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Objective: The present study aimed to assess seroprevalence of and risk factors for Toscana (TOSV) and Sicilian (SFSV) virus infections in a sample of Sicilian subjects., Methods: A cross-sectional seroepidemiological study was conducted on 271 individuals. Each participant completed a self-administrated questionnaire and provided a serum sample which was analyzed for the presence of IgG specific anti-TOSV and anti-SFSV viruses., Results: Overall, 90 subjects (33.2%) were positive for TOSV IgG, 25 (9.2%) were positive for SFSV IgG and 11 (4%) were positive for both the viruses. A higher risk for TOSV seropositivity was found in participants who were older (adjOR = 1.02 per year; 95% CI = 1.01-1.03), having a pet living outdoors (adjOR = 2.62; 95% CI = 1.42-4.83) and being obese (adjOR = 2.37; 95% CI = 1.06-5.30)., Conclusions: TOSV seroprevalence appears to be relatively high in Sicilian general population, especially in older adults, representing a potential public health concern. The observations that seropositivity for TOSV was not significantly associated with SFSV seropositivity, and none of the risk factors associated with TOSV were associated with SFSV seem to suggest that these two phleboviruses may have different ecology and transmission pathways., (Copyright © 2011 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Burkitt lymphoma is endemic in the Equatorial Belt of Africa, its molecular hallmark is an activated, MYC gene mostly due to a chromosomal translocation. Especially in its endemic clinical variant, Burkitt lymphoma is associated with the oncogenic Epstein-Barr virus (EBV), and holoendemic malaria acts as an amplifier. Environmental factors may also cooperate in Burkitt lymphomagenesis in the endemic regions, such as plants used as traditional herbal remedies. Euphorbia tirucalli, a plant known to possess EBV-activating substances, has a similar geographical distribution to endemic Burkitt's Lymphoma and is used as a hedge, herbal remedy and toy in the Lymphoma BeltI. In this study we aimed at determining if exposure to Euphorbia tirucalli could contribute to lymphomagenesis, and at which extent. Lymphoblastoid and cord blood-derived cell lines were treated with plant extracts, and the expression of EBV-coded proteins was checked, to assess EBV reactivation. The occurrence of chromosomal translocations was then investigated by FISH. Our preliminary results suggest that E. tirucalli is able to reactivate EBV and determine chromosomal alterations, which leads to c-MYC altered expression. The existence of genomic alterations might determine the accumulation of further genetic alteration, which could eventually lead to a transformed phenotype.

To assess whether arthropod bites promote Kaposi sarcoma (KS), we determined the seroprevalence of Sicilian (SFSV) and Toscana (TOSV) phlebovirus antibodies in 30 patients with classic KS and 100 controls in Sicily. Nine (6.9%) subjects, all controls, were positive for SFSV, whereas 41 (31.5%) were positive for TOSV. Seroprevalence with immunoglobulin (Ig) M or IgG against either virus was significantly higher in controls (43% vs 13.3% in case patients; P < .01). Adjusted for age, IgG seroprevalence was significantly lower in KS patients compared to controls (adjusted odds ratio, 0.22; 95% confidence interval, .07-.72). Low phlebovirus seroprevalence in patients with KS may reflect incapacity to produce robust, persistent antibody responses, and suggests that arthropod bites do not promote KS.

We report a case of West Nile virus (WNV) infection in a symptomatic woman living in Tuscany in 2007. A retrospective analysis on cerebrospinal fluids drawn from people affected by neurological diseases with unknown etiology allowed the identification of a case of WNV infection before the WNV outbreak in the Northeast Italy in 2008. This emphasizes the importance of maintaining a high level of epidemiological surveillance all over the Italian territory.

Toscana virus (TOSV; Phlebovirus, Bunyaviridae) is an important etiological agent of acute meningitis and meningoencephalitis in Mediterranean countries. Laboratory diagnosis has been carried out in serological studies using ELISA, immunofluorescence and/or neutralization tests that are not influenced by the virus viability; however, in the acute phase of the infection, nucleic acid amplification techniques are the methods of choice to diagnose viral meningitis from cerebrospinal fluid samples. Molecular methods are rapid and sensitive and, unlike traditional methods, such as virus isolation by cell culture, they are not influenced by the viability of the virus in the clinical specimen; however, the RNA integrity is crucial for the success of these methods. Real-time PCR is the most important molecular method used in laboratories worldwide, since it is less time-consuming and it reduces the risk of contamination. Therefore, a sensitive real-time PCR has been developed for diagnosis of suspected cases of TOSV infection either autochthonous and/or imported, since a new lineage of TOSV, divergent from the Italian prototype, has recently been reported in Spain.

Toscana virus (TOSV) infection is a frequent cause of meningitis in central Italy during summer. The disease generally has a benign course. Rarely, the infection produces a severe disease, with encephalitis and signs of systemic involvement, including lymphadenopathy. Since there is no clinical necessity of performing lymph node biopsy in such cases, the histopathological feature of TOSV-related lymphadenitis is not known. We herein present a case in which lymphadenopathy preceded the onset of meningitis. The excised lymph node showed a non-specific mixed-type lymphoid hyperplasia, with follicular hyperplasia, sinusal expansion and paracortical involvement. We also demonstrated the presence of viral protein and viral RNA in the lymph node tissue., (Copyright © 2010. Published by Elsevier GmbH.)

