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It is unknown whether components present in heart failure (HF) patients' serum provide an angiogenic stimulus. We sought to determine whether serum from HF patients affects angiogenesis and its major modulator, the Notch pathway, in human umbilical vein endothelial cells (HUVECs). In cells treated with serum from healthy subjects or from patients at different HF stage we determined: (1) Sprouting angiogenesis, by measuring cells network (closed tubes) in collagen gel. (2) Protein levels of Notch receptors 1, 2, 4, and ligands Jagged1, Delta-like4. We found a higher number of closed tubes in HUVECs treated with advanced HF patients serum in comparison with cells treated with serum from mild HF patients or controls. Furthermore, as indicated by the reduction of the active form of Notch4 (N4IC) and of Jagged1, advanced HF patients serum inhibited Notch signalling in HUVECs in comparison with mild HF patients' serum and controls. The circulating levels of NT-proBNP (N-terminal of the pro-hormone brain natriuretic peptide), a marker for the detection and evalutation of HF, were positively correlated with the number of closed tubes (r = 0.485) and negatively with Notch4IC and Jagged1 levels in sera-treated cells (r = -0.526 and r = -0.604, respectively). In conclusion, we found that sera from advanced HF patients promote sprouting angiogenesis and dysregulate Notch signaling in HUVECs. Our study provides in vitro evidence of an angiogenic stimulus arising during HF progression and suggests a role for the Notch pathway in it. J. Cell. Physiol. 231: 2700-2710, 2016. © 2016 Wiley Periodicals, Inc.

Novel biomaterials are of paramount importance for bone regrowth. In this study, we investigated human adipose stem cells (hASCs) for osteogenic, osteoconductivity, and osteoinductivity effects of an innovative collagen/hydroxylapatite hybrid scaffold. In hASCs that were grown on this scaffold, osteogenic genes were analyzed for their expression profiles, together with adhesion and extracellular matrix genes. In hASC integrins, basement membrane constituents and collagens were up-regulated, together with cell proliferation. In addition, expression of osteopontin and activated focal adhesion kinase was studied at the protein level. Our in vitro data indicate that hASCs, together with hybrid biomaterial, is an important model of study to investigate in vitro bone induction.-Mazzoni, E., D'Agostino, A., Manfrini, M., Maniero, S., Puozzo, A., Bassi, E., Marsico, S., Fortini, C., Trevisiol, L., Patergnani, S., Tognon, M. Human adipose stem cells induced to osteogenic differentiation by an innovative collagen/hydroxylapatite hybrid scaffold.

Pulsed electromagnetic fields (PEMFs) play a regulatory role on osteoblast activity and are clinically beneficial during fracture healing. Human mesenchymal stem cells (MSCs) derived from different sources have been extensively used in bone tissue engineering. Compared with MSCs isolated from bone marrow (BMSCs), those derived from adipose tissue (ASCs) are easier to obtain and available in larger amounts, although they show a less osteogenic differentiation potential than BMSCs. The hypothesis tested in this study was to evaluate whether PEMFs favor osteogenic differentiation both in BMSCs and in ASCs and to compare the role of PEMFs alone and in combination with the biochemical osteogenic stimulus bone morphogenetic protein (BMP)-2. Early and later osteogenic markers, such as alkaline phosphatase (ALP) activity, osteocalcin levels, and matrix mineralization, were analyzed at different times during osteogenic differentiation. Results showed that PEMFs induced osteogenic differentiation by increasing ALP activity, osteocalcin, and matrix mineralization in both BMSCs and ASCs, suggesting that PEMF activity is maintained during the whole differentiation period. The addition of BMP-2 in PEMF exposed cultures further increased all the osteogenic markers in BMSCs, while in ASCs, the stimulatory role of PEMFs was independent of BMP-2. Our results indicate that PEMFs may stimulate an early osteogenic induction in both BMSCs and ASCs and they suggest PEMFs as a bioactive factor to enhance the osteogenesis of ASCs, which are an attractive cell source for clinical applications. In conclusion, PEMFs may be considered a possible tool to improve autologous cell-based regeneration of bone defects in orthopedics.

