Mechanism of action and limited cross-resistance of new lipopeptide MX-2401.

MX-2401 is a semisynthetic calcium-dependent lipopeptide antibiotic (analogue of amphomycin) in preclinical development for the treatment of serious Gram-positive infections. In vitro and in vivo, MX-2401 demonstrates broad-spectrum bactericidal activity against Gram-positive organisms, including antibiotic-resistant strains. The objective of this study was to investigate the mechanism of action of MX-2401 and compare it with that of the lipopeptide daptomycin. The results indicated that although both daptomycin and MX-2401 are in the structural class of Ca²âº-dependent lipopeptide antibiotics, the latter has a different mechanism of action. Specifically, MX-2401 inhibits peptidoglycan synthesis by binding to the substrate undecaprenylphosphate (C55-P), the universal carbohydrate carrier involved in several biosynthetic pathways. This interaction resulted in inhibition, in a dose-dependent manner, of the biosynthesis of the cell wall precursors lipids I and II and the wall teichoic acid precursor lipid III, while daptomycin had no significant effect on these processes. MX-2401 induced very slow membrane depolarization that was observed only at high concentrations. Unlike daptomycin, membrane depolarization by MX-2401 did not correlate with its bactericidal activity and did not affect general membrane permeability. In contrast to daptomycin, MX-2401 had no effect on lipid flip-flop, calcein release, or membrane fusion with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG) liposomes. MX-2401 adopts a more defined structure than daptomycin, presumably to facilitate interaction with C55-P. Mutants resistant to MX-2401 demonstrated low cross-resistance to other antibiotics. Overall, these results provided strong evidence that the mode of action of MX-2401 is unique and different from that of any of the approved antibiotics, including daptomycin.

Recombinant expression and neutralizing activity of an MHC class II binding epitope of toxic shock syndrome toxin-1.

Toxic shock syndrome (TSS) is caused by the staphylococcal superantigen, TSST-1. The MHC class II binding domain of TSST-1 containing a conserved sequence with other related staphylococcal enterotoxins, comprising TSST-1 residues 47-64 [(T(47-64)], was expressed as a fusion protein with either glutathione-S-transferase (GST(47-64)), filamentous phage coat protein (pIII(47-64)), or E. coli outer membrane porin protein (OprF(47-64)), or synthesized as a peptide conjugated to bovine serum albumin, BSA(47-64). GST(47-64), OprF(47-64) and BSA(47-64), but not pIII(47-64), all induced high-titer T(47-64)-specific antibodies in Balb/c mice. However, only anti-GST(47-64) antibodies inhibited (125)I-TSST-1 binding to MHC class II and abrogated TSST-1-induced T cell mitogenesis and TNFalpha secretion in human peripheral blood mononuclear cells. Purified GST(47-64) also inhibited (125)I-TSST-1 binding in a dose-dependent manner. These findings suggest that GST(47-64) may have potential as a recombinant peptide vaccine or TSST-1 receptor inhibitor against TSS.

Responsiveness of human skin mast cells to repeated activation: an in vitro study.

To assess human mast-cell (MC) behavior after repetitive activation, we cocultured human foreskin MC (SMC) with human foreskin fibroblasts (F). Under these conditions, we have previously demonstrated that SMC keep their viability and functional activity for up to 8 days. SMC were presensitized with atopic serum and repeatedly activated by consecutively increasing concentrations of anti-IgE antibodies (alpha-IgE, 0.0002-0.1%). This treatment, which mimics the "rush desensitization" procedure, led to complete SMC unresponsiveness to activation by alpha-IgE at optimal concentrations, as evaluated by histamine release. However, presensitization of SMC with IgE antibodies before exposure to alpha-IgE restored their sensitivity to this stimulus. These data indicate that desensitization was probably due to lack of membrane-bound IgE rather than to downregulation of intracellular mechanisms. In fact, SMC challenged by an optimal concentration of alpha-IgE could release histamine upon a second activation by 2 h after the first activation, if the cells had been presensitized before the second challenge. SMC incubation with increasing concentrations of compound 48/80 (0.2-10 micrograms/ml) led to MC unresponsiveness to an optimal concentration of this stimulus. Furthermore, SMC activated by an optimal concentration of compound 48/80 and rechallenged with the same agent were insensitive to the second activation for at least 24 h. In summary, we have shown that it is possible to induce "desensitization" in SMC to both IgE-dependent and IgE-independent stimuli by incubating the cultures with consecutively increasing concentrations of the activator. SMC can release histamine when reactivated with alpha-IgE antibodies after presensitization by 2 h after the first challenge, while they reacquire their susceptibility to reactivation with compound 48/80 in only 2-3 days.

Regulation of the functional activity of mast cells and fibroblasts by mononuclear cells in murine and human chronic graft-vs.-host disease.

