Abstract: BACKGROUND: As 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. RESULTS: In 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. CONCLUSION: These newly identified CTL epitopes should be considered in the preclinical evaluation of T cell responses induced by Gag-based vaccines in mice.

Abstract: Here we report the synthesis and biological properties of peptide-based molecules bearing constrained analogues of phenylalanine at the C-terminal. Compounds were tested as proteasome subunits' inhibitors. Dehydro-peptides showed good inhibition, in particular against trypsin-like (T-L) proteasome activity while some C-terminal Tic-derivatives inhibit only caspase-like activity in enzymatic beta1 subunits with a certain degree of efficacy. The best analogues of the series demonstrated good resistance to proteolysis and a capacity to permeate the cell membrane.

Abstract: 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.

Abstract: The 20S proteasome is a multicatalytic protease complex responsible for the degradation of many proteins in mammalian cells. Specific inhibition of proteasome enzymatic subunits represents a topic of great interest for the development of new drug therapies. Following our previous development of a new class of peptide-based inhibitors bearing a C-terminal vinyl ester residue as a pharmacophoric unit that are able to interact with the catalytic threonine, we report here the synthesis and biological properties of a new series of vinyl ester cyclopeptide analogues. Some of these derivatives were shown to inhibit the chymotrypsin-like activity of the proteasome at nanomolar concentration and their potency was found to depend on the size of the tetrapeptidic cyclic portion.

Abstract: The development of a vaccine against HIV/AIDS capable of inducing broad humoral and cellular responses at both systemic and mucosal sites, able to stop or reduce viral infection at the portal of entry, represents the only realistic way to control the infection caused by HIV world-wide. The promising results obtained with the HIV-1 Tat-based vaccines in preclinical and clinical settings, the evidence that a broad immunity against HIV correlates with reduced viral load or virus control, as well as the availability of novel gp140 V2-loop deleted HIV-1 Env (DeltaV2Env) immunogens capable of inducing cross-reactive neutralizing antibodies, have led to the design of new vaccine strategies based on the combination of non-structural and structural proteins. In this study, we demonstrate that immunization with a biologically active HIV-1 Tat protein in combination with the oligomeric HIV-1 gp140 DeltaV2Env and/or SIV Gag proteins, delivered intranasally with the detoxified LTK63 mucosal adjuvant, whose safety has been recently shown in humans, elicits long-lasting local and systemic antibody and cellular immune responses against the co-administered antigens in a fashion similar to immune responses induced by vaccination with Tat, DeltaV2Env and Gag proteins alone. The results indicate lack of antigen interference implying that HIV-1 Tat is an optimal co-antigen for combined vaccine strategies employing DeltaV2Env and/or Gag proteins.

Abstract: In Burkitt's lymphoma cells, Epstein Barr virus (EBV) latency products interact with the ubiquitin-proteasome system to promote episomal maintenance and immunological evasion while the tripeptidylpeptidase II (TPPII) functions as an alternative protease. In the present study, we have examined the activities and levels of the proteasome and TPPII complex in Raji and in Akata cells after induction of EBV lytic cycle. The results show that the chymotrypsin-like and caspase-like activities of the proteasome were substantially reduced in Raji and Akata cells. Similarly, TPPII activity was diminished in both cell lines but was recovered in Akata cells at longer time after induction. Protein levels of the alpha/beta subunits of the 20S proteasome and TPPII concentration decreased to different extents after EBV activation, whereas the ubiquitin binding S6' subunit of the 19S regulatory complex increased three to fourfold along with the levels of ubiquitin-conjugates. Collectively, these observations demonstrate impairment of two major cellular proteolytic systems at the onset of EBV lytic infection.

Abstract: Here we report the study of a new series of peptide-based proteasome inhibitors with a vinyl ester moiety at C-terminal. The presence of Tic, a rigid analogue of phenylalanine, in the central portion of some derivatives is not favourable for the activity. The best analogue of the series shows a potent and selective inhibition for the beta2 subunit and good enzymatic stability.

Abstract: The platinum mixed-phosphine complexes (SP-4,2)-[PtCl(8-MTT)(PPh3)(PTA)] (2) and cis-[Pt(8-MTT)2(PPh3)(PTA)] (3) (MTTH2 = 8-(methylthio)theophylline, PTA = 1,3,5-triaza-7-phosphaadamantane) have been prepared from the precursor cis-[PtCl2(PPh3)(PTA)] (1), which has been fully characterized by X-ray diffraction determination. Antiproliferative activity tests indicated that the presence of one lipophilic PPh3 and one hydrophilic PTA makes 1-3 more active than the analogues bearing two PPh3 or two PTA. The reactivity of cis-[PtCl2(PPh3)2], cis-[PtCl2(PTA)2], and cis-[PtCl2(PPh3)(PTA)] with the bis(thiopurines) bis(S-8-thiotheophylline)methane (MBTTH2), 1,2-bis(S-8-thiotheophylline)ethane (EBTTH2), and 1,3-bis(S-8-thiotheophylline)propane (PBTTH2) has also been investigated. New binuclear complexes have been prepared and identified by spectroscopic techniques and their antiproliferative activities on T2 and SKOV3 cell lines evaluated.

Abstract: According to recent estimates, 39.5 million people have been infected with HIV and 2.9 million have already died. The effect of HIV infection on individuals and communities is socially and economically devastating. Although antiretroviral drugs have had a dramatically beneficial impact on HIV-infected individuals who have access to treatment, it has had a negligible impact on the global epidemic. Therefore, the need for an efficacious HIV/AIDS vaccine remains the highest priority of the world HIV/AIDS agenda. The generation of a vaccine against HIV/AIDS has turned out to be extremely challenging, as indicated by > 20 years of unsuccessful attempts. This review discusses the major challenges in the field and key experimental evidence providing a rationale for the use of non-structural HIV proteins, such as Rev, Tat and Nef, either in the native form or expressed by viral vectors such as a replicating adeno-vector. These non-structural proteins alone or in combination with modified structural HIV-1 Env proteins represent a novel strategy for both preventative and therapeutic HIV/AIDS vaccine development.

Abstract: The human herpesvirus 8 (HHV-8) is a gamma 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.

Abstract: The Tat protein is the transcriptional activator of HIV-1 gene expression, which is not only essential for viral replication, but also important in the complex HIV-induced pathogenesis of AIDS, as both an intracellular and an extracellular released protein. Accordingly, Tat is able to profoundly affect cellular gene expression, regulating several cellular functions, also in non-infected cells. We showed recently that Tat induces modification of immunoproteasomes in that it up-regulates LMP7 (low-molecular-mass polypeptide 7) and MECL1 (multicatalytic endopeptidase complex-like 1) subunits and down-modulates the LMP2 subunit, resulting in a change in the generation and presentation of epitopes in the context of MHC class I. In particular, Tat increases presentation of subdominant and cryptic epitopes. In the present study, we investigated the molecular mechanism responsible for the Tat-induced LMP2 down-regulation and show that intracellular Tat represses transcription of the LMP2 gene by competing with STAT1 (signal transducer and activator of transcription 1) for binding to IRF-1 (interferon-regulatory factor-1) on the overlapping ICS-2 (interferon consensus sequence-2)-GAS (gamma-interferon-activated sequence) present in the LMP2 promoter. This element is constitutively occupied in vivo by the unphosphorylated STAT1-IRF-1 complex, which is responsible for the basal transcription of the gene. Sequestration of IRF-1 by intracellular Tat impairs the formation of the complex resulting in lower LMP2 gene transcription and LMP2 protein expression, which is associated with increased proteolytic activity. On the other hand, extracellular Tat induces the expression of LMP2. These effects of Tat provide another effective mechanism by which HIV-1 affects antigen presentation in the context of the MHC class I complex and may have important implications in the use of Tat for vaccination strategies.

