Alessandro Pavan

Distinct functional significance of Akt and mTOR constitutive activation in mantle cell lymphoma

Functional characterization of signaling pathways that critically control mantle cell lymphoma (MCL) cell growth and survival is relevant to designing new therapies for this lymphoma. We herein demonstrate that the constitutive activation of Akt correlates with the expression of the phosphorylated, inactive form of PTEN. Phosphatidyl-inositol-3 kinase (PI3-K)/Akt or mammalian target of rapamycin (mTOR) inhibition decreased the growth of both primary MCL cultures and established cell lines and antagonizes the growth-promoting activity of CD40 triggering and IL-4. These effects are mediated by nuclear accumulation of the p27(Kip1) inhibitor induced by down-regulation of the p45(Skp2) and Cks1 proteins, which target p27(Kip1) for degradation. Moreover, Akt inhibition down-regulated cyclin D1 by promoting its proteasome-dependent degradation driven by GSK-3. Intriguingly, mTOR inhibition affected cyclin D1 proteolysis only in MCL cells in which GSK-3 is under the direct control of mTOR, suggesting that different MCL subsets could be differently responsive to mTOR inhibition. Finally, PI3-K/Akt inhibitors, but not rapamycin, induced variable levels of caspase-dependent apoptosis and reduced telomerase activity. These results indicate that Akt and mTOR activation have distinct functional relevance in MCL and suggest that targeting Akt may result in more effective therapeutic effects compared with mTOR inhibition.

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.

Spontaneous T cell responses to Epstein-Barr virus-encoded BARF1 protein and derived peptides in patients with nasopharyngeal carcinoma: bases for improved immunotherapy.

Immunotherapy approaches targeting Epstein‐Barr virus (EBV)‐encoded antigens induce objective clinical responses only in a fraction of patients with undifferentiated nasopharyngeal carcinoma (UNPC). In the present study, we have characterized the immunogenicity of the EBV‐encoded BARF1 oncogene with the aim to assess whether this protein could constitute a new target antigen for immunotherapy in this setting. Spontaneous CD4+ and CD8+ T cell responses specific for the recombinant p29 BARF1 protein were detected by IFNγ‐ELISPOT in both EBV‐seropositive donors and UNPC patients, but not in EBV‐seronegative individuals. Using immunoinformatic prediction tools, we have selected 5 different candidate BARF1 T cell epitopes presented by HLA‐A*0201. Although only one of these peptides was able to bind HLA‐A2 with low affinity in the T2 stabilization assay, all 5 BARF1 nonamers readily elicited specific CD8+ T cell responses in EBV‐seropositive HLA‐A*0201+ donors and UNPC patients. Notably, the magnitude of CD8+ T cell responses to the whole BARF1 protein and derived A*0201 peptides was significantly higher in UNPC patients than in healthy donors. Moreover, cytotoxic T lymphocytes specific for the p2–10, p23–31, or p49–57 BARF1 peptides were easily obtained from HLA‐A*0201+ donors. These cultures were not only able to lyse autologous targets loaded with the antigenic peptide, but also recognized tumor cells endogenously expressing BARF1 in an antigen‐specific and HLA‐A2‐restricted manner. These findings, indicate that BARF1 is a particularly attractive antigen with immunogenic properties in most UNPC patients and provide valuable information to develop new strategies to improve the efficacy of EBV‐targeting immunotherapy of UNPC patients.

Recent prognostic factors in diffuse large B-cell lymphoma indicate NF-κB pathway as a target for new therapeutic strategies

The diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoid cancer. The classical chemotherapy regimen given to these patients was the CHOP (Cyclophosphamide, Hydroxydaunorubicin or Adriamycin, Oncovin or Vincristine, Prednisone or Prednisolone), but recently rituximab with CHOP (R-CHOP) increased the number of cases responding to first line therapy. DLBCL classification identified three principle subgroups. The first one, named germinal centre B cell-like (GCB), responds to both CHOP and R-CHOP treatment and it is mainly characterised by the expression of markers like Bcl-6 and CD10. The second, the activated B-cell like (ABC), has a worse prognosis in comparison with GCB, and is mainly characterised by the expression of IRF-4, PRDM1 and NF-κB. It is interesting to notice that IRF-4 and PRDM1 are under the transcriptional control of NF-κB, whose high activation level is associated with a worse prognosis. The third one, mediastinal large B-cell lymphoma (PMBCL) is an uncommon subtype characteristically found in young females. Gene expression profiling suggests that this disease resembles Hodgkin lymphoma more than other types of DLBCL. The impact of rituximab on the outcome of patients with PMBCL has still not been fully assessed. It was seen that rituximab inhibits NF-κB pathway in vitro. However, the clinical significance of this finding is still unknown, because both ABC and GCB DLBCL show a significant improvement of overall survival after R-CHOP treatment. In this review, the NF-κB pathway is suggested as a target for new chemotherapy strategies based on the association of CHOP with molecules more effective than rituximab in this pathway inhibition.

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.

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.

Clonal CD27+ CD19+ B cell expansion through inhibition of FCγIIR in HCV+ cryoglobulinemic patients: a model.

Persistent HCV infection may be associated with extrahepatic manifestations such as type II mixed cryoglobulinemia (II‐MC), a clonal B cell proliferative disorder. In persistent HCV infection without II‐MC, an increase in serum immunoglobulins (Ig) is commonly observed. This increase is polyclonal and is determined primarily by increased levels of IgG which include both HCV‐specific and nonspecific antibodies. Nonetheless, memory CD27+ B cells do not accumulate. This paradoxical phenomenon depends on heightened sensitivity of memory B cells to BCR‐independent noncognate T cell help, which speeds up their terminal differentiation into antibody‐secreting cells and makes them more prone to apoptosis. In persistent HCV infection with II‐MC, serum Ig elevation is also a general occurrence, and characteristically includes IgM antibodies with rheumatoid factor activity, which are essential for the development of circulating, cryoprecipitable immune complexes. Hypergammaglobulinemia is sustained by a peripheral expansion of IgM+k+IgDlow/negCD21lowCD27+ B cells. These cells exhibit marked VH, JH, and VK gene segment usage restriction, indicating that a limited number of antigens drive their proliferation through BCR interaction. Recently, two epitopes, one of the human IgG and the second of the HCVNS3 protein, had been identified and demonstrated able to link the BCR exposed on II‐MC subjects. Based on the above findings, we propose a model whereby BCR binding the IgM/IgG/HCVNS3 immune complexes deprives FcγIIR of its natural ligand. This takes the brake off RF+CD27+ B cell proliferation and promotes their selective accumulation, which is otherwise prevented by increased apoptosis susceptibility in persistent HCV infection without II‐MC.

Two-dimensional gel proteome reference map of human small intestine

BackgroundThe 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.ResultsThe 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.ConclusionThis 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.