Mast cell (MC)-fibroblast-immunocompetent cell interactions may play a role in the inflammatory and fibrotic processes present in chronic graft-vs.-host disease (cGVHD). Interactions between these cell types were examined in both murine and human cGVHD models. To this purpose, cell supernatants from mice or humans with cGVHD and from controls were incubated for 6 days with either rat peritoneal MCs cocultured with 3T3 fibroblasts or with 3T3 fibroblasts alone. Supernatants in the murine model were of splenocytes from either mice with cGVHD or syngeneic controls (B-->B). Supernatants in the human model were of peripheral blood mononuclear cells (PBMC) from cGVHD patients. Two groups of controls were used in the human model-patients who had undergone bone marrow transplantation (BMT) without developing cGVHD and patients with hematological malignancies who had not undergone bone marrow transplantation (pre-BMT). Histamine release was measured in MC/fibroblast cocultures incubated with the cell supernatants. Prostaglandin E2 (PGE2) and [3H]-thymidine incorporation were measured in both MC/fibroblast cocultures or 3T3 fibroblasts alone, incubated with the cell supernatants. In the murine model, the cGVHD supernatant caused significantly more histamine release from MCs than the syngeneic supernatant or medium alone. Moreover, cGVHD and syngeneic supernatants, compared with medium alone, inhibited 3T3 fibroblast proliferation. PGE2 production by 3T3 fibroblasts was higher after incubation with the cGVHD supernatant than with the syngeneic supernatant or in medium alone. Incubation of fibroblasts with supernatants and indomethacin decreased PGE2 production and increased [3H]-thymidine incorporation. In humans, PBMC supernatants from cGVHD patients, as well as from BMT and pre-BMT controls, also displayed histamine releasing activity when cocultured with rat MCs. As with the murine cGVHD splenocyte supernatant, the human cGVHD supernatant decreased fibroblast [3H]-thymidine uptake, but the presence of MCs in the culture abrogated this inhibitory effect. In addition, the human cGVHD supernatant was found to contain high levels of PGE2 and interleukin-1 beta (IL-1 beta). The addition of neutralizing anti-IL-1 beta antibodies to the cGVHD supernatant partially inhibited its histamine-releasing activity. Skin biopsies of involved areas in cGVHD patients revealed significantly reduced numbers of MCs and showed signs of MC degranulation compared with biopsies from pre-BMT controls. Immunocompetent cell supernatants from both mice and humans with cGVHD increased basal histamine release by MCs and reduced fibroblast proliferation. The murine cGVHD supernatant also enhanced PGE2 production by 3T3 fibroblasts. Our findings indicate that complex interactions between immunocompetent cells, MCs, and fibroblasts probably play a role in cGVHD pathogenesis.

Inhibition of proliferation of psoriatic and healthy fibroblasts in cell culture by selected Dead-sea salts.

The effect of five selected minerals abundant in the Dead-sea brine was studied on proliferation of fibroblasts grown from psoriatic and healthy skin biopsy specimens in cell culture. The reason for carrying out this study was looking for the mechanism of the antiproliferative effect of selective Dead-sea minerals in improving the psoriatic condition. Psoriatic skin shave biopsy specimens (both from involved and uninvolved areas of the body) as well as healthy skin (obtained from amputated limbs) were incubated in tissue culture, and their outgrowing fibroblasts were used for this study. The number of cells and their cyclic AMP content were used as parameters for cell division and for proving the selective involvement of magnesium salts in the antiproliferative effect. It is shown that the inhibitory effects of magnesium bromide and magnesium chloride on cell growth were significantly stronger than those of their corresponding potassium salts or of sodium chloride. These results were obtained with both psoriatic and healthy skin fibroblasts, indicating that the inhibitory effect of the selected Dead-sea minerals is present in healthy and psoriatic skin cells.

Interleukin-2 inhibits 3T3 fibroblast proliferation.

In order to clarify the role played by interleukin-2 (IL-2) in the regulation of fibroblast function, we investigated the effect of rat IL-2 and human recombinant IL-2 on 3T3 fibroblast proliferation and collagen synthesis. Fibroblasts were incubated with various concentrations of IL-2 for different periods of time. IL-2 was found to decrease in time- and dose-dependent manner the proliferation of 3T3 fibroblasts. This effect correlated with ability of IL-2 to enhance PGE2 production by 3T3 fibroblasts. When 3T3 fibroblasts were cocultured with rat peritoneal mast cells (MC), the growth-inhibiting effect of IL-2 was significantly less pronounced. Treatment of the cultures with IL-2 had no effect on collagen production by both 3T3 fibroblasts and fibroblasts cocultured with MC. In conclusion, in this study we provide evidence that IL-2, the key cytokine in T-cell growth and differentiation, can affect fibroblast functions.

Modulations of histamine release from mast cells by interleukin-2 is affected by nedocromil sodium.