Abstract: 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 microg) 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.

Abstract: Two small libraries of tripeptidic-based vinyl ester derivative proteasome inhibitors were synthesized and tested, starting with the Hmb-Val-Gln-Leu-VE prototype. The P3 and P4 positions were investigated with a complete set of amino acid residues, some of which showed remarkable selective inhibition of the trypsin-like (beta2) subunit. In both positions, aromatic and hydrophobic residues were preferred.

Abstract: Here we report the synthesis and biological activities of new tripeptidic-based vinyl ester derivative proteasome inhibitors. Starting from Hmb-Val-Ser-Leu-VE prototype, we investigated P2 position and N-terminal substitution. The more effective inhibitors of the series showed remarkable inhibition and selectivity for the trypsin-like (beta2) subunit and were revealed to be specific for the proteasome. In vitro metabolic stability studies of the new vinyl ester analogues are also reported here.

Abstract: 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.

Abstract: The proteasome is a multicatalytic proteinase complex which plays a central role in intracellular protein degradation. We report here the synthesis and biological activities of a new class of specific proteasome inhibitors selective for trypsin-like activity. These tripeptide-based compounds bearing a C-terminal vinyl ester are nontoxic, and do not affect cell proliferation, but are able to modulate the generation and presentation of immunogenic peptides presented by MHC class I molecules.

Abstract: The promising results obtained with the HIV-1 Tat-based vaccines in mice, monkeys and humans, a better understanding of Tat immunomodulatory functions, as well as evidence that vaccination with trimeric V2 loop-deleted HIV-1 Env induces cross-clade neutralizing antibodies led to the rational design of a novel vaccine based on the combination of Tat and V2-deleted Env.

Abstract: A 2-kDa synthetic derivative of the macrophage-activating lipopeptide (MALP-2) from Mycoplasma fermentans is a potent inducer of monocytes/macrophages and improves the immunogenicity of antigens co-administered by systemic and mucosal routes. Dendritic cells (DC) are the most potent antigen-presenting cells, which are able to prime naive T cells in vivo. To elucidate the underlying mechanisms of MALP-2 adjuvanticity, we analyzed its activity on bone marrow-derived murine DC. In vitro stimulation of immature murine DC with MALP-2 resulted in the induction of maturation with up-regulated expression of MHC class II, costimulatory (CD80, CD86) and adhesion (CD40, CD54) molecules. MALP-2 also enhances the secretion of cytokines (IL-1alpha, IL-6 and IL-12), and increases DC stimulatory activity on naive and antigen-specific T cells. Further studies demonstrated that MALP-2 treatment of DC results in a dose-dependent shift from the protein pattern of proteasomes to immunoproteasomes (up-regulation of LMP2, LMP7 and MECL1), which correlates with an increased proteolytic activity. Thus, the adjuvanticity of MALP-2 can be mediated, at least in part, by the stimulation of DC maturation, which in turn leads to an improved antigen presentation. Therefore, MALP-2 is a promising molecule for the development of immune therapeutic or prophylactic interventions.

Abstract: 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.

Abstract: The 26S proteasome is a multicatalytic protease complex that plays an essential role in intracellular protein degradation. We have synthesized and tested a series of arecoline peptide derivatives where the peptide portion derives from a screening of tripeptide sequences, and the arecoline moiety has been considered as a potential substrate for catalytic threonine. Derivatives 17-19 are the best compounds of the series, showing chymotryptic-like (beta5) inhibition (IC(50) congruent with 1 microM) and favorable pharmacokinetic properties.

Abstract: Complexes [Pt(mu-N,S-8-TT)(PPh(3))(2)](2) (1), [Pt(mu-S,N-8-TT)(PTA)(2)](2) (2), [Pt(8-TTH)(terpy)]BF(4) (3), cis-[PtCl(8-MTT)(PPh(3))(2)] (4), cis-[Pt(8-MTT)(2)(PPh(3))(2)] (5), cis-[Pt(8-MTT)(8-TTH)(PPh(3))(2)] (6), cis-[PtCl(8-MTT)(PTA)(2)] (7), cis-[Pt(8-MTT)(2)(PTA)(2)] (8), and trans-[Pt(8-MTT)(2)(py)(2)] (9) (8-TTH(2) = 8-thiotheophylline; 8-MTTH = 8-(methylthio)theophylline; PTA = 1,3,5-triaza-7-phosphaadamantane) are presented and studied by IR and multinuclear ((1)H, (31)P[(1)H]) NMR spectroscopy. The solid-state structure of 4 and 9 has been authenticated by X-ray crystallography. Growth inhibition of the cancer cells T2 and SKOV3 induced by the above new thiopurine platinum complexes has been investigated. The activity shown by complexes 4 and 9 was comparable with cisplatin on T2. Remarkably, 4 and 9 displayed also a valuable activity on cisplatin-resistant SKOV3 cancer cells.

Abstract: Two novel classes of biocompatible core-shell anionic microspheres, composed of an inner hard insoluble core, either made of poly(styrene) (PS) or poly(methyl methacrylate) (PMMA), and a soft outer tentacular shell made of long soluble negatively charged arms derived from the steric stabilizer, hemisuccinated poly(vinyl alcohol) or Eudragit L100/55, respectively, were prepared by dispersion polymerization and characterized. Five types of these novel microspheres, two made of poly(styrene) and hemisuccinated poly(vinyl alcohol) (A4 and A7), and three made of poly(methyl methacrylate) and Eudragit L100/55 (1D, 1E, H1D), differing for chemical composition, size, and surface charge density were analyzed for the delivery of the HIV-1 Tat protein for vaccine applications. All microspheres reversibly adsorbed the native biologically active HIV-1 Tat protein preventing Tat from oxidation and maintaining its biological activity, therefore increasing the shelf-life of the Tat protein vaccine. The microspheres efficiently delivered Tat intracellularly, and were not toxic in vitro nor in mice, even after multiple administrations. These results indicate that these novel microparticles are safe and represent a promising delivery system for vaccination with Tat, as well as for other subunit vaccines, particularly when a native protein conformation is required.

Abstract: 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.

Abstract: Over the last two decades most of the efforts in HIV vaccine development have been based on the use of the HIV Env with the goal to induce sterilizing immunity. However, as a result of Env variability disappointing results have been obtained in preclinical and phase III clinical trials. Although the objective of a preventive immunity still remains a priority, secondary endpoints (e.g. block of virus replication and disease onset) are being considered at the present as more achievable end-points in HIV vaccine development. This is based on accumulating evidence indicating that low viral load correlates with maintenance of immune functions and slow progression to disease, and that cell-mediated immunity plays a major protective role in the absence of sterilizing immunity. The promising results obtained in non-human primates with a vaccine based on a native Tat protein (B-clade), which is an early regulatory protein key for HIV replication and AIDS pathogenesis, highlights the importance of targeting the virus very early after infection. In particular, the immune response against Tat appears to modify the virus-host interactions at the very beginning of infection, thus containing the depletion of critical immune cells and the progression of infection. Moreover, since Tat targets and induces maturation of dendritic cells, has immunomodulatory activities and drives Th-1 and CTL responses, immunization with Tat may drive or increase these immune responses also against other HIV antigens to support an effective, long-lasting and hopefully even sterilizing antiviral immunity. Finally, Tat B-clade is similarly recognized by sera from individuals infected by different virus clades (A, B, C, D) supporting the concept of a cross-clade vaccine. Therefore, the Tat-vaccine should contain virus replication protecting from disease progression (non-sterilizing immunity) or even favoring an abortive infection. Although only a phase III clinical trial will establish the efficacy of this vaccine strategy, the Tat-vaccine has recently entered preventive and therapeutic phase I clinical testing in Italy to establish safety (primary-end-point) and immunogenicity (secondary end-point) and phase II studies are being prepared.

