J. Thivolet

In vitro and post-transplantation differentiation of human keratinocytes grown on the human type IV collagen film of a bilayered dermal substitute☆

Using human type IV and type I + III collagens and a new, nontoxic cross-linking procedure, we have developed a cell-free bilayered human dermal substitute for organotypic culture and transplantation of human skin keratinocytes. We have studied the formation of the basement membrane, and the differentiation of keratinocytes grown on the type IV collagen layer of this dermal substitute, in vitro and after grafting onto nude mice. These studies demonstrated the formation of essential constituents of the basement membrane in culture: hemidesmosomes and deposition of extracellular matrix on the top of the type IV collagen were observed as early as 6 days after plating of human keratinocytes. Although the keratinocytes formed a well-organized multilayered epithelium, they exhibited limited differentiation when grown submerged in liquid medium. However, the multilayered sheet obtained after 14 days in submerged culture was composed of a regular basal cell layer, several nucleated suprabasal cell layers containing granular cells, and several dense, anucleated cell layers. The grafting experiments have shown a good biocompatibility of the dermal substitute. It is repopulated by fibroblasts, newly synthesized collagen, vessels, and a few mononuclear cells. At Day 14 after grafting, the type IV collagen layer was still present and very dense, and the basement membrane appeared as in culture, with numerous well-structured hemidesmosomes and deposition of extracellular matrix resembling lamina densa. At Day 55 after transplantation, even if the epidermal graft did not exhibit all the characteristics of the normal epidermis in vivo, it was very close to it. At this stage, the basement membrane was complete, with structures clearly indicative of anchoring fibrils. This new dermal substitute offers many advantages. It is stable and easy to handle. Its production is standardized. The oxidation induced by periodic acid led to a nontoxic cross-linked matrix. This dermal substitute is the first one entirely composed of human collagens. The type I + III collagen underlayer is reorganized when grafted. It supports a type IV collagen top layer which offers an excellent substrate for keratinocytes, favors their anchorage, and favors the formation of the basement membrane in vitro. This dermal substitute could be useful for wound coverage or as an in vitro model for toxicological and pharmacological studies.

Keratin polypeptides distribution in normal and diseased human epidermis and oral mucosa

Immune sera against total keratin and keratin polypeptide subunits were induced in guinea pigs, using the different bands of SDS polyacrylamide gel electrophoresis of fibrous proteins of stratum corneum, derived from normal human epidermis. The distribution of the different polypeptides was studied in numerous human biopsies of normal epidermis, normal oral mucosa and epidermal and mucosal inflammatory, premalignant and malignant lesions using the indirect immunoperoxidase method. Antisera against total keratin (TK) and against the keratin polypeptide of M.W. 55,000 dalton (55K) labelled all keratinocytes in normal and pathological conditions. These antisera may be useful for the histodifferentiation in diagnostic pathology. Atisera against the keratin polypeptides of M.W. 67,000 (67K) and 62,000 dalton (62K) identified only keratin antigens in the spinous, granular and keratinized layer of normal epidermis and oral mucosa. No labelling of the basal layer was achieved with these immune sera. However, there were important differences in the distribution of these keratin antigens in altered epithelia which may be of value in the differential diagnosis of inflammatory, premalignant and malignant lesions of the skin and oral mucosa.

Experimental Production of Antibodies Against Stratum Corneum Keratin Polypeptides

