Biomarkers of disease differentiation: HCV recurrence versus acute cellular rejection

The wound-healing process induced by chronic hepatitis C virus (HCV) infection triggers liver damage characterized by fibrosis development and finally cirrhosis. Liver Transplantation (LT) is the optimal surgical treatment for HCV-cirrhotic patients at end-stage liver disease. However, acute cellular rejection (ACR) and HCV recurrence disease represent two devastating complications post-LT. The accurate differential diagnosis between both conditions is critical for treatment choice, and similar histological features represent a challenge for pathologists. Moreover, the HCV recurrence disease severity is highly variable post-LT. HCV recurrence disease progression is characterized by an accelerated fibrogenesis process, and almost 30% of those patients develop cirrhosis at 5-years of follow-up. Whole-genome gene expression (WGE) analyses through well-defined oligonucleotide microarray platforms represent a powerful tool for the molecular characterization of biological process. In the present manuscript, the utility of microarray technology is applied for the ACR and HCV-recurrence biological characterization in post-LT liver biopsy samples. Moreover, WGE analysis was performed to identify predictive biomarkers of HCV recurrence severity in formalin-fixed paraffin-embedded liver biopsies prospectively collected

Krüppel-Like Factor 6 Expression Changes during Trophoblast Syncytialization and Transactivates ßhCG and PSG Placental Genes

BACKGROUND: Krüppel-like factor-6 (KLF6) is a widely expressed member of the Sp1/KLF family of transcriptional regulators involved in differentiation, cell cycle control and proliferation in several cell systems. Even though the highest expression level of KLF6 has been detected in human and mice placenta, its function in trophoblast physiology is still unknown. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we explored KLF6 expression and sub-cellular distribution in human trophoblast cells differentiating into the syncytial pathway, and its role in the regulation of genes associated with placental development and pregnancy maintenance. Confocal immunofluorescence microscopy demonstrated that KLF6 is expressed throughout human cytotrophoblast differentiation showing no evident modifications in its nuclear and cytoplasmic localization pattern. KLF6 transcript and protein peaked early during the syncytialization process as determined by qRT-PCR and western blot assays. Overexpression of KLF6 in trophoblast-derived JEG-3 cells showed a preferential nuclear signal correlating with enhanced expression of human β-chorionic gonadotropin (βhCG) and pregnancy-specific glycoprotein (PSG) genes. Moreover, KLF6 transactivated βhCG5, PSG5 and PSG3 gene promoters. Deletion of KLF6 Zn-finger DNA binding domain or mutation of the consensus KLF6 binding site abolished transactivation of the PSG5 promoter. CONCLUSIONS/SIGNIFICANCE: Results are consistent with KLF6 playing a role as transcriptional regulator of relevant genes for placental differentiation and physiology such as βhCG and PSG, in agreement with an early and transient increase of KLF6 expression during trophoblast syncytialization

BAFF Mediates Splenic B Cell Response and Antibody Production in Experimental Chagas Disease

Chagas disease, caused by the protozoan Trypanosoma cruzi, is endemic in Central and South America. It affects 20 million people and about 100 million people are at risk of infection in endemic areas. Some cases have been identified in non-endemic countries as a consequence of blood transfusion and organ transplantation. Chagas disease presents three stages of infection. The acute phase appears one to two weeks after infection and includes fever, swelling around the bite site, enlarged lymph glands and spleen, and fatigue. This stage is characterized by circulating parasites and many immunological disturbances including a massive B cell response. In general, the acute episode self-resolves in about 2 months and is followed by a clinically silent indeterminate phase characterized by absence of circulating parasites. In about one-third of the cases, the indeterminate phase evolves into a chronic phase with clinically defined cardiac or digestive disturbances. Current knowledge suggests that the persistence of parasites coupled with an unbalanced immune response sustain inflammatory response in the chronic stage. We believe that an effective treatment for chronic Chagas disease should combine antiparasitic drugs with immunomodulators aimed at reducing inflammation and autoreactive response. Our findings enlighten a new role of BAFF-BAFF-R signaling in parasite infection that partially controls polyclonal B cell response but not parasitespecific class-switched primary effectors B cells

Demographic and clinicopathological characteristics of HCV-cirrhotic patient groups.

<p>(*)Histopathological examination identified one incidental HCC viable unique lesion (size: 1.7 cm) in the explanted liver from one HCV-cirrhosis case of the independent patients’ set. <i>P</i>-values were calculated among HCV-cirrhotic groups for each study set.</p

Schematic representation of the study design.

