Evaluation of predictable irritative power of surfactant mixtures by human fibroblast culture and its correlation to physico-chemical parameters.

Summary The predictable toxic effects of some surfactants, their blends and some preserving agents on human fibroblast cultures were investigated with in vitro tests, with the aim of finding a possible correlation between the biologic evaluations and some physical characteristics of detergent solutions. Lactate dehydrogenase release into the medium was used as a marker of the plasma membrane integrity, while the amount of (3)H-radiolabelled proteins in the fibroblasts was measured in order to assess the cell biosynthetic machinery function. Disodium-alkyl-semi-sulphosuccinate induced membrane damage in the lactate dehydrogenase test and decreased the protein synthesis, with an EC 50 around 1mm, while sodium lauryl ether sulphate had an EC 50 at about 100 mum, indicating that this compound is ten-fold more toxic, when measured by this method. An ethoxylated glyceride, on the contrary, was completely harmless on the plasma membrane and, surprisingly, activated fibroblast protein synthesis in a dose-response way up to two-fold. Mixtures of the three surfactants evidenced the protective effect of the non-ionic against the cellular functionality damage. Parabens do not influence this type of evaluation, while some influence was shown by the formaldehyde releaser 2-bromo-2-nitro-propandiol at the highest concentration. The comparison between critical micellar concentration measures of the different surfactants and their in vitro detected irritative power shows, for the two anionics, that in vitro toxicity is proportionally bound to the amount of micelles even if the structural differences between the two types of molecules are reflected into different damage values, while the non-ionic compound shows a not defined CMC and a very low toxicity profile. Blends of anionics with the non-ionic show an increased CMC and a reduced toxicity profile. Toxicity evaluations of complex finished foaming formulations, carried out with human fibroblast cultures evaluation show that a relationship between micelles amount and cell toxicity seems to exist, mainly when multiple surfactants blends are tested.

Fibroblasts increase adhesion to neutrophils after stimulation with phorbol ester and cytokines.

In our research there was spontaneous adhesion between resting fibroblasts and neutrophils in vitro which could be increased by stimulating either the coculture of cells or each cell type separately with various stimulants. Interferon-gamma, interleukin-1, and interleukin-6 significantly increased adhesion; however, the highest adhesive response was obtained when cocultures were treated with phorbol myristate acetate (PMA). As PMA-stimulated fibroblasts show the highest adhesion to resting neutrophils, it was suggested that adhesion was primarily due to an effect on fibroblasts. Without Mg2+ PMA did not stimulate fibroblast adhesion, whereas in the absence of Ca2+ the response was only partially reduced. Spontaneous adhesion was independent of both neutrophil integrins and fibroblast ICAM-1, whereas cytokine-stimulated adhesion was blocked by mAbs against ICAM-1; PMA-stimulated adhesion was not affected by mAbs anti-ICAM-1, but was partially inhibited by mAbs anti-beta 2 integrins. These results suggested the presence of mechanisms able to modulate the adhesive fibroblast-neutrophil interaction in inflammatory and wound healing processes.

Modulation of neutrophil functions by activated platelet release factors.

Platelets activated with physiological agonists, such as thrombin, ADP, or collagen, released products able to modulate neutrophil functions. In particular, platelet supernatant contained an inhibitor of superoxide anion generation induced by phorbol ester and a chemotactic factor for human neutrophils. The proteolytic digestion of platelet supernatant completely abrogated chemotactic activity without interfering with the inhibitory effect, indicating the presence of different molecules involved in the modulation of different neutrophil functions. This was further confirmed by the pretreatment of platelets with aromatic benzamidine which abolished chemotactic activity, but did not affect superoxide inhibition of neutrophils. This report provides evidence for interaction of platelets and inflammatory cells, suggesting that platelets are able to induce accumulation of neutrophils and control their respiratory burst, which also has a critical role in tissue damaging in inflammation.

[Cell biology and cosmetology]. / Biologie cellulaire et cosmétologie.

