Treatment with rapamycin ameliorates clinical and histological signs of protracted relapsing experimental allergic encephalomyelitis in Dark Agouti rats and induces expansion of peripheral CD4+CD25+Foxp3+ regulatory T cells.

We have presently evaluated the effects of the immunomodulatory drug rapamycin on the course of protracted relapsing experimental allergic encephalomyelitis (PR-EAE) in Dark Agouti (DA) rats, which serves as a preclinical model of multiple sclerosis. The data show that the oral administration of rapamycin at 3 mg/kg for 28 consecutive days significantly ameliorated the course of PR-EAE in DA rats. The rats that received the medication had significantly lower clinical cumulative scores and shorter duration of the disease than did the control rats treated with the vehicle. The milder course of the disease was associated with a reduction of the histopathological signs associated to EAE: increased percentage of splenic CD4+CD25 + Foxp3+ Tregs and concomitant reduction of splenic CD8+T cells. These data suggest that rapamycin has pharmacological potential worthy of consideration in the treatment of MS patients.

Evaluation of circulating CD4+CD25+ and liver-infiltrating Foxp3+ cells in HCV-associated liver disease.

In hepatitis C virus (HCV)-associated liver disease, the immune system is unable to clear the viral infection. Previous studies have raised the possibility of an involvement of regulatory T cells (Tregs). In this study, we analysed the peripheral blood from 30 patients with HCV-associated chronic liver disease and 20 healthy controls by flow cytometry for the evaluation of the Treg population [CD4⁺CD25hi forkhead box protein 3 (Foxp3)⁺], as well as the activated/effector CD4⁺ T cells (CD4⁺CD25low) and IFN-γ-secreting cells. We also analysed liver biopsies of the patients by immunohistochemical evaluation of Foxp3⁺ cells. Our results showed higher proportions of CD4⁺CD25low and IFN-γ⁺ cells in the patients than in the controls. By contrast, the proportions of peripheral CD4⁺CD25hi cells did not significantly differ. The 11 patients displaying Foxp3⁺ cells in the liver infiltrates showed significantly higher proportions of peripheral CD4⁺CD25low cells. Moreover, we found lower serum transaminase levels in the patients than in the controls, as shown by Foxp3⁺ immunohistochemistry, although these results were only statistically significant as regards alanine transaminase (ALT). In conclusion, these data suggest that the presence of Tregs infiltrating the liver is associated with high levels of activated/effector T cells in the peripheral blood and lower activity of hepatitis. Therefore, liver-infiltrating Tregs may play a role in limiting tissue damage and may thus support an effective immune response against HCV.

Nectin like-5 overexpression correlates with the malignant phenotype in cutaneous melanoma.

NECL-5 is involved in regulating cell-cell junctions, in cooperation with cadherins, integrins and platelet-derived growth factor receptor, that are essential for intercellular communication. Its role in malignant transformation was previously described. It has been reported that transformation of melanocytes is associated with altered expression of adhesion molecules suggesting the potential involment of NECL-5 in melanoma development and prognosis. To shed light on this issue, the expression and the role of NECL-5 in melanoma tissues was investigated by bioinformatic and molecular approaches. NECL-5 was up-regulated both at the mRNA and the protein levels in WM35, M14 and A375 cell lines compared with normal melanocytes. A subsequent analysis in primary and metastatic melanoma specimens confirmed "in vitro" findings. NECL-5 overexpression was observed in 53 of 59 (89.8%) and 12 of 12 (100%), primary melanoma and melanoma metastasis, respectively; while, low expression of NECL-5 was detected in 12 of 20 (60%) benign nevi. A significant correlation of NECL-5 overexpression was observed with most of known negative melanoma prognostic factors, including lymph-node involvement (P = 0.009) and thickness (P = 0.004). Intriguingly, by analyzing the large series of melanoma samples in the Xu dataset, we identified the transcription factor YY1 among genes positively correlated with NECL-5 (r = 0.5). The concordant computational and experimental data of the present study indicate that the extent of NECL-5 expression correlates with melanoma progression.

Relationship between circulating interleukin-10 and histological features in patients with chronic C hepatitis.