Toscana virus (TOSV) is a phlebotomus-transmitted virus that belongs to the family Bunyaviridae and causes widespread infections in humans; about 30 % of these cases result in aseptic meningitis. In the present study, it was shown that TOSV is an inducer of beta interferon (IFN-β), although its non-structural protein (NSs) could inhibit the induction of IFN-β if expressed in a heterologous context. A recombinant Rift Valley fever virus expressing the TOSV NSs could suppress IFN-β expression in infected cells. Moreover, in cells expressing NSs protein from a cDNA plasmid, IFN-β transcripts were not inducible by poly(I : C). Unlike other members of the family Bunyaviridae, TOSV appears to express an NSs protein that is a weak antagonist of IFN induction. Characterization of the interaction of TOSV with the IFN system will help our understanding of virus-host cell interactions and may explain why the pathogenesis of this disease is mostly mild in humans.

Respiratory syncytial virus (RSV) is the major pathogen causing respiratory disease in young infants and it is an important cause of serious illness in the elderly since the infection provides limited immune protection against reinfection. In order to explain this phenomenon, we investigated whether healthy adults of different age (20-40; 41-60 and > 60 years), have differences in central and effector memory, RSV-specific CD8+ T cell memory immune response and regulatory T cell expression status. In the peripheral blood of these donors, we were unable to detect any age related difference in term of central (CD45RA-CCR7+) and effector (CD45RA-CCR7-) memory T cell frequency. On the contrary, we found a significant increase in immunosuppressive regulatory (CD4+25+FoxP3+) T cells (Treg) in the elderly. An immunocytofluorimetric RSV pentamer analysis performed on these donors' peripheral blood mononuclear cells (PBMCs), in vitro sensitized against RSV antigen, revealed a marked decline in long-lasting RSV specific CD8+ memory T cell precursors expressing interleukin 7 receptor α (IL-7Rα), in the elderly. This effect was paralleled by a progressive switch from a Th1 (IFN-γ and TNF-α) to a Th2 (IL-10) functional phenotype. On the contrary, an increase in Treg was observed with aging. The finding of Treg over-expression status, a prominent Th2 response and an inefficient RSV-specific effector memory CD8+ T cell expansion in older donors could explain the poor protection against RSV reinfection and the increased risk to develop an RSV-related severe illness in this population. Our finding also lays the basis for new therapeutic perspectives that could limit or prevent severe RSV infection in elderly.

Human respiratory syncytial virus (hRSV) is the main viral cause of severe respiratory infections in children and a common cause of morbidity in the elderly. The nucleocapsid (N) and fusion (F) proteins of hRSV were expressed in insect cells and used as antigens in two independent enzyme-linked immunosorbent assays (ELISAs) to measure the serum antibody response in two populations at high risk of hRSV infection, children and the elderly. Fifty-seven serum specimens from children aged from 1 to 10 years old and 91 sera from adults over 60 years old were tested. The ELISA results were compared with those obtained by an immunofluorescence assay (IFA) based on hRSV-infected cells, which was considered as the reference technique. Sensitivity and specificity were 94% and 85% for the N-ELISA and 86% and 81% for the F-ELISA, respectively. When the immune responses of the two groups of individuals were compared, it appeared that almost 100% of the elderly had antibodies against the N or F protein whereas only 50% of the sera from children had antibodies against either of the two viral proteins. In conclusion, the F and N ELISAs can be used successfully for detecting a specific antibody response to hRSV., (Copyright 2010 Elsevier B.V. All rights reserved.)

Toscana virus (TOSV) is an arthropod-borne virus which is transmitted to humans by Phlebotomus spp sandflies. Infection is the cause of brain injuries, such as aseptic meningitis and meningoencephalitis, in Italy mainly during the summer. More recently some unusual clinical manifestations due to TOSV with severe sequelae, such as ischemic complications and hydrocephalus, have been reported. TOSV represents an important emerging pathogen and its presence is being investigated in several European countries on the Mediterranean basin, including Italy, France, Spain, Portugal and Cyprus. Phylogenetic analysis has distinguished two genotypes of TOSV, A and B; the first is circulating mainly in Italy and the second in Spain, indicating a different geographic distribution possibly related to the vector. This distribution, evolving with the climate, globalization and habitat modification, has implications for the epidemiology of TOSV.

Respiratory syncytial virus (RSV) has been demonstrated to cause substantial disease in elderly and immunocompromised subjects. The relationship of serum antibody to RSV infection and the risk of infection in elderly subjects is controversial, thus we evaluated the presence of neutralizing antibodies to RSV in healthy people of different age groups and the correlation with viral protection. Baseline blood samples from 197 subjects aged 20-80 years were analysed for the presence of anti-RSV antibodies either by indirect immunofluorescence and microneutralization test. The percentage of people who had neutralizing antibodies to RSV was significantly higher (P=0.001) in the youngest group (92.51%) compared to the frail group (36.21%). The RSV antibody level tends to wane in some older people; this factor could determine proneness to RSV re-infections in the elderly who are at a greater risk of developing severe respiratory disease.

In order to estimate the antibody prevalence rates for Toscana virus (TOSV) among children and adults, we evaluated the seroprevalence of TOSV in a population (n = 2,737) living in Tuscany during the period of 1999 to 2006. The seroprevalence rate was 19.8% in adults and 5.8% in children, showing an age-dependent increase in TOSV-specific immunity. Meningitis due to TOSV infection was more frequent in adults than in children.

Toscana virus (TosV), a sandfly fever virus, is one of the main causes of the aseptic meningitis that occurs during the summer in some Mediterranean regions, and whose epidemiology is largely unknown. We used a Bayesian Markov Chain Monte Carlo approach and a relaxed molecular clock to estimate the demographic history of the TosV infection in a series of isolates sampled between 1980 and 2003. The estimated mean evolutionary rate was 2.5 x 10(-4) substitutions per site per year (95% HPD: 0.31-5.44 x 10(-4)subs/site/year). Bayesian skyline plot revealed a sharp decline in the effective number of infections over the last 30 years. In conclusion, our data suggest that continuous and prolonged perturbations of vector/phlebovirus interactions due to the relatively recent climate changes may have contributed to gradually reducing the viral population in endemic areas.