We have previously demonstrated that histamine release from immunologically activated mast cells (MC) is enhanced by their preincubation (1 h) with interleukin-2(IL-2), and that IL-2 induces slow-chronic histamine release by MC in long-term cultures (6 days). In the present study we assessed whether nedocromil sodium can interfere with IL-2 modulation of MC histamine release. IL-2 enhancing effects nedocromil sodium activity were studied in cocultures of rat peritoneal MC with 3T3 fibroblasts (MC/3T3). MC/3T3 were preincubated for 1 h with IL-2 (50 micrograms/ml) and activated with either rabbit anti-rat IgE or compound 48/80. In chronic experiments MC/3T3 were long-term (5-6 days) incubated with IL-2 (50 micrograms/ml). Nedocromil sodium was used at 10(-5) M. MC activation both when added during the preincubation period (no tachyphylaxis was present) and when added together with the MC activators (30-50% inhibition). Washing out IL-2 before addition of the anti-IgE antibodies did not affect its histamine-release enhancing activity. Removal of nedocromil sodium before addition of the stimulus completely abrogated its effect. Continuous presence of IL-2 in the culture medium enhanced spontaneous histamine release by 37% and this effect was completely abolished in the presence of nedocromil sodium. Furthermore, nedocromil sodium decreased MC basal histamine release by 23% in long-term cocultures. Since IL-2 is known to be elevated in some pathological conditions, our results show that nedocromil sodium inhibits MC activation in an in vitro system which may represent a close resemblance to the in vivo allergic response.

Activated mast cells are fibrogenic for 3T3 fibroblasts.

The extent of mast cell direct involvement in fibrosis is not defined as yet. In the present study we assessed whether long-term co-culture (up to 7 d) of functionally active rat peritoneal mast cells with 3T3 mouse fibroblasts and mass cell activation can affect fibroblast proliferation and collagen production. Co-culture of subconfluent 3T3 fibroblasts with resting mast cells or with mast cells stimulated by alpha IgE (1:35) or repeatedly activated by low concentrations of compound 48/80 (0.25-0.75 microgram/ml) did not alter fibroblast proliferation. However, fibroblast proliferation was increased significantly (100-130%) when mast cells were repeatedly activated with higher concentrations of compound 48/80 (1-3 micrograms/ml). Repeated mast cell activation by compound 48/80 (0.25 microgram/ml) caused a twofold increase in collagen production and this was reduced by 63% by the mast cell stabilizer nedocromil sodium (10(-5) M). At the same time, co-culture of 3T3 fibroblasts with unstimulated or immunologically activated mast cells did not modulate their collagen production. In conclusion, we have demonstrated that mast cell activation, under certain conditions, can enhance significantly 3T3 fibroblast proliferation and collagen production, thus indicating a direct mast cell involvement in the fibrotic processes.

Human foreskin mast cell viability and functional activity is maintained ex vivo by coculture with fibroblasts.

The aim of the present study was to develop long-term culture conditions for foreskin-derived mast cells (HSMC) as we have previously done for rodent peritoneal mast cells (MC) and human lung-derived MC. HSMC were obtained by proteolytic treatment of foreskins from 8-day-old babies (4.8 +/- 2.0% purity) and seeded onto a confluent monolayer of human foreskin-derived fibroblasts (HF) in enriched culture medium. HSMC biochemical and functional properties were studied up to 8 days in these cocultures. Twenty-four hours after seeding the cell suspensions from the proteolytic-digested foreskins, all the cells which did not adhere to the HF were washed out. At this time point approximately 30% of the seeded HSMC were found to adhere to the HF monolayer. The only contaminating cells were endothelial cells (< 8%). Cocultured HSMC maintained their normal resting morphology and histamine content (3.1 +/- 0.7 pg/cell) up to 8 days in these cocultures. When challenged with compound 48/80 on Days 1, 2, 4, and 8 of coculture, HSMC released similar percentages of histamine which were comparable to the one released by freshly isolated HSMC in suspension (approximately 30%). Similarly, HSMC, sensitized with IgE antibodies and challenged at various time of coculture with anti-IgE antibodies, released throughout the experiment comparable percentages of this mediator (approximately 50%). Thus, coculture of HSMC with HF fibroblasts provides a suitable in vitro system for long-term studies on HSMC functional and biochemical properties in a microenvironment which mimics the physiologic one.

Mast cells and fibroblasts: two interacting cells.

Mast cell (MC) fibroblast interactions may have a role in health and disease. We analyzed the relationships between these cells by utilizing our in vitro model in which mucosal (MMC) and connective tissue (CTMC) type MC were cocultured long-term with different fibroblasts. Mouse 3T3 fibroblasts were used to provide a normal microenvironment for MC, while fibroblasts derived from mouse with chronic graft-versus-host disease (cGVHD) provided a fibrotic one. We found that both 3T3 and cGVHD fibroblasts maintain CTMC viability, phenotype and functional activity. When MMC were cocultured with 3T3 or cGVHD fibroblasts, they changed their phenotype towards that of CTMC. On the other hand, MCs were found to affect fibroblast properties. Coculture of MMC on 3T3 monolayers was shown to increase Forsmann antigen production and collagen synthesis and stimulate fibroblast proliferation. Resting CTMC or CTMC activated by anaphylactic stimuli induced 3T3 and cGVHD fibroblasts to proliferate more. In addition, CTMC activation increased collagen production by 3T3 fibroblasts. In conclusion, fibroblasts were found to regulate MC survival and differentiation, whereas MCs were shown to affect the biochemical properties of fibroblasts, which can lead to fibrosis.