Consultancy and surveillance of post-immunisation adverse events in the Veneto region of Italy for 1992-2008.

Prevention and control of adverse events following immunization (AEFI) are fundamental activities of successful immunisation programs. AEFI reporting, investigation and analysis, integrated by consultancy for subjects needing a specialized evaluation, represent an ideal model for vaccine safety surveillance. In the Veneto Region of Italy the Green Channel Centre has been created by the local Public Health authority, to offer a consultancy activity for vaccinations at risk of adverse events and to ensure an efficient AEFI surveillance system with regular feedback data for vaccine personnel. This report updates the overall activity provided by the Green Channel between 1992 and 2008, concerning consultations for previous AEFI and contraindications to vaccinations and analysis of AEFI reports. After 1280 consultancy cases, 998 (78%) subjects were found eligible for vaccination, with personalized precautions suggested in 42% of cases. Of a total of 724 patients actually vaccinated as per the Green Channel instructions, only 55 subjects (7.6%) reported mild symptoms and one (0.3%) a moderate allergic reaction. Since 1993, a total of 5,006 AEFI reports have been collected and evaluated by the Green Channel against more than 20 millions of vaccine doses administered with an estimate mean AEFI rate of 2.3 x 10.000 doses per year. The majority of them (94%) were found in causal relationship with vaccines; of these, 267 reports (5,6% - 0.1/10,000 doses) were serious and 9 of these subjects, affected by a neurological event, were not recovered or were still on therapy at follow up. This regional activity has proven efficacious in evaluating and managing individual cases at potential risk of AEFI and integrating the national passive surveillance system.

Differential response of human basophil activation markers: a multi-parameter flow cytometry approach.

BACKGROUND: Basophils are circulating cells involved in hypersensitivity reactions and allergy but many aspects of their activation, including the sensitivity to external triggering factors and the molecular aspects of cell responses, are still to be focused. In this context, polychromatic flow cytometry (PFC) is a proper tool to investigate basophil function, as it allows to distinguish the expression of several membrane markers upon activation in multiple experimental conditions. METHODS: Cell suspensions were prepared from leukocyte buffy coat of K2-EDTA anticoagulated blood specimens; about 1500-2500 cellular events for each tested sample, gated in the lymphocyte CD45dim area and then electronically purified as HLADRnon expressing/CD123bright, were identified as basophilic cells. Basophil activation with fMLP, anti-IgE and calcium ionophore A23187 was evaluated by studying up-regulation of the indicated membrane markers with a two-laser six-color PFC protocol. RESULTS: Following stimulation, CD63, CD13, CD45 and the ectoenzyme CD203c up-regulated their membrane expression, while CD69 did not; CD63 expression occurred immediately (within 60 sec) but only in a minority of basophils, even at optimal agonist doses (in 33% and 14% of basophils, following fMLP and anti-IgE stimulation respectively). CD203c up-regulation occurred in the whole basophil population, even in CD63non expressing cells. Dose-dependence curves revealed CD203c as a more sensitive marker than CD63, in response to fMLP but not in response to anti-IgE and to calcium ionophore. CONCLUSION: Use of polychromatic flow cytometry allowed efficient basophil electronic purification and identification of different behaviors of the major activation markers. The simultaneous use of two markers of activation and careful choice of activator are essential steps for reliable assessment of human basophil functions.

Novel prion protein conformation and glycotype in Creutzfeldt-Jakob disease.

OBJECTIVE: To describe a novel molecular and pathological phenotype of Creutzfeldt-Jakob disease. Patient A 69-year-old woman with behavioral and personality changes followed by rapidly evolving dementia. RESULTS: Postmortem examination of the brain showed intracellular prion protein deposition and axonal swellings filled with amyloid fibrils. Biochemical analysis of the pathological prion protein disclosed a previously unrecognized PrP(Sc) tertiary structure lacking diglycosylated species. Genetic analysis revealed a wild-type prion protein gene. The prion agent responsible for this atypical phenotype was successfully passaged to bank voles. CONCLUSION: To our knowledge, our results define a new human prion disorder characterized by intracellular accumulation of a novel type of pathological prion protein.

Immune regulation by mesenchymal stem cells derived from adult spleen and thymus.

