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Biomedical subjects

L Dubertret

Publications and source records attributed to L Dubertret.

At least 271 records · Page 15Linked to original sources
PubMed1988

Biosynthesis of paf-acether factor-acether by human skin fibroblasts in vitro.

The synthesis and release of paf-acether by fibroblasts from normal human skin was investigated in vitro. When fibroblasts in suspension (1 X 10(6) cells) were stimulated with 2 microM Ca1+ ionophore A23187 (Io), they synthesized a material that aggregated aspirin-treated washed rabbit platelets and was identified as paf because 1) the platelet aggregation it induced was inhibited by BN 52021, an antagonist of paf putative receptors; 2) the factor was inactivated by phospholipase A2 but was insensitive to lipase from Rhizopus arrhizus; 3) it exhibited the same retention time as synthetic paf during standard and reverse phase HPLC elution. Paf production by fibroblasts occurred as soon as the first min of Io stimulation (287 +/- 92 pg/1 X 10(6) cells), reached a maximum at 5 min (369 +/- 85 pg/1 X 10(6) cells) and decreased thereafter. Half of the fibroblast-produced paf was recovered in supernatants. Addition of exogenous 1-O-alkyl-sn-glycero-3-phosphocholine (lyso-paf) at 0.1 microM and/or acetyl-coenzyme A at 0.1 mM to fibroblasts during Io stimulation enhanced paf production by two- and three-fold, respectively. The paf precursors, i.e., 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-alkyl-2-acyl-GPC) and lyso-paf, were detected in fibroblasts either stimulated with Io or not. These precursors exhibited 80% hexadecyl and 20% octadecyl chains at the sn-1 position of the molecules, as determined by reverse phase HPLC and gas chromatography analysis. The present results are the first to demonstrate the synthesis and release of paf by fibroblasts from normal human skin. Such production within the dermis might account for the development of cutaneous inflammation and for the pathogenesis of many skin disorders.

Adult↗
PubMed1988

[Skin cultures].

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Culture Techniques↗
PubMed1988

Inhibitory effect of oral cetirizine on in vivo antigen-induced histamine and PAF-acether release and eosinophil recruitment in human skin.

The use of a noninvasive skin chamber technique in vivo in pollen-sensitive patients allowed us to quantify the time-course release of histamine and the recruitment of inflammatory cells (i.e., neutrophils, monocytes, and eosinophils) in skin sites challenged with pollen, histamine, and compound 48/80. The new H1-receptor antagonist, cetirizine 2 HCl, orally administered with 10 mg once a day to pollen-sensitive patients in a double-blind, crossover study versus placebo, induced a significant decrease in the wheal-and-flare cutaneous reaction induced by intradermal injection of pollen, histamine, and compound 48/80. It also significantly inhibited the immediate histamine release occurring in skin chambers after pollen introduction, whereas it did not significantly inhibit the late release. In patients receiving placebo, we detected platelet-activating factor-acether in media collected at the sixth hour from chambers filled with pollen. With cetirizine 2 HCl treatment, platelet-activating factor-acether was not detected in chamber media. Interestingly, cetirizine 2 HCl significantly reduced the eosinophil recruitment observed on the superficial dermis 24 hours after pollen challenge.

Administration, Oral↗
PubMed1988

Phototherapeutic, photobiologic, and photosensitizing properties of khellin.

Khellin, whose chemical structure closely resembles that of psoralen, is reported to be an efficient drug for treating vitiligo when combined with ultraviolet A irradiation. Photobiological activity on yeast is found to be much lower than that of bifunctional psoralens such as 5-methoxypsoralen. In vitro experiments reveal that khellin is a poor photosensitizer. It behaves as a monofunctional agent with respect to DNA photoaddition. It does not photoinduce cross-links in DNA in vitro or in Chinese hamster cells in vivo. This behavior may explain the low photogenotoxicity in yeast and the lack of phototoxic erythemal response when treating vitiligo with khellin.

