Methods to manufacture regulatory T cells for cell therapy.

Regulatory T cell (Treg ) therapy has shown promise in early clinical trials for treating graft-versus-host disease, transplant rejection and autoimmune disorders. A challenge has been to isolate sufficiently pure Tregs and expand them to a clinical dose. However, there has been considerable progress in the development and optimization of these methods, resulting in a variety of manufacturing protocols being tested in clinical trials. In this review, we summarize methods that have been used to manufacture Tregs for clinical trials, including the choice of cell source and protocols for cell isolation and expansion. We also discuss alternative culture or genome editing methods for modulating Treg specificity, function or stability that could be applied to future clinical manufacturing protocols to increase the efficacy of Treg therapy.

Comprehensive microRNA expression profiling of the hematopoietic hierarchy.

The hematopoietic system produces a large number of highly specialized cell types that are derived through a hierarchical differentiation process from a common stem cell population. miRNAs are critical players in orchestrating this differentiation. Here, we report the development and application of a high-throughput microfluidic real-time quantitative PCR (RT-qPCR) approach for generating global miRNA profiles for 27 phenotypically distinct cell populations isolated from normal adult mouse hematopoietic tissues. A total of 80,000 RT-qPCR assays were used to map the landscape of miRNA expression across the hematopoietic hierarchy, including rare progenitor and stem cell populations. We show that miRNA profiles allow for the direct inference of cell lineage relations and functional similarity. Our analysis reveals a close relatedness of the miRNA expression patterns in multipotent progenitors and stem cells, followed by a major reprogramming upon restriction of differentiation potential to a single lineage. The analysis of miRNA expression in single hematopoietic cells further demonstrates that miRNA expression is very tightly regulated within highly purified populations, underscoring the potential of single-cell miRNA profiling for assessing compartment heterogeneity.

Reciprocal modulation of adult beta cell maturity by activin A and follistatin.

AIMS/HYPOTHESIS: The functional maturity of pancreatic beta cells is impaired in diabetes mellitus. We sought to define factors that can influence adult beta cell maturation status and function. METHODS: MIN6 cells labelled with a Pdx1 monomeric red fluorescent protein-Ins1 enhanced green fluorescent protein dual reporter lentivirus were used to screen candidate growth and/or differentiation factors using image-based approaches with confirmation by real-time RT-PCR and assays of beta cell function using primary mouse islets. RESULTS: Activin A strikingly decreased the number of mature beta cells and increased the number of immature beta cells. While activins are critical for pancreatic morphogenesis, their role in adult beta cells remains controversial. In primary islets and MIN6 cells, activin A significantly decreased the expression of insulin and several genes associated with beta cell maturity (e.g. Pdx1, Mafa, Glut2 [also known as Slc2a2]). Genes found in immature beta cells (e.g. Mafb) tended to be upregulated by activin A. Insulin secretion was also reduced by activin A. In addition, activin A-treated MIN6 cells proliferated faster than non-treated cells. The effects of endogenous activin A on beta cells were completely reversed by exogenous follistatin. CONCLUSIONS/INTERPRETATION: These results suggest that autocrine and/or paracrine activin A signalling exerts a suppressive effect on adult beta cell maturation and function. Thus, the maturation state of adult beta cells can be modulated by external factors in culture. Interventions inhibiting activin or its signalling pathways may improve beta cell function. Understanding of maturation and plasticity of adult pancreatic tissue has significant implications for islet regeneration and for in vitro generation of functional beta cells.

Differential cytokine effects on primitive (CD34+CD38-) human hematopoietic cells: novel responses to Flt3-ligand and thrombopoietin.

A high proportion of the CD34+CD38- cells in normal human marrow are defined as long-term culture-initiating cells (LTC-IC) because they can proliferate and differentiate when co-cultured with cytokine-producing stromal feeder layers. In contrast, very few CD34+CD38- cells will divide in cytokine-containing methylcellulose and thus are not classifiable as direct colony-forming cells (CFC), although most can proliferate in serum-free liquid cultures containing certain soluble cytokines. Analysis of the effects of 16 cytokines on CD34+CD38- cells in the latter type of culture showed that Flt3-ligand (FL), Steel factor (SF), and interleukin (IL)-3 were both necessary and sufficient to obtain an approximately 30-fold amplification of the input LTC-IC population within 10 d. As single factors, only FL and thrombopoietin (TPO) stimulated a net increase in LTC-IC within 10 d. Interestingly, a significantly increased proportion of the CFC produced from the TPO-amplified LTC-IC were erythroid. Increases in the number of directly detectable CFC of > 500-fold were also obtainable within 10 d in serum-free cultures of CD34+CD38- cells. However, this required the presence of IL-6 and/or granulocyte/colony-stimulating factor and/or nerve growth factor beta in addition to FL, SF, and IL-3. Also, for this response, the most potent single-acting factor tested was IL-3, not FL. Identification of cytokine combinations that differentially stimulate primitive human hematopoietic cell self-renewal and lineage determination should facilitate analysis of the intracellular pathways that regulate these decisions as well as the development of improved ex vivo expansion and gene transfer protocols.

