Diagnostic Markers for Schistosome-Mediated Liver Disease

<p>Chronic schistosomiasis presents with either a moderate or a severe form, termed intestinal (INT) or hepatosplenic (HS) schistosomiasis, respectively. The Schistosoma mansoni-associated hepatomegaly is estimated in 8.5 million people and ultimately results from liver granulomas induced by trapped parasitic eggs. The CBA/J mouse model replicates these two human disease forms and was used to understand the progressive pathology that leads to HS and to identify potential biomarkers. In this model 20% of infected mice spontaneously develop hypersplenomegaly syndrome (HSS) by 20 weeks of infection while the remaining 80% develop moderate splenomegaly syndrome (MSS). Using this model, we compared the liver protein patterns of control mice and mice infected for 6, 8, 12, or 20 (MSS and HSS) weeks. Two-dimensional differential in gel electrophoresis (2D-DIGE) was used to identify protein pattern variations and protein spots were identified using matrix adsorption laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry. In the first experiment, we found 124 protein spot unique changes for MSS and HSS compared to control mice of which 80 were identified and 35 changes were specific for HSS. In the second experiment, comparison between various time points with control mice revealed 76 significant protein spot changes of which 44 were identified using MALDI-TOF mass spectrometry. Importantly, we found that the abundance of liver keratin D, transferrin isoforms, collagen isoforms, hydroxyproline and Schistosoma mansoni-phosphoenolpyruvate carboxykinase increased while epoxide hydrolase isoforms, peroxiredoxin 6 and major urinary protein (MUP) isoforms decreased significantly with infection. To verify the changes in the liver 2D-DIGE analysis, candidate liver protein markers were measured in mouse serum using targeted biochemical assays. The mouse serum analysis showed MUP levels were decreased, while transferrin, connective tissue growth factor (CTGF), keratin D, hydroxyproline were increased in HSS mice compared to control mice or MSS mice supporting the liver 2D-DIGE analysis. Using targeted assays, serum samples from INT and HS patients were tested for the candidate liver protein markers: keratin D, CTGF, hydroxyproline and transferrin. The human serum analysis showed keratin D levels increased for HS compared to INT and normal sera. The CTGF levels were high in INT compared to HS and normal sera, while transferrin remained unchanged in INT and HS similar to normal sera. Additionally, in severe HS disease, serum hydroxyproline emerged as a strong indicator of fibrosis. We believe that these findings will have direct value in the development of diagnostic tools for early detection of hepatosplenic schistosomiasis in humans.</p>

Diagnostic Markers for Schistosome-Mediated Liver Disease

<p>Chronic schistosomiasis presents with either a moderate or a severe form, termed intestinal (INT) or hepatosplenic (HS) schistosomiasis, respectively. The Schistosoma mansoni-associated hepatomegaly is estimated in 8.5 million people and ultimately results from liver granulomas induced by trapped parasitic eggs. The CBA/J mouse model replicates these two human disease forms and was used to understand the progressive pathology that leads to HS and to identify potential biomarkers. In this model 20% of infected mice spontaneously develop hypersplenomegaly syndrome (HSS) by 20 weeks of infection while the remaining 80% develop moderate splenomegaly syndrome (MSS). Using this model, we compared the liver protein patterns of control mice and mice infected for 6, 8, 12, or 20 (MSS and HSS) weeks. Two-dimensional differential in gel electrophoresis (2D-DIGE) was used to identify protein pattern variations and protein spots were identified using matrix adsorption laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry. In the first experiment, we found 124 protein spot unique changes for MSS and HSS compared to control mice of which 80 were identified and 35 changes were specific for HSS. In the second experiment, comparison between various time points with control mice revealed 76 significant protein spot changes of which 44 were identified using MALDI-TOF mass spectrometry. Importantly, we found that the abundance of liver keratin D, transferrin isoforms, collagen isoforms, hydroxyproline and Schistosoma mansoni-phosphoenolpyruvate carboxykinase increased while epoxide hydrolase isoforms, peroxiredoxin 6 and major urinary protein (MUP) isoforms decreased significantly with infection. To verify the changes in the liver 2D-DIGE analysis, candidate liver protein markers were measured in mouse serum using targeted biochemical assays. The mouse serum analysis showed MUP levels were decreased, while transferrin, connective tissue growth factor (CTGF), keratin D, hydroxyproline were increased in HSS mice compared to control mice or MSS mice supporting the liver 2D-DIGE analysis. Using targeted assays, serum samples from INT and HS patients were tested for the candidate liver protein markers: keratin D, CTGF, hydroxyproline and transferrin. The human serum analysis showed keratin D levels increased for HS compared to INT and normal sera. The CTGF levels were high in INT compared to HS and normal sera, while transferrin remained unchanged in INT and HS similar to normal sera. Additionally, in severe HS disease, serum hydroxyproline emerged as a strong indicator of fibrosis. We believe that these findings will have direct value in the development of diagnostic tools for early detection of hepatosplenic schistosomiasis in humans.</p>

