Anna Pia Riviera

Improved method for carbohydrate-deficient transferrin determination in human serum by capillary zone electrophoresis

Carbohydrate-deficient transferrin (CDT) is a reliable marker of chronic or repeated alcohol abuse. It indicates a group of isoforms of human transferrin (Tf), the main iron transport serum protein, deficient in sialic acid residues (asialo-, monosialo- and disialo-Tf) in comparison to the main isotransferrin which contains four sialic acid groups (tetrasialo-Tf). The aim of the present work was to develop a capillary electrophoretic method suitable for rapid determination of CDT components in serum. Serum samples (0.1 ml) were saturated with iron by incubation with 10 mM FeCl3 (2 microl) and 500 mM NaHCO3 (3 microl) for 30 min, then diluted 1:10 in water and injected by positive pressure (0.5 p.s.i. for 10 s). Separation was performed with a capillary zone electrophoretic method using bare fused-silica capillaries (57 cm x 20 microm I.D.) and a buffer composed of 100 mM sodium tetraborate adjusted with 6 M HCl to pH 8.3 added with 1.5 mM diaminobutane. Applied voltage was 20 kV and temperature 25 degrees C. Detection was by UV absorption at 200 nm wavelength. Under the described conditions, asialo-, monosialo-, disialo-, trisialo- and tetrasialo-transferrin were baseline separated. The limit of detection (signal-to-noise ratio of 2) was about 0.3% for disialo-Tf, and 0.5% of trisialo-Tf, expressed as percentages of the terasialo-Tf peak area. Day-to-day RSDs of relative migration times were < or = 0.2%. Quantitation showed day-to-day RDSs < or = 6.9% and < or = 10.9% for disialo- and trisialo-Tf, respectively. The results from 79 control subjects, including social drinkers, and 23 alcoholics showed disialo- and trisialo-Tf significantly increased in patients (P<0.0001 and <0.01, respectively). A clear interference from trisialo-Tf in an immunoassay for CDT was demonstrated. The present method is suitable for confirmation of CDT immunoassays by independent technique.

IFN-γ-mediated upmodulation of MHC class I expression activates tumor-specific immune response in a mouse model of prostate cancer

De novo expression of B7-1 impaired tumorigenicity of TRAMP-C2 mouse prostate adenocarcinoma (TRAMP-C2/B7), but it did not elicit a protective response against TRAMP-C2 parental tumor, unless after in vitro treatment with IFN-gamma. TRAMP-C2 cells secrete TGF-beta and show low MHC-I expression. Treatment with IFN-gamma increased MHC-I expression by induction of some APM components and antagonizing the immunosuppressant activity of TGF-beta. Thus, immunization with TRAMP-C2/B7 conferred protection against TRAMP-C2-derived tumors in function of the IFN-gamma-mediated fine-tuned modulation of either APM expression or TGF-beta signaling. To explore possible clinical translation, we delivered IFN-gamma to TRAMP-C2 tumor site by means of genetically engineered MSCs secreting IFN-gamma.

Constitutive Activation of p38 and ERK1/2 MAPKs in Epithelial Cells of Myasthenic Thymus Leads to IL-6 and RANTES Overexpression: Effects on Survival and Migration of Peripheral T and B Cells

Myasthenia gravis (MG) is an autoimmune disease of neuromuscular junctions where thymus plays a pathogenetic role. Thymectomy benefits patients, and thymic hyperplasia, a lymphoid infiltration of perivascular spaces becoming site of autoantibody production, is recurrently observed. Cytokines and chemokines, produced by thymic epithelium and supporting survival and migration of T and B cells, are likely to be of great relevance in pathogenesis of thymic hyperplasia. In thymic epithelial cell (TEC) cultures derived “in vitro” from normal or hyperplastic age-matched MG thymuses, we demonstrate by gene profiling analysis that MG-TEC basally overexpress genes coding for p38 and ERK1/2 MAPKs and for components of their signaling pathways. Immunoblotting experiments confirmed that p38 and ERK1/2 proteins were overexpressed in MG-TEC and, in addition, constitutively activated. Pharmacological blockage with specific inhibitors confirmed their role in the control of IL-6 and RANTES gene expression. According to our results, IL-6 and RANTES levels were abnormally augmented in MG-TEC, either basally or upon induction by adhesion-related stimuli. The finding that IL-6 and RANTES modulate, respectively, survival and migration of peripheral lymphocytes of myasthenic patients point to MAPK transcriptional and posttranscriptional abnormalities of MG-TEC as a key step in the pathological remodelling of myasthenic thymus.