Mechanoreceptors in the Anterior Horn of the Equine Medial Meniscus: an Immunohistochemical Approach.

Lameness due to stifle and especially meniscal lesions is frequent in equine species. In humans, mechanoreceptors involved in proprioceptive function are well studied. Given the high incidence of meniscal injuries in horses, and the lack of information concerning them in equine menisci, our objective was to study these corpuscles in six healthy anterior horns of the equine medial meniscus, which is the most common localisation reported for equine meniscal injuries. Immunohistochemical stainings were performed using antibodies against high molecular weight neurofilaments and glial fibrillary acidic proteins. From a purely fundamental point of view, our work highlights for the first time the presence of Ruffini, Pacini and Golgi corpuscles in equine meniscus. They were found, isolated or in clusters and always located at the vicinity of blood vessels, at the level of the anterior horn of the equine medial meniscus. This morphological approach could serve as a basis for clinical studies, to evaluate the impact of these corpuscles on the poor sportive prognosis in equine meniscal tears.

Mammalian cell culture processes.

Over the past year, mammalian cell culture research has been aimed at investigating the influence of culture conditions on viability, productivity and the consistency of post-translational modifications. Studies of the effect of medium conditions and the development of kinetic models are being made in relation to current efforts to develop fed-batch strategies that will optimize recombinant protein production processes. Recent advances have included novel biosensor and bioreactor developments. New technologies have also been applied to investigate high cell density bioreactor and culture conditions.

Advances in hematopoietic stem cell culture.

Recent advances in our understanding of the earliest stages of hematopoietic cell differentiation, and how these may be manipulated under defined conditions in vitro, have set the stage for the development of robust bioprocess technology applicable to hematopoietic cells. Sensitive and specific assays now exist for measuring the frequency of hematopoietic stem cells with long-term in vivo repopulating activity from human as well as murine sources. The production of natural or engineered ligands through recombinant DNA and/or combinatorial chemistry strategies is providing new reagents for enhancing the productivity of hematopoietic cell cultures. Multifactorial and dose-response analyses have yielded new insight into the different types and concentrations of factors required to optimize the rate and the extent of amplification of specific subpopulations of primitive hematopoietic cells. In addition, the rate of cytokine depletion from the medium has also been found to be dependent on the types of cell present. The discovery of these cell-type-specific parameters affecting cytokine concentrations and responses has introduced a new level of complexity into the design of optimized hematopoietic bioprocess systems.

Characterization of collagen fibrils after equine suspensory ligament injury: an ultrastructural and biochemical approach.

Suspensory ligament (SL) injuries are an important cause of lameness in horses. The mechanical properties of connective tissue in normal and pathological ligaments are mainly related to fibril morphology, as well as collagen content and types. The purpose of this study was to evaluate, using biochemical and ultrastructural approaches, the alterations in collagen fibrils after injury. Eight Warmblood horses with visible signs of injury in only one forelimb SL were selected and specimens were examined by transmission electron microscope (TEM). Collagen types I, III and V were purified by differential salt precipitation after collagen extraction with acetic acid containing pepsin. TEM revealed abnormal organization as well as alterations in the diameter and shape of fibrils after SL injury. The bands corresponding to types I, III and V collagen were assessed by densitometry after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis indicated that the proportions of type III and type V collagen were higher (P < 0.001) in damaged tissues compared with normal tissues with a mean increase of 20.9% and 17.3%, respectively. Concurrently, a decrease (P < 0.001) in type I collagen within damaged tissues was recorded with a mean decrease of 15.2%. These alterations could be the hallmark of a decrease in the tissue quality and mechanical properties of the ligament. The findings provide new insight for subsequent research on tissue regeneration that may lead to the development of future treatment strategies for SL injury.

