNE1, MMP9, MYL4 and TREML4, are differentially expressed in both phases of this study. CONCLUSIONS: Transcriptomic analysis by microarray technology demonstrated differential expression during a 48 h time course suggesting a potential use of some of these genes as biomarkers for very early stages of ACS, as well as for monitoring early cardiac ischemic recovery.

Cloning and prokaryotic expression of rat homolog of Serpina3n and its expression change during liver regeneration.

A strikingly upregulated expressed sequence tag was screened from regenerating rat liver at 8 h in a 0-4-8-12 h short-interval successive partial hepatectomy model from a previous study. In the present study, a full-length open reading frame (ORF) corresponding to this expressed sequence tag was predicted through electronic cloning and was subsequently cloned from an 8-h rat regenerating liver and deposited in GenBank (accession No. HM448398). Sequence analysis of HM448398 and the predicted ORF revealed that the two ORFs may be different transcripts of a gene. The sequence of HM448398 was highly homologous to that of rat Serpina3n, suggesting that it may be a homolog of Serpina3n. The pGEX-2TK prokaryotic expression vector for this ORF was constructed, and the result of sodium dodecyl sulfate polyacrylamide gel electrophoresis manifested that the recombinant expression vector could express the glutathione-S-transferase-fused rat homolog of Serpina3n in an insoluble form in BL21. The target fusion protein was purified with affinity chromatography and was used as antigen to immunize rabbits for the production of polyclonal antibodies. Immunohistochemistry and real-time reverse transcription polymerase chain reaction analysis revealed that the gene was highly expressed in the priming and termination phases of liver regeneration. These findings lay a solid foundation for further study of roles of HM448398 using knock-in and RNA interference methods during liver regeneration.

A new strategy for mannose-binding lectin gene haplotyping.

The mannose-binding lectin 2 (MBL2) gene is polymorphic and codes for a protein with an important role in the innate immune response, whose variants have been associated with a great number of diseases. Point variations have been described in the 5' regulatory region at positions -550 (MBL2*H or *L) and -221 (*X or *Y), in the 5' untranslated sequence at position +4 (*P or *Q), and in the coding sequence of exon 1 at codons 52, 54, and 57 (MBL2*A or D, A or B, and A or C, respectively). These can be in cis or in trans configuration. The different haplotypes influence the immunological phenotype of the individual, which makes MBL2 haplotyping very important. Previously described MBL2-typing methods do not present adequate haplotype resolution or are too complex and costly. We have developed a new MBL2-typing strategy that is economical and renders rapid and reliable results without ambiguities. We typed 202 individuals of European, 32 of African, and 16 of Oriental descent. Only five to six reactions from 10 possible PCR-SSPs (sequence-specific polymerase chain reactions) were sufficient to genotype one individual unambiguously. The reactions were specific for amplification of the variants located upstream of the coding sequence. The results were associated to the results of hybridizations of the amplified products with eight sequence-specific oligonucleotide probes (SSOP). The strategy led to identification of eight alleles: MBL2*HYPA, HYPD, LYPA, LYPB, LYPD, LYQA, LYQC, and LXPA. Their frequencies in each of the groups were similar to those of other populations studied to date, with MBL2*LYPD (g.[-550G>C; -221C>G; 4T>C; 223C>T; 230A>G; 239A>G]) being novel. All samples were found to be in Hardy-Weinberg equilibrium.

Lipopolysaccharide directly affects pancreatic acinar cells: implications on acute pancreatitis pathophysiology.

We have explored whether lipopolysaccharide (LPS, endotoxin) induces pancreatic injury on pancreatic acinar cells both in vivo and in vitro. Wistar male rats were treated with four intraperitoneal injections of 10 mg/kg LPS, and AR4-2J cells were exposed to increasing doses of LPS. Expression of pancreatitis-associated-protein (PAP) mRNA was strongly induced in AR4-2J cells exposed to LPS, while amylase mRNA was reduced. LPS also induced apoptosis and expression of TNF-alpha, IL-1beta, and IL-8 mRNA in AR4-2J cells. The in vivo effect of LPS showed structural signs of cellular damage, including numerous cytoplasmic vacuoles, severe nuclear alterations, and high expression of PAP mRNA. This study demonstrated that LPS induced pancreatic damage by directly affecting the pancreatic acinar cells. The role of LPS in the pathophysiology of acute pancreatitis may be partly due to the effect LPS has on the acinar cell.