Cofilin 2 Acts as an Inflammatory Linker Between Chronic Periodontitis and Alzheimer’s Disease in Amyloid Precursor Protein/Presenilin 1 Mice

Increasing evidence has shown a correlation between chronic periodontitis (CP) and Alzheimer’s disease (AD). Nevertheless, there is still a lack of direct evidence, and especially key molecules to connect the two diseases. This study aims to investigate potential protein links between CP and AD within the inflammatory aspect. The hippocampus of CP model mice and controls were collected, and changes in protein expression were evaluated using two-dimensional differential in-gel electrophoresis (2D-DIGE) analysis combined with liquid chromatography tandem mass spectrometry. A total of 15 differentially expressed proteins were identified in CP model mice, as compared with the controls. Among them, S100-A9, transthyretin, Cofilin 2, peroxiredoxin 2, and lipocalin-2 were validated by Western blot according to their dual function both in inflammation and AD. Based on 2D-DIGE analysis, CP animal model had higher levels of S100-A9, Cofilin 2, peroxiredoxin 2, and lipocalin-2 compared to controls. The level of Cofilin 2, one of the well-established proteins in the pathology of AD, was strongly correlated with the time course of CP pathology, indicating a specific molecular correlation between CP and AD. Moreover, the in vivo results showed the level of Cofilin 2 increased significantly along with a prominent increase of the phosphorylation of protein phosphatase 2 (PP2A) and tau protein in the cell lysates of Porphyromonas gingivalis (P.g-LPS)-treated SK-N-SH APPwt cells. Cofilin 2 inhibition resulted in a sharp decrease in PP2A dependent of tau phosphorylation. Furthermore, tumor growth factor (TGF)-β1 was one of the most important inflammatory cytokines for the Pg-LPS-induced Cofilin 2 upregulation in SK-N-SH APPwt cells. These results showed inflammation served as the bond between CP and AD, whereas inflammatory related proteins could be the key linkers between the two diseases. Determining the association between CP and AD at the molecular mechanism will not only hold the direct evidence of the association between the two diseases but also provide a new way of preventing and treating AD: the effective prevention and treatment of CP could serve as a useful method to alleviate the development of AD.

Circadian Analysis of the Mouse Cerebellum Proteome

The cerebellum contains a circadian clock, generating internal temporal signals. The daily oscillations of cerebellar proteins were investigated in mice using a large-scale two-dimensional difference in gel electrophoresis (2D-DIGE). Analysis of 2D-DIGE gels highlighted the rhythmic variation in the intensity of 27/588 protein spots (5%) over 24 h based on cosinor regression. Notably, the rhythmic expression of most abundant cerebellar proteins was clustered in two main phases (i.e., midday and midnight), leading to bimodal distribution. Only six proteins identified here to be rhythmic in the cerebellum are also known to oscillate in the suprachiasmatic nuclei, including two proteins involved in the synapse activity (Synapsin 2 [SYN2] and vesicle-fusing ATPase [NSF]), two others participating in carbohydrate metabolism (triosephosphate isomerase (TPI1] and alpha-enolase [ENO1]), Glutamine synthetase (GLUL), as well as Tubulin alpha (TUBA4A). Most oscillating cerebellar proteins were not previously identified in circadian proteomic analyses of any tissue. Strikingly, the daily accumulation of mitochondrial proteins was clustered to the mid-resting phase, as previously observed for distinct mitochondrial proteins in the liver. Moreover, a number of rhythmic proteins, such as SYN2, NSF and TPI1, were associated with non-rhythmic mRNAs, indicating widespread post-transcriptional control in cerebellar oscillations. Thus, this study highlights extensive rhythmic aspects of the cerebellar proteome.