Evaluation of the suitability of archival Bouin-fixed paraffin-embedded tissue specimens to proteomic investigation.

Bouin's solution has been used for over a century as a common fixative in several pathology laboratories worldwide. Therefore, a considerable number of Bouin-fixed paraffin-embedded (BFPE) tumor samples of various origin are available in hospital repositories as a powerful information mine for clinical investigations. To date, however, such archived tissues have not been subjected to a systematic study aimed to evaluate their potential use in proteomics. In this report, we investigated whether archival BFPE tissue specimens could be exploited for proteomic studies, upon application of protein extraction and proteomic analysis methods previously optimized for formalin-fixed samples. As a result, gastric BFPE protein extracts exhibited poor suitability for 2D-PAGE analysis, whereas over 300 unique proteins could be successfully detected when extracts were subjected to SDS-PAGE followed by LC-MS/MS (GeLC-MS/MS). Among these, several known markers for gastric cancer and normal gastric functionality were identified, indicative of biological and clinical significance of proteomic data mined from BFPE tissues. A quantitative and qualitative comparison of FFPE and BFPE tissue proteomes was also performed, and results are reported. In conclusion, we demonstrated that BFPE specimens can be analyzed by means of a proteomic approach such as GeLC-MS/MS. Although considerable molecular biases and technical constraints exist, BFPE tissue archives can be fruitfully exploited for gathering proteomic data from particularly precious samples.

2-D gel electrophoresis: constructing 2D-gel proteome reference maps.

Two-dimensional gel electrophoresis (2-DE) is the most popular and versatile method of protein separation among a rapidly growing array of proteomic technologies. Based on two independent biochemical characteristics of proteins, it combines isoelectric focusing, which separates proteins according to their isoelectric point (pI), and SDS-PAGE, which separates them further according to their molecular mass. An evolution of conventional 2-DE is represented by the 2D-Difference in Gel Electrophoresis (2D-DIGE) that allows sample multiplexing and achieving more accurate and sensitive quantitative proteomic determinations. The 2-DE separation permits the generation of protein maps of different cells or tissues and the study, by differential proteomics, of protein expression changes associated to the different states of a biological system. In order to identify the molecular bases of pathological processes, it is also useful to characterize the physiological protein homeostasis in healthy cells or tissues. On these grounds, the availability of detailed 2D reference maps could be very useful for proteomic studies. The protocol described in this chapter is based on the 2D-DIGE technology and has been applied to obtain the first 2-DE reference map of the human small intestine.

Hepatitis C virus-induced oxidative stress and mitochondrial dysfunction: a focus on recent advances in proteomics.

The natural history of chronic hepatitis C virus (HCV) infection presents two major aspects. On one side, the illness is by itself benign, whereas, on the other side, epidemiological evidence clearly identifies chronic HCV infection as the principal cause of cirrhosis, hepatocellular carcinoma, and extrahepatic diseases, such as autoimmune type II mixed cryoglobulinemia and some B cell non-Hodgkin's lymphomas. The mechanisms responsible for the progression of liver disease to severe liver injury are still poorly understood. Nonetheless, considerable biological data and studies from animal models suggest that oxidative stress contributes to steatohepatitis and that the increased generation of reactive oxygen and nitrogen species, together with the decreased antioxidant defense, promotes the development of hepatic and extrahepatic complications of HCV infection. The principal mechanisms causing oxidative stress in HCV-positive subjects have only been partially elucidated and have identified chronic inflammation, iron overload, ER stress, and a direct activity of HCV proteins in increasing mitochondrial ROS production, as key events. This review summarizes current knowledge regarding mechanisms of HCV-induced oxidative stress with its long-term effects in the context of HCV-related diseases, and includes a discussion of recent contributions from proteomics studies.

Proteomic analyses lead to a better understanding of celiac disease: focus on epitope recognition and autoantibodies.