Abstract: Cytotoxic T cell responses are key to the control of intracellular pathogens including HIV-1. In particular, HIV-1 vaccines based on regulatory proteins, such as Tat, are aimed at controlling HIV-1 replication and at blocking disease development by inducing cytotoxic T cell responses. Naked DNA is capable of inducing such responses but it requires several inoculations of high amounts of DNA, and/or prime-boost regimens. Here, we show that a novel class of cationic block copolymers protect the DNA from DNAse I digestion, and improve DNA delivery to antigen-presenting cells (APCs) after intramuscular (i.m.) vaccination. In particular, three cationic block copolymers (K1, K2 and K5) were used to deliver the HIV-1 pCV-tat DNA vaccine in BALB/c mice. The results indicate that vaccination with a very low dose (1 microg) of pCV-tat delivered by the cationic block copolymer K2 is safe and, as compared to naked DNA (up to 30 microg), greatly increases the CTL response against Tat, which was detected in all animals in the absence or in the presence of re-stimulation.

Abstract: 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 10 microg 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 5 microg 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.

Abstract: 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.

Abstract: The human herpesvirus 8 (HHV-8) is a human gamma2-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.

Abstract: In order to improve the immunotherapeutical potential of H-Cys-Leu-Gly-Gly-Leu-Leu-Thr-Met-Val-OH (CLG) peptide, an Epstein-Barr virus (EBV) subdominant epitope derived from the membrane protein LMP2, we have synthesized and tested CLG analogues containing cis- and/or trans-4-aminocyclohexanecarboxylic acid (ACCA) replacing Gly-Gly and/or Thr-Met dipeptide units. All pseudopeptides were tested for metabolic stability and for their capacity to bind HLA-A2 molecules and to sensitize target cells to lysis. All new compounds exhibited higher enzymatic resistance compared to the original CLG and some trans-ACCA-derivatives were able to associate HLA-A2 and to efficiently stimulate CTL responses directed against the CLG natural epitope.

Abstract: EBV-infected cells and EBV-associated tumors may evade CTL recognition by defective antigen processing, resulting in poor presentation of CTL epitopes. Since the proteasome is the major source of MHC class I-presented peptides, we analyzed the effect of proteasome inhibitors on the expression of surface HLA class I and the generation of EBV-derived CTL epitopes presented by the HLA-A2 and HLA-A11 alleles. Treatment with covalent and reversible inhibitors of the proteasome partially reduced the total and allele-specific expression of surface HLA class I in EBV-carrying LCLs. HLA-A2 expression was also decreased by treatment with leupeptin and bestatin, while HLA-A11 expression was affected by treatment with phenanthroline. Despite their general inhibitory effect on HLA class I expression, all proteasome inhibitors tested enhanced the presentation of 2 subdominant HLA-A2 epitopes from EBV LMP1 and LMP2, while the presentation of the immunodominant HLA-A11-restricted epitope from EBNA4 was inhibited by MG132 and lactacystin and increased by ZL(3)VS. Treatment with ZL(3)VS restored the presentation of endogenously expressed EBNA4 in 1 HLA-A11-positive BL cell line. These findings suggest that specific inhibitors of the proteasome may be used to increase the antigenicity of virus-infected and malignant cells that are per se inefficient at generating particular CTL target epitopes.

Abstract: Copper complex formation equilibria of glycyl-L-histidyl-L-lysine (Gly-His-Lys, GHK) and of two synthetic analogues, where the histidine residue was replaced with a synthetic amino acid (L-spinacine or L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid), have been carefully investigated using different experimental techniques: potentiometry, solution calorimetry, UV-VIS spectrophotometry, circular dichroism and electron paramagnetic resonance spectroscopies. All the ligands formed complexes having different stoichiometries and stabilities; evidence for the formation of binuclear species is also shown. The structures of the main complexes are discussed. It is suggested that the lateral lysine amino group participates in complex formation, but only at alkaline pH values: at physiological pH this group is protonated and available for possible interactions with cellular receptors. The above tripeptides have been tested for their enzymatic stability in human serum: the synthetic compounds showed no significant degradation for at least 3 h. Finally, their activity as growth factor has been studied in vitro. The two synthetic analogues showed an activity comparable to or even higher than that of GHK, thus suggesting their possible use as additives in cell culture media, even in the presence of serum. Relevant information on the GHK action mechanism as cell growth factor has been obtained: the formation of copper complexes, driven by the first (Gly) residue, appears necessary while the second residue (His) does not appear to play a specific role; the presence of the free side chain of the third residue (Lys) appears to be of fundamental importance.

Abstract: Burkitt's lymphoma (BL) is a highly malignant B-cell tumour characterized by chromosomal translocations that constitutively activate the c-myc oncogene. Here we show that BL cells are resistant to apoptosis and do not accumulate ubiquitin conjugates in response to otherwise toxic doses of inhibitors of the proteasome. Deubiquitinating enzymes and the cytosolic subtilisin-like protease tripeptidylpeptidase II are upregulated in BLs, and could be rapidly induced by the overexpression of c-myc in normal B cells carrying oestrogen-driven recombinant Epstein-Barr virus. Apoptosis was induced by inhibiting tripeptidylpeptidase II, suggesting that the activity of this protease may be required for the survival of BL cells. We thus show that there is a regulatory link between c-myc activation and changes in proteolysis that may affect malignant transformation.

Abstract: H-Cys-Leu-Gly-Gly-Leu-Leu-Thr-Met-Val-OH (CLG) peptide is an EBV subdominant epitope that represents the target of HLA-A2 restricted CTL responses. The CLG peptide has low affinity for HLA-A2 and does not produce stable complexes, both factors that determine weak CTL responses. In contrast, the [Tyr(1), Ala(3)]CLG (YLA) analogue showed high affinity for HLA-A2 molecules and efficiently stimulated CLG-specific CTL precursors. Nevertheless, this modified epitope showed low enzymatic stability. To further improve the immunotherapeutical potential of this "improved epitope", we have synthesized and tested YLA analogues containing different modifications next to the scissile peptide bond. Among the analogues we found three peptides, with higher enzymatic resistance, that efficiently stimulate CTL responses. These peptides may be used for EBV-specific immunotherapies.

Abstract: The latent membrane protein 2 (LMP2) is expressed in EBV-associated tumours. LMP2 is a target of HLA-A2 restricted EBV-specific CTL responses and consequently it may represent a good target for specific CTL-based immunotherapies. However, the efficacy of such therapy is limited by the poor immunogenicity of the protein that induces weak cytotoxic T lymphocyte (CTL) responses directed against the CLGGLLTMV (CLG) epitope. Indeed, the CLG peptide presents low affinity for HLA-A2 and does not produce stable complexes. Therefore we synthesized and tested CLG-dimeric analogues with the purpose of characterizing new compounds with the capacity to bind HLA-A2 molecules. By these studies we have identified a few peptides which, compared to the natural epitope, showed higher affinity for HLA-A2 molecules and superior capacity to form a complex. These dimeric peptides may have the potential to induce efficient CTL responses directed to the natural epitope.