Guiding the interaction of single cells acting as partners in heterotypic interactions (e.g., effectors and targets of immune lysis) and monitoring the outcome of these interactions are regarded as crucial biomedical achievements. In this study, taking advantage of a dielectrophoresis (DEP)-based Laboratory-on-a-chip platform (the DEPArray), we show that it is possible to generate closed DEP cages entrapping CTLs and NK cells as either single cells or clusters; reversibly immobilize a single virus-presenting or tumor cell within the chip at a selected position; move cages and their content to predetermined spatial coordinates by software-guided routing; force a cytotoxic effector to physically interact with a putative target within a secluded area by merging their respective cages; generate cages containing effector and target cells at predetermined E:T ratios; accurately assess cytotoxicity by real-time quantitation of the release kinetics of the fluorescent dye calcein from target cells (>50 lytic events may be tested simultaneously); estimate end points of calcein release within 16 min of initial E:T cell contact; simultaneously deliver Ab-based phenotyping and on-chip lysis assessment; and identify lytic and nonlytic E:T combinations and discriminate nonlytic effector phenotypes from target refractoriness to immune lysis. The proof of principle is provided that DEPArray technology, previously used to levitate and move single cells, can be used to identify highly lytic antiviral CTLs and tumor cells that are particularly refractory to NK cell lysis. These findings are of primary interest in targeted immunotherapy.

We have investigated the blood levels of sub-classes of stem cells (SCs) [mesenchymal stem cells (MSCs), haematopoietic stem cells (HSCs), endothelial progenitor cells/circulating endothelial cells (EPCs/CECs) and tissue-committed stem cells (TCSCs)] in heart failure (HF) patients at different stage of pathology and correlated it with plasmatic levels of proangiogenic cytokines. Peripheral blood level of SCs were analysed in 97 HF patients (24 in NYHA class I, 41 in class II, 17 in class III and 15 in class IV) and in 23 healthy controls. Plasmatic levels of PDGF-BB, bFGF, HGF, vascular endothelial growth factor (VEGF), SDF-1α, TNF-α and NTproBNP were also measured. Compared with healthy individuals, MSC, and in particular the sub-classes CD45(-) CD34(-) CD90(+) , CD45(-) CD34(-) CD105(+) and CD45(-) CD34(-) CXCR4(+) were significantly enhanced in NYHA class IV patients (16.8-, 6.4- and 2.7-fold, respectively). Level of CD45(-) CD34(-) CD90(+) CXCR4(+) cells progressively increased from class II to class IV (fold increases compared with controls: 8.5, 12 and 21.5, respectively). A significant involvement of CXCR4(+) subpopulation of HSC (CD45(+) CD34(+) CD90(+) CXCR4(+) , 1.4 versus 13.3 cells/μl in controls and NYHA class III patients, respectively) and TCSC (CD45(-) CD34(+) CXCR4(+) , 1.5 cells/ μl in controls versus 12.4 and 28.6 cells/μl in NYHA classes II and IV, respectively) were also observed. All tested cytokines were enhanced in HF patients. In particular, for PDGF-BB and SDF-1α we studied specific ligand/receptors pairs. Interestingly, the first one positively correlated with TCSCs expressing PDGFR (r = 0.52, P = 0.001), whereas the second one correlated with TCSCs (r = 0.34, P = 0.005) and with MSCs CD90(+) expressing CXCR4 (r = 0.39, P = 0.001). HF is characterized by the increase in the circulating levels of different MSC, HSC, EPC and TCSC subsets. Both the entity and kinetic of this process varied in distinct cell subsets. Specifically, differently from HSCs and EPCs/CECs, MSCs and TCSCs significantly increased with the progression of the disease, suggesting a possible distinct role of these cells in the pathophysiology of HF.