Proteomic technologies are being used with increasing frequency in the scientific community. In this review, we have highlighted their use in celiac disease (CD). The available techniques, which include two-dimensional (2D) gel electrophoresis, mass spectrometry, and antibody and tissue arrays, have been used to identify proteins or changes in protein expression specific to gut tissue from patients with CD. A number of studies have employed proteomic methodologies to determine the diagnostic biomarkers in body fluids or to examine changes in protein expression and posttranslational modifications during signaling. A fast technological development of these methods, along with the combination of classic techniques with proteomics, will lead to new discoveries, which will consent a better understanding of the pathogenesis of CD.

PPAR signaling pathway and cancer-related proteins are involved in celiac disease-associated tissue damage.

Celiac disease (CD) is an immune-mediated disorder triggered by the ingestion of wheat gliadin and related proteins in genetically predisposed individuals. To find a proteomic CD diagnostic signature and to gain a better understanding of pathogenetic mechanisms associated with CD, we analyzed the intestinal mucosa proteome alterations using two dimensional difference gel electrophoresis (2D-DIGE) coupled with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF ms) of CD patients with varying degrees of histological abnormalities defined by Marsh criteria and controls. Our results clearly evidenced the presence of two groups of patients: Group A, including controls and Marsh 0-I CD patients; and Group B, consisting of CD subjects with grade II-III Oberhuber-Marsh classification. Differentially expressed proteins were involved mainly in lipid, protein and sugar metabolism. Interestingly, in Group B, several downregulated proteins (FABP1, FABP2, APOC3, HMGCS2, ACADM and PEPCK) were implicated directly in the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Moreover, Group B patients presented a deregulation of some proteins involved in apoptosis/survival pathways: phosphatidylethanolamine-binding protein 1 (PEBP1), Ras-related nuclear protein (Ran) and peroxiredoxin 4 (PRDX4). PEBP1 downregulation and RAN and PRDX4 upregulation were associated with more severe tissue damage. Likewise, IgMs were found strongly upregulated in Group B. In conclusion, our results indicate that a downregulation of proteins involved in PPAR signaling and the modulation of several cancer-related proteins are associated with the highest CD histological score according to Oberhuber-Marsh classification.

Differential proteomic analysis of hepatocellular carcinoma.

The principal aim of the present study consisted in the identification of the disregulated proteins associated with the development of hepatocellular carcinoma (HCC). The differences in protein expression between hepatocellular carcinoma (HCC) and the corresponding non-HCC liver tissues were investigated in a cohort of 20 patients using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS). The up- and down-regulated protein spots that exhibited 1.5-fold difference signal intensity with statistical significance (p<0.05, t-test, confidence intervals 95%) were excised from the gel and identified by peptide mass fingerprinting using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Thirty-six protein spots corresponding to 29 different disregulated proteins, belonging to heterogeneous metabolic pathways, have been identified. Down-regulated proteins (n=23) were found superior in number than the up-regulated proteins (n=6). Detoxification, carbohydrate metabolism and amino acid biotrasformation represented the main disregulated pathways in HCC. Up-regulation of aldo-keto reductase 1C2, thioredoxin and transketolase, involved in metabolic and regulatory cellular processes including proliferation, differentiation and carcinogenesis were remarkable. These proteins could represent useful biomarkers to provide new insights into global pathophysiologic changes of HCC and for the development of new pharmacological approaches to HCC therapy.

Characterization of antibodies directed against the immunoglobulin light kappa chain variable chain region (VK) of hepatitis C virus-related type-II mixed cryoglobulinemia and B-cell proliferations.

Autoimmune type-II cryoglobulinemia (II-MC) is sustained by hepatitis C virus (HCV) infection and B-cell (oligo)clones. This is the reason why the disease may be considered an "indolent B-cell lymphoma (NHL)." B clones show a restricted use of immunoglobulin variable genes (BCR), in particular in the use of the variable kappa (VK)3-20/15 light chain, and show a homology between their BCR functional regions and those of autoimmune rheumatoid factors. We underlined the BCR unique repertoire with frequent rheumatoid factor activity also observed in other autoimmune disorders associated with NHL. The immunoglobulin idiotype is a clonal B-cell marker and an ideal target for immunotherapy. Five monoclonal antibodies were produced in our laboratory toward the VK3-20 of a subject with HCV infection and a II-MC-associated NHL. Epitope determination was performed using the epitope excision approach. Monoclonal antibody reactivity was tested in vitro in ELISA, Western blot, and cytofluorimetry. Data confirmed that a panel of antibodies, reactive against shared idiotypes, can be produced from patients with HCV-associated B-cell lymphoproliferative diseases, thus obviating the need to produce an anti-idiotype antibody for each patient.

