Roberto Tomatis

Effect of interferon-α 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‐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.

New features of the δ opioid receptor: Conformational properties of deltorphin I analogues

Deltorphin I is an opioid peptide of sequence H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, recently isolated from the skin of Phyllomedusa bicolor. Its enormous selectivity towards the delta opioid receptor and the similarity of the conformation of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-he-Gly-Tyr-Pro-Ser-NH2), a mu selective peptide, prompted the synthesis, biological evaluation and comparative conformational study of four analogs. A 1H-NMR study showed that the conformational preferences of the N-terminal sequences of all peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects in the interaction with the membrane and at the receptor site and of hydrophobicity of the C-terminal part, when structured in a folded conformation.

Interaction of deltorphin with opioid receptors: molecular determinants for affinity and selectivity.

Opioid receptor analyses of deltorphin A (H-Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) analogues indicated the following: (a) increased negativity differentially affected affinities (Ki) and selectivity (Ki mu/Ki delta); (b) shifted sequence heptapeptides, [Asp5,Leu6,Met-NH2(7)] and [Asp4,His5,Leu6,Met-NH2(7)], reversed selectivity (delta-->mu); (c) substitutions at positions 4, 5, and 6 diminished selectivity, with changes in residue 5 being the most detrimental; (d) C-terminal deletions differentially effected Ki. These are the first data to demonstrate a reversal of delta selectivity in heptapeptides containing a negative charge and indicate that modifications in affinity occur through changes in both anionic and hydrophobic properties of residues at specific positions in the peptide. Deltorphin analogues might also be applied to differentiate between opioid receptor subsites.

Conformational properties of deltorphin: new features of the δ-opioid receptor

Deltorphin is an opioid peptide with the sequence H‐Tyr‐D‐Met‐Phe‐His‐Leu‐Met‐Asp‐NH2, recently isolated from the skin of Phyllomedusa sauvagei. Its enormous selectivity towards the δ‐opioid receptor and the similarity of the N‐terminal part of the sequence with that of dermorphin (H‐Tyr‐D‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH2), a μ selective peptide isolated from the same natural source, prompted a comparative conformational study. A 1H‐NMR study in two different solvent systems showed that the conformational preferences of the N‐terminal sequences of the two peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects. Besides a general trend consistent with the role of the membrane in the preselection of the peptides, the present study demonstrates the crucial role played by charged residues in the interaction inside the receptors.

Polymorphonuclear neutrophils pulsed with synthetic peptides efficiently activate memory cytotoxic T lymphocytes

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‐A11–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. J. Leukoc. Biol. 60: 207–213; 1996.

Dermorphin inhibits spinal nociceptive flexion reflex in humans

Dermorphin (D) is a potent opiate-like peptide isolated from the skin of some species of frogs. Experimental studies in animals indicate that D has a potent antinociceptive effect, while no investigation exists about its analgesic properties in humans. Our study shows that i.v. infusion of 0.16 mg/kg D induces a marked and long-lasting increase in the threshold of nociceptive flexion reflex in healthy volunteers. This effect is also evident in a complete chronic spinal subject, showing that D depresses the nociceptive transmission mainly acting at spinal level. Naloxone, while fully antagonizing the effects of morphine and enkephalin analogue, is able to reverse only partly (ca. 50%) the depressive effect of D on nociceptive spinal reflex. This fact may suggest that D interacts with different spinal opiate receptor populations in inducing analgesia.

Selective amino acid substitutions of a subdominant Epstein-Barr virus LMP2-derived epitope increase HLA/peptide complex stability and immunogenicity: implications for immunotherapy of Epstein-Barr virus-associated malignancies

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.

Synthesis and activity of 3-pyridylamine ligands at central nicotinic receptors

A series of thirty 2-(3-pyridylaminomethyl)azetidine, pyrrolidine and piperidine analogues as nicotinic acetylcholine receptor (nAChR) ligands was explored. In general, pyrrolidinyl and many azetidinyl compounds were found to bind with enhanced affinity relative to the piperidines. In the three series, the parallel structural changes (stereochemistry, N-methylation and/or chloro substitution) do not consistently lead to parallel shifts in affinity. The more active compounds (K(i) affinity values ranging from 8.9 to 90 nM) were about as analgesic as nicotine in a tail-flick assay in mice after subcutaneous injections.

Reversed-phase HPLC study on the in vitro enzymic degradation of dermorphin

A high-performance liquid chromatographic (HPLC) method for the separation of the opioid heptapeptide dermorphin and related fragments has been developed. The chromatographic system was applied in the study of the kinetics of degradation of dermorphin (Der) in various tissues. Der was found to be extremely resistant to human and rat plasma (T 1/2 greater than 180 min). Upon incubation with homogenates of rat brains and kidneys, Der was cleaved with a half-life of 20.8 +/- 2.2 min and 2.4 +/- 0.3 min respectively. The catabolite formed was identified, in both tissues, as the N-terminal tetrapeptide H-Tyr-D-Ala-Phe-Gly-OH. The stability to rat kidney and brain of the N-terminal hexa- and pentapeptides and of the [4 psi 5, NHCO] Der analogue was also investigated. The nature of the enzyme systems involved in the in vitro degradations is discussed.

A 500 MHz study of peptide T in a DMSO solution

Peptide T, an octapeptide of sequence ASTTTNYT that binds to human T cells, was studied as a zwitterion in DMSOd6 solution by means of proton NMR spectroscopy at 500 MHz. The unusual dispersion of the resonances of residues of the same type (T) makes it possible to assign all resonances to specific residues by means of several 2D techniques. The non‐random nature of the conformation is substantiated by the observation of sequential nuclear Overhauser enhancements (NOEs). The low value of the temperature coefficient of the chemical shift of the NH of T8 and a diagnostic NOE between the NHs of T7 and T8 hint that a β‐turn including T5, N6, Y7 and T8 is a prominent conformational feature in solution. The ring current high field shifts of the methyl group and of the NH of T8 are consistent with an interaction with the side‐chain of Y7, favoured by the β‐turn.