Impact of recently discovered viruses on epidemiology of acute respiratory tract infections (ARTI) is still unclear. We studied the impact of recently discovered human metapneumovirus (hMPV), human bocavirus (HBoV), and new coronaviruses (HCoV-NL63 and HKU1) on the global epidemiology of ARTI. From October 2006 to April 2007, 237 pediatric patients affected by ARTI were enrolled in our study. Specimens were tested for respiratory viruses by polymerase chain reaction. One hundred twenty-four out of 237 samples (52.3%) were positive for one or more viruses. Picornaviruses were the most prevalent viruses (n = 61, 43.6%), followed by respiratory syncytial virus (n = 34, 24.3%) and Adenovirus (n = 25, 17.9%); hMPV (n = 9, 6.4%) was the fourth most common virus detected. HBoV and HCoV showed a low prevalence (respectively 2.9% and 2.1%). RSV was the prevalent agent of LRTI (38%). Viruses were identified in more than 50% of the studied ARTI, providing useful information on clinical features and epidemiology of specific agents affecting children in cold months. Although routine surveillance of respiratory viruses does not seem cost-effective, continuous monitoring of ARTI etiology could be a useful tool for planning resources for the development of new vaccines and antiviral agents., ((c) 2009 Wiley-Liss, Inc.)

Purpose: GOLFIG chemoimmunotherapy regimen proved to be a safe and very active chemoimmunotherapy regimen in advanced colon cancer patients. We have thus investigated the immunobiological feedback to the treatment and its possible correlation with the clinical outcome of these patients., Experimental Design: This clinical and immunologic study involved 46 patients, 27 males and 19 females, enrolled in the GOLFIG-1 phase II trial who received gemcitabine (1,000 mg/m(2) on days 1 and 15), oxaliplatin (85 mg/m(2) on days 2 and 16), levofolinic acid (100 mg/m(2) on days 1, 2, 15, and 16), and 5-fluorouracil (400 mg/m(2) as a bolus, and 800 mg/m(2) as a 24-hour infusion on days 1, 2, 15, and 16) followed by s.c. granulocyte macrophage colony-stimulating factor (100 mug, on days 3-7) and interleukin 2 (0.5 x 10(6) IU twice a day on days 8-14 and 17-29)., Results: The regimen was confirmed to be safe and very active in pretreated patients with metastatic colorectal cancer. A subgroup analysis of these patients revealed a prolonged time to progression and survival in six patients who developed late signs of autoimmunity. A multivariate analysis validated the occurrence of autoimmunity signs as an independent predictor of favorable outcome. A parallel immunologic study detected in the peripheral blood mononuclear cells of these patients a progressive increase in lymphocyte and eosinophil counts, amplification in central memory, a marked depletion of immunosuppressive regulatory T cells, and activation of colon cancer-specific cytotoxic T cells., Conclusions: Our results suggest that immunity feedback to GOLFIG regimen and its antitumor activity are tightly correlated.

Toscana virus (TOSV), a member of the Bunyaviridae family, is an important etiologic agent of neurologic infection transmissible to humans by bites of the Phlebotomus spp. In consideration of the variations in the antigenic properties of Bunyaviruses and their potential genetic variability, we analysed a large region (2500nt) of the Toscana virus M segment coding for the non-structural protein (NSm) and the G(N) and G(C) glycoproteins in several strains isolated from patients with meningitis from 1998 to 2004 in the region of Tuscany in Italy. The sequences were compared with the reference strain of Toscana virus isolated from phlebotomus (ISS Phl. 3) and revealed some changes in amino acids, particularly in the G(C) protein, that are probably involved in recognition and binding to the cell receptor. The analyses were aimed at identifying the amino acid changes commonly to all of the clinical isolates potentially related to TOSV virulence.

Toscana virus (TOSV) is an emerging virus, circulating in the Mediterranean area, that is responsible for aseptic meningitis, meningoencephalitis, and encephalitis. The development of a vaccine that could provide complete protection from TOSV infection is needed. In this study we investigated the capacity of TOSV structural proteins, nucleocapsid protein N and the two Gc and Gn glycoproteins, produced as recombinant proteins, in an animal model. In particular, we investigated their role in inducing specific and protective immune responses against virus infection. Mice were immunized intraperitoneally using TOSV antigens singly or in combination. The results show that only the N-Gc combination was able to protect 100% of animals from a lethal challenge with a neurovirulent strain of TOSV. This potential vaccine induces high serum antibody titres with neutralizing activity and it is safe for animals. Moreover, immunization induces a virus specific cell-mediated immune response, in particular a CD8+ T cell response associated with a marked expression of interferon gamma. These results indicate that the N+Gc viral antigen combination could be useful for future development of a vaccine controlling the spread of this emerging virus that could pose a new threat for humans.

Background: C-IRIV/PTR-4 is a novel anticancer vaccine construct composed of immune-reconstituted influenza virosomes (IRIV) assembled with the PTH-rP derived peptide (PTR)-4, a synthetic CTL epitope with HLA-A(*)02.01 amino acid binding motifs. This peptide is able to generate a human PTH-rP specific CTL response with anti-tumor activity in vitro and in mice., Materials and Methods: We have investigated the immunological and preventive anti-tumor activity of C-IRIV/PTR-4 compared with the soluble PTR-4 peptide, in HHD mice inoculated with autologous PTH-rP+ tumor cells., Results: Peptide vaccination with either a soluble and an IRIV formulation showed similar immunological activity and the ability to purge the tumor tissue of tumor cell clones able to produce the target antigen (PTR-rP). The most efficient protection from tumor growth was however observed in animals vaccinated with C-IRIV/PTR-4 in which an additional IRIV related anti-angiogenetic effect was detected in the tumor tissue., Conclusions: These results confirm the immunological activity of PTR-4 vaccination and suggest a more efficacious therapeutic potential of C-IRIV/PTR-4 against bone metastases and malignancies like breast, prostate and lung which very often over-express PTH-rP.

