Plasma, the soluble component of the human blood, is believed to harbor thousands of distinct proteins, which originate from a variety of cells and tissues through either active secretion or leakage from blood cells or tissues. The dynamic range of plasma protein concentrations comprises at least nine orders of magnitude. Proteins involved in coagulation, immune defense, small molecule transport, and protease inhibition, many of them present in high abundance in this body fluid, have been functionally characterized and associated with disease processes. For example, protein sequence mutations in coagulation factors cause various serious disease states. Diagnosing and monitoring such diseases in blood plasma of affected individuals has typically been conducted by use of enzyme-linked immunosorbent assays, which using a specific antibody quantitatively measure only the affected protein in the tested plasma samples. The discovery of protein biomarkers in plasma for diseases with no known correlations to genetic mutations is challenging. It requires a highly parallel display and quantitation strategy for proteins. We fractionated blood serum proteins prior to display on two-dimensional electrophoresis (2-DE) gels using immunoaffinity chromatography to remove the most abundant serum proteins, followed by sequential anion-exchange and size-exclusion chromatography. Serum proteins from 74 fractions were displayed on 2-DE gels. This approach succeeded in resolving approximately 3700 distinct protein spots, many of them post-translationally modified variants of plasma proteins. About 1800 distinct serum protein spots were identified by mass spectrometry. They collapsed into 325 distinct proteins, after sequence homology and similarity searches were carried out to eliminate redundant protein annotations. Although a relatively insensitive dye, Coomassie Brilliant Blue G-250, was used to visualize protein spots, several proteins known to be present in serum in < 10 ng/mL concentrations were identified such as interleukin-6, cathepsins, and peptide hormones. Considering that our strategy allows highly parallel protein quantitation on 2-DE gels, it holds promise to accelerate the discovery of novel serum protein biomarkers.

Interferon (IFN) consensus sequence-binding protein/IFN regulatory factor 8 (IRF8) is a transcription factor that regulates the differentiation and function of macrophages, granulocytes, and dendritic cells through activation or repression of target genes. Although IRF8 is also expressed in lymphocytes, its roles in B cell and T cell maturation or function are ill defined, and few transcriptional targets are known. Gene expression profiling of human tonsillar B cells and mouse B cell lymphomas showed that IRF8 transcripts were expressed at highest levels in centroblasts, either from secondary lymphoid tissue or transformed cells. In addition, staining for IRF8 was most intense in tonsillar germinal center (GC) dark-zone centroblasts. To discover B cell genes regulated by IRF8, we transfected purified primary tonsillar B cells with enhanced green fluorescent protein–tagged IRF8, generated small interfering RNA knockdowns of IRF8 expression in a mouse B cell lymphoma cell line, and examined the effects of a null mutation of IRF8 on B cells. Each approach identified activation-induced cytidine deaminase (AICDA) and BCL6 as targets of transcriptional activation. Chromatin immunoprecipitation studies demonstrated in vivo occupancy of 5′ sequences of both genes by IRF8 protein. These results suggest previously unappreciated roles for IRF8 in the transcriptional regulation of B cell GC reactions that include direct regulation of AICDA and BCL6.

Detection of serum markers for pancreatic cancer has been elusive. Although CA 19-9 is most commonly used, its sensitivity and specificity are modest. We used large-scale proteomics to identify potential serum markers for pancreatic cancer. Samples were analyzed using high-resolution twodimensional gel electrophoresis to identify differentially expressed proteins in 32 normal and 30 pancreatic cancer patients. Up to 1,744 protein spots were resolved for each serum sample. Candidate proteins were identified using mass spectrometry. ANOVA was used to identify proteins that could discriminate cancer from normal sera. Serum fibrinogen level was also measured using enzymatic assay. Immunohistochemistry was used to detect fibrinogen in resected pancreatic cancers. One hundred fifty-four proteins were commonly overexpressed in all pancreatic cancers. Nine protein spots ( four with identifications by mass spectrometry) could effectively separate cancer from normal controls using cross-validation. These proteins successfully discriminated all pancreatic cancer samples (30 of 30) and 94% of normal (30 of 32) samples. Prominent among these candidates was fibrinogen ;, which was subsequently confirmed to be overexpressed in pancreatic cancer sera by enzymatic analysis (54.1 F 64.1 versus 0.0 F 0.0 mg/dL, P < 0.05) and tissue by immunohistochemistry (67% versus 29%, P < 0.05) relative to normal pancreas. Proteomic analysis combining two-dimensional gel electrophoresis and mass spectrometry successfully identified 154 potential serum markers for pancreatic cancer. Of these, fibrinogen ;, a protein associated with the hypercoagulable state of pancreatic cancer, discriminated cancer from normal sera. Fibrinogen is a potential tumor marker in pancreatic cancer. (Cancer Res 2006; 66(5): 2592-9)

Hepatic cirrhosis is the end-stage of chronic liver diseases. The majority of patients with hepatic cirrhosis die from life-threatening complications occurring at their earlier ages. Liver transplantation has been the most effective treatment for these patients. Since liver transplantation is critically limited by the shortage of available donor livers, searching for an effective alternative therapy has attracted great interest in preclinical studies. The transplantation of autologous bone marrow-derived mesenchymal stem cells holds great potential for treating hepatic cirrhosis. Mesenchymal stem cells can differentiate to hepatocytes, stimulate the regeneration of endogenous parenchymal cells, and enhance fibrous matrix degradation. Experimental and clinical studies have shown promising beneficial effects. This review is intended to translate the bench study results to the patients' bedside. The potential interventions of mesenchymal stem cells on cirrhosis are illustrated in terms of the cellular and molecular mechanisms of hepatic fibrogenesis.

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