Cellular biology can become the natural support of research in the field of cosmetics because it is able to provide alternative experimental models which can partially replace the massive use of laboratory animals. Cultures of human skin cells could be used in tests investigating irritation of the skin. We have developed an "in vitro" experimental model that allows to evaluate the damage caused by the free radicals to the fibroblasts in culture and to test the protective action of the lipoaminoacids. Experimenting on human cell cultures presents the advantage of eliminating the extrapolation between the different species, of allowing a determination of the biological action of a substance and of evaluating its dose/response effect. This does not mean that "in vitro" experimenting could completely replace experimenting on living animals, but the "in vitro" model can be introduced in the realisation of preliminary screenings.

Chemotactic response of human monocytes to pentapeptide analog derived from immunodeficiency virus protein gp 120.

The octapeptide sequence of peptide T is contained within the envelope of HIV and seems to mediate the viral binding to CD4 expressing cells, including monocytes. The biological activity of the alpha-aminobutyric acid pentapeptide derived from the C-terminal sequence of peptide T, in which the polar side chain of threonine in position 4 is substituted by a hydrophilic group, is measured by the monocyte chemotaxis assay. The chemotactic activity of human monocytes is assessed by determining the concentration at which the pentapeptide analog is maximally active and the effectiveness at that concentration, in comparison with peptide T and two shorter homologs, the pentapeptide and tetrapeptide. These experiments suggest that the synthetic analog is a potent chemotactic factor active at picomolar concentrations and that it competes with peptide T for the monocyte binding site.

Formyl peptide chemoattractants. Synthesis and chemotactic activity.

The tetrapeptides CHO-Met-Leu-Phe-CO-NH-(CH2)n-COOMe (n = 1-5) have been synthesized. These peptides containing an omega-amino acid residue in position 4 exhibit a very high chemotactic activity. Like the chemotactic peptide CHO-Met-Leu-Phe-OMe, these tetrapeptides in solution undoubtedly adopt an "active" conformation which allows a strong interaction with the receptor on the human polymorphonuclear leukocyte surface.

Response of human neutrophils to formyl-peptide modified at the terminal amino and carboxyl groups.

Two analogs of chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine were examined for their capacity to activate several functions of human neutrophils. The C-terminus methyl ester derivative of the chemotactic peptide was found to possess strong biological activity and was able to induce levels of chemotaxis, enzyme secretion, and superoxide generation comparable to those observed with the same concentrations of N-formyl-L-methionyl-L-leucyl-L-phenylalanine. The analog containing a tert-butyloxycarbonyl group at the N-terminus, as well as the C-terminal methyl ester, was completely devoid of activity towards neutrophils. From these results, it appears that the free carboxyl group is not necessary for biological function. In contrast, the substituent at the N-terminus may play a critical role in the induction of the cellular response.

[Indomethacin and oxamethacin as modulators of the production of superoxide anion in human leukocytes]. / Indometacina ed ossametacina come modulatori della produzione di anione superossido dei leucociti umani.

Treatment of human neutrophils with the two chemoattractants formyl-methyonyl-leucyl-phenylalanine and opsonized zymosan or with phorbol myristate acetate induce the production of superoxide anion (O-.2), which plays a role in the inflammatory reaction and microbicidal activity. The time-course of O-.2 release and the extent to which the anion is produced are related to the nature of the stimulating agents. Anti-inflammatory non steroid drugs, such as indomethacin and oxamethacin, exert a variable depression of the oxidative burst, which does not seem correlated to the inhibition of prostaglandin synthesis, but to the lipophilic character of the drug molecule.

[An analog of formyl-met-leu-phe effects the movement of human leukocytes]. / Analogo del formil-met-leu-phe influenza il movimento dei leucociti umani.

Two tripeptides, related to the chemotactic formyl-peptide, are tested for its ability to affect random and directional locomotion and to induce chemotaxis of polymorphonuclear leukocytes. The results indicate that the methyl ester of formyl-methyonyl-leucyl-phenylalanine possesses a biological activity towards human phagocytes, including a chemotactic potency, comparable to that of unmodified peptide. Therefore, the free carboxyl group does not seem essential to generate active leukoattractant. On the contrary, the replacement of the formyl group by the butyloxycarbonyl results in a drastic loss of biological activity. Our data may indicate that the two formyl-peptides interact with the same binding site on the cell membrane.