Cytotoxic T lymphocytes eliminate tumor cells expressing antigenic peptides in the context of MHC-I molecules. Peptides are generated during protein degradation by the proteasome and resulting products, surviving cytosolic amino-peptidases activity, may be presented by MHC-I molecules. The MHC-I processing pathway is altered in a large number of malignancies and modulation of antigen generation is one strategy employed by cells to evade immune control. In this study we analyzed the generation and presentation of a survivin-derived CTL epitope in HLA-A2-positive colon-carcinoma cells. Although all cell lines expressed the anti-apoptotic protein survivin, some tumors were poorly recognized by ELTLGEFLKL (ELT)-specific CTL cultures. The expression of MHC-I or TAP molecules was similar in all cell lines suggesting that tumors not recognized by CTLs may present defects in the generation of the ELT-epitope which could be due either to lack of generation or to subsequent degradation of the epitope. The cells were analyzed for the expression and the activity of extra-proteasomal peptidases. A significant overexpression and higher activity of TPPII was observed in colon-carcinoma cells which are not killed by ELT-specific CTLs, suggesting a possible role of TPPII in the degradation of the ELT-epitope. To confirm the role of TPPII in the degradation of the ELT-peptide, we showed that treatment of colon-carcinoma cells with a TPPII inhibitor resulted in a dose-dependent increased sensitivity to ELT-specific CTLs. These results suggest that TPPII is involved in degradation of the ELT-peptide, and its overexpression may contribute to the immune escape of colon-carcinoma cells.

We have previously shown that the biologically active Tat protein targets and efficiently enters dendritic cells, and increases the proteolytic activities of the immunoproteasome, thereby favoring the generation and presentation of the subdominant MHC-I binding CTL epitopes of heterologous antigens. In the present study, we demonstrate that Tat broadens in vivo epitope-specific T cell responses directed to heterologous antigens including HIV structural proteins. Specifically, co-immunization of mice with OVA and Tat proteins induces CTL responses against subdominant and cryptic OVA-derived epitopes, which are not detected in mice vaccinated with OVA alone. Similarly, mice vaccinated with the HIV-1 Gag, Env or V2-deleted Env antigens in combination with Tat show Th1-type and CTL responses directed to a larger number of T cell epitopes, as compared to mice vaccinated with these proteins in absence of Tat. In contrast, Tat did not affect Th2-type responses to these structural HIV proteins. These results indicate that Tat is not only an antigen but also a novel Th1-type adjuvant capable of broadening in vivo the spectrum of epitopes recognized by T cells, and suggest that Tat can be considered an optimal co-antigen in the development of novel vaccination strategies against AIDS.

The immunotherapeutic potential of biologically active HIV-1 Tat protein coupled to autologous red blood cells (RBCs) was evaluated in a mouse model. HIV-1 Tat expressed in Escherichia coli and purified to homogeneity was found to be active in viral trans activation and efficiently internalised by monocyte-derived dendritic cells (MDDCs). The product of HIV-Tat biotinylation and coupling to RBCs by means of a biotin–avidin–biotin bridge, (RBC-Tat), showed no trans activation activity and was still efficiently internalized by MDDCs as compared to uncoupled Tat. Balb/c mice were then immunized with 10g of soluble Tat in complete Freund’s adjuvant or with 40 ng of Tat coupled on RBCs surface and boosted at week 3, 6 and 25 with 5g soluble Tat in incomplete Freund’s adjuvant or with 20 ng of RBC-coupled Tat, respectively. Anti-Tat antibody response was similar in both groups; however, 2/6 animals immunized with soluble Tat and 6/6 animals immunized with RBC-Tat developed anti-Tat neutralizing antibodies. In addition, at week 28 cytolytic anti-Tat CTLs were detected in all animals although they were slightly higher in mice immunized with RBC-Tat. These results indicate that RBC-mediated delivery of HIV-1 Tat, in amounts 250 times lower than soluble Tat, is safe and induces specific CTL responses and neutralizing antibodies. © 2002 Elsevier Science Ltd. All rights reserved.