Animals↗
PubMed1988

Cantharide acantholysis: endogenous protease activation leading to desmosomal plaque dissolution.

Using a method which allowed us to study the morphological consequences of the expression and the inhibition of proteases in living tissues, we demonstrated that the primary detectable cellular event in cantharide acantholysis is the dissolution of the dense plaque, leading to the detachment of tonofilaments from desmosomes. This process is inhibited by neutral serine protease inhibitors. This suggests that the desmosome-tonofilament complex, more precisely the desmosomal dense plaque, is the primary target of activated proteases during cantharide acantholysis, and can be disrupted by a specific epidermal protease-anti protease system. Cantharide acantholysis may be useful model for studying desmosomal turnover.

Acantholysis↗
PubMed1988

Characterization of specific leukotriene C4 binding sites on cultured human keratinocytes.

Peptidoleukotriene C4 (LTC4) and leukotriene B4 (LTB4) are both suspected of being inflammatory mediators and epidermal mitogenic agents in cutaneous psoriatic lesions. In the present study an LTC4 specific binding site was characterized in membranes from cultured human keratinocytes (Kd, 8.7 nmol/l; Bmax, 1.2 pmol/mg protein). In contrast LTB4 did not show any high affinity binding which could account for its biological effects. These data suggest that LTC4, unlike LTB4, acts on epidermal cells through a receptor-mediated mechanism.

Binding Sites↗
PubMed1988

Growth of melanocytes in a skin equivalent model in vitro.

We have developed a pigmented human skin equivalent by inserting a punch biopsy of human infant foreskin as a source of epidermis into a collagen lattice (dermal equivalent). Using a conventional epidermal culture medium and stimulation with UVB irradiation or 8-MOP + UVA treatment, melanocytes were found to grow out from the biopsy with the epidermal sheet. In this newly formed epidermis, melanocytes and keratinocytes were maintained in an architectural relationship similar to that present in vivo and melanocyte outgrowth could be quantitatively evaluated. Consequently, this pigmented human skin equivalent is a useful model for investigating the biology and photobiology of human skin pigmentation.

Child↗
PubMed1988

The mixed epidermal cell lymphocyte-reaction is the most predictive factor of acute graft-versus-host disease in bone marrow graft recipients.

Risk factors for acute graft-versus-host disease (GvHD) remain controversial. We performed uni- and multivariate statistical analyses on a series of 37 patients receiving a non-depleted allogeneic bone marrow transplant from an HLA-identical sibling donor for a haematological malignancy, in order to identify risk factors for GvHD. Three factors were associated with development of moderate to severe GvHD: a positive mixed epidermal cell-lymphocyte reaction (MECLR) between donor and recipient, previous pregnancies in female donors and chronic myeloid leukaemia diagnosis. The MECLR was the most important predictive factor, selected in first rank by the stepwise linear discriminant analysis. Combining these three prognostic factors in the jackknifed procedure, we could correctly classify 33/37 patients in two groups: grade O-I versus grade II-IV acute GvHD. These results should apply to donor selection and to predict donor/recipient pairs at high risk of GvHD who might benefit of bone marrow T-cell depletion and those at low risk for whom depletion could be avoided.

Adolescent↗
PubMed1988

Autoradiographic study of the effects of psoralen and ultraviolet light on DNA, RNA and protein synthesis in cultured keratinocytes and psoriatic skin.

DNA, RNA and protein synthesis were studied by autoradiography in cultured keratinocytes, immediately, 24 and 48 h after 8-methoxypsoralen and ultraviolet light, (PUVA) treatment. Using the same technique, the immediate and long-term effects of PUVA therapy on DNA, RNA and protein synthesis were analysed in skin biopsies from psoriatic patients. In the cultures, immediately after irradiation, DNA and RNA syntheses were similarly inhibited in a dose-dependent manner, while protein synthesis was slightly affected only for the highest dose. After 24 h, RNA synthesis recovered whereas DNA synthesis was more severely inhibited suggesting that other cell components may be damaged by PUVA. In patients, DNA and RNA syntheses decreased immediately after PUVA sessions. During all the sessions until the psoriatic plaques had cleared, an impairment of DNA synthesis was observed in comparison with the synthesis in involved and uninvolved skin before treatment. These results suggest that the therapeutic efficiency of PUVA is based on the inhibition of DNA replication due to direct effects on nucleic acids but also to photoreactions with other cell components.