The effects of anticancer chemotherapy on antigen-specific cytotoxic T lymphocytes (CTLs) are mostly unknown. We tested the effects of cytotoxic drugs such as 5-fluorouracil, gemcitabine, and oxaliplatin on the functional activity of antigen-specific CTL cultures derived from the peripheral blood mononuclear cells of human donors. We found that a biweekly drug-exposure of human HLA-A(*)02.01+ CTLs derived from bulk cultures led to completely different effects if occurring early (day second) or late (day thirteenth) after the in vitro stimulations with the cognate peptides. In the first case, there was a significant CTL inhibition, whereas in the second, there was a marked enhancement of the antigen-specific cytolytic activity. Results of immunocytofluorimetric studies and CTL/natural killer inhibition assays suggested that the latter effect could be related to a more selective drug-mediated inhibition of cohabitant T regulatory (reg) cells. These results were translated in an in vivo therapeutic mouse model where humanized HLA-A(*)02.01 transgenic mice inoculated with EL-4/humanized HLA-A(*)02.01 transgenic mice showed a prolonged survival and the greatest rate of cure when receiving a combined treatment with a thymidylate synthase-specific peptide vaccine and a multidrug chemotherapy regimen administered late after immunization. Tumor samples derived from this group of mice showed a reduced expression of the target thymidylate synthase antigen, a marked reduction of T(reg)s, and a noteworthy infiltration of C8+ T cells. These results may have clinical implications for the design of new translational anticancer regimens aimed at combining chemotherapy and immunotherapy.

Virus-specific cytotoxic T lymphocytes (CTLs) are crucial for the control of respiratory syncytial virus (RSV) infection. This study has identified CTL epitopes of the RSV N protein in healthy subjects. We screened the primary structure of the N protein for HLA-A 0201-binding amino acid consensus motifs, identifying three peptides designated as N-RSV1, N-RSV2, and N-RSV3. These peptides were used to generate CTL lines by stimulating human HLA-A 02.01 peripheral blood mononuclear cells (PBMCs) in vitro. These CTL lines were then characterized by performing CTL chromium release assays and IFN-gamma secretion detection by intracellular cytokine staining. N-RSV1 and N-RSV3 peptides elicited the strongest cytolytic activity against RSV-infected cells and they could be useful epitopes for the analysis of CTL responses to RSV and for understanding immune-induced disease pathogenesis.

Background: Human bocavirus (HBoV) is a newly discovered human parvovirus. HBoV was detected in respiratory samples by PCR, but its aetiologic role in the pathogenesis of acute respiratory infectious diseases is still unclear., Results: In this report, we describe an atopic child affected by pneumonia, with a past history of wheezing. A panel of bacteria and respiratory viruses were searched in the nasopharyngeal swab, only human bocavirus was detected by PCR., Conclusions: Detection of HboV, as the only microbial agent, in samples from children with wheezing and acute respiratory diseases supports the assumption that this emerging virus could have an aetiologic role in the pathogenesis of respiratory diseases.

A cDNA vaccine (pVax1/pet-neu) was designed to encode 12 different Her-2/ErbB-2-derived, HLA-A*0201-restricted dominant and high-affinity heteroclitic cryptic epitopes. Vaccination with pVax1/pet-neu triggered multiple and ErbB-2-specific CTL responses in HLA-A*0201 transgenic HHD mice and in HLA-A*0201 healthy donors in vitro. Human and murine CTL specific for each one of the 12 ErbB-2 peptides recognized in vitro both human and murine tumor cells overexpressing endogenous ErbB-2. Furthermore, vaccination of HHD mice with pVax1/pet-neu significantly delayed the in vivo growth of challenged ErbB-2-expressing tumor (EL4/HHD/neu murine thymoma) more actively when compared with vaccination with the empty vector (pVax1) or vehicle alone. These data indicate that the pVax1/pet-neu cDNA vaccine coding for a poly-ErbB-2 epitope is able to generate simultaneous ErbB-2-specific antitumor responses against dominant and cryptic multiple epitopes.

Advanced colorectal cancer is a common disease with an high mortality rate. For four decades, pharmacological treatment of the advanced disease was based on the use of 5-fluorouracil alone or in combination with biomodulators such as folinic acid and intereferon alpha. In the last 5 years, response to therapy has been considerably ameliorated thanks to the discovery of new drugs such as oxaliplatin and CPT-11. These agents, in combination with 5-fluorouracil, according to various schedules of treatment, have reached a significant improvement of palliation, response rate and survival. Immunotherapy is an uprising modality of treatment for human cancer including colorectal carcinoma. Its rationale is based on the knowledge that tumour cells are genetically unstable and produce molecular structures which allow their recognition and destruction by the immune-surveillance system. Therefore, humoral as well as cellular compartments of the immune system can be utilized according to a "passive" strategy (e.g. monoclonal antibody administration and adoptive immunotherapy) or an "active" approach, by using different modalities of vaccine therapy. In this context, monoclonal antibodies (mAbs) and cancer vaccines are being tested for the treatment of advanced colorectal cancer. Due to their genetic instability and extraordinary adaptative potential, tumour cells may acquire resistance to the immune effectors and mAbs exactly as they do for cytotoxic drugs. To improve the results of both immunological and chemical modality of cancer treatment, an increasing number of authors is starting to combine chemo and immunotherapy in the attempt to circumvent the limitations of both strategies. This report tries to review the possible rationale of the chemo-immunotherapy combination, illustrating preliminary results of preclinical and clinical studies.