We show here that human and mouse mesenchymal stem cells (MSCs) can be obtained not only from bone marrow (BM), but also from adult spleen and thymus. In vitro, both human and mouse spleen- and thymus-derived MSCs exhibit immunophenotypic characteristics and differentiation potential completely comparable to BM-MSCs. In addition, they can inhibit immune responses mediated by activated T lymphocytes with efficiency comparable to BM-MSCs. In vivo, mouse MSCs from BM, spleen, and thymus, if injected together with a genetically modified tumor cell vaccine, can equally prevent the onset of an anti-tumor memory immune response, thus leading to tumor growth in normally resistant mice. Our data suggest that not only do spleen and thymus have a stem cell reservoir to build up their stromal architecture, but also contain microenviromental immunoregulatory cells with the same properties of BM-MSCs.

In celiac disease, a subset of autoantibodies against transglutaminase binds toll-like receptor 4 and induces activation of monocytes.

BACKGROUND: Celiac disease is a small intestine inflammatory disorder with multiple organ involvement, sustained by an inappropriate immune response to dietary gluten. Anti-transglutaminase antibodies are a typical serological marker in patients with active disease, and may disappear during a gluten-free diet treatment. Involvement of infectious agents and innate immunity has been suggested but never proven. Molecular mimicry is one of the mechanisms that links infection and autoimmunity. METHODS AND FINDINGS: In our attempt to clarify the pathogenesis of celiac disease, we screened a random peptide library with pooled sera of patients affected by active disease after a pre-screening with the sera of the same patients on a gluten-free diet. We identified a peptide recognized by serum immunoglobulins of patients with active disease, but not by those of patients on a gluten-free diet. This peptide shares homology with the rotavirus major neutralizing protein VP-7 and with the self-antigens tissue transglutaminase, human heat shock protein 60, desmoglein 1, and Toll-like receptor 4. We show that antibodies against the peptide affinity-purified from the sera of patients with active disease recognize the viral product and self-antigens in ELISA and Western blot. These antibodies were able to induce increased epithelial cell permeability evaluated by transepithelial flux of [(3)H] mannitol in the T84 human intestinal epithelial cell line. Finally, the purified antibodies induced monocyte activation upon binding Toll-like receptor 4, evaluated both by surface expression of activation markers and by production of pro-inflammatory cytokines. CONCLUSIONS: Our findings show that in active celiac disease, a subset of anti-transglutaminase IgA antibodies recognize the viral protein VP-7, suggesting a possible involvement of rotavirus infection in the pathogenesis of the disease, through a mechanism of molecular mimicry. Moreover, such antibodies recognize self-antigens and are functionally active, able to increase intestinal permeability and induce monocyte activation. We therefore provide evidence for the involvement of innate immunity in the pathogenesis of celiac disease through a previously unknown mechanism of engagement of Toll-like receptor 4.

Induction of human immunodeficiency virus neutralizing antibodies using fusion complexes.

Human immunodeficiency virus-1 (HIV-1) infects cells by membrane fusion that is mediated by the envelope proteins gp120/gp41 and the cellular receptors CD4 and CCR5. During this process, some conserved viral epitopes are temporarily exposed and may induce a neutralizing antibody response when fixed in the fusogenic conformation. These transient structures are conserved and may be effective antigens for use in an anti-HIV-1 vaccine. In this study we tested different conditions of preparation of fusion complexes inducing neutralizing antibodies against both R5 and X4 tropic HIV-1 strains. Cell lines expressing HIV-1 gp120/gp41 and CD4-CCR5 were prepared and conditions for producing fusion complexes were tested. Complexes produced at different temperature and fixative combinations were used to immunize mice. Results indicated that (a) fusion complexes prepared at either 21 degrees C, 30 degrees C or 37 degrees C were immunogenic and induced neutralizing antibodies against both R5 and X4 HIV-1 heterologous isolates; (b) after extensive purification of antibodies there was no cytotoxic effect; (c) complexes prepared at 37 degrees C were more immunogenic and induced higher titers of neutralizing antibodies than complexes prepared at either 21 degrees C or 30 degrees C; (d) the fixative used did not affect the titer of neutralizing antibodies except for glutaraldehyde which was ineffective; (e) the neutralizing activity was retained after CD4-CCR5 antibody removal. The production of higher titers of neutralizing antibody with fusion complexes prepared at 37 degrees C, as compared to lower temperatures, may be related to the induction of antibodies against many different conformation intermediates that subsequently act synergistically at different steps in the fusion process.