Cell cycle distribution of primitive haematopoietic cells stimulated in vitro and in vivo.

A novel approach is used to study the proliferating behaviour of primitive haematopoietic cell populations in response to different stimuli. A mathematical model based on the average proportion of apoptotic, dividing and quiescent cells in primitive haematopoietic cell populations is developed to describe the mitotic history of 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester-labelled cells. The cell cycle distributions in different cytokine-supplemented cultures of primitive human and mouse bone marrow cells are determined and compared with those found in vivo. The results indicate that a combination of flt-3 ligand, Steel factor and interleukin-11 or hyper-interleukin-6 provide a level of mitogenic stimulation similar to that existing in vivo after a myeloablative radiation dose. The comparison of the cell cycle distribution obtained for different cultures of human bone marrow CD34(+)(45RA/71)(-) cells demonstrates that the addition of flt-3 ligand in these cultures decreases apoptosis significantly but does not reduce quiescence. In addition, in vivo and in vitro, it was found that more than 3 days of stimulation are required to recruit a maximum number of quiescent cells into active cell cycle. These kinetics of cell cycle activation are found to be similar to those identified for the haematopoietic stem cells compartment in the same cultures. This mathematical analysis provides a useful tool for the development of haematopoietic stem cell culture processes for clinical applications.

The restriction mapping of c gene deletions in Streptomyces bacteriophage phi C31 and their use in cloning vector development.

In addition to 20 previously mapped restriction sites in the DNA of phi C31, we have determined eight sites for SphI, four for EcoRV, and two for SstII; there are none for BglII or SstI. Nine sites were in a 12-kb segment of DNA containing no previously mapped sites. Deletions causing clear-plaque morphology were located in this part of the DNA, in a 3-kb interval between an EcoRV and an SphI site at the centre of the DNA molecule. One of the deletions (delta C3) was obtained in a previously described phi C31c+::vph (viomycin phosphotransferase) derivative containing two PstI sites separated by 3.9-kb of inessential DNA. After in vitro PstI treatment, plaque-forming phages lacking the 3.9-kb fragment were obtained from the c+ phage but not from its delta C3 derivative. Thus a 36.2-kb genome, but not one of 34.4 kb, was able to give infectious virions. PstI-generated DNA fragments of up to 8 kb can be inserted in vitro into the delta C3 derivative with retention of the vph selective marker. With the insertion of a 6.03-kb PstI fragment of plasmid SCP2, the latter phage became a potential vector (with loss of vph) for BamHI-generated DNA fragments of up to 9 kb. In the course of this work, several ClaI sites in phi C31::pBR322 bifunctional replicons were shown to be lost when the DNA was propagated in a dam+ Escherichia coli strain. This will allow the use of such replicons for the cloning of ClaI-generated DNA fragments of up to 6.7 kb.

Hyperactivity of cathepsin B and other lysosomal enzymes in fibroblasts exposed to azithromycin, a dicationic macrolide antibiotic with exceptional tissue accumulation.

Azithromycin accumulates in lysosomes where it causes phospholipidosis. In homogenates prepared by sonication of fibroblasts incubated for 3 days with azithromycin (66 microM), the activities of sulfatase A, phospholipase A1, N-acetyl-beta-hexosaminidase and cathepsin B increased from 180 to 330%, but not those of 3 non-lysosomal enzymes. The level of cathepsin B mRNA was unaffected. The hyperactivity induced by azithromycin is non-reversible upon drug withdrawal, prevented by coincubation with cycloheximide, affects the Vmax but not the Km, and is not reproduced with gentamicin, another drug also causing lysosomal phospholipidosis. The data therefore suggest that azithromycin increases the level of lysosomal enzymes by a mechanism distinct from the stimulation of gene expression but requiring protein synthesis, and is not in direct relation to the lysosomal phospholipidosis.

Sodium lauryl sulfate, a microbicide effective against enveloped and nonenveloped viruses.

