Rebeca Moreira

Transcriptomics of In Vitro Immune-Stimulated Hemocytes from the Manila Clam Ruditapes philippinarum Using High-Throughput Sequencing

Background The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases. Methodology and Principal Findings High-throughput deep sequencing of R. philippinarum using 454 pyrosequencing technology yielded 974,976 high-quality reads with an average read length of 250 bp. The reads were assembled into 51,265 contigs and the 44.7% of the translated nucleotide sequences into protein were annotated successfully. The 35 most frequently found contigs included a large number of immune-related genes, and a more detailed analysis showed the presence of putative members of several immune pathways and processes like the apoptosis, the toll like signaling pathway and the complement cascade. We have found sequences from molecules never described in bivalves before, especially in the complement pathway where almost all the components are present. Conclusions This study represents the first transcriptome analysis using 454-pyrosequencing conducted on R. philippinarum focused on its immune system. Our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and the study of gene expression as well as for the identification of genetic markers. The discovery of new immune sequences was very productive and resulted in a large variety of contigs that may play a role in the defense mechanisms of Ruditapes philippinarum.

Urinary neutrophil gelatinase-associated lipocalin predicts kidney outcome and death in patients with cirrhosis and bacterial infections

BACKGROUND & AIMS Infections in cirrhosis are frequently complicated by kidney dysfunction that entails a poor prognosis. Urinary biomarkers may be of potential clinical usefulness in this setting. We aimed at assessing the value of urinary neutrophil gelatinase-associated lipocalin (uNGAL), a biomarker overexpressed in kidney tubules during kidney injury, in predicting clinical outcomes in cirrhosis with infections. METHODS One-hundred and thirty-two consecutive patients hospitalized with infections were evaluated prospectively. Acute kidney injury (AKI) was defined according to AKIN criteria. uNGAL was measured at infection diagnosis and at days 3 and 7 (ELISA, Bioporto, DK). RESULTS Patients with AKI (n=65) had significantly higher levels of uNGAL compared to patients without AKI (203 ± 390 vs. 79 ± 126 μg/g creatinine, p<0.001). Moreover, uNGAL levels were significantly higher in patients who developed persistent AKI (n=40), compared to those with transient AKI (n=25) (281 ± 477 vs. 85 ± 79 μg/g creatinine, p<0.001). Among patients with persistent AKI, uNGAL was able to discriminate type-1 HRS from other causes of AKI (59 ± 46 vs. 429 ± 572 μg/g creatinine, respectively; p<0.001). Moreover, the time course of uNGAL was markedly different between the two groups. Interestingly, baseline uNGAL levels also predicted the development of a second infection during hospitalization. Overall, 3-month mortality was 34%. Independent predictive factors of 3-month mortality were MELD score, serum sodium, and uNGAL levels at diagnosis, but not presence or stage of AKI. CONCLUSIONS In patients with cirrhosis and infections, measurement of urinary NGAL at infection diagnosis is useful in predicting important clinical outcomes, specifically persistency and type of AKI, development of a second infection, and 3-month mortality.

Analysis of a Urinary Biomarker Panel for Clinical Outcomes Assessment in Cirrhosis

Background Biomarkers are potentially useful in assessment of outcomes in patients with cirrhosis, but information is very limited. Given the large number of biomarkers, adequate choice of which biomarker(s) to investigate first is important. Aim Analysis of potential usefulness of a panel of urinary biomarkers in outcome assessment in cirrhosis. Patients and Methods Fifty-five patients with acute decompensation of cirrhosis were studied: 39 had Acute Kidney Injury (AKI) (Prerenal 12, type-1 HRS (hepatorenal syndrome) 15 and Acute Tubular Necrosis (ATN) 12) and 16 acute decompensation without AKI. Thirty-four patients had Acute-on-chronic liver failure (ACLF). A panel of 12 urinary biomarkers was assessed, using a multiplex assay, for their relationship with ATN, ACLF and mortality. Results Biomarker with best accuracy for ATN diagnosis was NGAL (neutrophil-gelatinase associated lipocalin): 36 [26-125], 104 [58-208] and 1807 [494-3,716] μg/g creatinine in Prerenal-AKI, type-1 HRS and ATN, respectively; p<0.0001 (AUROC 0.957). Other attractive biomarkers for ATN diagnosis were IL-18, albumin, trefoil-factor-3 (TFF-3) and glutathione-S-transferase-π (GST-π) Biomarkers with less accuracy for ATN AUCROC<0.8 were β2-microglobulin, calbindin, cystatin-C, clusterin and KIM-1 (kidney injury molecule-1). For ACLF, the biomarker with the best accuracy was NGAL (ACLF vs. No-ACLF: 165 [67-676] and 32 [19-40] μg/g creatinine; respectively; p<0.0001; AUROC 0.878). Interestingly, other biomarkers with high accuracy for ACLF were osteopontin, albumin, and TFF-3. Biomarkers with best accuracy for prognosis were those associated with ACLF. Conclusions A number of biomarkers appear promising for differential diagnosis between ATN and other types of AKI. The most interesting biomarkers for ACLF and prognosis are NGAL, osteopontin, albumin, and TFF-3. These results support the role of major inflammatory reaction in the pathogenesis of ACLF.

