Umbilical cord blood (UCB) is a source of hematopoietic progenitor cells and is used as an alternative to the bone marrow or peripheral blood for treatment of several onco-hematological diseases. Because of the limited number of CD34+ hematopoietic stem cells present in UCB units and of the elevated costs of cryopreservation, it is of paramount importance to select the UCB units that are clinically useful before storage and optimize banking efficiency by designing reliable procedures to process and freeze the selected units. Among the different parameters characterizing UCB, nucleated cell (NC) and CD34+ cell content provides useful criteria to select UCB units since clinical data documented that the infused cell load (both NC and CD34+ cells) plays an important role in the successful outcome of transplants. By evaluating volume, CD34+ cell content, NC total amount, and NC density of 117 UCB units, we found a significant association between CD34+ cell content and NC density and total amount, indicating these parameters as useful to decide UCB clinical utility. Furthermore, we set up a fast procedure to process UCB units for storage. A system for NC separation and volume reduction of UCB samples in a dedicated, germ-free, closed circuit was developed, where plasma and red blood cells (RBC) depletion was obtained by sedimentation in the presence of a 3.5% Polygeline solution. By this separation system, both RBC depletion and high NC and CD34+ cell recoveries were achieved in 60 min, and the yield was comparable to the one obtained by other separation methods. Since Polygeline has been clinically used as a plasma expander and no toxic effects on patients were reported, the protocol can be applied in the large-scale banking of UCB.

BMAP-28, a bovine antimicrobial peptide of the cathelicidin family, induces membrane permeabilization and death in human tumor cell lines and in activated, but not resting, human lymphocytes. In addition, we found that BMAP-28 causes depolarization of the inner mitochondrial membrane in single cells and in isolated mitochondria. The effect of the peptide was synergistic with that of Ca(2+) and inhibited by cyclosporine, suggesting that depolarization depends on opening of the mitochondrial permeability transition pore. The occurrence of a permeability transition was investigated on the basis of mitochondrial permeabilization to calcein and cytochrome c release. We show that BMAP-28 permeabilizes mitochondria to entrapped calcein in a cyclosporine-sensitive manner and that it releases cytochrome c in situ. Our results demonstrate that BMAP-28 is an inducer of the mitochondrial permeability transition pore and that its cytotoxic potential depends on its effects on mitochondrial permeability.

UCB ex-vivo expansion has been proposed as a method to increase the number of cells available for transplantation. We investigated total NCs, CD34+ and CD34+CD38- pluripotent non committed cell fold increase after ex-vivo expansion of CD34+ purified cells in the presence of different human growth factors combinations. Results show that the addition or the withdrawal of IL-3 to the early-acting cytokines flt3-L and TPO, is crucial to influence the total NC fold increase and the ratio of immature versus committed cells. We therefore propose the ezpansion of each UCB sample in two aliquots containing different cytokines; this protocol allows high NC proliferation as well as high number of non committed pluripotent cells in the final product.

An antibody was raised against a synthetic oligopeptide, corresponding to the 19–31 interval of the polypeptide chain of soluble lipoxygenase 1 (LOX 1) from soybean seeds. Cross-reactivity of this antibody towards proteins of a soybean cotyledon plasma membrane (PM) fraction was detected. The anti-LOX 1 antibody cross-reacted with a protein of approximately 94 kDa, when the membrane proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The same protein, separated in non-denaturing conditions, exhibited LOX activity with an optimum at pH 9.0. The cross-reactivity of the 94 kDa protein was maintained also in Na2CO3 or NaCl-washed membranes, confirming that the enzyme appears to be tightly bound. Finally, the protein, showing LOX activity, was separated by immunoprecipitation using the anti-LOX 1 antibody bound to protein A-Sepharose CL-4B. Again, reactivity with the protein of 94 kDa was detected. These findings show that isolated soybean PMs exhibit a LOX activity, which depends on the presence of the LOX 1 isoform. It is suggested that soluble LOX 1 may be in a dynamic equilibrium with a part of the enzyme, which appears to be linked to PMs.