The induction of effective cellular and humoral immune responses against antigens is the major goal in vaccination strategies against infectious diseases and cancer. In this review, we will discuss influenza virosomes as a versatile delivery system for molecules of a different nature, such as proteins, peptides and nucleic acids. These molecules can be administered in different ways, such as nasal, muscular or dermal routes, also facilitating the uptake of the antigen by antigen-presenting cells (APC) and inducing a specific humoral and cell-mediated immune response, particularly a CTL response. Influenza virosomes, due to their applicability in different medical fields, are a promising tool in vaccinology and immunotherapy applications.

Purpose: Tumor cell killing by anticancer drugs may be supported by their immuno- and pharmacologic effects. Chemotherapy is in fact able to (A) upregulate tumor-associated antigen expression, including carcinoembryonic antigen (CEA) or other target molecules such as thymidylate synthase (TS); and (B) downregulate tumor cell resistance to the death signals induced by tumor antigen-specific cytotoxic T lymphocytes. This provides the rationale for combining chemo- and immunotherapy., Materials and Methods: We describe the results of a translational phase II trial designed to evaluate the toxicity, antitumor activity and immunologic effects of gemcitabine + FOLFOX-4 (oxaliplatin, fluorouracil, and folinic acid) polychemotherapy followed by the subcutaneous administration of granulocyte macrophage colony-stimulating factor and low-dose interleukin-2 in colorectal carcinoma patients. The study involved 29 patients (16 males and 13 females with a mean age of 69 years), 21 of whom had received a previous line of treatment, and 19 had liver involvement., Results: The treatment was well tolerated and induced very high objective response (68.9%) and disease control rates (96.5%), with an average time to progression of 12.5 months. An immunologic study of peripheral blood mononuclear cells (PBMCs) taken from 20 patients showed an enhanced proliferative response to colon carcinoma antigen and a significant reduction in suppressive regulatory T lymphocytes (CD4+CD25T-reg+). A cytofluorimetric study of the PBMCs of five HLA-A(*)02.01+ patients who achieved an objective response showed an increased frequency of cytolytic T lymphocyte precursors specific for known CEA- and TS-derived epitopes., Conclusion: The results show that our regimen has strong immunologic and antitumor activity in colorectal cancer patients and deserves to be investigated in phase III trials.

Gemcitabine, oxaliplatin, leucovorin, and 5-fluorouracil (GOLF) is a novel multidrug regimen inducing high levels of necrosis and apoptosis in colon carcinoma cells. This regimen is also able to promote a process of Ag remodeling including up-regulation of immunotherapy targets like carcinoembryonic Ag (CEA), thymidylate synthase (TS). We have conducted a preclinical study aimed to investigate whether these drug-induced modifications would also enhance colon cancer cell immunogenicity. Several CTL lines were thus generated by in vitro stimulating human HLA-A(*)02.01(+) PBMCs, from normal donors and colon cancer patients, with autologous dendritic cells cross-primed with cell lysates of colon cancer cells untreated, irradiated, or previously exposed to different drug treatments including the GOLF regimen. Class I HLA-restricted cytolytic activity of these CTL lines was tested against colon cancer cells and CEA and TS gene transfected target cells. These experiments revealed that CTLs sensitized with GOLF-treated cancer cells were much more effective than those sensitized with the untreated colon carcinoma cells or those exposed to the other treatments. CTL lines sensitized against the GOLF-treated colon cancer cells, also expressed a greater percentage of T-lymphocyte precursors able to recognize TS- and CEA-derived peptides. These results suggest that GOLF regimen is a powerful antitumor and immunomodulating regimen that can make the tumor cells a suitable means to induce an Ag-specific CTL response. These results suggest that a rationale combination of GOLF chemotherapy with cytokine-based immunotherapy could generate a chemotherapy-modulated Ag-specific T-lymphocyte response in cancer patients able to destroy the residual disease survived to the cytotoxic drugs.

The correct interaction of a costimulatory molecule such as CD40L with its contrareceptor CD40 expressed on the membrane of professional APCs, provides transmembrane signaling that leads to APC activation. This process can be exploited to significantly improve the efficacy of cancer vaccines and the outcome of a possible cancer vaccine-induced, Ag-specific CTL response. Therefore, we investigated whether a novel intranasal delivery of immune-reconstituted influenza virosomes (IRIV), assembled with the CD40L gene (CD40L/IRIV), could be used to improve protective immunity and the Ag-specific CTL response against carcinoembryonic Ag (CEA) generated with a novel vaccine constituted of IRIV assembled with the CEA gene (CEA/IRIV). Our results suggest that CD40L/IRIV was able to augment CEA-specific CTL activity and CEA-specific protective immunity induced by CEA/IRIV most likely through the induction of a CTL response associated with a Th1 phenotype. In conclusion, we provide evidence that CD40L/IRIV, by acting through the CD40L/CD40 signaling pathway, acts as an immune-adjuvant that could increase the efficacy of a CEA-specific cancer vaccine, which could provide an efficacious new strategy for cancer therapy.