SummaryAnti-keratin polypeptide sera (K.P.S.) were obtained by immunizing guinea pigs with fibrous proteins from stratum corneum, which were acquired from normal human epidermis by means of S.D.S. polyacrylamide gel electrophoresis. After absorption with red blood cells and liver powder the sera were tested by indirect immunofluorescence technique on different substrates. Antibodies against polypeptides P1 and P2 of M.W. 67,000 and 62,000 dalton, respectively, were directed toward cytoplasmic Ag of keratinocytes of spinous and granular layer of normal human and rabbit epidermis. No labeling could be detected in the basal cell layer. This finding is in favor of various differentiation stages of the keratinizing cells.P3 of M.W. 53,000 dalton induced low titre antibodies which labelled the whole epidermis, including the basal cell layer. The fourth polypeptide of M.W. 49,000 dalton seemed not to be immunogenic in such experiences.In tumors, such as basal cell carcinoma, squamous cell carcinoma, and warts, the expression of keratin antigens is markedly diminished.No analogy could be drawn between experimental keratin polypeptide antibodies and the human epidermal cytoplasmic antibodies which were detected in some patient sera.ZusammenfassungAntikeratin-Polypeptidseren (K.P.S.) wurden durch Immunisierung von Meerschweinchen mit fibrösen Proteinen des Stratum corneum erhalten, welches über die S.D.S. Polyacrylamid-Gel-Elektrophorese von normaler menschlicher Epidermis gewonnen wurde. Nach Absorption mit Erythrocyten und Leberpuder wurden diese Sera mit Hilfe der indirekten Immunfluorescenztechnik an verschiedenen Substraten getestet. Es wurden Antikörper gegen Polypeptide P1 und P2 mit einem Molekulargewicht von 67 000 und 62 000 Dalton nachgewiesen, die gegen cytoplasmatische Antikörper der Keratinocyten des Stratum spinosum und des Stratum granulosum der normalen menschlichen und Kaninchenepidermis gerichtet war. Ein Nachweis in den Basalzellagen konnte nicht erbracht werden. Dadurch wurden die verschiedenen Differenzierungsstadien der Zellen belegt, die sich zu Keratinocyten entwickeln und die oberhalb der Basalschicht lokalisiert sind.Polypeptide P3 des Molekulargewichts 53 000 Dalton induzieren niedrigere Antikörpertiter, die die gesamte Epidermis markieren einschließlich der Basalzellage. Polypeptide mit einem Molekulargewicht 49 000 Dalton schienen keine immunogenetische Wirkung in diesen Experimenten zu haben.Tumoren, wie Basalzellcarcinoma und Bindezellcarcinom, wie auch Warzen, wiesen eine deutlich verminderte Expression des Keratinantigens auf. Es wurde keine Analogie zwischen experimentell erzeugten Keratinpolypeptid-Antikörper und cytoplasmatischen Antikörpern der menschlichen Epidermis.

Anti-CD4 monoclonal antibody therapy in severe psoriasis

We report here the treatment of psoriasis, a chronic inflammatory skin disease characterized by uncontrolled keratinocyte proliferation, with BB14, a CD4 murine IgG1 antibody. Three patients with severe psoriasis were treated with anti-CD4 mAb infusions (0.2 mg/kg/day for the first patient, 0.4 mg/kg/day for 2 days and 0.8 mg/kg/day during the following days for the 2 others) for 7 or 8 days, without other therapy. Rapid clinical improvement, with major reduction of the Psoriasis Area Severity Index, was observed during 1 month after treatment. Moderate decreases in CD4+ blood cells occurred in the last two patients but not in the first one. Circulating T cells coated with anti-CD4 mAb were detectable during the first 48 h in the first patient and from day 1/2 to day 7/8 in the two others. The density of CD4 molecules on the surfaces of peripheral blood lymphocytes was decreased in all patients and remained low as long as anti-CD4 mAb was detectable in patient serum. The maximal 24 h residual mAb levels ranged from 0.3 microgram/ml in the first patient to 3.8 and 7.0 microgram/ml in the two others. The three patients produced IgM antibodies against the anti-CD4 mAb at day 7/8 or 15 and two patients had IgG antibodies at day 15. Lesional skin samples demonstrated (1) gradual improvement in parakeratosis, papillomatosis and acanthosis, (2) decreased expression of ICAM-1 and HLA-DR by keratinocytes, (3) an increase in CD1a+ Langerhans cell number, (4) partial decrease in epidermal T cell infiltrate and (5) no major change in the dermal infiltrate composed of CD3+, TcR alpha beta+, CD45Ro+, HLA-DR+ T cells. We conclude that anti-CD4 mAb administration can induce a rapid and major improvement in psoriatic lesions, with immunohistochemical changes different from those induced by cyclosporin A or 8-methoxypsoralen plus long wave UV light (PUVA) therapy. Our data provide strong evidence for a critical role of CD4+ lymphocytes in psoriasis.

Inhibition of PMN leukocytes chemotaxis by thalidomide.

SummaryThe effects of thalidomide on chemotaxis of normal human peripheral blood PMN leukocytes have been studied in vitro. The chemotaxis factor was generated by interacting normal human serum with bovine gamma globulin-antibovine-gamma globulin immune complexes. At concentrations of 1, 10, and 100 μg/ml, thalidomide failed to inhibit the chemotactic factor. At the same concentrations, erythromycin caused a marked inhibition of chemotaxis. Pre-incubation of PMNs with thalidomide or erythromycin caused a marked, dose-independent inhibition of chemotaxis. Random mobility did not appear to be affected.Inhibition of PMN chemotactic ability by thalidomide may account for its ability to improve inflammatory dermatoses, such as aphtosis.ZusammenfassungDie Wirkung von Thalidomid auf die Chemotaxis normaler menschlicher gelapptkerniger Leukocyten im peripheren Blut wurde in vitro untersucht. Der Chemotaxis-Faktor wurde durch Interaktion eines normalen menschlichen Serums mit Rinder-Gammaglobulin-Anti-Rind-Gamma-globulin-Immunkomplexen erzeugt. Bei Konzentrationen von 1, 10 und 100 μg/ml wurde durch Thalidomid keine Inhibition des Chemotaxis-Faktors hervorgerufen. Bei denselben Konzentrationen inhibierte Erythromycin die Chemotaxis deutlich. Eine Präinkubation polymorphkerniger Leukocyten mit Thalidomid oder Erythromycin verursachte eine starke dosisunabhängige Inhibition der Chemotaxis. Die Zufallsmobilität schien dabei nicht betroffen zu sein.Die Inhibition der chemotaktischen Fähigkeit polymorphonucleärer Leukocyten, verursacht durch Thalidomid, könnte für die Potenz dieses Medikamentes sprechen, entzündliche Dermatosen, wie die Aphtose, günstig zu beeinflussen.