Analysis of mammalian scrapie protein by novel monoclonal antibodies recognizing distinct prion protein glycoforms: an immunoblot and immunohistochemical study at the light and electron microscopic levels.

The availability of specific monoclonal antibodies (mAbs) recognizing the aberrant form (PrP(Sc)) of the cellular prion protein (PrP(C)) in different mammalian species is important for molecular diagnostics, PrP(Sc) typing and future immunotherapy. We obtained a panel of anti-PrP monoclonal antibodies in PrP(0/0) knock-out mice immunized with recombinant human PrP(23-231). Two mAbs, recognizing PrP epitopes in the alpha-helix 1 (mAb SA65) and alpha-helix 2 (mAb SA21) regions, immunoreacted with PrP(C) and PrP(Sc) and its proteolytic product, PrP27-30, from human, murine, bovine, caprine and ovine brains by Western blot. Remarkably, mAb SA21 recognized unglycosylated and monoglycosylated PrP with the second site occupied by glycan moieties, but not monoglycosylated PrP with the first consensus site occupied or highly glycosylated species. Immunoblots with mAb SA21 disclosed that PrP glycosylated at the second site accounted for the slower migrating form of the customary monoglycosylated PrP doublet. mAb SA65 immunolabelled all PrP glycoforms by Western blot and was highly efficient in detecting tissue PrP by immunohistochemistry in light microscopy and in immunoelectron microscopy. These novel anti-PrP mAbs provide tools to investigate the subcellular site of PrP deposition in mammalian prion diseases and may also contribute to assess the role of different PrP glycoforms in human and animal prion diseases.

Myelin basic protein epitopes secreted by human T cells encounter natural autoantibodies in the serum.

A previously isolated and characterized IgM monoclonal antibody (mAb 1H6.2) specific to myelin basic protein (MBP) and to MBP epitopes expressed by nonneural cells was used to immunoprecipitate and investigate the expression of MBP epitopes by human T cells. Peripheral T lymphocytes secreted MBP epitopes, and secretion increased in time after mitogen stimulation. Conversely, thymocytes secreted these proteins independently on mitogen stimulation. Specific antibody reactivity (primarily due to IgG3) towards immunoprecipitated MBP epitopes was found in all tested sera from healthy donors and from multiple sclerosis patients as well as in sera from normal human cord blood. Collectively, these data provide insights into the immunological mechanisms leading to central and peripheral tolerance to MBP products.

Reductive activation of ricin and ricin A-chain immunotoxins by protein disulfide isomerase and thioredoxin reductase.

Intracellular activation of ricin and of the ricin A-chain (RTA) immunotoxins requires reduction of their intersubunit disulfide(s). This crucial event is likely to be catalyzed by disulfide oxidoreductases and precedes dislocation of the toxic subunit to the cytosol. We investigated the role of protein disulfide isomerase (EC 5.3.4.1, PDI), thioredoxin (Trx), and thioredoxin reductase (EC 1.8.1.9, TrxR) in the reduction of ricin and of a ricin A-chain immunotoxin by combining enzymatic assays, SDS-PAGE separation and immunoblotting. We found that, whereas PDI, Trx, and TrxR used separately were unable to directly reduce ricin and the immunotoxin, PDI and Trx in the presence of TrxR and NADPH could reduce both ricin and immunotoxin in vitro. PDI functioned only after pre-incubation with TrxR and the reductive activation of ricin was more efficient in the presence of glutathione. Similar results were obtained with microsomal membranes or crude cell extracts. Pre-incubation with the gold(I) compound auranofin, which irreversibly inactivates TrxR, resulted in a dose-dependent inhibition of ricin and immunotoxin reduction. Reductive activation of ricin and immunotoxin decreased or was abolished in microsomes depleted of TrxR and in cell extracts depleted of both PDI and Trx. Pre-incubation of U-937, Molt-3, Jurkat, and DU145 cells with auranofin significantly decreased ricin cytotoxicity with respect to mock-treated controls (P<0.05). Conversely, auranofin failed to protect cells from the toxicity of pre-reduced ricin which does not require intracellular reduction of disulfide between the two ricin subunits. We conclude that TrxR, by activating disulfide reductase activity of PDI, can ultimately lead to reduction/activation of ricin and immunotoxin in the cell.

Immunotoxins and other conjugates: preparation and general characteristics.

Targeted toxins represent an invaluable tool offering a wide range of potential applications, both in experimental models and in the clinics. Here we will review several aspects related to the preparation and properties of carrier molecule-toxin heteroconjugates and fusion toxins.