Toscana virus (TOSV) has recently been recognized as an emerging virus transmitted by phlebotomus vectors, responsible for acute neurological diseases in Mediterranean countries. In our study, we demonstrated that adult Balb/c mice were susceptible to TOSV when infected intracerebrally (i.c.) or subcutaneously (s.c.) with a neuroadapted strain of the virus. We have shown that by performing serial passages of a wild type human isolate of TOSV in mouse brains, selection occurs for a highly virulent variant which replicates efficiently in the central nervous system (CNS) of i.c.-injected mice, causing acute encephalitis and death. Immunohistochemical analysis and TUNEL assay of post-mortem organs showed that TOSV replication was highly restricted to neurons in which it induced apoptotic death; however, virus antigen-positivity was also observed in the spleen and lymph nodes. In s.c.-injected mice, virus was detectable in the spleen and lymph nodes, whereas only few meningeal cells and neurons were affected, allowing for the mouse survival the infection. The presence of TOSV in spleen and lymph node cells in both s.c.- and i.c.-treated mice suggests their possible involvement in the diffusion of the infection. This animal model may be helpful for the development of prophylactic measures against TOSV infections.

Immune-based approaches of cell therapy against viral pathogens such as the human immunodeficiency virus type 1 (HIV-1) could be of primary importance for the control of this viral infection. Here, we designed a chimeric cell surface receptor (105TCR) to provide primary human T-lymphocytes with antibody-type specificity for the HIV-1 envelope glycoprotein. This receptor includes the single chain Fv domain of the neutralizing anti-gp120 human monoclonal antibody F105, CD8alpha hinge and the transmembrane and the cytoplasmic domains of TCRzeta. Our results show that 105TCR is expressed at the cellular surface and is capable of recognizing the HIV-1 envelope glycoprotein inducing highly efficient effector T-cell responses, including extracellular signal-regulated kinase phosphorylation and cytokine secretion. Moreover, human primary CD8+ T-lymphocytes transduced by oncoretroviral and lentiviral vectors containing the 105TCR gene are able to mediate in vitro-specific cytolysis of envelope-expressing cells and HIV-1-infected CD4+ T-lymphocytes. These findings suggest that 105TCR is particularly suited for in vivo efficacy studies.

In an attempt to enhance the immunological efficacy of DNA-based vaccines, we have investigated a new biological means for delivering target gene DNA directly to professional antigen presenting cells (APC), such as the dendritic cells (DC), which are ultimately responsible for the antigen presentation and the primary activation of the immune system. For this purpose we investigated influenza virosomes (IRIV) with assembled DNA as a possible biological carrier for targeting the APC in vivo and in vitro. By cytofluorimetric analysis of the draining lymph nodes of Balb/c mice which had received (by intranasal (in.) administration) FITC-labeled DNA assembled with IRIV, we detected a significant labeled DNA uptake in a subset of lymph node deriving cells expressing DC surface markers. Subsequent mRNA analysis of these lymph nodes showed that the trans-gene delivered by the virosomes was effectively expressed as mRNA. Finally, a further cytofluorimetric analysis performed on human DC-enriched-PMBC, infected in vitro with labeled DNA/IRIV lead to the conclusion that the majority of APC (DC, B lymphocytes and CD16+ cells) are able to incorporate the labeled DNA transported by the construct. These findings suggest that the virosome is an efficient delivery system for testing infectious, as well as anti-cancer, DNA-based vaccine research.

Toscana virus (Bunyaviridae family, Phlebovirus genus) is a sandfly fever virus responsible for human neurological infections. Sandfly viruses are transmitted by insect vectors (Phlebotomus species) and the infection is present in climatic areas that allow the life cycle of the vector. The arthropode-borne Toscana virus is the etiologic agent of meningitis, meningoencephalitis, and encephalitis. The frequency of this neuropathic infection increases in the summer months, peaking in August in the endemic Mediterranean areas (Italy, Portugal, Spain, and Cyprus). Infection diagnosis is carried out by molecular assays and immunoenzymatic tests, which are rapid and sensitive. Recent studies have investigated the antigenic properties of the viral proteins (nucleoprotein N and surface glycoproteins G1 and G2), to better understand their immunogentic role.

Parathyroid hormone-related protein (PTH-rP), a secreted protein produced by prostate carcinoma and other epithelial cancers, is considered a key agent for the development of bone metastases. We investigated the construct GC90/IRIV, composed of immunopotentiating reconstituted influenza virosomes (IRIV) containing PTH-rP gene plasmids (GC90), as a potential tool for human anticancer immunotherapy into humanised mice transgenic for HLA-A(*)02.01, the human-beta2 microglobulin, and the human CD8alpha molecule. Intranasal administration of GC90/IRIV resulted in the induction of a PTH-rP-specific multiepitope cytotoxic T-cell (CTL) response. Cytotoxic T cells derived from vaccinated mice were capable of lysing in vitro syngenic murine PTH-rP transfectants and human HLA-A((*))02.01(+)/PTH-rP(+) prostate carcinoma LNCaP cells as well. The immune response capacity and the absence of any sign of toxicity and/or autoimmunity in vivo suggest the GC90/IRIV vaccine as a valid tool for active specific immunotherapy of human cancers and metastases overexpressing PTH-rP.

Toscana virus is the most important agent responsible for meningitis in central Italy. We report a serosurveillance study, using an immunoenzymatic assay, of 360 serum samples harvested from a high-risk population occupationally exposed to Toscana virus in two regions of Italy, Tuscany and Piedmont. The results indicates a seroprevalence of Toscana virus of 77.2% in the forestry workers, particularly in the Tuscany region. This fact is strictly correlated with the ecological niches specific for the survival of Toscana virus arthropod vector.