To obtain long-term engraftment and hematopoiesis in myeloablated patients, the cell population used for hematopoietic reconstitution should include a sufficient number of early pluripotent hematopoietic stem cells (HSCs), along with committed cells from the various lineages. For this purpose, the small subset of CD34+ cells purified from different sources must be expanded ex vivo. Since cytokines may induce both proliferation and differentiation, expansion would provide a cell population comprising committed as well as uncommitted cells. Optimization of HSC expansion methods could be obtained by a combination of cytokines able to sustain renewal of pluripotent cells yet endowed with poor differentiation potential. We used variations of the combinations of cytokines describedby Brugger et al. [W. Brugger, S. Heimfels, R. J. Berenson, R. Mertelsmann, and L. Kanz (1995) N. Engl. J. Med. 333, 283–287] andPiacibello et al. [W. Piacibello, F. Sanavio, L. Garetto, A. Severino, D. Bergandi, J. Ferrario, F. Fagioli, M. Berger, and M. Aglietta (1997) Blood 89, 2644–2653] to expand UCB CD34+ cells and monitored proliferation rate and phenotype after 14 days of culture. Several hematopoietic lineage-associated surface antigens were evaluated. Our data show that flt3L and thrombopoietin in combination with IL-3, while sustaining a high CD34+ proliferation rate, provide a relatively low enrichment in very early uncommitted CD34+/CD38- cells. Conversely, in the absence of IL-3, they are less effective in inducing proliferation yet significantly increase the number of CD34+/CD38- cells. A combination of the above protocols, applied simultaneously to aliquots of the same sample, would allow expansion of both committed and pluripotent HSC. This strategy may represent a significant improvement for clinical applications.

To explore the effect of fluctuating glucose on endothelial cells, human umbilical vein endothelial cells were incubated for 14 days in media containing different glucose concentrations: 5 mmol/l, 20 mmol/l, or a daily alternating 5 or 20 mmol/l glucose. Apoptosis was studied by different methods: viability assay, cell cycle analysis, DNA fragmentation, and morphological analysis. Furthermore, the levels of Bcl-2 and Bax, well known proteins involved in apoptosis, were evaluated. Stable high glucose induced apoptosis in human umbilical vein endothelial cells, a phenomenon accompanied by a significant decrease of Bcl-2 and a simultaneous increase of Bax expression. However, apoptosis was enhanced in human umbilical vein endothelial cells exposed to intermittent, rather than constant, high glucose concentration. In this condition, Bcl-2 was not detectable, whereas Bax expression was significantly enhanced. These findings suggest that variability in glycemic control could be more deleterious to endothelial cells than a constant high concentration of glucose.

Cathelicidin-derived antimicrobial peptides are a component of the peptide-based host defense of neutrophils and epithelia, with a widespread distribution in mammals. We recently reported the cDNA sequences of three putative horse myeloid cathelicidins, named eCATH-1, -2, and -3. A Western analysis was performed to investigate their presence in neutrophils and processing to mature peptides. eCATH-2 and eCATH-3, but not eCATH-1, were found to be present in uncleaved forms in horse neutrophils. The corresponding mature peptides were detected in inflammatory sites, suggesting that processing of the propeptides takes place upon neutrophil activation. A functional characterization was then performed with synthetic eCATH peptides. Circular dichroism measurements indicated an amphipathic alpha-helical conformation of these peptides in an anisotropic environment, and in vitro assays revealed a potent activity and a broad spectrum of antimicrobial activity for eCATH-1 and a somewhat more restricted spectrum of activity for eCATH-2. Conversely, a strong dependence on salt concentration was observed when the activity of eCATH-3 was tested. This peptide efficiently killed bacteria and some fungal species, i.e., Cryptococcus neoformans and Rhodotorula rubra, in low-ionic-strength media, but the activity was inhibited in the presence of physiological salt medium. This behavior could be modified by modulating the amphipathicity of the molecule. In fact, the synthetic analogue LLK-eCATH-3, with a slightly modified sequence that increases the hydrophobic moment of the peptide, displayed a potent activity in physiological salt medium against the strains resistant to eCATH-3 under these conditions.