Animals↗
PubMed1988

Prostaglandin D2 generation in mouse bone marrow-derived mast cells exposed to dexamethasone is associated with endogenous peroxidase activity.

The appearance of fixative-sensitive peroxidase activity in the nuclear envelope and endoplasmic reticulum of bone marrow-derived mast cells (BMMC) cultured in the presence of 1 microM dexamethasone (DM) for up to 14 days and its relationship with immunologic release of prostaglandin D2 (PGD2) by these cells were studied. Endogenous peroxidase activity, previously shown as a marker of arachidonic acid metabolism in various cell types, was visualized by cell incubation in 3,3' diaminobenzidine-containing solution before glutaraldehyde fixation. PGD2 release was induced by passive sensitization of BMMC with an optimal dose of monoclonal IgE and subsequent challenge with specific a antigen. We found that 4-week-old BMMC, used as the starting population of the present study, exhibited immature morphologic features, did not present peroxidase activity when cytochemically processed, and released minute amounts of PGD2 in response to IgE-dependent stimulation. When such BMMC were exposed to DM during 24 hours, they showed aldehyde-inhibited peroxidase activity in the perinuclear envelope and a few endoplasmic reticulum segments. As compared with untreated cells, 24-hour DM-exposed BMMC released higher amounts of PGD2 upon immunologic stimulation. After an additional 14-day period of DM exposure, an intense peroxidase activity was detected in the perinuclear envelope and the endoplasmic reticulum of BMMC, which, under immunologic stimulation, released as much as 42.4 +/- 14.7 ng of PGD2/1 x 10(6) cells. Aminotriazole (20 and 50 mM) extinguished both peroxidase activity and PGD2 release from BMMC whereas indomethacin (1 microM) suppressed PGD2 production, but did not alter endogenous peroxidase activity. Previous cell fixation with glutaraldehyde totally inhibited endogenous peroxidase reaction in DM-exposed BMMC. Moreover, 14-day DM-exposed BMMC exhibited morphologic characteristics of mature mast cells and possessed alcian blue+/safranin+ granules. Therefore, the present data suggest that appearance of peroxidase activity in the nuclear envelope and the endoplasmic reticulum of DM-exposed BMMC is associated with the ability of the cells to synthetize PGD2 and appears as a cytochemical marker of the in vitro maturation of mouse bone marrow-derived mast cells.

Aldehydes↗
PubMed1988

Reconstructed human epidermis: absence of Langerhans cells and failure to stimulate allogeneic lymphocytes in vitro.

Whole human skin can be reconstructed in vitro, using dermal equivalents made of fibroblasts in a collagen matrix. We recently described a new method of epidermalization of dermal equivalents, based on the insertion of punch biopsies and the migration of epidermal cells (EC) on the reconstructed dermis. In the present study, we show that no MHC class II or T6 positive Langerhans cells (LC) can be detected in this new epidermis. Functional studies with EC of this reconstructed epidermis show that these EC completely fail to induce proliferation of allogeneic lymphocytes in mixed epidermal cell lymphocyte reactions and to raise an allogeneic T cell response. In contrast, fresh EC from the same donors induce a strong proliferative and cytotoxic response of the same effector cells. Moreover, the addition of fresh LC-containing EC autologous to effector lymphocytes does not restore an allogeneic proliferative and cytotoxic response directed against class I different EC of the new epidermis. Such a non-immunogenic whole skin model composed of two compartments, dermis and epidermis, completely devoid of class II-bearing antigen presenting cells, is thus a very promising technique for allogeneic skin grafting in the treatment of burns.

