Identification of CD8+ T cell epitopes within lytic antigens of human herpes virus 8.

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.

A 'pure' chemoattractant formylpeptide analogue triggers a specific signalling pathway in human neutrophil chemotaxis.

As it has not yet been established whether the second messengers involved in the neutrophil response have identical or specific signalling requirements for each physiological function, protein kinase C (PKC) isoforms and mitogen activated protein kinases (MAPKs) were studied in human chemotaxis triggered by the full agonist for-Met-Leu-Phe-OMe (fMLP-OMe) and the 'pure' chemoattractant for-Thp-Leu-Ain-OMe [Thp1,Ain3] analogue. Experiments were performed in the presence or absence of extracellular Ca2+, known to be an important modulator of second messengers. Our data demonstrate that specific PKC beta1 translocation and p38 MAPK phosphorylation are strongly associated with the chemotactic response of the neutrophils triggered by both peptides, while Ca2+ is not necessary for chemotaxis to occur. PKC and MAPK inhibitors were used in Western blotting assays and in cell locomotion experiments to investigate if the MAPK signalling pathway was controlled by PKC activation. The most important finding emerging from this study is that PKC and MAPK activate the chemotactic function of human neutrophils by two independent pathways.

N-Benzyl-2-chloroindole-3-carboxylic acids as potential anti-inflammatory agents. Synthesis and screening for the effects on human neutrophil functions and on COX1/COX2 activity.

The synthesis of N-benzyl-2-chloroindole-3-carboxylic acids related to indomethacin is reported. These compounds were tested on in vitro human neutrophil activation. Some of them, more soluble in water, were tested to define the influence on prostaglandin biosynthesis via inhibition of cyclooxygenases (COX1 and COX2) by a chemiluminescent method suitable for fast screening. Several derivatives showed inhibitory effects and in some cases were more active than the parent compound.

Formylpeptides trigger selective molecular pathways that are required in the physiological functions of human neutrophils.

For-Met-Delta(z)Leu-Phe-OMe ([Delta(z)Leu(2)]) is a conformationally restricted for-Met-Leu-Phe-OMe (fMLP-OMe) analogue able to discriminate between different responses of human neutrophils. In contrast, [Delta(z)Leu(2)] significantly activates the transduction pathways-involving Ca(2+), inositol phosphate, and cyclic AMP (cAMP) enhancement, as is the case with the full agonist fMLP-OMe. Here, we have studied the specific involvement of protein kinase C (PKC) isoforms and mitogen activated protein kinases (MAPKs) in the presence or absence of extracellular Ca(2+), being the cation clearly involved in the activation of neutrophils by fMLP. A strong correlation has been found between PKC isoforms, MAPKs and the selective physiological functions by [Delta(z)Leu(2)]-activated neutrophils. In a calcium-free condition, our data suggest that the failure of PKC beta1 translocation and of p38 MAPK phosphorylation by the analogue refers to its inability to induce chemotaxis, and that the failure by both fMLP-OMe and [Delta(z)Leu(2)] to evoke extracellular response kinase 1 and 2 (ERK1/2) phosphorylation would suggest a reduction in superoxide anion production.

Identification of cytotoxic T lymphocyte epitopes of human herpesvirus 8.

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.

Probing structural requirements of fMLP receptor: on the size of the hydrophobic pocket corresponding to residue 2 of the tripeptide.

The conformationally constrained f-L-Met-Ac(n)c-L-Phe-OMe (n = 4,9-12) tripeptides, analogues of the chemoattractant f-L-Met-L-Leu-L-Phe-OH, were synthesized in solution by classical methods and fully characterized. These compounds and the published f-L-Met-Xxx-L-Phe-OMe (Xxx = Aib and Ac(n)c where n = 3, 5-8) analogues were compared to determine the combined effect of backbone preferred conformation and side-chain bulkiness at position 2 on the relation of 3D-structure to biological activity. A conformational study of all the analogues was performed in solution by FT-IR absorption and 1H-NMR techniques. In parallel, each peptide was tested for its ability to induce chemotaxis, superoxide anion production and lysozyme secretion from human neutrophils. The biological and conformational data are discussed in relation to the proposed model of the chemotactic receptor on neutrophils, in particular of the hydrophobic pocket accommodating residue 2 of the tripeptide.

C- and N-terminal residue effect on peptide derivatives' antagonism toward the formyl-peptide receptor.

The biological action of several X-Phe-D-Leu-Phe-D-Leu-Z (X=3',5'-dimethylphenyl-ureido; Z=Phe, Lys, Glu, Tyr) analogues was analysed on human neutrophils to evaluate their ability to antagonize formyl-peptide receptors. X-Phe-D-Leu-Phe-D-Leu-Phe analogues obtained as C-terminal olo or amido derivatives and T-Phe-D-Leu-Phe-D-Leu-Phe analogues (T=thiazolyl-ureido) were also analysed. The activities of pentapeptide derivatives were compared with those of X-Phe-D-Leu-Phe-D-Leu-Phe chosen as reference antagonist. Our results demonstrate that X-Phe-D-Leu-Phe-D-Leu-Phe-olo, X-Phe-D-Leu-Phe-D-Leu-Glu and X-Phe-D-Leu-Phe-D-Leu-Tyr are more active antagonists than X-Phe-D-Leu-Phe-D-Leu-Phe. The presence of Lys (X-Phe-D-Leu-Phe-D-Leu-Lys) seems, instead, to inhibit the formyl-peptide receptor antagonist properties. The presence of the N-terminal thiazolyl-ureido group seems to considerably contribute to the receptor antagonist properties of T-Phe-D-Leu-Phe-D-Leu-Phe-OH. The introduction of the C-terminal methyl ester (T-Phe-D-Leu-Phe-D-Leu-Phe-OMe) or amido group (X-Phe-D-Leu-Phe-D-Leu-Phe-NH2) appears detrimental for the affinity and formyl-peptide receptor antagonist properties of the Phe-D-Leu-Phe-D-Leu-Phe derivatives. The examined peptides inhibit superoxide anion production and lysozyme release more efficaciously than neutrophil chemotaxis.

Effect of interferon-alpha therapy on epitope-specific cytotoxic T lymphocyte responses in hepatitis C virus-infected individuals.

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.