The number of individuals infected with human immunodeficiency virus (HIV) and other pathogens causing sexually transmitted diseases (STDs) is growing dramatically worldwide. Globally, heterosexual transmission may account for as much as 85-90% of new cases of HIV infection. Latex condoms represent an effective barrier against sexually transmitted pathogens, but unfortunately, their use is not generalized. Therefore, there is an urgent need to develop safe and potent topical microbicides under the control of women to efficiently reduce the spread of sexually transmitted infections. Sodium lauryl sulfate (SLS), an anionic surfactant with protein denaturing potency, is a potent inhibitor of the infectivity of several enveloped (Herpes simplex viruses, HIV-1, Semliki Forest virus) and nonenveloped (papillomaviruses, reovirus, rotavirus and poliovirus) viruses. The mechanism of action of SLS involves the solubilization of the viral envelope and/or the denaturation of envelope and/or capsid proteins. Studies have shown that SLS is not toxic for cultured cell lines of different origins at concentrations that inactivate HIV-1, herpes and human papillomavirus in vitro. In addition, intravaginal pretreatment of mice with a gel formulation containing SLS, completely protected animals against Herpes simplex virus type-2 infection. The gel formulation containing SLS was also well-tolerated following repeated intravaginal administrations to rabbits. Taken together, these data suggest that SLS represents a potential candidate for the use as a topical microbicide to prevent the sexual transmission of HIV-1, herpes, human papillomavirus and possibly other sexually transmitted pathogens. The impact of such a preventive tool on public health can be enormous.

Immobilized mammalian cell cultivation in hollow fiber bioreactors.

Cultivation of animal cells for the production of recombinant proteins is an important method for manufacturing complex proteins requiring posttranslational processing. One of the often considered methods for cultivation is by immobilization of the cells in hollow fiber bioreactors (HFBRs). These systems allow the cells to grow to high densities in a shear protected environment; furthermore the product can be accumulated in high concentration in the case of ultrafiltration HFBRs. Operation and scale-up are constrained by nutrient and product transport with oxygen transfer to growing cells being the most critical parameter. Mathematical models describing HFBRs have proved to be useful in quantitating and understanding the constraints and guiding the scale-up of this approach to animal cell cultivation.

Effect of substrate organization on the activity and on the mechanism of gentamicin-induced inhibition of rat liver lysosomal phospholipase A1.

Aminoglycoside antibiotics, such as gentamicin, induce a lysosomal phospholipidosis in the kidney cortex of experimental animals and humans. In vitro, gentamicin binds to negatively charged phospholipids, such as phosphatidylinositol, and decreases the activity of lysosomal phospholipases towards a neutral phospholipid (phosphatidylcholine) included in lipid vesicles. The mechanism of such an inhibition was not unequivocally established. On one hand Mingeot-Leclercq et al. (Biochem Pharmacol 37: 591-599, 1988) observed that the activity of phospholipase A1 is modulated by the negative charges of the bilayer and that the inhibitory potency of gentamicin is inversely related to the phosphatidylinositol content of the vesicles, and therefore proposed that inhibition is due to charge neutralization. On the other hand, Hostetler and Jellison (J Pharmacol Exp Ther 254: 188-191, 1990) observed that the activity of phospholipase A1 is not modulated by the negative charges of the vesicles and that the inhibitory potency of gentamicin is directly related to the phosphatidylinositol content of the bilayer, and therefore proposed that inhibition is due to substrate depletion. However, the experimental designs of these two models differed in several respects such as the source (liver versus kidney) and nature of the enzyme (native lysosomal extract versus purified delipidated phospholipase A1), and the composition of lipid vesicles (those containing constant amounts of phosphatidylcholine and cholesterol, and inversely varying amounts of phosphatidylinositol and sphingomyelin versus those containing inversely related amounts of phosphatidylcholine and phosphatidylinositol only). In order to assess the nature of the differences between these models, we compared the activity of phospholipase A1 and its inhibition by gentamicin using only one source of enzyme, the rat liver lysosomal extract, and the two types of lipid vesicles as used in the above models. Our results showed that both models are true within the frame work of their respective experimental designs. However, since the composition of the lipid vesicles as well as the nature of the enzyme preparation (whole lysosomal extract) in the "charge neutralization" model is closer to in vivo conditions, we suggest that this model may be more relevant to the in vivo situation.

Effect of acidic phospholipids on the activity of lysosomal phospholipases and on their inhibition by aminoglycoside antibiotics--I. Biochemical analysis.