Abstract: Single amino acid substitutions at TCR contacts may transform a natural peptide Ag in CTL ligands with partial agonist, antagonist, or null activity. We obtained peptide variants by changing nonanchor amino acid residues involved in MHC class I binding. These peptides were derived from a subdominant HLA-A2-presented, latent membrane protein 2-derived epitope expressed in EBV-infected cells and in EBV-associated tumors. We found that small structural changes produced ligands with vastly different activities. In particular, the variants that associated more stably to HLA-A2/molecules did not activate any CTL function, behaving as null ligands. Interestingly, T cell stimulations performed with the combination of null ligands and the natural epitope produced significantly higher specific CTL reactivation than reactivation of CTLs induced by the wild-type epitope alone. In addition, these particular variants activated memory CTL responses in the presence of concentrations of natural epitope that per se did not induce T cell responses. We show here that null ligands increased ZAP-70 tyrosine kinase activation induced by the natural epitope. Our results demonstrate for the first time that particular peptide variants, apparently behaving as null ligands, interact with the TCR, showing a supra-agonist activity. These variant peptides did not affect the effector T cell functions activated by the natural epitope. Supra-agonist peptides represent the counterpart of antagonists and may have important applications in the development of therapeutic peptides.

Abstract: In the present study, we examined the structural requirements of peptide Ags for productive interactions with the TCR of CTL. For this purpose, we used as a model a previously identified immunodominant epitope that represents the target of EBV-specific HLA-A11-restricted CTL responses. By the use of peptides having minimal sequence homology with the wild-type epitope, we demonstrated that it is possible to selectively expand and reactivate memory CTL precursors without triggering the lytic mechanisms of wild-type specific effectors. In fact, stimulation of PBL from EBV-seropositive donors by polyalanine analogues, sharing only the putative TCR contact residue with the natural epitope, exclusively induced clonal expansion and reactivation of EBV-specific memory CTL precursors. Interestingly, these polyalanine peptides failed to trigger the cytotoxic function of CTLs specific for the wild-type viral epitope. This clearly indicates that reactivation of memory CTL precursors and triggering of the cytotoxic function have different requirements. The same phenomenon was observed using as stimulators naturally occurring peptides carrying the appropriate TCR contact residue. These data strongly suggest that cross-reactive peptides may play an important role in the expansion and reactivation of CTL clones from the memory T cell pool, and may be involved in long-term maintenance of T cell memory.

Abstract: The latent membrane protein 2 is an immunogenic antigen expressed in Epstein-Barr virus (EBV)-associated tumors and consequently it may represent a target for specific cytotoxic T lymphocyte (CTL)-based immunotherapies. However, the efficacy of such a therapy is limited by the poor immunogenicity of the protein that induces weak CTL responses directed to the CLGGLLTMV (CLG) epitope only in the minority of EBV-seropositive donors. We have now demonstrated that selective peptide stimulation of peripheral blood lymphocytes induced CLG-specific CTL in all donors, suggesting that this epitope can be a suitable target for specific immunotherapies. We found that the CLG peptide has a low affinity for HLA-A*0201 and does not produce stable complexes, both factors that are likely to determine the strength of CTL responses to this epitope. Therefore, we synthesized and tested CLG analogues carrying single or combined amino acid substitutions to increase HLA/peptide stability. Among the analogues tested we identified two peptides which, compared to the natural epitope, showed higher affinity for HLA-A*0201 molecules, and produced stable complexes. These peptides demonstrated a potent, specific stimulatory capacity and could be used for selective CTL-based therapies.

Abstract: Major histocompatibility complex (MHC)/peptide association and stability are determined by specific amino acid interactions between peptide antigens and the MHC groove, and are regarded as a critical feature in ensuring efficient monitoring by T cells. In this investigation we examined the relationship between MHC/peptide stability and the immunostimulatory capacity of MHC/peptide complexes. For this purpose we compared synthetic peptide analogues derived from the immunodominant HLA-A11-presented IVTDFSVIK (IVT) epitope, for their capacity to reactivate IVT-specific memory cytotoxic T-lymphocyte (CTL) responses. The analogues differentiated from the wild-type epitope by single amino acid substitution at position 2. All peptides showed similar affinity for HLA-A11 molecules and were recognized by IVT-specific CTL clones, but induced HLA-A11 complexes at the cell surface with different lifespan. This model offered the possibility of comparing the capacity of an immunogenic epitope to stimulate a unique population of T-cell precursors depending on the lifespan of its presentation at the cell surface. We demonstrated that stable HLA-A11/peptide complexes efficiently stimulate IVT-specific CTL responses, while HLA-A11/peptide complexes with short lifespan do not. The precise identification of the role of amino acid residues in the formation of stable MHC/peptide complexes may be relevant for the design of wild-type-derived epitopes with high immunogenicity. These analogues may have important applications in the immunotherapy of infectious diseases and immunogenic tumours.

Abstract: Peptides binding to HLA-A11 contain a hydrophobic or a small polar amino acid at position 2 and a lysine at the carboxy terminus. Synthetic peptides carrying natural and unnatural amino acids in position 2 were used to determine the requirements for formation of stable HLA-A11/peptide complexes. By kinetic analysis we demonstrate that a stereospecific interaction between the side chain residue in position 2 and a subsite of pocket B is required to obtain stable HLA/peptide complexes. This specific interaction is mediated by a methyl group or by an ethyl group bound to the asymmetric Cbeta atom with the correct configuration. Experiments performed with different peptide sequences suggest that the presence of adequate anchor residues may be sufficient to produce stable HLA/peptide complexes.

Abstract: The T cell receptor (TCR) repertoires of cytotoxic responses to the immunodominant and subdominant HLA A11-restricted epitopes in the Epstein-Barr virus (EBV) nuclear antigen-4 were investigated in four healthy virus carriers. The response to the subdominant epitope (EBNA4 399-408, designated AVF) was highly restricted with conserved Vbeta usage and identical length and amino acid motifs in the third complementarity-determining regions (CDR3), while a broad repertoire using different combinations of TCR-alpha/beta V and J segments and CDR3 regions was selected by the immunodominant epitope (EBNA4 416-424, designated IVT). Distinct patterns of interaction with the A11-peptide complex were revealed for each AVF- or IVT-specific TCR clonotype by alanine scanning mutagenesis analysis. Blocking of cytotoxic function by antibodies specific for the CD8 coreceptor indicated that, while AVF-specific TCRs are of high affinity, the oligoclonal response to the IVT epitope includes both low- and high-affinity TCRs. Thus, comparison of the memory response to two epitopes derived from the same viral antigen and presented through the same MHC class I allele suggests that immunodominance may correlate with the capacity to maintain a broad TCR repertoire.