Cancer vaccines directed against tumor associate antigen (TAA) have produced encouraging results in preclinical models but not in cancer patients. A major limitation of this strategy is the relative degree of tolerance to these antigens and the low and heterogeneous tumor cell expression of TAA and major histocompatibility complex (MHC). Previous studies have shown that 5-fluorouracil (5-FU) can upregulate the expression of membrane-associated carcino-embryonic antigen (CEA), and MHC molecules in colon and breast carcinoma cell lines. We have investigated whether this drug can also enhance their sensitivity to the lytic effects of CEA-peptide specific Cytotoxic T cell lymphocytes (CTL). The CEA peptide-specific CTLs generated in our laboratory from normal HLA-A(*)02.01(+) donor PBMCs, were able to kill HLA-A(*)02.01(+)/CEA(+) breast (MCF-7-T103) and colon (HLA-A(*)02.01 gene-transfected HT-29 and C22.20) carcinoma cells in HLA-A(*)02.01 restricted manner. The treatment of target cells with 5-FU, enhanced their CEA expression and susceptibility to CTL-mediated lysis. Cold competition assays confirmed these results, thus supporting the hypothesis that immune target cell lysis and 5-FU mediated enhancement were dependent on CEA peptide presentation by cancer cells. 5-FU treatment of functionally "mature" CTL after in vitro expansion, did not reduce their cytolytic activity against MT-2 target cells but, when the anti-metabolite was added during the immune-sensitization phase, CTL generation was significantly inhibited. These results provide a rationale for investigating a possible new role of 5-FU as an immuno targeting amplifier agent in breast and colorectal cancer patients immunized with CEA-directed cancer vaccines., (Copyright 2003 Wiley-Liss, Inc.)

Parathyroid hormone-related protein (PTH-rP), a protein produced by prostate carcinoma and other epithelial cancers, is a key agent in the development of bone metastases. We investigated whether the protein follows the self-tolerance paradigm or can be used as a target Ag for anticancer immunotherapy by investigating the immunogenicity of two HLA-A(*)02.01-binding PTH-rP-derived peptides (PTR-2 and -4) with different affinity qualities. PTH-rP peptide-specific CTL lines were generated from the PBMC of two HLA-A(*)02.01(+) healthy individuals, stimulated in vitro with PTH-rP peptide-loaded autologous dendritic cells and IL-2. The peptide-specific CTLs were able to kill PTH-rP(+)HLA-A(*)02.01(+) breast and prostate carcinoma cell lines. The two peptides were also able to elicit a strong antitumor PTH-rP-specific CTL response in HLA-A(*)02.01 (HHD) transgenic mice. The vaccinated mice did not show any sign of side effects due to cell-mediated autoimmunity or toxicity. In this study we describe two immunogenic and toxic-free PTH-rP peptides as valid candidates for the design of peptide-based vaccination strategies against prostate cancer and bone metastases from the most common epithelial malignancies.

In the present study we investigated the efficacy of a new potential vaccine constituted of the respiratory syncytial virus (RSV)-F protein associated with influenza virosomes (RSV-F/IRIV) in combination with the mucosal adjuvant Escheriagen (Escherichia coli heat-labile toxin), administered intranasally (i.n.) to BALB/c mice. After an intramuscular "priming" with influenza virus vaccine, group A of mice was i.n. immunized with of RSV-F/IRIV+heat-labile toxin (HLT), groups B and C were inoculated i.n. with F-RSV+HLT and IRIV+HLT, respectively. The results showed that the virosomal delivery system greatly potentiate immune responses in animals. All mice immunized with the RSV-F/IRIV+HLT developed a mucosal IgA response and a high level of serum IgG. A balanced Th1/Th2 cytokine profile was observed in mice immunized with RSV-F/IRIV+HLT, while a Th2 response was observed in mice immunized with RSV-F+HLT. Histological analysis of lung tissue of RSV challenged mice did not reveal a vaccine-enhanced pulmonary eosinophilia. These results show that i.n. immunization of BALB/c mice with RSV-F/IRIV in combination with HLT can be considered a promising approach for the development of an efficacious human vaccine.

Acute meningitis is the most common neurologic disease that involves the central nervous system. The spectrum of infectious agents that cause neurologic infection is remarkably broad and numerous viruses are the most frequent cause of the aseptic meningitis syndrome. We applied a multiplex one-step method for the rapid detection of the genomic RNA of different neurotropic viruses: particles in the genus Enterovirus and Toscana virus, which are the most representative aetiologic agents in our country during the spring-summer period. We have evaluated the sensitivity and the specificity of the multiplex one-step test on positive controls and on RNA extracted from clinical samples harvested from 475 patients with meningitis hospitalized during the 1996-2001 period. The multiplex one-step RT-nPCR protocol allows for the detection of enterovirus and Toscana virus RNA in a single sample, by using, at the same time, a very small clinical sample volume. In our study we were able to diagnose 192 cases of meningitis by Toscana virus and 31 cases by enteroviruses out of 475 cases of meningitis utilizing the described one-step multiplex method.

We investigated influenza virosomes as a TAA-gene delivery system for use in TAA-directed anti-cancer vaccine therapy. An engineered plasmid (GC90) expressing the parathyroid hormone-related peptide (PTH-rP), a protein secreted by prostate and lung carcinoma cells, was included in influenza virosomes (GC90V). The ability of GC90V to elicit a PTH-rP-specific cytotoxic T cell (CTL) response was demonstrated in BALB/c mice immunised with intranasal (i.n.) GC90V+/-adjuvant subcutaneous (s.c.) interleukin-2 (IL-2). A PTH-rP-specific CTL response with antitumour activity was also demonstrated in human peripheral blood mononuclear cells (PBMC) stimulated in vitro with GC90V infected autologous dendritic cells (DC). These results provide a rationale for investigating GC90V in clinical trials of anticancer vaccine therapy.