Galectin-10, eosinophils, and celiac disease.

Celiac disease (CD) is a chronic intestinal disease caused by intolerance to dietary wheat gluten in genetically susceptible individuals. There are a number of important open questions that impede the full explanation of the pathogenesis of this disease. We analyzed protein expression pattern in gut biopsies of CD subjects. Patients were selected and grouped according to histological inflammatory degree. Groups consisted of nine individuals with CD: three patients had a Marsh 0, three a Marsh I-II, and three a Marsh III. All CD patients showed a human leukocyte antigen DQ2/8 variant. Controls were three individuals with an excluded CD diagnosis. For the first time, galectin-10 expression was found related to the histological grade (P = 0.0092) and with the number of eosinophils in the lesion (P = 0.0040). Results suggest galectin-10 is a novel marker for evaluating CD tissue damage and eosinophils as a possible target for therapeutic approaches. Moreover, our data provide insights into alterations associated with CD tissue damage and pathogenesis.

Identification of proteins associated to multi-drug resistance in LoVo human colon cancer cells.

Multi-drug resistance (MDR) limits the effectiveness of chemotherapy. P-glycoprotein encoded by the MDR1 gene, is known to be implicated in MDR phenotype, but other factors could be determinant in MDR. The aim of this study was to investigate new molecular factors potentially associated with the MDR phenotype using a proteomic approach. Two dimensional fluorescence difference gel electrophoresis (2D-DIGE) and MALDI-TOF peptide mass fingerprinting were used to determine differentially expressed proteins between LoVo human colon carcinoma cell line and one of its MDR sublines (LoVo-R1). Thirty-four differentially expressed proteins were identified. They were classified into five groups based on their biological functions: i) proteins involved in energy request pathways, ii) in detoxification pathways, iii) in cell survival activity, iv) in drug transport and v) in chaperone functions. Among these proteins, endothelin 1 and proteasome subunit beta2 regulations were validated by immunofluorescence and Western blotting, respectively, showing complete consistency with 2D-DIGE results. In conclusion, the proteomic approach indicates that multiple mechanisms are simultaneously involved in MDR. These might be useful in the search for new forms of interventional therapeutic approaches for MDR reversal.

Two-dimensional gel proteome reference map of human small intestine.

BACKGROUND: The small intestine is an important human organ that plays a central role in many physiological functions including digestion, absorption, secretion and defense. Duodenal pathologies include, for instance, the ulcer associated to Helicobacter Pylori infection, adenoma and, in genetically predisposed individuals, celiac disease. Alterations in the bowel reduce its capability to absorb nutrients, minerals and fat-soluble vitamins. Anemia and osteopenia or osteoporosis may develop as a consequence of vitamins malabsorption. Adenoma is a benign tumor that has the potential to become cancerous. Adult celiac disease patients present an overall risk of cancer that is almost twice than that found in the general population. These disease processes are not completely known.To date, a two dimensional (2D) reference map of proteins expressed in human duodenal tissue is not yet available: the aim of our study was to characterize the 2D protein map, and to identify proteins of duodenal mucosa of adult individuals without duodenal illness, to create a protein database. This approach, may be useful for comparing similar protein samples in different laboratories and for the molecular characterization of intestinal pathologies without recurring to the use of surgical material. RESULTS: The enrolled population comprised five selected samples (3 males and 2 females, aged 19 to 42), taken from 20 adult subjects, on their first visit at the gastroenterology unit for a suspected celiac disease, who did not turn to be affected by any duodenal pathology after gastrointestinal and histological evaluations. Proteins extracted from the five duodenal mucosal specimens were singly separated by 2D gel electrophoresis. After image analysis of each 2D gel, 179 protein spots, representing 145 unique proteins, from 218 spots tested, were successfully identified by MALDI-TOF ms analysis. Normalized volumes, for each protein, have been reported for every gel. Proteins have been grouped according to their biological/metabolic functions. CONCLUSION: This study represents to date the first detailed and reproducible 2D protein map of human duodenum. Spots identifications, reported in a database, will be helpful to identify the variability in protein expression levels, in isoforms expression, or in post-translational modifications associated to pathology or to a therapy.

