Esculentin‐1 is a potent anti‐microbial peptide present in minute amounts in skin secretions of Rana esculenta. It contains 46 amino‐acid residues and a C‐terminal disulfide bridge. We have explored ...the possibility of producing analogues of this peptide by recombinant expression in Escherichia coli of a fusion protein which is sequestered in inclusion bodies. The peptide of interest has been inserted at the N‐terminus of the protein, from which it can be released by cyanogen bromide cleavage. The anti‐microbial activities of the recombinant peptide as well as that of a mutant linear form devoid of the disulfide bridge are presented. The recombinant analogues retain the biological activity of the natural peptide, as tested with an inhibition zone assay against a variety of microorganisms. However, experiments on the rate of bacterial killing show that gram‐negative bacteria are more sensitive to the peptides than the gram‐positive bacterium, the effect of the cyclic peptide being in all cases faster than that of the linear molecule. Moreover, the activity against gram‐negative bacteria for both peptides is not affected by salts, whereas the activity against Staphylococcus aureus is lost at high salt concentration.
Umbilical cord blood (UCB) progenitor cells have been demonstrated to possess significant advantages over bone marrow (BM), in terms of proliferative capacity and immunologic reactivity. Therefore, ...UCB has been recently considered an attractive potential alternative to BM as a source of hematopoietic progenitor cells for clinical applications. Since several programs throughout the world are currently evaluating the feasibility of large-scale UCB banking for unrelated transplants, it was the aim of this study to evaluate whether cryopreservation procedures might heavily impair the clonogenic capacity, the feasibility of CD34+ selection and the ex vivo expansion potential of UCB progenitor cells. UCB samples were collected and cryopreserved as unseparated (n = 21) or mononuclear (MNC) cells (n = 15) within 12 h from delivery, and evaluated for viability, immunophenotype, cell and progenitor numbers after a minimum stay in liquid nitrogen of 6 months (range 6-14 months). Viability was always > 97% and no statistically significant difference was detected by flow cytometric analysis. Clonogenic recovery from unseparated cells was 80-87% for HPP-CFC, CFU-GEMM, BFU-E and CFU-GM, and from MNC cells ranged from 82 to 91% for LTC-IC, CFU-GEMM, BFU-E and CFU-GM. CD34+ selection (n = 8) was performed on fresh and cryopreserved MNC cells using the MiniMACS immunomagnetic separation device, showing no difference in yield (68 +/- 7% vs 57 +/- 4%, P < or = 0.4) or in purity (89 +/- 2% vs 81 +/- 6%, < or = 0.4), for fresh in comparison to cryopreserved MNC cells. After 14 days of liquid culture in the presence of different combinations of SCF, IL-3, IL-6 and G-CSF no statistically significant difference was detected in CFC fold-expansion for fresh or cryopreserved MNC cells and for CD34+ cells, either selected and cultured from fresh or cryopreserved MNC cells. In conclusion we can state that UCB is a potential source of primitive progenitor cells that can be cryopreserved unmanipulated or after physical separation without major losses in clonogenic capacity and immunophenotypic composition. Moreover, CD34+ selection from cryopreserved MNC cells is feasible and ex vivo expansion is not impaired. These results have important implications in the large scale UCB banking, in view of the potential applications of ex vivo expanded hematopoietic progenitor cells for the engraftment of adult patients.
In amphibian skin secretions, a peptidylprolyl
cis/trans isomerase activity was detected. A
Xenopus laevis skin cDNA coding for this protein was cloned, sequenced and over-expressed in
Escherichia ...coli. The primary structure of the protein shows extensive similarity with members of the cyclophilin A family. Catalytic parameters of the recombinant protein are similar to those of the human enzyme. The enzymatic activity is inhibited by cyclosporin A. Data suggesting that peptidylprolyl isomerization influences the biological activity of antibacterial peptides of amphibian origin are presented, and its putative role in the defence mechanism discussed.
A cDNA library from the skin of Rana temporaria has been screened using a cDNA fragment probe that encodes the signal peptide of the precursor of esculentin from the skin secretion of Rana esculentu. ...With this approach, the cDNAs encoding the precursors of three peptides were isolated. Subsequently, the peptides predicted from the sequence of the cloned cDNAs as well as several structurally related peptides could be isolated from the skin secretion of R. temporuria. These peptides, which were named temporins, have a length of 10–13 residues and show some sequence similarity to hemolytic peptides isolated from Vespu venom Argiolas, A. & Pisano, J. J. (1984) J. Biol. Chem. 259, 10106–10111. Natural and synthetic temporins have antibacterial activity against gram‐positive bacteria, but they are not hemolytic. Temporins are the smallest antibacterial peptides hitherto found in nature.
The aim of this multicenter randomized study was to compare conventional therapy with conventional plus high-dose therapy (HDT) and autologous bone marrow transplantation (ABMT) as front-line ...treatment for poor-prognosis non-Hodgkin's lymphoma (NHL).
Between October 1991 and June 1995, 124 patients, aged 15 to 60 years, with diffuse intermediate- to high-grade NHL (Working Formulation criteria), stages II bulky (> or = 10 cm), III, or IV were enrolled. Sixty-one patients were randomized to receive etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin (VACOP-B) for 12 weeks and cisplatin, cytarabine, and dexamethasone (DHAP) as a salvage regimen (arm A), and 63 to receive VACOP-B for 12 weeks plus HDT and ABMT (Arm B).