The majority of hepatitis C virus (HCV)-infected individuals fail to resolve the infection and become chronically infected despite the presence of HCV-specific CTL responses directed to different HCV-derived peptide antigens. Only a minority of individuals is able to clear the virus by mounting efficient CTL responses early after acute infection, but at present it is not clear whether viral clearance is associated with CTL responses of defined specificity. To elucidate those responses associated with improvement of the disease, we analyzed CTL responses to 16 different HLA-A2-presented, HCV-derived epitopes in 12 chronically infected patients, 14 chronically infected patients treated with interferon-α, and in one patient with acute symptomatic disease. We show here that the majority of chronically infected individuals present CTL responses directed to an NS4-derived peptide antigen (amino acids 1789–1797). Treated patients presented stronger HCV-specific CTL responses and therapy-induced changes in CTL target choice. In particular, 13 out of 14 individuals responded to an NS3-derived epitope (amino acids 1073–1081). By longitudinal analysis we show that five individuals responding to IFN-α therapy with decreases in alanine aminotransfrase levels presented a strong CTL activity directed to the NS3-derived epitope. One patient that spontaneously resolved the infection presented a generally strong CTL activity specific for HCV-derived epitopes with a dominant response to the NS3-derived peptide antigen. This suggests that CTL responses directed to this NS3-derived antigen may be beneficial for the control of HCV infection. Improvement of these responses may represent a therapeutic intervention in chronic HCV infection.

Pulsed electromagnetic fields (PEMFs) play a regulatory role on osteoblast activity and are clinically beneficial during fracture healing. Human mesenchymal stem cells (MSCs) derived from different sources have been extensively used in bone tissue engineering. Compared with MSCs isolated from bone marrow (BMSCs), those derived from adipose tissue (ASCs) are easier to obtain and available in larger amounts, although they show a less osteogenic differentiation potential than BMSCs. The hypothesis tested in this study was to evaluate whether PEMFs favor osteogenic differentiation both in BMSCs and in ASCs and to compare the role of PEMFs alone and in combination with the biochemical osteogenic stimulus bone morphogenetic protein (BMP)-2. Early and later osteogenic markers, such as alkaline phosphatase (ALP) activity, osteocalcin levels, and matrix mineralization, were analyzed at different times during osteogenic differentiation. Results showed that PEMFs induced osteogenic differentiation by increasing ALP activity, osteocalcin, and matrix mineralization in both BMSCs and ASCs, suggesting that PEMF activity is maintained during the whole differentiation period. The addition of BMP-2 in PEMF exposed cultures further increased all the osteogenic markers in BMSCs, while in ASCs, the stimulatory role of PEMFs was independent of BMP-2. Our results indicate that PEMFs may stimulate an early osteogenic induction in both BMSCs and ASCs and they suggest PEMFs as a bioactive factor to enhance the osteogenesis of ASCs, which are an attractive cell source for clinical applications. In conclusion, PEMFs may be considered a possible tool to improve autologous cell-based regeneration of bone defects in orthopedics.

Circulating mesenchymal cells increase in heart failure (HF) patients and could be used therapeutically. Our aim was to investigate whether HF affects adipose tissue derived mesenchymal cell (adMSC) isolation, functional characteristics and Notch pathway.We compared 25 patients with different degrees of HF (11 NYHA classes I and II and 14 NYHA III and IV) with 10 age and gender matched controls. 100% adMSC cultures were obtained from controls, while only 72.7% and 35.7% from patients with mild or severe HF (p0.0001). adMSC from HF patients showed higher markers of senescence (p16 positive cells: 14±2.3% in controls and 35.6±5.6% (p0.05) and 69±14.7% (p0.01) in mild or severe HF; γ-H2AX positive cells: 3.7±1.2%, 19.4±4.1% (p0.05) and 23.7±3.4% (p0.05) respectively), lower proliferation index (Ki67 positive cells: 21.5±4.9%, 13.2±2.8% and 13.7±3.2%, respectively), reduced pluripotency-associated genes (Oct4 positive cells: 86.7±4.9%, 55±12% (p0.05) and 43.3±8.7% (p0.05), respectively; NANOG positive cells: 89.8±3.7%, 39.6±14.4% (p0.01) and 47±8.1%, respectively), and decreased differentiation markers (α-sarcomeric actin positive cells: 79.8±4.6%, 49±18.1% and 47±12.1% (p0.05) and CD31-positive endothelial cells: 24.5±2.9%, 0.5±0.5% (p0.001) and 2.3±2.3% (p0.001), respectively). AdMSC from HF patients also showed reduced Notch transcriptional activity (lowered expression of Hey 1 and Hey 2 mRNAs). Stimulation with TNF-α of adMSC isolated from controls affected the transcription of several components of the Notch pathway (reduction of Notch 4 and Hes 1 mRNAs and increase of Notch 2 and Hey 1 mRNAs).In HF yield and functionality of adMSC are impaired and their Notch signaling is downregulated.

