Maria Paola Simula

HCV-NS3 and IgG-Fc crossreactive IgM in patients with type II mixed cryoglobulinemia and B-cell clonal proliferations.

We demonstrate that in three cases of MC (two with immunocytoma), the IgM-RF+ component of their cryoprecipitated represents the circulating counterpart of the B-cell receptor (BCR) of the monoclonal overexpanded B-cell population. These IgMs were isolated and used to demonstrate a crossreactivity against both hepatitis C virus (HCV) NS3 antigen and the Fc portion of IgG. Epitopes were identified in a fraction of exemplary samples by using epitope excision approach (NS31250–1334 and IgG Fc345–355). The same phenomenon of crossreactivity has been shown to occur in vivo after immunization of a mouse with the NS31251−1270 peptide. To verify if the same reaction was also present in MC samples characterized by an oligo/polyclonal B-cell proliferation, IgM crossreactivity was tested in 14 additional samples. Five out of the 14 were reactive against HCV NS3 and 11 out of 14 were reactive against IgG-Fc peptide. The data support the role of HCV NS3 antigen in a subset of patients with MC, whereas the high frequency of the IgG-Fc epitope suggests that these B cells originate from precursors strongly selected for auto-IgG specificity. We suggest that engagement of specific BCRs by NS3 (or NS3-immunocomplex) antigen could explain the prevalence of IgM cryoglobulins in these patients

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.

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‐Hodgkins 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.

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.

Role of the HLA class II: HCV-related disorders

Abstract:  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, charateristics 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.

Characterization of antibodies directed against the immunoglobulin light κ 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 κ (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.

Proteins specifically hyperexpressed in a coeliac disease patient with aberrant T cells

An aberrant T cell population is the basis for diagnosis of refractory coeliac disease and determines the risk of enteropathy‐associated T cell lymphoma. This disease is serious with a poor survival. Pathogenetic mechanisms sustaining aberrant T cell proliferation remain unknown. Recently, alemtuzumab has been proposed as a promising new approach to treat these patients. Only few single cases have been tested at present; nevertheless, in all the cases a clinical improvement was observed. However, whether intraepithelial lymphocytes have been targeted effectively by alemtuzumab is still debated. This study reports, using two‐dimensional difference gel electrophoresis (2D DIGE), hyperexpressed proteins associated specifically with aberrant T cells found in a patient with coeliac disease by comparison of the protein expression of this sample with that of patients with coeliac disease and polyclonal T cells or with control subjects. The data demonstrated a significantly higher expression of IgM, apolipoprotein C‐III and Charcot–Leyden crystal proteins in a duodenal biopsy specimen of the patient with clonal T cells compared with that of other patients. These preliminary results allow hypothesizing different clinical effects of alemtuzumab in patients with coeliac disease and aberrant T cell proliferation, because as well as the probable effect on T cells, alemtuzumab could exert its effect by acting on inflammatory associated CD52+ IgM+ B cells and eosinophil cells, known to produce IgM and Charcot–Leyden crystal proteins, that we demonstrated to be altered in this patient. The results also emphasize the possible association of apolipoprotein with aberrant T cell proliferation.

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.

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.

Comment re: Ran-GTP Control of Tumor Cell Mitosis

In Response: In a recent work, Xia and colleagues ( [1][1]) found that the small GTPase Ran is broadly overexpressed in cancer. In accordance with the emerging evidence that the Ran-GTP signaling pathway may be preferentially exploited in cancer, the authors show that it becomes essential for cell