Aminoglycoside antibiotics accumulate in lysosomes of kidney and cultured cells and cause an impairment of phospholipid catabolism which is considered to be an early and significant step in the development of their toxicity. Using liposomes, wer previously demonstrated that the activity of lysosomal phospholipases A1 and A2 towards phosphatidylcholine was markedly enhanced by the inclusion of phosphatidylinositol in the bilayer, and that gentamicin impaired this activity by binding to phosphatidylinositol. Since gentamicin-induced inhibition was inversely related to the amount of phosphatidylinositol included in the liposomes, we proposed that gentamicin impairs activity of phospholipases by decreasing the quantity of available negative charges carried by the bilayer surface (Mingeot-Leclercq et al., Biochem Pharmacol 37: 591-599, 1988). We now extend these observations to phosphatidylserine and phosphatidic acid, and compare the inhibition caused by gentamicin, amikacin and streptomycin towards lysosomal phospholipases on the hydrolysis of phosphatidylcholine in the presence of each of these acidic phospholipids. Inclusion of phosphatidic acid in liposomes, and, to a lesser extent, phosphatidylserine, caused a larger increase in phospholipases activity than phosphatidylinositol. In parallel, the three aminoglycosides tested were found less inhibitory towards phospholipases activity measured on phosphatidic acid-or phosphatidylserine-containing liposomes than was previously observed with phosphatidylinositol, even though equilibrium dialysis experiments failed to demonstrate significant difference in binding parameters of the drug towards each of these liposomes populations. Yet, as for phosphatidylinositol-containing liposomes, the inhibition was inversely related to the amount of phosphatidic acid or phosphatidylserine included in the bilayer and the inhibitory potency of the three drugs was consistently gentamicin greater than amikacin greater than streptomycin with the three types of negatively-charged liposomes used. We conclude that impairment of lysosomal phospholipases activity towards phosphatidylcholine included in negatively-charged membranes by aminoglycoside antibiotics is dependent upon drug binding to the bilayer, but that it is modulated by the nature of the acidic phospholipid that binds the drug as well as by that of the drug itself. A companion paper (Mingeot-Leclercq et al., Biochem Pharmacol 40: 499-506, 1990) will examine by computer-aided conformational analysis the parameters (drug-phospholipid energy of interaction, position of the drug in a monolayer and its accessibility to the aqueous phase) which may be important for these effects.

Effect of acidic phospholipids on the activity of lysosomal phospholipases and on their inhibition induced by aminoglycoside antibiotics--II. Conformational analysis.

In a companion paper (Mingeot-Leclercq et al. Biochem Pharmacol 40: 489-497, 1990), we showed that the inhibitory potency of gentamicin on the activity of lysosomal phospholipases, measured towards phosphatidylcholine included in negatively-charged liposomes, is markedly influenced by the nature of the acidic phospholipid used (phosphatidylinositol, phosphatidylserine, phosphatidic acid), whereas the binding of the drug to the three types of liposomes is similar. This result challenged previous conclusions pointing to a key role exerted by drug binding to phospholipid membranes and presumably charge neutralization, for phospholipases inhibition (Carlier et al. Antimicrob Agents Chemother, 23: 440-449, 1983; Mingeot-Leclercq et al., Biochem Pharmacol 37:591-599, 1988). Conformational analysis of mixed monolayers of gentamicin and each of the three acid phospholipids shows that gentamicin systematically adopts an orientation largely parallel to the hydrophobic-hydrophilic interface, but that (i) the energies of interaction are largely different (phosphatidylinositol greater than phosphatidylserine greater than phosphatidic acid), and (ii) the apparent accessibility of the bound drug to water varies in an inverse relation with the energies of interaction. Amikacin, a semisynthetic derivative of kanamycin A with a lower inhibitory potential towards phospholipases than gentamicin in the three types of liposomes used, also showed similar differences in energies of interaction and accessibility to water, but constantly exhibited an orientation perpendicular to the hydrophobic-hydrophilic interface. We conclude that impairment of lysosomal phospholipase activities towards phosphatidylcholine included in negatively-charged membranes by aminoglycoside antibiotics is indeed dependent upon drug binding to the bilayer, but is also modulated by (i) the nature of the acidic phospholipid, which influences the energy of interaction and the accessibility of the drug with respect to the hydrophilic phase, and (ii) the orientation of the drug, which it itself related to its chemical structure. Inasmuch as phospholipases inhibition is related to aminoglycoside nephrotoxicity, these findings may help in better defining the molecular determinants and mechanisms responsible for this adverse effect.

Interdependence of gene expression for early steps of cephalosporin synthesis in Streptomyces clavuligerus.