Estrogens play a protective role in coronary artery disease. The mechanisms of action are still poorly understood, although a role for estrogens in stimulation of angiogenesis has been suggested. In several cell types, estrogens modulate the Notch pathway, which is involved in controlling angiogenesis downstream of vascular endothelial growth factor A (VEGF-A). The goal of our study was to establish whether estrogens modulate Notch activity in endothelial cells and the possible consequences on angiogenesis. Human umbilical vein endothelial cells (HUVECs) were treated with 17β-estradiol (E2) and the effects on Notch signalling were evaluated. E2 increased Notch1 processing as indicated by i) decreased levels of Notch1 transmembrane subunit ii) increased amount of Notch1 in nuclei iii) unaffected level of mRNA. Similarly, E2 increased the levels of the active form of Notch4 without altering Notch4 mRNA. Conversely, protein and mRNA levels of Notch2 were both reduced suggesting transcriptional repression of Notch2 by E2. Under conditions where Notch was activated by upregulation of Delta-like ligand 4 (Dll4) following VEGF-A treatment, E2 caused a further increase of the active form of Notch1, of the number of cells with nuclear Notch1 and of Hey2 mRNA. Estrogen receptor antagonist ICI 182.780 antagonized these effects suggesting that E2 modulation of Notch1 is mediated by estrogen receptors. E2 treatment abolished the increase in endothelial cells sprouting caused by Notch inhibition in a tube formation assay on 3D Matrigel and in mouse aortic ring explants. In conclusion, E2 affects several Notch pathway components in HUVECs, leading to an activation of the VEGF-A-Dll4-Notch1 axis and to a modulation of vascular branching when Notch signalling is inhibited. These results contribute to our understanding of the molecular mechanisms of cardiovascular protection exerted by estrogens by uncovering a novel role of E2 in the Notch signalling-mediated modulation of angiogenesis.

The recent increase in human lifespan, coupled with unhealthy diets and lifestyles have led to an unprecedented increase in cardiovascular diseases. Even in the presence of a wide range of therapeutic options with variable efficacy, mortality due to heart failure is still high and there is a need to identify new therapeutic targets. Genetic and in vitro studies have implicated the Notch signalling in the development and maintenance of the cardiovascular system through a direct effect on biological functions of vascular cells (endothelial and vascular smooth muscle cells) and cardiomyocytes. Notch signalling is also involved in the modulation of inflammation, which plays a major role in causing and exacerbating cardiovascular diseases. The Notch pathway could represent a new therapeutic target for the treatment of cardiovascular diseases.

The equilibrium between endothelial apoptosis and endothelial renewal is altered in acute coronary syndromes and may be related to differences in the beneficial effects of angiotensin-converting enzyme inhibitors and angiotensin II receptor antagonists (angiotensin receptor blockers).We evaluated the effect of treatment on endothelial function in post-myocardial infarction (MI) patients treated with perindopril (group 2, n = 16) or valsartan (group 3, n = 17) at baseline and after 7, 15, and 30 days and in normal controls (group 1, n = 20). Endothelial apoptosis was determined by cultivating serum samples in vitro with human umbilical vein endothelial cells (HUVECs), while endothelial renewal was assessed by mobilization of CD34+ bone marrow cells.At baseline, post-MI patients had significantly elevated rates of apoptosis (16.6 ± 5.0% and 16.5 ± 8.4% in groups 2 and 3, respectively [both p = 0.01] vs 1.6 ± 0.7% in group 1), which declined in group 2 (10.5 ± 4.4% at 30 days, p = 0.04), but not in group 3. Similar results and trends were found for the Bax/Bcl-2 ratio. CD34+ mobilization was significantly increased in group 2 (3.0 ± 1.0 at baseline to 6.2 ± 1.6 at 15 days, p = 0.03), whereas in group 3 CD34+ mobilization did not change significantly. The findings in group 2 were accompanied by an increase in vascular endothelial growth factor at 15 days, and a reduction in tumor necrosis factor-α and its soluble receptors, versus no change in group 3. Similar findings were observed for angiotensin II and bradykinin.Our results indicate that perindopril, but not valsartan, reduces the proapoptotic effect of serum on the endothelium and increases endothelial renewal in patients with acute coronary syndromes.