<p>HCV-cirrhotic tissue samples were divided in two main groups: 1) microarray hybridization (n = 80), and 2) independent validation set (n = 27), represented in gray squares. Pairwise comparisons among woHCC, wHCC, and iHCC groups of hybridized microarrays were performed for molecular pathway analyses. L₁-penalized regression model was fit for woHCC and wHCC groups with hybridized microarrays (training set). The iHCC group was the test set for the identified prediction model. The independent set (n = 27) validated the expression of 9 randomly selected genes from the prediction model.</p

Identification of HCV-cirrhosis with and without HCC by qPCR validated genes.

<p>Canonical plot derived from applying quadratic discriminate analysis to nine validated genes from the best L1-penalized regression model in an independent set of HCV-cirrhotic samples. The 95% confidence ellipses of HCV-cirrhosis with (wHCC) and without HCC (woHCC) are illustrated in red and blue ovals, respectively. Individual samples are indicated by red (wHCC) and blue (woHCC) squares.</p

Abstract 11550: Human Mesenchymal Stem Cell-Derived Microvesicles Mitigate Aortic Smooth Muscle Cell Activation via miR-147 and Attenuate Aortic Aneurysm Formation

Objective: Mesenchymal stem cells (MSCs) can release microvesicles (MVs) but their effects on aortic aneurysm (AA) formation are unknown. We hypothesize that MSC-derived MVs can inhibit AA formation via modulation of specific microRNAs (miRs). Methods: An elastase-perfusion abdominal AA model was used with 8- to 12- week old male mice. The abdominal aorta was perfused for 5 minutes with porcine pancreatic elastase and treated with or without human umbilical cord MSC-derived MVs (1x10 6 given intravenously on day 1). On day14 following perfusion, the abdominal aortic diameter was measured by video micrometry and expressed as percentage increase over baseline. miRNA microarray was performed on aortic tissue using Affymetrix GeneChip hybridization. MVs were transfected with either miR-147b mimic or inhibitor (25nM, Invitrogen, CA) using Lipofectamine RNAiMAX and co-cultured with aortic smooth muscle cells (AoSMCs) followed by treatment with transient elastase (5 minutes) or recombinant IL-17 (10 ng/ml). Cytokines were analyzed in cell culture supernatants after 24 hrs. Groups were compared by ANOVA followed by Bonferroni post hoc test. Results: Treatment of elastase-perfused WT mice with MVs caused a significant attenuation of aortic diameter compared to elastase-perfused mice alone (102.4±6 vs. 162.6±17.9% respectively; mean +/- S.E.; n=5, p=0.03). A significant mitigation of pro-inflammatory cytokines (IL-17, HMGB1, MCP-1, TNF-α and KC), inflammatory cell infiltration (CD3+ T cells, macrophages and neutrophils) and decrease in elastic fiber disruption occurred in aortic tissue from elastase-perfused mice treated with MVs compared to elastase-perfused mice alone. miRNA analysis of aortic tissue demonstrated a significant upregulation of miR-147b (10-fold; p&lt;0.001), miR-19a (9-fold; p&lt;0.001) and a downregulation of miR-98 (1.2 fold; p&lt;0.05) in mice with AA compared to controls (n=5 mice/group). Treatment of AoSMCs with elastase or IL-17 induced cytokines (IL-6, RANTES, MCP-1, TNF-α and KC) which were attenuated by co-cultures with MVs transfected with miR-147 inhibitor. Conclusions: MSC-derived MVs attenuate aortic inflammation and inhibit AoSMC activation via miR-147 suggesting a novel protective mechanism in AA pathobiology. </jats:p

Best fitting prediction model for HCC identification in HCV-cirrhotic patients.

<p>L1-penalized regression model identified 17 differentially expressed Psets as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040275#pone-0040275-t002" target="_blank"><b>Table 2</b></a>. <b>A)</b> Three-dimensional plot derived from applying classical multidimensional scaling (MDS) to the gene expression dataset for those genes identified by the L1-penalized regression model. Individual samples are represented by a colored square for wHCC (green), woHCC (blue), and iHCC (red). <b>B)</b> Two-way supervised hierarchical clustering and heatmap using ward’s method including all training set samples and Psets identified by the best fitting model.</p