Gene expression analysis of clams Ruditapes philippinarum and Ruditapes decussatus following bacterial infection yields molecular insights into pathogen resistance and immunity

The carpet shell clam (Ruditapes decussatus) and Manila clam (Ruditapes philippinarum), which are cultured bivalve species with important commercial value, are affected by diseases that result in large economic losses. Because the molecular mechanism of the immune response of bivalves, especially clams, is scarce and fragmentary, we have examined all Expressed Sequence Tags (EST) resources available in public databases for these two species in order to increase our knowledge on genes related with the immune function in these animals. After automatic annotation and classification of the 3784 not-annotated ESTs of R. decussatus and 4607 of R. philippinarum found in GenBank, 424 ESTs of R. decussatus and 464 of R. philippinarum were found to be putatively involved in immune response. These were carefully reviewed and reannotated. As a result, 13 immune-related ESTs were selected and studied to compare the immune response of R. decussatus and R. philippinarum following a Vibrio alginolyticus challenge. Quantitative PCR was performed, and the expression of each EST was determined. The results showed that, in R. philippinarum, the immune response seems to be faster than that in R. decussatus. Additionally, expression of NF-κB activating genes in R. decussatus did not seem to be sufficient to promote an immune response after Vibrio infection. R. philippinarum, however, was able to trigger and efficiently regulate the transcriptional activity of NF-κB, even when low expression values were reported.

Neutrophil gelatinase-associated lipocalin is a biomarker of acute-on-chronic liver failure and prognosis in cirrhosis

BACKGROUND & AIMS Acute-on-chronic liver failure (ACLF) is a syndrome that occurs in cirrhosis characterized by organ failure(s) and high mortality rate. There are no biomarkers of ACLF. The LCN2 gene and its product, neutrophil gelatinase-associated lipocalin (NGAL), are upregulated in experimental models of liver injury and cultured hepatocytes as a result of injury by toxins or proinflammatory cytokines, particularly Interleukin-6. The aim of this study was to investigate whether NGAL could be a biomarker of ACLF and whether LCN2 gene may be upregulated in the liver in ACLF. METHODS We analyzed urine and plasma NGAL levels in 716 patients hospitalized for complications of cirrhosis, 148 with ACLF. LCN2 expression was assessed in liver biopsies from 29 additional patients with decompensated cirrhosis with and without ACLF. RESULTS Urine NGAL was markedly increased in ACLF vs. no ACLF patients (108(35-400) vs. 29(12-73)μg/g creatinine; p<0.001) and was an independent predictive factor of ACLF; the independent association persisted after adjustment for kidney function or exclusion of variables present in ACLF definition. Urine NGAL was also an independent predictive factor of 28day transplant-free mortality together with MELD score and leukocyte count (AUROC 0.88(0.83-0.92)). Urine NGAL improved significantly the accuracy of MELD in predicting prognosis. The LCN2 gene was markedly upregulated in the liver of patients with ACLF. Gene expression correlated directly with serum bilirubin and INR (r=0.79; p<0.001 and r=0.67; p<0.001), MELD (r=0.68; p<0.001) and Interleukin-6 (r=0.65; p<0.001). CONCLUSIONS NGAL is a biomarker of ACLF and prognosis and correlates with liver failure and systemic inflammation. There is remarkable overexpression of LCN2 gene in the liver in ACLF syndrome. LAY SUMMARY Urine NGAL is a biomarker of acute-on-chronic liver failure (ACLF). NGAL is a protein that may be expressed in several tissues in response to injury. The protein is filtered by the kidneys due to its small size and can be measured in the urine. Ariza, Graupera and colleagues found in a series of 716 patients with cirrhosis that urine NGAL was markedly increased in patients with ACLF and correlated with prognosis. Moreover, gene coding NGAL was markedly overexpressed in the liver tissue in ACLF.