There was no significant difference in terms of complete remissions (CRS) in the two groups: 75% in arm A, and 73% in arm B. The median follow-up observation time was 42 months. The 6-year survival probability was 65% in both arms. There was no difference in disease-free survival (DFS) or progression-free survival (PFS) between the two groups. DFS was 60% and 80% (P = .1) and PFS was 48% and 60% (P = .4) for arms A and B, respectively. Procedure feasibility was the major problem. In arm B, 29% of enrolled patients did not undergo HDT and ABMT. A statistical improvement in terms of DFS (P = .008) and a favorable trend in terms of PFS (P = .08) for intermediate-/high- plus high-risk group patients assigned to HDT and ABMT was observed.
In this study, conventional chemotherapy followed by HDT and ABMT as front-line therapy seems no more successful than conventional treatment in terms of overall results. However, our results suggest that controlled studies of HDT plus ABMT should be proposed for higher risk patients.
Leishmaniasis encompasses a wide range of infections caused by the human parasitic protozoan species belonging to the Leishmania genus. It appears frequently as an opportunistic disease, especially ...in virus-infected immunodepressed people. Similarly to other pathogens, parasites became resistant to most of the first-line drugs. Therefore, there is an urgent need to develop antiparasitic agents with new modes of action. Gene-encoded antimicrobial peptides are promising candidates, but so far only a few of them have shown anti-protozoa activities. Here we found that temporins A and B, 13-amino acid antimicrobial peptides secreted from the skin of the European red frog Rana temporaria, display anti-Leishmania activity at micromolar concentrations, with no cytolytic activity against human erythrocytes. To the best of our knowledge, temporins represent the shortest natural peptides having the highest leishmanicidal activity and the lowest number of positively charged amino acids (a single lysine/arginine) and maintain biological function in serum. Their lethal mechanism involves plasma membrane permeation based on the following data. (i) They induce a rapid collapse of the plasma membrane potential. (ii) They induce the influx of the vital dye SYTOX™ Green. (iii) They reduce intracellular ATP levels. (iv) They severely damage the membrane of the parasite, as shown by transmission electron microscopy. Besides giving us basic important information, the unique properties of temporins, as well as their membranolytic effect, which should make it difficult for the pathogen to develop resistance, suggest them as potential candidates for the future design of antiparasitic drugs with a new mode of action.
Gene-encoded peptide antibiotics are widespread in insects, plants and vertebrates and confer protection against bacterial and fungal infections. NF-κB is an important transcription factor for many ...immunity-related mammalian proteins and also for insect immune genes. The activity of NF-κB is regulated by the interaction with an inhibitor, IκB. It was recently demonstrated that glucocorticoids induce the synthesis of IκB in human cell lines. So far, all genes for peptide antibiotics have promoter motifs with NF-κB binding sites, but its actual function in peptide regulation has been studied only in insects. Here we show that glucocorticoid treatment of the frog
Rana esculenta inhibits the transcription of all genes encoding antibacterial peptides by inducing the synthesis of IκBα. These results suggest that also in vertebrates peptide-mediated innate immunity is controlled by NF-κB-regulated transcription.
Chronic lymphocytic leukemia (CLL) is an indolent disease characterized by an abnormal proliferation of monoclonal lymphocytes in the bone marrow and lymphoid tissues. Most of the cases (> 90%) ...belong to the B-lymphocyte lineage and the course of the disease is variable depending on the presence of poor prognostic factors at diagnosis. Therefore optimal treatment is still questioned; presently there are no proven cures for CLL, but the natural history of the disease and the advanced age of the majority of patients makes prolongation of survival a reasonable therapeutic goal in most cases. The traditional therapeutic approach has been based on the activity of alkylating agents and corticosteroid, while patients resistant have been treated with nucleoside analogs. However, patients ultimately relapse and the choice of salvage therapy by conventional methods does not offer many chances. Particularly in younger patients, with poor prognostic factors, the therapeutic options may substantially change in the near future, based on alternative and innovative approaches aimed at achieving cure or long disease-free-survival. The results of high-dose therapy followed by reinfusion of hematopoietic stem cells, either from bone marrow or peripheral blood, will be presented and discussed.
Ten adult patients with Ph-positive chronic myelogenous leukemia (CML) received autologous transplantation using marrow treated ex vivo with mafosfamide. At transplant, 7 patients were in chronic ...phase (5 in first, 2 in second) and 3 in accelerated phase. The median time to achieve 500 x 10(6)/l neutrophils was 32 days (range 17-72 days). A platelet count of 20 x 10(9)/l was achieved at a median of 40 days (range 24-151 days). After transplant, cytogenetic analysis revealed 100% Ph-negative marrow metaphases in 6 of 9 analyzable patients with a median duration of Ph-negative hematopoiesis of 6.5 months. After a median follow-up of 16 months (range 3-31 months), five patients evolved into blast crisis, two died of non-hematological causes, one is Ph-negative in chronic phase at +4 and one is in chronic phase, but Ph-positive, at +22. In conclusion, this pilot study demonstrates that: (1) engraftment can occur from Ph-negative stem cells selected by mafosfamide, (2) mafosfamide purging may induce a transient period of Ph-negative hematopoiesis, and (3) modifications of the purging procedure and post-transplant manipulations of the immune-hematopoietic system are required to prolong cytogenetic remission.
Six novel 9,10-dihydrophenanthrene metabolites have been isolated from the aquatic plant Juncus effusus, together with the already known juncusol, juncunol and effusol. The structures of the ...compounds have been defined on spectroscopic grounds. Two of them showed a good antitumour activity.
PDF