HCV inhibits antigen processing and presentation and induces oxidative stress response in gastric mucosa.

Productive hepatitis C virus (HCV) infection appears to be primarily confined to the liver. However, a wide variety of extrahepatic disease manifestations are associated with the infection and HCV RNA has been frequently detected in gastric mucosa. The present study aims to determine molecular alterations present in vivo in the stomach where HCV expression does not induce a carcinoma but a lymphoma, thus extending the knowledge of alterations in intracellular pathways consequent to HCV infection. We compared, by 2-D DIGE, the gastric protein expression profile from six HCV positive and six HCV negative samples lacking neoplastic or dysplastic conditions. In HCV positive tissue we observed a down regulation of proteins involved in MHC maturation and assembly, antigen processing and presentation and ER stress, in addition to an up regulation of proteins involved in cellular oxidative stress responses. Ubiquinol-cytochrome-C-reductase (UQCRFS1), part of the mitochondrial respiratory chain complex-III, was identified as the most up regulated protein. Data were confirmed by Western blot and immunohistochemistry. Our results demonstrate a HCV negative influence on the different pathways that determine antigen processing and presentation via MHC-I and the cellular attempts to counteract HCV induced oxidative stress. Both these processes facilitate immune escape and cell survival and probably contribute to HCV chronicization.

HCV-related immunocytoma and type II mixed cryoglobulinemia-associated autoantigens.

Hepatitis C virus (HCV) is a hepatotropic virus causing hepatocellular damage and chronic liver inflammation that progressively can lead to cirrhosis and hepatocellular carcinoma (HCC). HCV is also lymphotropic, as demonstrated by its capacity to replicate in lymphocytes, by the recurrent detection of organ- and non-organ-specific autoantibodies in HCV-infected patients, and by the strong association found between HCV infection and type II mixed cryoglobulinemic syndrome (MC-II). Moreover, accumulating data ascribe an etiopathogenetic role in the development of B cell non-Hodgkin's lymphomas (NHL) to HCV. All these findings account for the profound effect of HCV infection in the host's immune system. The unique virus-host interactions that culminate in the generation and sustained production of autoantibodies and cryoglobulins have not been delineated. It appears that chronic antigenic stimulation could cause the emergence of specific B cell clones that produce cryoglobulins; moreover, B cell activation and/or deregulation could originate as a result of HCV binding to CD81 tetraspanin or as a consequence of its ability to replicate in B cells. In a previous study we demonstrated that, in MC-II HCV-positive patients, cryoprecipitated monoclonal IgMs, and B cell receptors (BCR) of overexpanded B cell clones share the same combinatory region. Moreover, these IgMs were reactive against both the Fc region of human IgG and the HCV-NS3 antigen. NS3 and Fc epitopes have been idengified by epitope excision approach. One of the idengified NS3 epitopes has been used to immunize a mouse and the monoclonal antibody obtained showed the same cross-reactivity as patients' IgMs. The characterization of antigenic specificity of this antibody may be useful to idengify antigens that can stimulate B cell proliferation in HCV-infected individuals.

Protein expression profile of celiac disease patient with aberrant T cell by two-dimensional difference gel electrophoresis.