Abstract: Polymorphonuclear neutrophils (PMNs), traditionally considered effector cells in the inflammatory response, have recently been regarded as potential regulators of the immune response. In the present study we investigate whether PMNs are efficient antigen-presenting cells for reactivation of memory cytotoxic T lymphocytes (CTLs). PMNs were pulsed with synthetic peptides derived from Epstein-Barr virus (EBV) antigens. We have used the IVTDFSVIK (IVT) peptide derived from the Epstein-Barr virus-encoded nuclear antigen 4 protein, corresponding to the immunodominant epitope of HLA-AII-restricted CTL responses, and the CLGGLLTMV (CLG) peptide derived from the latent membrane protein 2 antigen, representing a subdominant epitope of HLA-A2-restricted CTL responses. The data indicate that peptide-pulsed PMNs selectively activate specific CTL responses to both immunodominant and subdominant epitopes. The efficiency of CTL induction by PMNs was comparable to that observed with the conventional method of EBV-specific CTL reactivation with the autologous lymphoblastoid cell line, as well as with peptide-pulsed monocyte-enriched adherent cells. On the contrary, unactivated peptide-pulsed lymphocytes failed to induce an epitope-specific CTL response. These results demonstrate that PMNs efficiently present antigens to memory virus-specific CTLs and suggest that they may have a role as antigen-presenting cells.

Abstract: The capacity of MHC class I to protect target cells from NK is well established, but the mechanism by which these molecules influence NK recognition and the physical properties associated with this function remain poorly defined. We have examined this issue using as a model the HLA-A11 allele. HLA-A11 expression correlated with reduced susceptibility to NK and interferon-activated cytotoxicity in transfected sublines of the A11-defective Burkitt's lymphoma WW2-BL and the HLA class I A,B-null C1R cell line. Protection was also achieved by transfection of HLA-A11 in the peptide processing mutant T2 cells line (T2/A11), despite a very low expression of the transfected product at the cell surface. Induction of surface HLA-A11 by culture of T2/A11 cells at 26 degrees C or in the presence of beta 2m did not affect lysis, whereas NK sensitivity was restored by culture in the presence of HLA-All-binding synthetic peptides derived from viral or cellular proteins. Acid treatment rendered T2/A11 and C1R/A11 cells sensitive to lysis, but protection was restored after preincubation with peptide preparations derived from surface stripping of T2/A11 cells. Similar peptide preparations from T2 cells had no effect. The results suggest that NK protection is mediated by HLA-A11 molecules carrying a particular set of peptides that are translocated to the site of MHC class I assembly in the ER in a TAP-independent fashion.

Abstract: Cytotoxic T lymphocytes (CTL) recognize antigens as short peptides selected for presentation by their ability to bind to MHC class I molecules. Polyclonal Epstein-Barr virus (EBV)-specific memory CTL responses, reactivated from blood lymphocytes of HLA-A11-positive individuals by stimulation with the autologous EBV-transformed lymphoblastoid cell line (LCL), are often dominated by reactivites directed to the peptide epitope IVTDFSVIK (IVT), corresponding to amino acids 416-424 of EBV nuclear antigen-4 (EBNA4). We now report the selective activation of IVT-specific CTL by stimulation of lymphocytes with the corresponding synthetic peptide. A more than 10-fold increase in frequency of CTL clones with this specificity (from 8% to 96%) was obtained when the peptide was presented by HLA-A11-transfected T2 cells (T2/A11). Titration of synthetic peptide in cytotoxic assay demonstrated that clones activated under these conditions are as efficient as clones activated by conventional LCL stimulations. Induction of memory CTL responses required low surface density of MHC: peptide complexes, since reactivation was achieved by stimulation with T2/A11 cells pulsed with concentrations of peptide that are suboptimal for induction of target cell lysis. This protocol of activation revealed the presence of IVT-specific CTL precursors in a donor that failed to mount an IVT-specific response upon stimulation with the autologous B95.8 virus-transformed LCL. The results suggest that stimulation with synthetic peptide epitopes can be efficiently used for induction of memory CTL responses, and may be particularly helpful for the selective expansion of subdominant CTL specificities.

Abstract: Human neutrophils are able to kill in vitro colorectal carcinoma cell line SW11-16 coated with mAb 17-1A, but they are not cytotoxic towards a non-immunized tumour target. Neutrophil exposure to the inflammatory cytokine, IL-8, produces a significant increase in antibody-dependent cellular cytotoxicity, which is related to IL-8 concentration. Oxyradical production is one of the lytic mechanisms used by phagocytes, and IL-8 is shown to activate this function, which does not occur if neutrophils are pretreated with the protein kinase C inhibitor, staurosporine, but is increased by R59022, a dyacylglycerol kinase inhibitor. The IL-8 effect is mediated by protein kinase C, which is potentiated by the calcium flux induced by the interaction between antibody coating tumour target and Fc gamma RIII on effector cells, as previously demonstrated. Data suggest a possible new role for IL-8 in tumour surveillance.

Abstract: MHC class I antigens bind peptides derived from endogenous proteins and present them to cytotoxic T lymphocytes. This binding is selective and shows high allele specificity. Peptides binding to HLA-A11 contain a hydrophobic or a small polar amino acid at position 2 and a lysine at the carboxy terminus. Peptide analogues, derived from previously identified high affinity peptides and carrying amino acid substitutions in position 2, were used to determine the requirements for formation of stable HLA-A11/peptide complexes. By kinetic analysis we were able to discriminate among apparent and true binders. Only analogues carrying in position 2 the amino acids valine, threonine and isoleucine formed stable complexes with HLA-A11 with a half life > or = 72 hours.

Abstract: 17-1A is a murine monoclonal antibody (MAb) specific for the tumor-associated antigen CO17-1A on colorectal carcinoma cells. One of the tumor cell destruction mechanisms induced by in vivo immunotherapy with MAb17-1A has been claimed to be antibody-dependent cellular cytotoxicity (ADCC) by monocytes and NK cells. In the present study we investigated whether human neutrophils (PMN) could be involved in colorectal carcinoma cell lysis and whether IFN-gamma influences this function. We showed that neutrophils are capable of tumor lysis mediated by MAb17-1A, although to a lesser extent than are the mononuclear cells (PBMC). Neutrophil ADCC was, however, markedly increased in the presence of IFN-gamma. Enhancement by IFN-gamma was also observed for PBMC. ADCC by PMN required the binding of MAb17-1A to Fc gamma RIII (CD16) since anti-Fc gamma RIII MAbs efficiently blocked tumor cell lysis. In contrast, in the presence of IFN-gamma the neutralization of Fc gamma RIII did not affect MAb17-1A-mediated cytotoxicity, suggesting that receptors other than Fc gamma RIII were involved in the process. PBMC cytotoxicity was also inhibited by anti-CD16 antibodies but IFN-gamma did not overcome this effect. Finally, the scavenger enzymes superoxide dismutase and catalase did not block ADCC by PMN or PBMC, indicating that oxidants are not key factors in MAb17-1A-mediated lysis; however, in IFN-gamma-activated PMN the oxygen-dependent mechanism was in part involved in tumor lysis.