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.

A comparative method for processing immunological parameters: developing an "Immunogram".

BACKGROUND: The immune system is a network of numerous cells that communicate both directly and indirectly with each other. The system is very sensitive to antigenic stimuli, which are memorised, and is closely connected with the endocrine and nervous systems. Therefore, in order to study the immune system correctly, it must be considered in all its complexity by analysing its components with multiparametric tools that take its dynamic characteristic into account. METHODS: We analysed lymphocyte subpopulations by using monoclonal antibodies with six different fluorochromes; the monoclonal panel employed included CD45, CD3, CD4, CD8, CD16, CD56, CD57, CD19, CD23, CD27, CD5, and HLA-DR. This panel has enabled us to measure many lymphocyte subsets in different states and with different functions: helper, suppressor, activated, effector, naïve, memory, and regulatory. A database was created to collect the values of immunological parameters of approximately 8,000 subjects who have undergone testing since 2000. When the distributions of the values for these parameters were compared with the medians of reference values published in the literature, we found that most of the values from the subjects included in the database were close to the medians in the literature. To process the data we used a comparative method that calculates the percentile rank of the values of a subject by comparing them with the values for others subjects of the same age. RESULTS: From this data processing we obtained a set of percentile ranks that represent the positions of the various parameters with regard to the data for other age-matched subjects included in the database. These positions, relative to both the absolute values and percentages, are plotted in a graph. We have called the final plot, which can be likened to that subject's immunological fingerprint, an "Immunogram". In order to perform the necessary calculations automatically, we developed dedicated software (Immunogramma) which provides at least two different "pictures" for each subject: the first is based on a comparison of the individual's data with those from all age-related subjects, while the second provides a comparison with only age and disease-related subjects. In addition, we can superimpose two fingerprints from the same subject, calculated at different times, in order to produce a dynamic picture, for instance before and after treatment. Finally, with the aim of interpreting the clinical and diagnostic meaning of a set of positions for the values of the measured parameters, we can also search the database to determine whether it contains other subjects who have a similar pattern for some selected immune parameters. CONCLUSIONS: This method helps to study and follow-up immune parameters over time. The software enables automation of the process and data sharing with other departments and laboratories, so the database can grow rapidly, thus expanding its informational capacity.

The puzzling uniqueness of the heterotrimeric G15 protein and its potential beyond hematopoiesis.

Heterotrimeric G proteins transduce the signals of the largest family of membrane receptors (G protein-coupled receptors, GPCRs) hence triggering the activation of a wide variety of physiological responses. G15 is a G protein characterized by a number of functional peculiarities that make its signaling exceptional: 1) it can couple a variety of Gs-, Gi/o-, and Gq-linked receptors to phospholipase C activation; 2) relatively to other G proteins, it is poorly affected by beta-arrestin-dependent desensitization, the general mechanism that regulates GPCR function and 3) at the protein level, its expression is only detected in highly specific cell types (hematopoietic and epithelial cells). G15 alpha-subunit displays unique structural and biochemical properties, and is phylogenetically the most recent and divergent component of the Galphaq/11 subfamily. All these aspects shed a mysterious light on G15 biological role, which remains substantially elusive. Thus, far, G15 signaling has been analyzed in the context of hematopoiesis. Here, we highlight observations supporting the view that G15 functions may extend further beyond the immune system. In addition, we describe puzzling aspects of G15 signaling that offer a novel perspective in the understanding of its physiological role.

IFN-gamma-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.

Expression of CD27 and CD23 on peripheral blood B lymphocytes in humans of different ages.

BACKGROUND: Due to the fact that the coexpression of CD23 and CD27 has been reported to occur in B lymphocytic leukaemic clones and that there is debate about CD23 expression on memory B cells, we evaluated the behaviour of naive B cells (CD23-/CD27-) and memory B cells (CD27+) in the peripheral blood of a large number of humans of all ages. B cells were also distinguished into B2 (CD5-) and B1-a cells (CD5+). METHODS: The cell surface expression of CD19, CD5, CD23 and CD27 was assessed on peripheral blood lymphocytes from 1,427 subjects of all ages undergoing peripheral blood immunophenotyping for a variety of reasons. RESULTS: The absolute number of B lymphocytes and the percentage of naive cells (CD23-/CD27-) decreased with age whereas there was an increase in memory cells (CD27+). A small subset of B cells co-expressing CD23 and CD27 was present in humans of all ages, although the majority of CD27+ cells were CD23-. The percentages and rate of increase with age of B1-a CD23+/CD27+ were slightly higher than those of B2 cell counterparts. CONCLUSIONS: On the basis of our data, age-associated changes in surface markers of B cells seem to be finely balanced and probably related to functional changes after antigen encounters, while the whole peripheral blood B-cell compartment undergoes a quantitative regression.