One complication of celiac disease (CD) is refractory CD. These patients frequently show aberrant intraepithelial T cell clones and an increasing risk of evolution into enteropathy-associated T cell lymphoma (EATL). There is debate in the literature whether these cases are actually a smoldering lymphoma from the outset. The mechanism inducing T cell proliferation and prognosis remains unknown. Recently, alemtuzumab has been proposed as a promising new approach to treat these patients. Only few single cases have been tested presently, nevertheless, in all of them a clinical improvement has been observed, while intraepithelial lymphocytes (IELs) effectively targeted by alemtuzumab are still a debated issue. Using 2D-DIGE, we found hyperexpressed proteins specifically associated with aberrant T cell in a patient with CD by comparing the protein expression with that of patients with CD and polyclonal T cell or with that of control subjects (patients with polyclonal T cell and no CD). Proteins with a higher expression in duodenal biopsy of the patient with aberrant T cell were identified as IgM, apolipoprotein C-III, and Charcot-Leyden crystal proteins. These preliminary data allow hypothesizing different clinical effects of alemtuzumab in patients with CD, since besides the probable effect of alemtuzumab on T cell, it could effect inflammatory-associated CD52(+) IgM(+)B cell and eosinophils cells, known to produce IgM and Charcot-Leyden crystal proteins, which we demonstrated to be altered in this patient. Results also emphasize the possible association of apolipoprotein with aberrant T cell proliferation.

Role of the HLA class II: HCV-related disorders.

The paper highlights the role of different HLA class II molecules in hepatic and lymphoproliferative HCV-related disorders. HLA molecules have been reviewed, according to an in silico cluster classification, based on the sequence, the biochemical structure of the pockets, and the functional characteristics of the HLA II molecules. Thus, by reducing the complexity of HLA II polymorphism, characteristics that unite different HLA molecules with specific HCV-associated pathologies may be recognized with greater case. Results show that HLA clusters associated with better dlimination of the virus are protective against HCC development, while the same clusters are associated with a higher risk of developing cryoglobulinemic syndrome and the concomitant NHL. These data added further acknowledgements on pathogenetic mechanisms associated with HCV infection. Results also highlight differences of NHL occurring in HCV-positive subjects, with or without a concomitant type II autoimmune cryoglobulinemic syndrome, suggesting that cryoglobulinemic background associated with NHL should be considered in the evaluation of the effectiveness of new therapies in the course of HCV-associated NHLs.

Mammalian ATPsynthase monomer versus dimer profiled by blue native PAGE and activity stain.

Studies into the effects of oligomerization on F(0)F(1)ATPsynthase function are contradictory. We optimized the in-gel ATPase assay to investigate the functional differences of monomers versus dimers. In Triton X-100 extracts of heavy bovine heart mitochondria (HBHM) and mitoplasts, but not submitochondrial particles (MgATP-SMP), dimers had greater specific activity than monomers: at 30 degrees C, the dimer/monomer activity ratios were 2.3, 1.4, and 1.0, respectively. These differences in HBHM and mitoplasts extracts were enhanced at 37 degrees C but lost at 20 degrees C. In mitoplasts but not in MgATP-SMP, dimers were selectively shielded from limited chymotrypsin degradation of F(1) alpha subunit, possibly due to interactions with other proteins or ligands in the native inner membrane. Despite these differences, all three preparations had similar percentages of dimers and similar contents of the native inhibitor IF(1) in Vm (monomer) and (dimer) Vd. These results suggest that, in native membrane, monomers and dimers are functionally distinct.

Identification of novel chimpanzee MHC class I and II alleles using an improved sequence-based typing strategy.

The chimpanzee (Pan troglodytes) is a valuable model for the study of infection with hepatitis C virus and hepatitis B virus, to which only humans and chimpanzees are susceptible. Because the cellular immune response plays a crucial role in host defense against these viruses, the analysis of major histocompatibility complex (MHC) (Patr) class I and II allele diversity in chimpanzees is essential for immune response analysis and vaccine development. In the present study, we report a novel, rapid, and sensitive sequence-based typing strategy to identify polymorphisms of the principal Patr class I (Patr-A and Patr-B) and Patr class II (Patr-DQB1 and Patr-DRB1) alleles. Using this method we identified seven novel Patr alleles in 17 chimpanzees, one of them present in 3 chimpanzees. Furthermore, we detected heterozygosity more rapidly and with higher sensitivity than was done with previous techniques that were based on reverse transcription and amplification of messenger RNA followed by molecular cloning and sequencing.