SMAP-29 is a cathelicidin-derived peptide deduced from sheep myeloid mRNA. The C-terminally amidated form of this peptide was chemically synthesized and shown to exert a potent antimicrobial activity. Antibiotic-resistant clinical isolates highly susceptible to this peptide include MRSA and VREF isolates, that are a major worldwide problem, and mucoid Pseudomonas aeruginosa associated with chronic respiratory inflammation in CF patients. In addition, SMAP-29 is also active against fungi, including Cryptococcus neoformans isolated from immunocompromised patients. SMAP-29 causes significant morphological alterations of the bacterial surfaces, as shown by scanning electron microscopy, and is also hemolytic against human, but not sheep erythrocytes. Its potent antimicrobial activity suggests that this peptide is an excellent candidate as a lead compound for the development of novel antiinfective agents.

Antimicrobial peptides are present in a wide range of species, from protozoa to man, as effector molecules of innate immunity. Several bovine precursors of antimicrobial peptides have recently been identified, as deduced from cDNA, and assigned to the cathelicidin family. Two of these are the proforms of the antimicrobial peptides BMAP-27 and BMAP-28, which share a similar amino acid sequence, structural conformation, and toxic activity toward several bacterial and fungal strains. Here we report that they are cytotoxic to human tumor cells and normal proliferating, but not resting, lymphocytes at concentrations comparable to those microbiocidal. This effect is primarily due to damage of plasma membrane integrity. A more detailed investigation of the U937 cell line revealed that a Ca2+ influx into the cytosol occurs in the early steps of permeabilization. The perturbation of the membrane structure and the Ca2+ influx are followed by programmed death. A similar apoptosis inducing effect is also observed on in vitro activated human lymphocytes.

Cathelicidins are a family of myeloid antimicrobial peptide precursors that have been identified in several mammalian species (Zanetti, M., Gennaro, R., and Romeo, D. (1995) FEBS Lett, 374, 1-5), Two novel bovine congeners have been deduced from cDNA, Their C-terminal sequences of 27 and 28 residues correspond to putative antimicrobial peptides with a cationic N-terminal region predicted to assume an amphipathic alpha-helical conformation followed by a hydrophobic C-terminal tail, Peptides corresponding to these sequences have been chemically synthesized and shown to exert a potent antimicrobial activity against Gram-negative and Gram positive bacteria, including methicillin-resistant Staphylococcus aureus, and fungi, Both peptides are also cytotoxic to human erythrocytes and neutrophils, although at higher than microbicidal concentrations, The target selectivity has been improved by synthesizing truncated analogues, comprising only the 18 N-terminal residues, which show a great reduction in cytotoxic, but not in antimicrobial activity, The involvement of the C-terminal hydrophobic tail in the cytotoxic activity has been further demonstrated by inducing a major loss of activity in an analogue after replacing highly hydrophobic residues with more hydrophilic ones.

During the last decade a variety of potent antimicrobial peptides have been isolated from both animals and plants. In contrast to the better known antibiotics, that are enzymatically synthesized in a complex fashion, the antimicrobial peptides are directly encoded by genes. Although all these peptides display a wide diversity in sequence, they show some common features such as a high content of basic residues and the tendency to adopt an amphipathic conformation. Most of the antibacterial peptides derive from proteolytic processing of larger precursor and can be grouped into families, based on their amino acid sequences and structural motifs. Recently, we have shown that in several antimicrobial peptide precursors from bovine neutrophils, a structurally varied C-terminal region, which displays antimicrobial activity after processing, is preceded by a highly conserved preproregion. In view of the presence of the highly conserved region, a study was undertaken to identify homologous precursors of novel antimicrobial peptides in porcine myeloid cells. By using a molecular biological approach based on amplification of cDNA ends containing the conserved nucleotide sequence, we have cloned the cDNAs of two known and three novel porcine members of this family. The novel polypeptides contain C-terminal regions which show diverse and unique sequences, ranging from 23 to 37 residues, with features consistent with antimicrobial activity. Peptides corresponding to these sequences have thus been chemically synthesized and shown to exert a potent in vitro antibacterial activity against gram-negative and gram-positive microorganisms.