Vaccine adverse event monitoring systems across the European Union countries: time for unifying efforts.

A survey conducted among 26 European Countries within the Vaccine European New Integrated Collaboration Effort (VENICE) project assessed the status of organization in prevention and management of adverse events following immunization (AEFI) and level of interconnection, with the aim at individuating points of strength and weakness. The emerging picture is for a strong political commitment to control AEFIs in Member States (MS), but with consistent heterogeneity in procedures, regulations and capacity of systems to collect, analyze and use data, although with great potentialities. Suggestions are posed by authors to promote actions for unifying strategies and policies among MS.

Heterotrimeric G proteins demonstrate differential sensitivity to beta-arrestin dependent desensitization.

G15 is a heterotrimeric G protein of the Gq/11 family. In this study, we describe its exceptional poor sensitivity to the general regulatory mechanism of G protein-coupled receptor (GPCR) desensitization. Enhancing beta2 adrenergic receptor desensitization by arrestin overexpression, did not affect signalling to G15. Similarly, increased levels of arrestin did not affect G15 signalling triggered by the activation of V2 vasopressin and delta opioid receptors. Furthermore, co-immunoprecipitation experiments showed that G15 alpha subunit (as opposed to Galphaq and Galphas) is recruited to a V2 vasopressin receptor mutant that is constitutively desensitized by beta-arrestin. Interestingly, co-expression of Galpha15 partially rescued cell surface localization and signalling capabilities of the same mutant receptor and reduced beta2 adrenergic receptor internalization. Taken together, these findings provide evidence for a novel mechanism whereby GPCR desensitization can be bypassed and G15 can support sustained signalling in cells chronically exposed to hormones or neurotransmitters.

The prostate specific membrane antigen regulates the expression of IL-6 and CCL5 in prostate tumour cells by activating the MAPK pathways.

The interleukin-6 (IL-6) and the chemokine CCL5 are implicated in the development and progression of several forms of tumours including that of the prostate. The expression of the prostate specific membrane antigen (PSMA) is augmented in high-grade and metastatic tumors. Observations of the clinical behaviour of prostate tumors suggest that the increased secretion of IL-6 and CCL5 and the higher expression of PSMA may be correlated. We hypothesized that PSMA could be endowed with signalling properties and that its stimulation might impact on the regulation of the gene expression of IL-6 and CCL5. We herein demonstrate that the cross-linking of cell surface PSMA with specific antibodies activates the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 in prostate carcinoma LNCaP cells. As downstream effects of the PSMA-fostered RAS-RAC1-MAPK pathway activation we observed a strong induction of NF-kappaB activation associated with an increased expression of IL-6 and CCL5 genes. Pharmacological blockade with specific inhibitors revealed that both p38 and ERK1/2 participate in the phenomenon, although a major role exerted by p38 was evident. Finally we demonstrate that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically and in a dose-dependent manner and that CCL5 functioned by receptor-mediated activation of the STAT5-Cyclin D1 pro-proliferative pathway. The novel functions attributable to PSMA which are described in the present report may have profound influence on the survival and proliferation of prostate tumor cells, accounting for the observation that PSMA overexpression in prostate cancer patients is related to a worse prognosis.

Changes of human B and B-1a peripheral blood lymphocytes with age.

In 2057 consecutive subjects admitted to the Department of Pathology, Section of Immunology of the Verona University Hospital, CD19+ and CD5/CD19 double positive cells were determined to assess the behaviour of total peripheral B-lymphocytes and B-1a (CD5+) compartments in humans during aging. We show that the absolute number of total B lymphocytes increases about three-fold from the baseline conditions in the first year of life and progressively decreases until adult age. A slower decrease was detected from the adult age onwards. A similar behaviour has been observed within the B-1a subset of B-lymphocytes, although the decrease after the adult age seems more pronounced. Possible physiological explanations and/or implications for the disease states are taken into account.