BACKGROUND AND OBJECTIVES: An imbalance in cytokine production may be involved in the pathogenesis of chronic C hepatitis. The aim of the study was to investigate circulating levels of interleukin-10 (IL-10) in a selected cohort of patients affected by chronic C hepatitis. DESIGN AND SETTING: Retrospective study based on consecutive hepatitis C virus patients, affected by chronic active hepatitis, attending the general hospital of hepatology unit from June to September 2009 PATIENTS AND METHODS: A total of 49 patients with chronic C hepatitis and 20 healthy control subjects similar in gender and age were examined. Circulating IL-10 was assessed by ELISA commercial kit (R and D Systems) in all investigated subjects. RESULTS: There was no significant difference in IL-10 values between controls and overall patients (P>.05). Nevertheless, among patients, subjects with more severe necroinflammation had higher values than others (P<.001). Moreover, a close relationship was found between IL-10 values and serum aspartate aminotransferase (r=0.61; P<.001). CONCLUSIONS: These findings suggest that IL-10 may be a useful additional marker to assess necroinflammation and to monitor the evolution of liver damage. They also argue for a potential pathophysiological role for IL-10 in the persistence and progression of hepatitis.

Understanding rituximab function and resistance: implications for tailored therapy.

The addition of anti-CD20 monoclonal antibody (rituximab) to chemotherapy has significantly improved survival in B-cell lymphoma. However, a substantial number of patients relapse after treatment with rituximab. Understanding of anti-CD20 antibody molecular function may facilitate the development of pharmacologic strategies to overcome resistance. Cell death have been demonstrated to be caused by rituximab binding to CD20 throughout direct and indirect mechanisms. The direct mechanism comprises growth inhibition, induction of apoptosis and sensitization of cells to chemotherapy. While, the indirect mechanisms to Rituximab include complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). However, these mechanisms are still poorly understood. To shed light on this issue, we have analyzed the most significant results showing the role of Rituximab as a signal-inducing antibody and as a chemosensitizing agent through negative regulation of major survival pathways. Mechanisms of resistance to Rituximab are also discussed. Additionally, studies here reported show that, cellular targets are modified after treatment with Rituximab and may become useful for novel therapeutic strategies in the treatment of patients resistant to standard therapy.

Liver immunolocalization and plasma levels of MMP-9 in non-alcoholic steatohepatitis (NASH) and hepatitis C infection.

Non-alcoholic steatohepatitis (NASH) is a progressive fibrotic disease. Many issues related to the pathogenesis of this disease remain unresolved. Because of NASH association with the activation of liver fibrogenesis, we examined the plasma levels and liver immunolocalization of matrix metalloprotease-9 (MMP-9), a molecule involved in the remodelling processes of fibrogenesis. In addition, patients with chronic hepatitis C (HCV) were analyzed. Plasma concentrations of MMP-9 were determined by ELISA from peripheral blood and immunohistochemistry of the same protein was carried out in formalin-fixed and paraffin wax-embedded liver specimens. The mean value of circulating concentrations of MMP-9 in healthy controls was 39.7 ng/ml (SD: +/-4.6). In NASH and HCV-infected patients, MMP-9 concentrations were higher: 69.0 ng/ml (SD: +/-14.5) and 61.7 ng/ml (SD: +/-11.0), respectively. In NASH livers, MMP-9 was mainly immunolocalized on neutrophils, whereas in HVC-infected livers it was mainly localized over biliary canaliculi, bile ducts and hepatocyte cytoplasm. The different MMP-9 immunolocalization patterns in the examined diseases suggest the presence of a different pathophysiological involvement of this protease in the fibrogenesis underlying these diseases.

Comparison of phenotype of gammadelta T cells generated using various cultivation methods.

It has been demonstrated, that gammadelta T cells play an important role in the development of immune responses to many pathogens. gammadelta T cells play a role in the clearance of viral and microbiological infections, anti-tumor responses, but also in autoimmune diseases. Many different protocols for the isolation and cultivation of gammadelta T cells can be found in the literature. Here we compare three common cultivation protocols for gammadelta T cells derived from peripheral blood with a newly developed protocol depending on SLAM (Signaling Lymphocyte Activation Molecule) stimulation. We demonstrate that the cultivation protocol chosen to raise gammadelta T cells has direct impact on the resulting gammadelta T cell phenotype. We show differences in gammadelta TCR composition, memory phenotype formation, CD8 receptor expression and the expression of NK cell markers depending on the stimulation protocol used. As such, the cultivation protocol chosen for a series of experiments might have significant impact on the outcome of the experiments and should be considered carefully.