Immune responses during the larval stages of Mytilus galloprovincialis: Metamorphosis alters immunocompetence, body shape and behavior

We investigated the development of the immune system during the larval stages of the mussel Mytilus galloprovincialis. The ability of trochophore and veliger larvae to phagocytose foreign particles (Escherichia coli and zymosan) was measured. Phagocytosis was detected as early as 24 h post-fertilization (hpf) using flow cytometry and fluorescence microscopy. However, although there was a high basal production of reactive oxygen and nitrogen species (ROS and NRS), the phagocytosis of zymosan did not trigger an associated increase in radical production. In addition, a panel of immune-related mussel genes (Myticin B, Myticin C, Mytilin B, Mytimycin precursor 1, Macrophage migration inhibition factor, lysozyme, C1q, membrane attack complex protein and fibrinogen-related protein) was selected for expression profile analysis throughout the different developmental stages (trochophore, veliger, metamorphosis, post-settlement and spat). The expression of these genes increased during the transition from trochophore to spat, and the level of expression was higher in oocytes than in trochophores, suggesting that gene expression during the first larval stages might be maternal in origin. Metamorphosis was identified as a crucial stage when larvae increased the expression of immune-related genes and responded to environmental signals. Whole-mount in situ hybridization studies showed the mantle edge as an important area in the development of immunocompetence in bivalve larvae. Larvae responded to both live and heat-inactivated bacteria by modulating expression of immune-related genes. Altogether, our results support that during the early stages of M. galloprovincialis development, immune mechanisms emerge to aid larvae in managing infections.

Gene expression profile analysis of Manila clam (Ruditapes philippinarum) hemocytes after a Vibrio alginolyticus challenge using an immune-enriched oligo-microarray

BackgroundThe Manila clam (Ruditapes philippinarum) is a cultured bivalve with worldwide commercial importance, and diseases cause high economic losses. For this reason, interest in the immune genes in this species has recently increased. The present work describes the construction of the first R. philippinarum microarray containing immune-related hemocyte sequences and its application to study the gene transcription profiles of hemocytes from clams infected with V. alginolyticus through a time course.ResultsThe complete set of sequences from R. philippinarum available in the public databases and the hemocyte sequences enriched in immune transcripts were assembled successfully. A total of 12,156 annotated sequences were used to construct the 8 ×15 k oligo-microarray. The microarray experiments yielded a total of 579 differentially expressed transcripts. Using the gene expression results, the associated Gene Ontology terms and the enrichment analysis, we found different response mechanisms throughout the experiment. Genes related to signaling, transcription and apoptosis, such as IL-17D, NF-κB or calmodulin, were typically expressed as early as 3 hours post-challenge (hpc), while characteristic immune genes, such as PGRPs, FREPs and defense proteins appeared later at 8 hpc. This immune-triggering response could have affected a high number of processes that seemed to be activated 24 hpc to overcome the Vibrio challenge, including the expression of many cytoskeleton molecules, which is indicative of the active movement of hemocytes. In fact functional studies showed an increment in apoptosis, necrosis or cell migration after the infection. Finally, 72 hpc, activity returned to normal levels, and more than 50% of the genes were downregulated in a negative feedback of all of the previously active processes.ConclusionsUsing a new version of the R. philippinarum oligo-microarray, a putative timing for the response against a Vibrio infection was established. The key point to overcome the challenge seemed to be 8 hours after the challenge, when we detected immune functions that could lead to the destruction of the pathogen and the activation of a wide variety of processes related to homeostasis and defense. These results highlight the importance of a fast response in bivalves and the effectiveness of their innate immune system.

Characterization of inflammatory response in acute-on-chronic liver failure and relationship with prognosis

ACLF is characterized by a systemic inflammatory response, but the cytokines involved in this process have not been well studied. The aim of this study was to characterize the systemic inflammatory response in patients with cirrhosis and ACLF and its relationship with prognosis. Fifty-five patients with cirrhosis, 26 with ACLF, were studied prospectively. Systemic inflammatory response was analyzed by measuring a large array of plasma cytokines by using a multiplex kit. A principal component analysis show noticeable differences between ACLF and decompensated cirrhosis without ACLF. Patients with ACLF had significant abnormal levels of 12 cytokines compared to those without ACLF, including: VCAM-1, VEGF-A, Fractalkine, MIP-1α, Eotaxin, IP-10, RANTES, GM-CSF, IL-1β, IL-2, ICAM-1, and MCP-1. Cytokines showing the most marked relationship with ACLF were VCAM-1 and VEGF-A (AUCROC 0.77; p = 0.001). There was a significant relationship between some of inflammatory mediators and 3-month mortality, particularly VCAM-1, ICAM-1, and GM-CSF (AUCROC>0.7; p < 0.05). Functional Enrichment Analysis showed that inflammatory markers differentially expressed in ACLF patients were enriched in leukocyte migration, particularly monocytes and macrophages, and chemotaxis pathways. In conclusion, ACLF is characterized by a marked inflammatory reaction with activation of mediators of adhesion and migration of leukocytes. The intensity of the inflammatory reaction correlates with prognosis.