Abstract: Epstein-Barr virus (EBV), a ubiquitous herpesvirus, induces potent HLA class I-restricted cytotoxic T-lymphocyte (CTL) responses. Analyses of target antigen choice have shown that the very strong CTL responses which are often observed through the HLA A11 allele map are due almost entirely to a single transformation-associated EBV protein, the nuclear antigen EBNA4. Here, we sought to determine the number and relative immunogenicities of HLA A11-restricted epitopes within this 938-amino-acid protein. An initial screening with a series of recombinant vaccinia virus vectors encoding progressively truncated forms of EBNA4 was followed by peptide sensitization experiments using overlapping 14- or 15-mers from the entire sequence. These two approaches allowed the identification of five epitope regions located between residues 101 and 115, 416 and 429, 396 and 410, 481 and 495, and 551 and 564 of the EBNA4 molecule. CTL preparations from all seven HLA A11-positive donors tested had demonstrable reactivities against the 416-to-429 peptide, whereas reactivities against the other epitopes either tended to be lost on serial passage or, for some of the donors, were never detected. The immunodominance of the 416-to-429 epitope was further supported by peptide dilution assays using polyclonal effectors and by CTL cloning experiments. Analysis of the 416-to-429 region identified the nanomer 416-424 (IVTDFSVIK) as the cognate peptide. This peptide was able to sensitize targets to lysis by A11-restricted CTL clones at concentrations as low as 5 x 10(-14) M.

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Abstract: T lymphocytes recognize their antigenic targets as peptides associated with major histocompatibility complex molecules. The HLA-A11 allele, a preferred restriction element for Epstein-Barr virus (EBV)-specific cytotoxic T-lymphocyte responses, presents an immunodominant epitope derived from the EBV nuclear antigen 4. Subpicomolar concentrations of a synthetic nonamer peptide, IVTDFSVIK, corresponding to amino acids 416-424 of the EBV nuclear antigen 4 sequence, can sensitize phytohemagglutinin-stimulated blasts to lysis by EBV-specific HLA-A11-restricted cytotoxic T-lymphocytes. We show that micromolar concentrations of this peptide induce assembly and surface expression of HLA-A11 in an A11-transfected subline of the peptide transporter mutant cell line T2. Using the IVTDFSVIK peptide and a series of synthetic nonamer peptides, differing from the original sequence by single amino acid substitutions, we have defined a motif for HLA-A11-binding peptides. This predicts the presence of a hydrophobic amino acid in position 2, amino acids with small side chains in positions 3 and 6, and a lysine in position 9. Using this motif, we have identified a peptide in the carboxyl-terminal end of wild-type p53, ELNEALELK, which is able to induce HLA-A11 assembly as efficiently as the IVTDFSVIK viral peptide.

Abstract: Cytotoxic T lymphocytes (CTLs) control viral infections by recognizing viral peptides presented by major histocompatibility complex (MHC) class I molecules. Human leukocyte antigen (HLA)-A11-restricted CTLs that recognize peptide residues 416 to 424 of the Epstein-Barr virus (EBV) nuclear antigen-4 frequently dominate EBV-induced responses in A11+ Caucasian donors. This epitope is conserved in type A EBV strains from Caucasians and central African populations, where A11 is relatively infrequent. However, strains from highly A11+ populations in New Guinea carry a lysine-to-threonine mutation at residue 424 that abrogates CTL recognition and binding of the peptide to nascent A11 molecules. The results suggest that evolution of a widespread and genetically stable virus such as EBV is influenced by pressure from MHC-restricted CTL responses.

Abstract: Platelets activated with physiological agonists, such as thrombin, ADP, or collagen, released products able to modulate neutrophil functions. In particular, platelet supernatant contained an inhibitor of superoxide anion generation induced by phorbol ester and a chemotactic factor for human neutrophils. The proteolytic digestion of platelet supernatant completely abrogated chemotactic activity without interfering with the inhibitory effect, indicating the presence of different molecules involved in the modulation of different neutrophil functions. This was further confirmed by the pretreatment of platelets with aromatic benzamidine which abolished chemotactic activity, but did not affect superoxide inhibition of neutrophils. This report provides evidence for interaction of platelets and inflammatory cells, suggesting that platelets are able to induce accumulation of neutrophils and control their respiratory burst, which also has a critical role in tissue damaging in inflammation.

Abstract: The CD69 glycoprotein is an early activation antigen of T and B lymphocytes and it is constitutively expressed on thymocytes and platelets. Here we report its presence on neutrophils and on bone marrow-derived myeloid precursors. Indeed, promyelocytic cells are CD69+ on the cell membrane, while in resting neutrophils this molecule is located inside the cell. However, intracellular CD69 molecules are rapidly mobilized to the cell surface upon activation by PMA or fMLP. This translocation is independent on a new protein synthesis, as it is not inhibited by cycloheximide; furthermore, CD69 molecules are likely stored in a trans-Golgi structure since their expression is not affected by brefeldin A, a drug that blocks molecular trafficking from ER to Golgi vesicles. Immunoprecipitation of CD69 molecules either from activated neutrophils or from bone marrow cells showed that this protein has the same molecular size (28-34 kDa) as observed in platelets, T and B lymphocytes, and thymocytes. This similarity is reflected also in the functional role played by this molecule: in neutrophils as well as in lymphocytes and platelets, CD69 stimulation induced Ca2+ influx through cellular membrane; furthermore, the perturbation of the CD69 antigen on PMA-activated neutrophils enhances the lysozyme release, suggesting a role of this molecule in the regulation of granule exocytosis, probably through a Ca(2+)-dependent mechanism.

Abstract: Epstein-Barr virus (EBV) positive Burkitt's lymphoma (BL) cells are markedly less sensitive to EBV-specific cytotoxic T cell (CTL) recognition than EBV-transformed lymphoblastoid cell lines of normal B cell origin. Three features of the BL cell phenotype might contribute to this reduced susceptibility: (i) low expression of cell adhesion molecules, (ii) low expression of HLA class I and selective down-regulation of particular alleles, and (iii) down-regulation of all transformation-associated EBV antigens except EBV-encoded nuclear antigen (EBNA)-1. This study assesses the individual importance of each of these features for immune escape. For this purpose the WW1-BL cell line was used which expresses all the known transformation-associated EBV antigens (EBNA-1 to -6 and latent membrane protein-1 and -2) but which is negative for HLA A11 and for the adhesion molecule leukocyte function associated antigen-3 (LFA-3). Using recombinant vectors, these deficiencies have been sequentially corrected and the cells have been tested for sensitivity to EBV (B95.8 strain)-induced CTL preparations recognizing epitope(s) of EBNA-4 in the context of HLA A11. Expression of HLA A11 alone or in combination with LFA-3 did not sensitize WW1-BL cells to these effectors. Lysis was only achieved when HLA A11 was co-expressed with the B95.8 virus-encoded EBNA-4 protein, and in these circumstances sensitization did not require LFA-3. These results indicate that reconstitution of the relevant HLA-EBV epitope target complex on the cell membrane is sufficient to render BL cells sensitive to virus-specific cytolysis. The requirement for EBNA-4 reconstitution to achieve lysis of the WW1-BL/A11 transfectant suggested that the resident WW1 virus-encoded EBNA-4 protein did not contain the relevant target epitope for HLA A11-restricted recognition. This was confirmed by transferring the WW1 virus isolate into another A11-positive B cell background.