Change in myasthenia gravis epidemiology in Trento, Italy, after twenty years.

Background: The recent literature suggests that the incidence and prevalence of myasthenia gravis (MG) are changing. We performed a population-based study of MG in the province of Trentino (524,826 inhabitants) and compared the results with those collected 20 years ago. Methods: Multiple sources of information (discharge diagnosis, antibody tests and anticholinesterase drugs) were analyzed. Incidence was calculated from 2005 to 2009. Prevalence was calculated on December 31, 2009. Comparison was made with descriptive statistics for 1981–1990 for the identical region. Results: Incidence and prevalence greatly increased in comparison with 1981–1990 data. The prevalence rate increased from 82.9 (95% confidence interval, CI, 58.4–114.3) in 1990 to 129.6 (95% CI 100.6–164.3) per million population, whereas the average annual incidence increased from 7.4 (95% CI 5–10.4) per million person-years in 1981–1990 to 14.8 (95% CI 10.5–20.3) in 2005–2009. This increase was mainly due to male patients with late-onset MG. Conclusions: The study confirms the increase in incidence and prevalence of late-onset MG in the same region 20 years apart. So we should consider MG also as a disease of the elderly.

Analysis of CIITA encoding AIR-1 gene promoters in insulin-dependent diabetes mellitus and rheumatoid arthritis patients from the Northeast of Italy: Absence of sequence variability

Qualitative and/or quantitative alterations in the expression of the MHC class II molecules affect the onset and maintenance of the immune response and may be the basis of a wide variety of disease states, such as autoimmunity and immunodeficiency.CIITA is a major physiological regulator of the expression of MHC class II genes. The availability of CIITA ap- pears generally essential for MHC class II gene expression, and hence its own transcriptional regulatory mechanisms result of fundamental importance for a correct homeostasis of the immune response. Therefore, it is possible to hypothesize that variability at the CIITA-encoding locus, AIR-1, could constitute an additional source of susceptible traits to autoimmune diseases. Mutations at AIR-1/CIITA promoters could modulate expression of CIITA. Variations in CIITA expression could influence the qualitative and quantitative expression of MHC class II molecules at cell surface. We have analyzed sequence variation at AIR-1/CIITA promoters by PCR-SSCP in 23 IDDM and 30 RA patients compared to a sample of 19 unaffected normal controls and 16 unaffected IDDM family members, for a total of 88 Caucasian subjects from the Northeast of Italy. No sequence difference was found at the four AIR-1/CIITA promoters between autoimmune patients and normal controls. Moreover, the promoters resulted invariant within the entire group of 88 subjects analyzed, comprising patients and controls. This finding suggests a possible selective advantage in maintaining CIITA upstream regulatory sequences invariant.

Thymic involvement in myasthenia gravis: Study by immunofluorescent and immunoperoxidase staining

Changes in the thymus are often encountered in myasthenia gravis (MG), together with neuromuscular pathology. In the present study, the cell population of 28 thymuses from patients with MG were analyzed by immunofluorescent and 49 thymuses by immunoperoxidase techniques. We were able to show the presence of sIg receptor positive, SpA reactive medium and large B lymphocytes of light specific density in the majority of thymuses, and to demonstrate a characteristic pattern of distribution in the gland. PAP analysis showed that the infiltrating B cells appeared in the interlobular septa, then reached the medulla, occasionally the cortex which mostly revealed signs of atrophy. These findings are consistent with an autoimmune reaction occurring against a thymic antigen; such an antigen could be acetylcholine receptor (AchR) of the thymic structures, since anti-AchR antibody or serum from patients with MG will react with 60% thymocytes and some epithelial cells and Hassalls corpuscles.