PUVA-induced repigmentation of vitiligo: a histochemical (split-DOPA) and ultrastructural study.

Repigmentation induced by oral photochemotherapy (8‐MOP+UV‐A) in four patients with vitiligo has been studied by histochemical and ultrastructural techniques. Hypertrophic melanocytes were demonstrated both in the middle and deep portions of the hair follicles in the centre of islands of repigmentation and also in the epidermis of the expanding repigmenting border. Mitosis of melanocytes was absent in these areas. Ultrastructural study showed that the melanocytes of repigmented areas were hyperactive. The melanosomes were larger than those of surrounding healthy skin, although the mode of packaging was unaltered. These observations suggest that melanocytes repigmenting vitiliginous skin under the influence of oral photochemotherapy are derived from a melanocytic reservoir localized in the hair follicles.

Growth and differentiation of human epidermal cultures used as auto‐ and allografts in humans

Human keratinocytes from small skin specimens were grown on mouse 3T3 cell feeder layers into epidermal sheets free from Langerhans cells and MHC class II antigen. These were found to be suitable for the permanent coverage of wounds when used as autografts or allografts. We report here the ultrastructural differentiation of this cultured epidermis after grafting onto autologous or allogeneic recipients. The cultured epidermis was a thin but multilayered Malpighian epithelium composed of keratinocytes at different stages of differentiation. The dermo‐epidermal basement membrane was newly synthesized during the first few days following transplantation onto de‐epidermized wounds. The analysis of keratins and examination of various keratinocyte membrane antigens by immunofluorescence indicated that full terminal epithelial differentiation was only achieved after in vivo transplantation of the cultured epidermis. Langerhans cells, absent in cultures, progressively colonized the grafts, while melanocytes, not detectable in sections of the cultures, were identified among the keratinocytes 2 weeks after grafting.

Precocious appearance of involucrin and epidermal transglutaminase during differentiation of psoriatic skin

We compared the distribution in psoriatic skin of three different markers usually found in the stratum granulosum of normal skin. Using dansylcadaverine, we demonstrate that epidermal transglutaminase activity can be detected in most of the suprabasal layers of involved psoriatic skin and that the epidermal transglutaminase activity closely matches involucrin distribution. The glycoprotein GP37 was not detected in involved psoriatic skin of stable lesions. These results suggest that the integrated control of several independent pathways of terminal differentiation is lost in psoriasis, resulting in the classical feature of parakeratosis with absence of the stratum granulosum.

Progressive Replacement of Human Cultured Epithelial Allografts by Recipient Cells as Evidenced by HLA Class I Antigens Expression

Human keratinocytes may be grown in vitro into living epithelia devoid of Langerhans cells and MHC class II antigens. These epithelia have been shown to be usable as epidermal allografts in patients with dermal wounds, without any apparent sign of rejection in the 12-month follow-up study. To evidence a progressive replacement by recipient cells of the grafted keratinocytes, we employed anti-MHC class I antigen monoclonal antibodies directed against tissue specificities expressed by either donors or recipients. At 2 and 4 weeks after grafting, some small epithelial cell islets from the recipient phenotype were clearly identified among cells from a donor origin by indirect immunofluorescence. At 6 months, all keratinocytes present at the grafted areas were labelled by antibodies directed toward recipient specificities only. This replacement may be related to the fact that, when placed on such superficial dermal wounds, the allografts are likely colonized by epithelial cells proliferating from residual recipient dermal appendages.

Oral photochemotherapy in the treatment of lichen planus (LP). Clinical results, histological and ultrastructural observations.

Seven patients with diffuse lichen planus were treated with oral photochemotherapy. Remission was effected in 6 cases. The only failure may be attributable to a low UVA dose. Histological examination after treatment showed disappearance of the superficial dermal lymphocytic infiltrate. Ultrastructural study revealed nuclear alterations of keratinocytes and nuclear and cytoplasmic changes in superficial dermal cells.