Abstract: Evasion from cytotoxic T-lymphocyte (CTL) surveillance may be an important step in the pathogenesis of Epstein-Barr virus (EBV)-carrying Burkitt lymphoma (BL) as suggested by the consistent down-regulation of all transformation-associated viral antigens, except EBV nuclear antigen 1 (EBNA-1), and of certain HLA class I alleles in BL biopsies and cell lines that maintain the tumor cell phenotype in vitro. The most common HLA class I defect recorded in BL lines is a selective down-regulation of HLA-A11. To gain some insight into the role of HLA-A11 down-regulation in pathogenesis of BL, we have investigated the target specificity of HLA-A11-restricted CTLs derived by stimulation of lymphocytes from three EBV-seropositive individuals with autologous EBV-transformed lymphoblastoid cell lines. Recombinant vaccinia viruses carrying the coding sequences for EBNA-1, -2A, -2B, -5, -3, -4, and -6 (also known as EBNA-1, -2A, -2B, -LP, -3a, -3b, and -3c, respectively) and EBV latent membrane protein 1 were used to induce high levels of expression of the relevant EBV antigen in fibroblasts derived from HLA class I-matched individuals. EBNA-4-expressing fibroblasts were the predominant target of HLA-A11-restricted CTLs in all three donors. A less pronounced and less regular EBNA-6-specific cytotoxic component was found in two of the donors.

Abstract: The expression of the complement receptor CR1 has been evaluated using an immunoalkaline phosphatase staining method on peripheral blood neutrophils and granulocyte precursors from 22 patients with chronic myeloid leukaemia (CML) and 15 healthy subjects. The immunocytochemical labelling pattern of CR1 was evaluated semiquantitatively on cell smears using three different anti-CR1 monoclonal antibodies. The scoring method showed that seven patients with CML had a marked reduction in CR1 expression which did not change with in vitro stimulation of neutrophils with phorbol-myristate-acetate (PMA) whereas control cells responded to PMA, increasing the receptor level two-fold. In addition, functional analysis of neutrophils with low CR1 expression from CML patients showed a very low cytolytic activity against K562 tumour target, suggesting a relationship between the cellular content of CR1 and neutrophil tumouricidal activity. The involvement of CR1 in neutrophil-mediated lysis is consistent with complete lack of tumour toxicity following receptor neutralization by anti-CR1 monoclonal antibodies. Interferon therapy improved CR1 expression and the cytolytic response of neutrophils in three out of five CML patients with a moderately low CR1 score. CML patients non-responding to interferon therapy and those with a very low CR1 score, independent of the clinical stage, progressed more rapidly into the advanced clinical stage and blastic crisis.

Abstract: HLA B51 specificity is strongly associated with Behcet's disease (BD) (for references see Baricordi et al., 1986), a multisystem vasculitis of unknown aetiology, for which an immunological pathogenesis has been proposed (O'Duffy et al., 1983; O'Duffy et al., 1990). Neutrophil abnormalities observed in BD patients even during clinical remission suggest prominent involvement of these phagocytic cells in the pathogenesis of the disease (Niwa et al., 1982). In order to clarify how HLA B51 antigen might confer susceptibility to BD, we have investigated neutrophil function in 13 B51-positive and 13 B51-negative healthy subjects. Lymphocyte spontaneous proliferation and circulating immune complexes were also evaluated. Whereas neutrophils from B51-positive subjects showed an increase in the chemotactic response toward casein (P = 0.003) and LPS (P = 0.033) and also in the PMA-induced superoxide production (P = 0.008) no evidence of enhanced lymphocyte activation emerged. These results suggest that the HLA region can exert a regulatory control on PMN functions.

Abstract: Previous studies have suggested that phorbol ester-activated neutrophils kill both antibody non-coated and antibody-coated K562 target cells. In this report the contribution of the receptors Fc gamma III (CD16) and CR3 (CD11b/CD18) in the lytic process was investigated. In neutrophils CD16 and CR3 are up-regulated by the phorbol ester up to 4 and 10 times, respectively. As expected, lysis of non-immunized K562 targets is not affected by the treatment of neutrophils with anti CD16, AB8.28, whereas lysis of immunized targets is decreased by 50%. In addition, the interaction of CD16 and AB8.28 induces calcium mobilization and increases granule secretion. Surprisingly, the simultaneous binding of AB8.28 and anti-CR3 OKM1 to neutrophils completely abolishes the lysis of antibody-coated targets. Unlike CD16, CR3 does not possess a functional role and binding of OKM1 to CR3 does not affect cytotoxicity of immunized K562 targets, but it blocks lysis of non-coated target almost completely, indicating a function as adhesion protein for CR3. These studies demonstrate a distinct role of CD16 and CR3 in mediating antibody-dependent and antibody-independent cellular cytotoxicity, respectively.

Abstract: Acquired immunodeficiency syndrome (AIDS) is initiated by the attachment of the human immunodeficiency virus (HIV) to a surface glycoprotein CD4 present on T4 helper/inducer lymphocytes, monocytes/macrophages and other cells. A simple octapeptide (H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, peptide T) seems to inhibit HIV infectivity and to activate human monocyte chemotaxis. In order to study in vitro metabolic stability and structure-activity relationships, peptide T and a number of analogues were prepared and tested on human monocytes by chemotactic assay. Peptide T and the shorter fragments T(3-8)-OH and T(4-8)-OH displayed potent bioactivity (maximal chemotactic activity in the range 10(-11)-10(-10) M). The C-terminal heptapeptide showed a reduction of potency, while further truncations at N-terminus of T(4-8)-OH abolished the biological action. In the octapeptide series, whereas the alpha-amino butyric acid (Abu) substitution for Thr4 was well tolerated, the same "slight" structural change at Thr5 or Thr8 was very detrimental. Finally, [D-Asn6]T(1-8)-OH analogue has low chemotactic activity. All these results indicate that i) the C-terminal pentapeptide is the minimum sequence required for bioactivity, ii) residues 5 to 8 appear to play a crucial biological role, iii) peptide T chemotaxis is mediated, at least in part, through the polar properties of Thr side chains at the critical positions 5 and 8, while the Thr4 does not interfere with biological characteristics of peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: The octapeptide sequence of peptide T is contained within the envelope of HIV and seems to mediate the viral binding to CD4 expressing cells, including monocytes. The biological activity of the alpha-aminobutyric acid pentapeptide derived from the C-terminal sequence of peptide T, in which the polar side chain of threonine in position 4 is substituted by a hydrophilic group, is measured by the monocyte chemotaxis assay. The chemotactic activity of human monocytes is assessed by determining the concentration at which the pentapeptide analog is maximally active and the effectiveness at that concentration, in comparison with peptide T and two shorter homologs, the pentapeptide and tetrapeptide. These experiments suggest that the synthetic analog is a potent chemotactic factor active at picomolar concentrations and that it competes with peptide T for the monocyte binding site.

Abstract: We have investigated the motility, superoxide anion production and tumor cell lysis of neutrophils from five patients affected by hairy cell leukemia. Random locomotion and chemotaxis towards denaturated casein and activated serum was normal as well as the superoxide production by opsonized zymosan. In contrast, chemotaxis and superoxide generation induced by FMLP and TPA were markedly reduced. The low responsiveness of neutrophils to TPA was also observed by evaluating cell lysis, against either non-immunized K562 target cells or antibody-coated tumor targets. In hairy cell leukemia neutrophils showed a selective reduced response to TPA and FMLP that, directly or indirectly, activate PKC, suggesting an impairment in the system of signal transduction.