BackgroundAs HIV-specific cytotoxic T cells play a key role during acute and chronic HIV-1 infection in humans, the ability of potential anti-HIV vaccines to elicit strong, broad T cell responses is likely to be crucial. The HIV-1 Gag antigen is widely considered a relevant antigen for the development of an anti-HIV vaccine since it is one of the most conserved viral proteins and is also known to induce T cell responses. In the majority of studies reporting Gag-specific cellular immune responses induced by Gag-based vaccines, only a small number of Gag T cell epitopes were tested in preclinical mouse models, thus giving an incomplete picture of the numerous possible cellular immune responses against this antigen. As is, this partial knowledge of epitope-specific T cell responses directed to Gag will unavoidably result in a limited preclinical evaluation of Gag-based vaccines.ResultsIn this study we identified new Gag CD8+ T cell epitopes in BALB/c mice vaccinated with the HIV-1 Gag antigen alone or in combination with the HIV-1 Tat protein, which was recently shown to broaden T cell responses directed to Gag. Specifically, we found that CTL responses to Gag may be directed to nine different CTL epitopes, and four of these were mapped as minimal CTL epitopes.ConclusionThese newly identified CTL epitopes should be considered in the preclinical evaluation of T cell responses induced by Gag-based vaccines in mice.

Dielectrophoresis (DEP)-based lab-on-a-chip devices represent a very appealing approach for cell manipulation and will enable laboratory testing to move from laboratories into nonlaboratory settings. DEP-based lab-on-a-chip platforms involve the miniaturization of sev- eral complex chemical and physical procedures in a single microchip-based device, allowing the identification and isolation of cell populations or single cells, separation of cells exhibiting dif- ferent DEP properties, isolation of infected from uninfected cells, and viable from nonviable cells. In addition, DEP-arrays allow cellomics procedures based on the parallel manipulation of thousands of cells. Arrayed lab-on-a-chip platforms are also suitable to study cell–cell interac- tions and cell targeting with programmed numbers of microspheres.

Novel biocompatible core-shell cationic nanoparticles, composed of an inner hard core of poly(methylmethacrylate) (PMMA) and a hydrophilic tentacular shell bearing positively charged groups and poly(ethyleneglycol) chains covalently bound to the core, were prepared by emulsion polymerization and characterized in vitro and in vivo for DNA vaccine applications. The nanoparticles reversibly adsorbed large amounts of DNA, mainly through electrostatic interactions, preserved its functional structure, efficiently delivered it intracellularly, and were not toxic in vitro or in mice. Furthermore, two intramuscular (i.m.) immunizations (4 weeks apart) with a very low dose (1 mu g) of the plasmid pCV-tat delivered by these nanoparticles followed by one or two protein boosts induced significant antigen-specific humoral and cellular responses and greatly increased Th1-type T cell responses and CTLs against HIV-1 Tat. (c) 2006 Elsevier Ltd. All rights reserved.

Tat, the trans activation protein of HIV, is produced early upon infection to promote and expand HIV replication and transmission. However, Tat appears to also have effects on target cells, which may affect Ag recognition both during infection and after vaccination. In particular, Tat targets dendritic cells and induces their maturation and Ag-presenting functions, increasing Th1 T cell responses. We show in this work that Tat modifies the catalytic subunit composition of immunoproteasomes in B and T cells either expressing Tat or treated with exogenous biological active Tat protein. In particular, Tat up-regulates latent membrane protein 7 and multicatalytic endopeptidase complex like-1 subunits and down-modulates the latent membrane protein 2 subunit. These changes correlate with the increase of all three major proteolytic activities of the proteasome and result in a more efficient generation and presentation of subdominant MHC-I-binding CTL epitopes of heterologous Ags. Thus, Tat modifies the Ag processing and modulates the generation of CTL epitopes. This may have an impact on both the control of virally infected cells during HIV-1 infection and the use of Tat for vaccination strategies.