Culture Techniques↗
PubMed1987

Immunoelectron microscopy in subepidermal autoimmune bullous diseases: a prospective study of IgG and C3 bound in vivo in 32 patients.

Thirty-two patients suffering from subepidermal autoimmune bullous disease were studied prospectively by clinical examination and immunoelectron microscopy. Clinically, 1 patient had herpes gestationnis, 14 typical bullous pemphigoid (BP), 3 epidermolysis acquisita (EBA), 3 cicatricial pemphigoid (CP), and 11 patients overlapping clinical diseases. These 11 patients shared clinical features of BP, EBA, or CP and a clinical diagnosis could not be done safely. Immunoelectron microscopy revealed diaminobenzidine deposits in 20 patients on the epidermal side of dermo-epidermal junction in the lamina lucida as in BP. In 5 patients, deposits located mostly under the anchoring fibril zone, in the floor of a sublamina densa dermoepidermal separation for 2 of them, were consistent with a diagnosis of EBA. In 6 patients, deposits were located mostly in the lamina densa, in the floor of a dermoepidermal separation occurring in the lamina lucida for 3 of them. This suggests that some of these 6 patients had neither EBA or BP, but another autoimmune bullous disease again, an uncharacterized component of dermoepidermal junction located in the lamina densa. Finally, a correlation exists between the sites of IgG and/or C3 components on epidermal or dermal side of dermoepidermal junction and the presence or absence of characteristic clinical features such as scar, milia formation, or mucosal involvement.

Adult↗
PubMed1987

Biologic properties of LTB4 and paf-acether in vivo in human skin.

Using an improved skin chamber technique, the consequences of prolonged contact of leukotriene B4 (LTB4) and platelet-activating factor (paf-acether) with human dermis were evaluated quantitatively and kinetically in vivo. Leukocyte chemotaxis, histoenzymologic alterations, and modifications in vascular permeability were studied in two sets of experiments. In a first set of experiments, the dose-effect relationship of LTB4 and paf-acether on leukocyte migration was studied. LTB4 (3 X 10(-8) M to 9 X 10(-7) M) in Hanks' balanced salt solution (HBSS) elicited an intense dose-dependent and time-dependent neutrophil migration. Paf-acether, at the same concentration range, induced a significant increase in cell migration only at 9 X 10(-7) M and when diluted in HBSS containing 0.25% serum albumin (HBSS-BSA). Histoenzymologic analysis demonstrated that LTB4 in vivo induced degranulation of most of the neutrophils migrating through the dermis. Paf-acether caused mild degranulation of neutrophils and induced the appearance of degranulated basophils in dermal vessels. A second set of experiments was designed to study simultaneously the modifications in vascular permeability and cell migration induced by LTB4 and paf-acether, with or without prostaglandin E2 (both at a concentration of 3 X 10(-7) M in HBSS). Since spontaneous protein diffusion in HBSS progressively declined up to a plateau reached after 20 h (1.2 +/- 0.15 mg of proteins/cm2/2 h), these experiments were carried out after a 20-h equilibration period. Leukotriene B4 induced a late and slight increase in vascular permeability. Paf-acether did so intensely and transiently. Prostaglandin E2 significantly enhanced protein diffusion and neutrophil migration induced by LTB4 and, to a lesser extent, by paf-acether. Interestingly, despite the reintroduction into the skin chambers of freshly prepared solutions containing the mediators, leukocyte migration and protein diffusion progressively decreased during the experiments. This suggests the local production of anti-inflammatory factors that inhibit local mediators and thus regulate the inflammatory response.

Capillary Permeability↗
PubMed1987

[Functions of neutrophils in vivo on the human skin. Applications to the study of psoriasis and anti-inflammatory agents].