5-Fluorouracil (5-FU) is a pyrimidine antimetabolite active against colorectal carcinoma and other malignancies of the digestive tract. Over-expression or mutation of thymidylate synthase (TS), the target enzyme of the 5-FU metabolite, 5-fluorodeoxyuridine monophosphate, is strictly correlated with cancer cell resistance to 5-FU. On this basis we investigated whether TS is a potential target for active specific immunotherapy of human colon carcinoma, which acquires resistance to 5-FU. Three TS-derived epitope peptides which fit defined amino acid consensus motifs for HLA-A2.1 binding were synthesized and investigated for their ability to induce human TS-specific cytotoxic T cell (CTL) responses in vitro. CTL lines specific for each peptide were established by stimulating peripheral blood mononuclear cells (PBMC) from an HLA-A2.1+ healthy donor with autologous dendritic cells loaded with TS peptide. Specific CTL lines showed HLA-A2.1-restricted cytotoxicity in vitro to HLA-A2.1+ target cells pulsed with the specific TS peptide and to HLA-class I matching colon carcinoma target cells over-expressing TS enzyme after exposure to 5-FU. Recognition by CTL lines suggests that these TS peptides may be potential candidates for use in a peptide-based vaccine against 5-FU resistant colon carcinoma.

The observation of many cases of mumps and mumps-associated CNS complications in vaccinees prompted us to perform an evaluation of the efficacy of four attenuated mumps virus (Urabe, Jeryl Lynn, Rubini and S12) vaccines. Two doses of vaccine were necessary to induce a good immunity in animals. The humoral and cell-mediated response induced in mice immunized intramuscularly or intranasally with these vaccines has been evaluated. Although the Urabe and Jeryl Lynn strains appear more immunogenic than the other strains and induce higher levels of IgG when administered intramuscularly, the S-12 strain administered intranasally induces a good IgG response. A marked specific CTL activity against mumps virus was observed in mice immunized intranasally with all the strains and, particularly, with the S12 strain. Thus, the intranasal immunization could be considered a possible alternative and efficient route of vaccination against mumps.

Experimental findings suggest that granulocyte-monocyte-colony stimulating factor (GM-CSF) synergistically interacts with interleukin-2 (IL-2) in generating an efficient antigen-specific immune response. We evaluated the toxicity, antitumour activity and immunobiological effects of human recombinant (hr)-GM-CSF and hr-IL-2 in 25 cancer patients who subcutaneously (s.c.) received hr-GM-CSF 150 microg/day for 5 days, followed by hrIL-2 s.c. for 10 days and 15 days rest. Two of the most common side-effects were bone pain and fever. Of the 24 patients evaluable for response, 3 achieved partial remission, 13 experienced stable disease, and 8 progressed. Cytokine treatment increased the number of monocytes, dendritic cells (DC), and lymphocytes (memory T cells) in the peripheral blood and enhanced the antigen-specific immunoreactivity of these patients. Our results show that the hr-GM-CSF and hr-IL-2 combination is active and well tolerated. Its biological activity may support tumour associated antigen (TAA)-specific anticancer immunotherapy by increasing antigen presenting cell (APC) activity and T cell immune competence in vivo.

The role of Toscana (TOS) virus in producing encephalitis without meningitis is uncertain. We studied 2 cases of TOS virus encephalitis without meningitis by means of nested polymerase chain reaction assay and DNA sequencing. Findings confirm that TOS virus may directly cause encephalitis and suggest the usefulness of DNA sequencing in investigating relationships between TOS virus molecular patterns and the spectrum of neurological involvement.

Four protein fragments which span the entire hemagglutinin-neuraminidase protein (HN) of mumps virus were expressed in HeLa cells and cell extracts were tested for their capability to induce neutralizing antibodies in mice. Fragment HN3 (aa 213-372) was able to induce the production of hemagglutination-inhibiting and neutralizing antibodies. When a subfragment of HN3, the synthetic peptide NSTLGVKSAREF (aa 329-340 of HN) was used for immunization, hemagglutination-inhibiting and neutralizing antibodies against mumps wild type virus but not against the Urabe Am9 vaccine virus were raised. The peptide could, therefore, contain a new epitope, which may be critical for protective host humoral immune response.

Samples of atherosclerotic tissue from 58 patients undergoing carotid surgery were analysed by tissue culture and PCR for Chlamydia pneumoniae; PCR was performed to detect Omp1, 16S rRNA and HSP-70 genes. To understand the active pathogenic role of C. pneumoniae, a reverse transcriptase-PCR (RT-PCR) assay was applied to detect the specific RNAs expressed either in the replicative form, or in the cryptic form found in chronic infection. The C. pneumoniae omp1 gene, encoding the major outer-membrane protein (MOMP), was detected in 13 of 58 samples. Among these, the result was confirmed in 11 samples after amplification of a further target, the 16S rRNA, and the presence of the HSP-70 gene, encoding heat-shock protein 70, was revealed in only five cases. All the samples were negative for evidence of specific RNAs by RT-PCR. The presence of genomic DNA and absence of specific RNAs in atherosclerotic tissue samples suggests a lack of an active metabolic or persistent infective role for C. pneumoniae. Thus, traces of C. pneumoniae DNA in these samples could be due to a degradative pathway of the host defensive cellular and biochemical mechanisms.

The envelope glycoproteins G1/G2 of Toscana virus (TOSV) seem to have the most important protective role in stimulating antibodies against the disease in humans, as well as antibodies against the Nucleoprotein (N), a partial neutralizing activity. Mice immunized with TOSV recombinant Nucleoprotein developed a strong humoral response to the TOSV that revealed the presence of neutralizing antibody than in vitro assay. The neutralizing antibody titre of mice immunized with the whole TOSV was analyzed before and after absorption of the sera with the recombinant N protein. A decrease of the neutralizing activity was observed in the treated sera. Similar results were obtained absorbing human anti-TOSV positive sera with the recombinant N protein. This study was designed to identify the nature of antibodies produced against the N protein of TOSV in mice and to establish correlation with antibodies produced in humans by natural infection.