Efficacy Assessment of Interferon-Alpha–Engineered Mesenchymal Stromal Cells in a Mouse Plasmacytoma Model

Bone marrow mesenchymal stromal cells (BM-MSCs) may survive and proliferate in the presence of cycling neoplastic cells. Exogenously administered MSCs are actively incorporated in the tumor as stromal fibroblasts, thus competing with the local mesenchymal cell precursors. For this reason, MSCs have been suggested as a suitable carrier for gene therapy strategies, as they can be genetically engineered with genes encoding for biologically active molecules that can inhibit tumor cell proliferation and enhance the antitumor immune response. We used BM-MSCs engineered with the murine interferon-alpha (IFN-α) gene (BM-MSCs/IFN-α) to assess in a mouse plasmacytoma model the efficacy of this approach toward neoplastic plasma cells. We found that IFN-α can be efficiently produced and delivered inside the tumor microenvironment. Subcutaneous multiple administration of BM-MSCs/IFN-α significantly hampered the tumor growth in vivo and prolonged the overall survival of mice. The antitumor effect was associated with enhanced apoptosis of tumor cells, reduction in microvessel density, and ischemic necrosis. By contrast, intravenous administration of BM-MSCs/IFN-α did not significantly modify the survival of mice, mainly as a consequence of an excessive entrapment of injected cells in the pulmonary vessels. In conclusion, BM-MSCs/IFN-α are effective in inhibiting neoplastic plasma cell growth; however, systemic administration of engineered MSCs needs to be improved to make this approach potentially suitable for the treatment of multiple myeloma.

Effects of dietary wheat germ deprivation on the immune system in Wistar rats: a pilot study

Bioactive molecules that can gain access to body tissues through the gastrointestinal tract may interact with immune regulatory circuits and effector functions. Among these are plant lectins, such as wheat germ (WG) agglutinin, which constitute common components of the human diet and target the immune system on a daily basis. Dietary bioactive molecules might be considered as immunomodulatory signals. To investigate the possible effects on the immune system of the long-term absence of such signals, two groups of rats were fed on a diet containing or deprived of WG. The WG-deprived diet induced a state of functional unresponsiveness in lymphocytes from primary and secondary lymphoid organs, as evaluated by in vitro stimulation with T cell mitogen phytohemoagglutinin (PHA) and B cell mitogen lypopolysaccarides (LPS). The unresponsive state of the immune cells could be reversed by injection of antigen emulsified in oil with inactivated mycobacteria (complete Freunds adjuvant, CFA) Dietary signals can thus interact with the immune system possibly influencing its shaping during ontogenesis.

Induction of an antitumour adaptive immune response elicited by tumour cells expressing de novo B7‐1 mainly depends on the anatomical site of their delivery: the dose applied regulates the expansion of the response

De novo expression of costimulatory molecules in tumours generally increases their immunogenicity, but does not always induce a protective response against the parental tumour. This issue was addressed in the mouse Sp6 hybridoma model, comparing different immunization routes (subcutaneous, intraperitoneal and intravenous) and doses (0·5 × 106 and 5 × 106 cells) of Sp6 cells expressing de novo B7‐1 (Sp6/B7). The results can be summarized as follows. First, de novo expression of B7‐1 rendered Sp6 immunogenic, as it significantly reduced the tumour incidence to ≤15% with all delivery routes and doses tested, whereas wild‐type Sp6 was invariably tumorigenic (100% tumour incidence). Second, long‐lasting protection against wild‐type Sp6 was mainly achieved when immunization with Sp6/B7 was subcutaneous: a dose of 0·5 × 106 Sp6/B7 cells elicited protection that was confined to sites in the same anatomical quarter as the immunizing injection. Repeated injections of the same dose extended protection against wild‐type Sp6 to other anatomical districts, as well as a single injection of a 10‐fold higher dose (5 × 106 cells). Finally, Sp6‐specific cytotoxic T‐lymphocyte activity was detected in draining lymph nodes, and the splenic expansion of Sp6‐specific cytotoxic T‐lymphocyte precursors quantitatively correlated with the dose of antigen. We conclude that activation of a protective immune response against Sp6 depends on the local environment where the immunogenic form of the ‘whole tumour cell antigen’ is delivered. The antigen dose regulates the anatomical extent of the protective response.

An electrophysiological study of the effects of myasthenia gravis sera and complement on rat isolated muscle fibres

The effect of human myasthenia gravis (MG) sera and complement on isolated adult rat muscle fibres was investigated. Heat-inactivated MG sera reduced the frequency of single acetylcholine receptor (AChR)-channel activity. One of the MG sera tested had a stronger effect on the extrajunctional type of AChRs than on the junctional type. The simultaneous addition of normal human serum (NHS), as source of complement, and MG serum to freshly dissociated muscle fibres caused contraction restricted to the endplate area and progressive depolarization of the muscle membrane, followed by contracture. An MG antibody-dependent complement-mediated damage of the muscle fibres is suggested.