Abstract: Human neutrophils can damage the non-immunized K562 cell line when stimulated by phorbol myristate acetate (PMA). The combination of the activation of protein kinase C by phorbol and the increase of free intracellular calcium by ionophore potentiates the lytic reaction. The OKT9-immunized target cells are not able to induce by themselves the lytic response in neutrophils, but by combining the two signals, the antigenic stimulus and PMA, a high level of cytolytic response is attained. The addition of EGTA does not affect neutrophil cytotoxicity against antibody-coated targets, while it markedly reduced the lytic reaction against non-immunized targets; in contrast, the addition of EGTA together with the ionophore ionomycin completely suppresses the lysis of immunized and non-immunized targets. The treatment of neutrophils with the protein kinase C inhibitor H-7 causes a dose-related inhibition of the lytic functions that is greater on unsensitized K562. Thus the interaction of Fc receptors with immunized targets is required for reaching the maximal cytolysis. The enhanced lytic activity that occurs in the presence of immunized targets is mediated by calcium flux, as detected by using the monoclonal antibody AB8.28 which binds to FcIII receptors (FcRIII), thus supporting that both signals are involved.

Abstract: Lymphocytes stimulated with alfa-interferon released a chemotactic factor for neutrophils. The process was not inhibited by cycloheximide, whereas mepacrine completely inhibited release of the chemotactic activity. The chemotactic factor was resistant to storage, heat treatment and proteolysis. Recombinant alfa-interferon did not stimulate lymphocytes to release a neutrophil chemotactic factor.

Abstract: Fifteen pentapeptide analogs of C-terminal fragment of peptide T, H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, were prepared and tested for human monocyte chemotaxis. Structure-activity studies suggest that the potent chemotactic activity of H-Thr-Thr-Asn-Tyr-Thr-OH is mediated through the polar properties of the C-terminal carboxyl group and Thr side chains at the critical positions 5 and 8, while the hydroxyl group of N-terminal Thr and its free amino function are not essential requirements for CD4 receptor interactions.

Abstract: The human erythroid myeloid leukaemia cell line K562 was used as target for human neutrophil cytotoxicity. Neutrophils demonstrated cytotoxicity against K562 only in the presence of a second stimulus, tetradecanoyl phorbol acetate (TPA), a result consistent with previous observations. We now demonstrate that antibody-coated K562 (using OKT9 and 345 monoclonal antibodies) are similarly only sensitive to neutrophils when TPA is added. The presence of both antibody and TPA in the cytotoxic assay resulted in significantly higher levels of cytotoxicity than in the absence of antibody; the result being consistent with a synergistic action between protein kinase C activation and Fc receptor perturbation in the neutrophil. The cytotoxicity against non-coated and antibody-coated targets was markedly inhibited, particularly against the former, by the protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine (H-7). There were marked differences in the extracellular calcium dependency of the two types of cytotoxicity reactions. TPA-activated respiratory burst was unaffected by the presence of non-coated and OKT9-coated targets, whereas TPA-induced lysosomal enzyme release was significantly increased by non-coated targets and a further increase occurred in the presence of OKT9-coated K562.

Abstract: We have studied the effect of the two antibodies, MF 25.1 and GF 26.7.3, on leucocyte function: these antibodies did not mimic the effect of cellular stimuli, but the pre-treatment of neutrophils with MF 25.1 and GF 26.7.3 modulated several cell activities. Both antibodies significantly reduced superoxide anion production in response to various stimuli. Despite similar cellular response MF 25.1 and GF 26.7.3 bound to different antigenic determinants, as shown both by immunoblotting studies and by treating neutrophils first with GF 26.7.3 and subsequently with MF 25.1, and vice-versa; the inhibition of superoxide production corresponded, in fact, to the total of that seen for each antibody. Moreover, in the absence of attractant, measures of locomotion of neutrophils treated with monoclonal antibody showed that 43% of the subjects responded to antibody binding by twofold activation of spontaneous movement. Chemotaxis and lysosomal enzyme release were not affected by antibody treatment.

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Abstract:

Abstract: Human neutrophils stimulated with phorbol myristate acetate were able to damage human erythroleukemic K562 cells, in the absence of specific antibody, as assessed by a two hour 51Cr release assay. Neutrophils treated with formyl-peptide fMet-Leu-Phe did not display tumoricidal response, but the addition of diacylglycerol kinase inhibitor R59022 together with formyl-peptide induced the cytotoxic capacity against tumor target cells. Phorbol ester is a potent activator of certain functions of neutrophils because of its ability to directly and irreversibly stimulate protein kinase C; formyl-peptide, on the contrary, activates protein kinase C by inducing a rapid and transient production of diacylglycerol, that is quickly metabolized. The addition of an inhibitor of diacylglycerol kinase, R59022, however potentiated the action of formyl-peptide. These results indicate that protein kinase C is involved in the tumoricidal activity of neutrophils against K562 cells, and that maximal activation of the enzyme is required to achieve the cytotoxic response.

Abstract:

Abstract: Monoclonal antibodies (mAbs) against cell surface antigens and receptors are instrumental in defining specific membrane markers. mAbs GF 26.7.3 and MF 25.1 against human neutrophils modulated the activation mechanism of superoxide anion production induced by formyl-peptide and PMA in all subject. However, treatment with mAb MF 25.1 of neutrophils from patients with rheumatoid arthritis did not have any effect. This may suggest that the antigen which MF 25.1 binds is absent in rheumatoid conditions. This confirms our previous data showing that defective expression of membrane components is associated with neutrophil dysfunction.

Abstract: Two analogs of chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine were examined for their capacity to activate several functions of human neutrophils. The C-terminus methyl ester derivative of the chemotactic peptide was found to possess strong biological activity and was able to induce levels of chemotaxis, enzyme secretion, and superoxide generation comparable to those observed with the same concentrations of N-formyl-L-methionyl-L-leucyl-L-phenylalanine. The analog containing a tert-butyloxycarbonyl group at the N-terminus, as well as the C-terminal methyl ester, was completely devoid of activity towards neutrophils. From these results, it appears that the free carboxyl group is not necessary for biological function. In contrast, the substituent at the N-terminus may play a critical role in the induction of the cellular response.

Abstract: Myelin basic protein, one of the major membrane protein component of the central nervous system, was used to probe the molecular mechanism of cellular activation. Pre-treatment of human neutrophils with myelin basic protein selectively inhibits the formyl-peptide-induced chemotaxis, without affecting chemotaxis evoked by casein and activated serum. Furthermore, both the degranulation and superoxide anion production stimulated by the chemotactic peptide are not modified by the prior treatment of the neutrophils with myelin basic protein.

Abstract: Treatment of human neutrophils with the two chemoattractants formyl-methyonyl-leucyl-phenylalanine and opsonized zymosan or with phorbol myristate acetate induce the production of superoxide anion (O-.2), which plays a role in the inflammatory reaction and microbicidal activity. The time-course of O-.2 release and the extent to which the anion is produced are related to the nature of the stimulating agents. Anti-inflammatory non steroid drugs, such as indomethacin and oxamethacin, exert a variable depression of the oxidative burst, which does not seem correlated to the inhibition of prostaglandin synthesis, but to the lipophilic character of the drug molecule.

Abstract: Two tripeptides, related to the chemotactic formyl-peptide, are tested for its ability to affect random and directional locomotion and to induce chemotaxis of polymorphonuclear leukocytes. The results indicate that the methyl ester of formyl-methyonyl-leucyl-phenylalanine possesses a biological activity towards human phagocytes, including a chemotactic potency, comparable to that of unmodified peptide. Therefore, the free carboxyl group does not seem essential to generate active leukoattractant. On the contrary, the replacement of the formyl group by the butyloxycarbonyl results in a drastic loss of biological activity. Our data may indicate that the two formyl-peptides interact with the same binding site on the cell membrane.