The early steps of cephamycin synthesis by S. clavuligerus are catalyzed sequentially by lysine epsilon-aminotransferase (LAT), delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthase (cyclase, IPNS). The genes (lat, pcbAB, and pcbC, respectively) are closely linked in the same order as the enzymes act in the biosynthetic pathway and are transcribed in the same direction. Four cephamycin non- (or low-) producing mutants are pleiotropic in that they have undetectable or markedly diminished levels of ACVS and cyclase; two mutants almost completely lack LAT activity. All four mutants are complemented in cephamycin formation by transformation with pNBR1, a plasmid containing a 7.2-kb genomic region of S. clavuligerus in vector pIJ702. The cloned DNA was found to possess no part of the cyclase gene, but instead it contained lat and the 5' upstream part of pcbAB. Doran et al. reported that the 31-bp region between pcbAB and pcbC contains no recognizable promoter or transcription termination sequences. We found that there are 153 bp between the lat ORF and the pcbAB start codon. A potential transcriptional terminator begins 4 to 6 bp downstream of the lat ORF. In the 111-bp segment between the end of the "terminator" and the pcbAB start codon, there are no Streptomyces-like or Escherichia coli-like promoter consensus sequences. However, upstream of the "terminator," that is, in the downstream portion of the lat ORF, are two regions resembling a Streptomyces consensus promoter. Promoter activity in gene fusion constructions was demonstrated in this region. A third potential promoter is upstream of the lat ORF, but only the--10 part is on the cloned DNA. The mechanism by which the cloned DNA (containing lat, the 5' part of pcbAB, and the intervening sequence) influences the expression of the downstream genes encoding ACVS and IPNS, even in strains that possess LAT activity, is an intriguing target of future investigation.

Characterization of human hematopoietic cells with short-lived in vivo repopulating activity.

Recent studies with purified hematopoietic stem cells in vitro support a model of stem cell self-renewal control that involves distinct mechanisms regulating permissiveness to and execution of lineage restriction. Such a model predicts the existence of phenotypically separable populations of hematopoietic cells that are pluripotent and either capable or incapable of extensive self-renewal. Such populations have been previously described in the mouse. We describe here the first evidence that such cells can now be identified in humans using different types of immunodeficient mice as hosts.

Introduction to stem cell biology in vitro. Threshold to the future.

Transplantable hematopoietic cells with multilineage reconstituting ability can be quantitated in suspensions of human or murine cells using similar assay procedures. The incorporation into these assays of stringently defined functional endpoints ensures a high degree of specificity for the cells detected. Application of these assays to stem cell-containing suspensions after they have been stimulated for several days with defined cytokines in vitro, or by a mixture of defined and/or undefined factors in vivo, has shown that net amplifications in these populations can be obtained under both circumstances. Such studies have allowed cytokine conditions that support stem cell self-renewal divisions to be identified and have also provided evidence that stem cell regeneration can be manipulated both in vitro and in vivo by altering the molecular milieu of the responding cells. These observations pave the way to future delineation of mechanisms that control the normal behavior, pathology and future clinical exploitation of hematopoietic stem cell populations.

Interaction of the macrolide azithromycin with phospholipids. I. Inhibition of lysosomal phospholipase A1 activity.

Azithromycin, the first clinically developed dicationic macrolide antibiotic, displays an exceptional accumulation in lysosomes of cultured cells. In fibroblasts incubated with 50 mg/l (66.6 microM), it induces a distinct phospholipidosis as evidenced by biochemical and ultrastructural criteria, which strikingly resembles alterations described previously with gentamicin, a pentacationic aminoglycoside antibiotic which inhibits the lysosomal catabolism of phospholipids. We show that both drugs inhibit, in an equimolar manner, the activity of phospholipase A1 (assayed for phosphatidylcholine, included in negatively charged liposomes), in a way consistent with the model of 'charge neutralization' proposed already for gentamicin (Mingeot-Leclercq et al., 1988, Biochem. Pharmacol. 37, 591). Both drugs bind to negatively charged liposomes. Yet, in spite of this binding, azithromycin does not induce aggregation or fusion of negatively charged vesicles, under conditions in which gentamicin (or spermine, a fully hydrophilic polycation) causes a massive aggregation, and bis(beta-diethylaminoethylether)hexestrol (a dicationic amphiphile) causes fusion. The molecular interactions of azithromycin with acidic phospholipids are further examined in a companion paper.