The present study investigates mRNA and protein levels of A3 adenosine receptors in resting (R) and activated (A) human lymphocytes. The receptors were evaluated by the antagonist radioligand [3H]5-N-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2(2furyl)-pyrazolo-[4,3e]-1,2,4-triazolo-[1,5-c]-pyrimidine ([3H]MRE 3008F20), which yielded Bmax values of 125 +/- 15 and 225 +/- 23 fmol/mg of protein and KD values of 1.79 +/- 0.30 and 1.85 +/- 0.25 nM in R and A cells, respectively. The protein seems to be induced with remarkable rapidity starting at 15 min and reaches a plateau at 30 min. Western blot assays revealed that the up-regulation of the A3 subtype after lymphocyte activation was caused by an increase in an enriched CD4+ cell fraction. Real-time reverse transcription-polymerase chain reaction experiments confirmed the rapid increase of A3 mRNA after T cell activation. Competition of radioligand binding by adenosine ligands displayed a rank order of potency typical of the A3 subtype. Thermodynamic data indicated that the binding is enthalpy- and entropy-driven in both R and A cells, suggesting that the activation process does not involve, at a molecular level, receptor alterations leading to modifications in the A3-related binding mechanisms. Functionally, the up-regulation of A3 adenosine receptors in A versus R cells corresponded to a potency increase of the A3 agonist N6-(3-iodo-benzyl)-2-chloro-adenosine-5'-N-methyluronamide in inhibiting cAMP accumulation (IC50=1.5 +/- 0.4 and 2.7 +/- 0.3 nM, respectively); this effect was antagonized by MRE 3008F20 (IC50=5.0 +/- 0.3 nM). In conclusion, our results provide, for the first time, an in-depth investigation of A3 receptors in human lymphocytes and demonstrate that, under activating conditions, they are up-regulated and may contribute to the effects triggered by adenosine.

The majority of hepatitis C virus (HCV)-infected individuals fail to resolve the infection and become chronically infected despite the presence of HCV-specific CTL responses directed to different HCV-derived peptide antigens. Only a minority of individuals is able to clear the virus by mounting efficient CTL responses early after acute infection, but at present it is not clear whether viral clearance is associated with CTL responses of defined specificity. To elucidate those responses associated with improvement of the disease, we analyzed CTL responses to 16 different HLA-A2-presented, HCV-derived epitopes in 12 chronically infected patients, 14 chronically infected patients treated with interferon-alpha, and in one patient with acute symptomatic disease. We show here that the majority of chronically infected individuals present CTL responses directed to an NS4-derived peptide antigen (amino acids 1789-1797). Treated patients presented stronger HCV-specific CTL responses and therapy-induced changes in CTL target choice. In particular, 13 out of 14 individuals responded to an NS3-derived epitope (amino acids 1073-1081). By longitudinal analysis we show that five individuals responding to IFN-alpha therapy with decreases in alanine aminotransferase levels presented a strong CTL activity directed to the NS3-derived epitope. One patient that spontaneously resolved the infection presented a generally strong CTL activity specific for HCV-derived epitopes with a dominant response to the NS3-derived peptide antigen. This suggests that CTL responses directed to this NS3-derived antigen may be beneficial for the control of HCV infection. Improvement of these responses may represent a therapeutic intervention in chronic HCV infection.