Severe acute kidney injury associated with non-steroidal anti-inflammatory drugs in cirrhosis: A case-control study

BACKGROUND & AIMS Non-steroidal anti-inflammatory drugs (NSAIDs) may cause impairment of kidney function in patients with cirrhosis. Investigational studies demonstrated reversibility of kidney dysfunction after drug withdrawal, but information based on clinical practice is lacking. The aim of the study was to investigate the characteristics and outcome of Acute Kidney Injury (AKI) developing in patients with cirrhosis treated with NSAIDs. METHODS Prospective cohort study in a tertiary referral center of all patients with NSAIDs-associated AKI seen from 2002 to 2014. For comparison, three control groups of patients with hypovolemic-induced AKI, type-1 HRS and ATN, respectively, were also evaluated. Urinary excretion of neutrophil gelatinase-associated lipocalin (uNGAL) was measured in a subset of patients. RESULTS Thirty patients with cirrhosis and NSAIDs-associated AKI were identified. In 19 patients (63%) AKI was transient and kidney function rapidly recovered (4±3 days) after NSAIDs withdrawal. In the remaining 11 patients (37%) AKI was more severe and persisted during hospitalization despite drug withdrawal. Patients with persistent AKI had remarkably higher uNGAL levels compared with those of patients with transient AKI (953±1,198 vs. 83±79 μg/g of creatinine, respectively, p=0.008). Moreover, seven of the 11 patients with persistent AKI (64%) died within three months compared with only one of the 19 (5%) patients with transient AKI (p=0.001). Mortality of persistent AKI was similar in NSAIDs patients compared to control groups. The only independent predictive factor of three-month mortality was persistent AKI. CONCLUSIONS Patients with cirrhosis treated with NSAIDs may develop severe AKI which may be irreversible and associated with poor short-term outcome.

Validation of a Staging System for Acute Kidney Injury in Patients With Cirrhosis and Association With Acute-on-Chronic Liver Failure

BACKGROUND & AIMS In patients with cirrhosis of the liver, acute kidney injury (AKI) is classified into 3 stages. Recent studies indicate that there are 2 subgroups of stage 1 disease, associated with different outcomes and serum levels of creatinine (SCr): stage 1A (SCr <1.5 mg/dL) and stage 1B (SCr ≥1.5 mg/dL). We performed a prospective study to validate, in a large series of patients with cirrhosis, the association between this new description and patient outcomes, and assess the relationship between AKI stage and the presence of acute‐on‐chronic liver failure. METHODS We collected data from 547 consecutive patients admitted for cirrhosis with acute decompensation to 2 tertiary hospitals (Italy and Spain), from February 2011 through June 2015. A total of 290 patients had AKI (53%; 197 had stage 1 disease); AKI stages were determined based on levels of SCr at diagnosis. Patients were followed up until death, liver transplantation, or for 90 days. The primary outcome was 90‐day survival; secondary outcomes were progression and resolution of AKI and association with acute‐on‐chronic liver failure. RESULTS Based on level of sCr at diagnosis, 58 patients had stage 1A disease and 139 had stage 1B disease. Of patients with stage 1A disease, 82% survived for 90 days; of patients with stage 1B disease, 55% survived for 90 days (P = .001). Hepatorenal syndrome and acute tubular necrosis were the most common causes of stage 1B AKI, and hypovolemia was the most common cause of stage 1A AKI. AKI progressed in a higher proportion of patients with 1B than 1A AKI (31% vs 15%; P = .017) and resolved in a higher proportion of patients with 1A disease (90% vs 52% of patients with stage 1B; P < .001). Stage 1B disease, but not 1A, was an independent predictor of AKI progression and mortality. ACLF developed in a significantly greater proportion of patients with stage 1B disease (76%) than stage 1A disease (22%; P < .001), which could account for the poor outcomes of patients with stage 1B disease. CONCLUSIONS In a large group of patients with decompensated cirrhosis, we validated the association between AKI stages IA and IB (based on level of sCR) with survival times and AKI progression. We also associated these subgroups of AKI with development of acute‐on‐chronic liver failure. These findings are important for management of patients with decompensated cirrhosis.