A reproducible method for sequential study of migration out of human skin, phagocytosis and bactericidal activity of neutrophils is described. Untreated psoriatics exhibit an early increase of chemotactic activity (0-8 h, p less than 0.02) and subsequently a strong inhibition of chemotaxis (8-24 h, p less than 0.01), a slight decrease of phagocytosis and a decrease in bactericidal activity (20 mn, p less than 0.02; 30 mn, p less than 0.003); 60 mn, p less than 0.001; 120 mn, p less than 0.001) as compared with controls. After clearing of skin lesions, the early increased chemotactic activity returned to normal values but the subsequent chemotactic inhibition remains as strong as before treatment. Phagocytosis increased to normal values (p less than 0.02) and bactericidal activity also increased but remained significantly low. The abnormalities were more evident in migrating than in circulating neutrophils, underlining the sensitivity of the described method. Using the same method we have shown that the aromatic retinoid etretinate (Ro 10-9359) and its main metabolite (Ro 10-1670) significantly inhibit the migration of neutrophils from the blood to tissues when applied into skin chambers (0.1 mg/ml) or when given orally (1 mg/kg/day) for 8 days to normal volunteers. This pharmacological property could be closely linked to the antipsoriatic properties of these drugs.

Adult↗
PubMed1986

In vitro interaction of the photoactive anticancer porphyrin derivative photofrin II with low density lipoprotein, and its delivery to cultured human fibroblasts.

Low density lipoprotein (LDL) doped with the anticancer mixture of hematoporphyrin derivatives Photofrin II (P2) competes with native LDL for binding to fibroblast receptors, despite a slight increase in the negative net charge related to the presence of acidic residues of porphyrins. P2 delivery to fibroblasts can be achieved by LDL, HDL3 or albumin doped with P2 (LDL-P2, HDL-P2 or A-P2, respectively). P2 delivery to cells assessed by fluorescence measurement, is much more efficient, at low protein concentrations (10-20 micrograms/ml) by LDL-P2 than by HDL-P2 or A-P2. Moreover, P2 delivery to cells by LDL-P2 as a function of protein concentration is a saturable process, whereas P2 delivery by HDL-P2 or A-P2 is a linear process. Finally, reduction of the LDL-receptor number by preincubation of fibroblasts in medium supplemented with lipoproteins results in a decrease of P2 delivery by LDL-P2. These results suggest a special role of the LDL-receptor pathway in P2 delivery to cells and could be of interest in cancer phototherapy by porphyrins.

Antineoplastic Agents↗
PubMed1986

A microspectrofluorometric study of the effect of anthralin, an antipsoriatic drug, on cellular structures and metabolism.

The microspectrofluorometric approach has been used to investigate in single living cells in culture fundamental questions raised by the use of anthralin, a potent antipsoriatic drug. This method allows fluorescence determinations on the intracellular fate of the drug as well as the recognition of structural and metabolic alterations induced by the drug. In the absence of demonstrable adduct formation with DNA, the antipsoriatic, i.e. antiproliferative effect of anthralin, has been attributed to its action at the level of mitochondria or at the level of glucose-6-phosphate dehydrogenase which initiates the pentose phosphate shunt (cf. its prominent role in nucleic acid synthesis). Upon addition of 2.3 to 23 microM anthralin to the L cell culture, the characteristic structure of the anthralin anion fluorescence spectrum is recognized almost immediately in the cytoplasm (much weaker in the nucleus) but disappears within minutes. The vital mitochondrial fluorescence probe dimethylaminostyryl-pyridinium-methyl-iodine reveals striking structural alterations of the mitochondria within 15 min after addition of the drug. At the same time, there is a stimulation of the transient NAD(P)+ reduction observed upon microinjection into the L cell of the Krebs' cycle substrate malate, or the pentose cycle substrate 6-phosphogluconate. Specially, the injection of the latter to anthralin-treated cells suggests that upon release of the mitochondrial control, there is a tremendous disruption of metabolic activity which could have profound consequences on the proliferative activity of the cell. These findings, while they open new possibilities for the intracellular evaluation of therapeutic agents, create also a challenge in understanding the complex and dynamic interrelationships between intracellular organelles and bioenergetic or biosynthetic pathways.

Animals↗