Summary The human herpesvirus 8 (HHV-8) is a human γ2-herpesvirus that is implicated in the development of Kaposi's sarcoma (KS), primary effusion lymphoma and Castelman's disease. Since the responses of cytotoxic T lymphocytes (CTL) play a key role in the control of herpesvirus infection, it is important to identify and to characterize the CTL target epitopes of HHV-8 viral antigens. In this study, using peptide-binding motifs, we selected potential human leucocyte antigen (HLA)-A2-binding peptides from kaposin A and glycoprotein H (gH), that are latent and lytic HHV-8 antigens, respectively. HLA-A2-binding peptides were tested for their capacity to induce CTL responses in HHV-8-negative healthy donors. By this approach, we found that the majority of individuals responded to two HHV-8-derived CTL epitopes, namely, VLLNGWRWRL (amino acids 16–25), which derives from kaposin A, and FLNWQNLLNV (amino acids 59–68), which derives from gH. In addition, memory CTL responses to these epitopes were detected in disease-free individuals infected by HHV-8 demonstrating that the two epitopes are relevant targets of CTL-mediated immunity in vivo. The identified epitopes may be investigated for the development of immunotherapeutic strategies against HHV-8-associated malignancies.

In the Ferrara district, an area south of the Po delta, four different variants of glucose-6-phosphate dehydrogenase (G6PD;E.C.1.1.49) have been described as a result of biochemical characterization of the enzyme protein: one was G6PD Mediterranean (G6PD Med) and three were local variants named Ferrara I, II, and III. The Ferrara I variant was recently analysed at the DNA level and shown to correspond to G6PD A376G/202A, while the mutations causing the variants II and III, still remain unknown. We analysed the G6PD coding region of 18 apparently unrelated G6PD deficient subjects, whose families have lived in the Ferrara district for at least three generations: 12 subjects had G6PD Med563T/1311T, 3, G6PD Santamaria376G/542T and 2, G6PD A-376G/202A. In one subject we found a new mutation, a G--A transition at nucleotide 242 causing an Arg--His amino-acid replacement at position 81. We named this new variant G6PD Lagosanto242 A. Phenotypically the enzyme has nearly normal kinetic properties and appears different from the variants Ferrara II and III.

In order to evaluate the criteria for discontinuing neonatal anticonvulsant treatment, 55 newborns with seizures have been studied. Clinical and EEG serial examinations were performed: soon after the first seizure, throughout the hospital course, and during the follow-up every 3 months until a year, and every 6 months later on. Anticonvulsant treatment with phenobarbital was discontinued (at 4 days-19 months; mean 104 days) on the basis of the following variables: type and number of seizures, time taken for their control, type and persistence of EEG abnormalities, initial neurological features, and seizure etiology. At the follow-up (12 months-8 years; mean 36 months) only 4 children had relapsed, 3 of them with a single short seizure without EEG abnormalities. The results obtained by means of the correlation between the length of anticonvulsant treatment and the clinical and EEG variables provide evidence of the value of the criteria employed. Of these, the duration of persistence of EEG abnormalities was the most important for planning the maintenance of anticonvulsant treatment and its discontinuation.

The human herpesvirus 8 (HHV-8) is a γ herpesvirus with oncogenic potential which establishes a chronic infection that is normally controlled by the immune system of healthy individuals. In particular, CTL responses seem to play a key role in control of the infection. In this study, we characterized epitope-specific CTL responses in healthy HHV-8-seropositive individuals against four HHV-8 lytic Ags: open reading frames (ORF) 26, 70, K3, and K5. We found that the majority of subjects responded to at least one HHV-8 lytic Ag-derived epitope, and some of these epitopes represented dominant targets, suggesting that they could be relevant targets of CTL-mediated immunity in vivo, and may be involved in host control of HHV-8. Specifically, we identified three CTL epitopes from ORF 26, which are presented by HLA-A2, six CTL epitopes from ORF 70 presented by HLA-A2 (three epitopes), -A24 (two epitopes), and -B7 (one epitope), three CTL epitopes from ORF K3 presented by HLA-A2 (two epitopes) and -B7 (one epitope), and one HLA-A2 presented epitope derived from ORF K5. The identified epitopes may be regarded as useful tools for understanding the role of CTL responses to lytic Ags in individuals affected by HHV-8-associated disorders, and for the development of immunotherapies for the treatment/prevention of HHV-8-associated malignancies.