p53 gene mutations in human epithelial skin cancers.

In the present study we analysed 38 epithelial skin cancers, 19 basal cell carcinomas (BCCs), 13 squamous cell carcinomas (SCCs) and six Bowen diseases (BwDs), using a combination of polymerase chain reaction (PCR) and single-stranded conformation polymorphism (SSCP) techniques for the presence of p53 and RAS gene mutations. Whereas 48% (9/19) of the BCCs tested presented a mutated p53 gene, the frequency was lower (15%, 2/13) in our series of SCCs and negative in the BwDs. Nine of the 11 characterized mutations were single-nucleotide substitutions and, interestingly, seven of these involved CC dimers, where a C was changed into a T or a G (three C-->T transitions and four C-->G transversions). This mutational pattern, added to the fact that all the mutated tumors occurred at sun-exposed body sites, implicates UV light in their genesis. Furthermore, we observed two internal deletions of 6 and 24 bp whose flanking sequences contained two or three Cs on either strand. In addition to molecular detection, we searched for p53 protein accumulation, by immunocytochemical staining, in a subset of 23 epithelial skin tumors (nine bearing a mutation, 14 which scored negative in our assay). Three commercially available anti-p53 antibodies (PAb CM1, mAbs DO7 and 1801) were used, and 3/23 (all showing a mutated p53 gene) presented specific nuclear staining. In contrast to other reported data we could not detect any activating RAS gene mutation in our series of human skin cancers.

The proto-oncogene c-fos increases the sensitivity of keratinocytes to apoptosis.

In human skin, most studies have suggested a role of c-fos or c-fos related genes in keratinocyte differentiation. The aim of our work was to more directly address this question by transfecting more or less differentiated keratinocyte cell lines (A431 and HaCaT) with constitutive expression vectors for c-Fos or c-Fos + c-Jun. Our results showed that c-Fos expression decreased keratinocyte growth, yet addition of c-Jun seemed to revert this c-Fos induced growth inhibition. Whereas no obvious differentiation program was turned on by c-Fos or c-Fos + c-Jun expression in our tissular model, apoptotic figures were observed and confirmed by in situ DNA fragmentation studies. These results do not rule out a role of c-Fos in keratinocyte differentiation but may indicate that the cell lines we used have reached an irreversible state of transformation so that they no longer respond to differentiation signals and rather die from apoptosis. These data add further evidence in favor of a role of c-Fos in epidermal homeostasis.

Comparative analysis of cellular and tissular expression of c-fos in human keratinocytes: evidence of its role in cell differentiation.

Recent studies on normal and pathological skin have suggested a role of the c-fos proto-oncogene in keratinocyte differentiation. To further elucidate this question we have used keratinocyte and skin culture models to study in vitro regulation of c-fos expression and attempted to correlate it with the keratinocyte maturation process. Our results show that c-fos expression is prolonged in keratinocyte monolayers both at the mRNA and protein level. Extracellular calcium which stimulate keratinocyte differentiation is able to induce c-fos expression in the presence of growth factors. However this c-fos expression cannot be maintained by these factors as seen in normal human skin in vivo. Conversely, spontaneous expression of c-fos can be seen in reconstituted skin when the neo-epidermis has completed its differentiation. All these data strongly support a role of c-fos as a switch between the early and late phases of keratinocyte differentiation allowing them to be definitively committed to their elimination process. Additionally, a differential regulation of c-fos seems to exist between keratinocyte culture and reconstituted epidermis, suggesting that tissular and serum factors are involved in the prolonged c-fos expression observed in human epidermis.

[Mycosis fungoides with predominant periorificial and mucous involvement]. / Mycosis fongoïde avec atteintes périorificielle et muqueuses dominantes.

INTRODUCTION: Specific involvement of the mucous membranes is possible during the course of mycosis fungoides but has seldom been reported, except in postmortem series. A single mucous membrane is most often involved, mainly in the mouth. Such mucous lesions are generally ominous with regard to the general outcome of the disease. OBSERVATION: A 74 year-old woman was investigated for mycosis fungoides complicated with lesions around the mouth and of the mucosa, involving the tongue and esophagus, featuring ulcerated nodules with specific chorion infiltration and epidermotropism. This progression was rapidly followed by a fatal outcome, in spite of various systemic treatments. DISCUSSION: This case report of mycosis fungoides displaying multiple and predominant oral and mucosal involvement of mycosis fungoides is unique. The rapidly unfavorable outcome confirms the ominous prognosis of mucous lesions, whereas no patent visceral extension was detected. The mechanisms underlying the mucous membranes involvement is discussed.

[Capillary malformations associated with cerebral cavernous malformation]. / Malformations capillaires associées à un angiome caverneux cérébral.

INTRODUCTION: Capillary malformations such as benign hereditary telangiectasia are a familial affection, of dominant autosomal transmission, characterized by the progressive development of cutaneous telangiectasia during childhood. The association with cutaneous vascular, arteriovenous or lymphatic malformations is exceptional and has only recently been described. CASE REPORT: A 5 year-old girl presented with widespread erythematous, predominantly telangiectasic, congenital and acquired macules when she was one year-old. Her history was marked by right temporal cerebral hemorrhage at the age of 4, revealing a right temporal cavernoma-like vascular malformation. The familial history of telangiectasic macules and clinical and histological examination led to the diagnosis of benign hereditary telangiectasia. DISCUSSION: This case report raises doubt on the exclusively cutaneous nature of benign hereditary telangiectasic-type capillary malformations. Moreover, it illustrates the possibility of a particular clinical form of this affection, associating classical telangiectasia and post-wine stain-type macules. The recent localization of the locus implied in this affection in 5q14 in the same chromosomic space as the CMC1 locus incriminated in familial capillary malformations, suggests that these two affections are in fact phenotype variations of a single and same clinical entity.

[Connubial contact dermatitis to an inhaled corticosteroid]. / Eczéma de contact par procuration à un corticoïde inhalé.

BACKGROUND: Inhaled corticosteroids are widely used in allergic asthma and rhinitis. They are most often used alone or sometimes in association. Allergic side-effects of inhaled corticosteroids are less frequent than those of topical corticosteroids. We report a case of a connubial dermatitis to a budesonide spray. OBSERVATION: A 3-year old boy was treated for asthma by budesonide (Pulmicort) and terbutaline (Bricanyl) aerosols with an inhalation chamber (Babyhaler). From the fourth day of treatment onwards, his mother had swollen and itchy lesions on the face with conjunctivitis several hours after the administration of the corticosteroids using the inhalation chamber. The last eruptions were marked by extensive lesions. The patient reported a worsening of her eruption when she was treated with a desonide cream (Tridesonit). Prick-tests conducted later on confirmed the contact allergy to budesonide and Pulmicort spray. They were also positive for Tridesonit cream and triamcinolone acetonide. Repeated open application tests with a 17-butyrate hydrocortisone cream (Locoid) for three weeks remainded negative. DISCUSSION: Our observation is original: allergic contact dermatitis to inhaled corticosteroids is rare, the clinical presentation mimicked angioedema although it was a delayed-type hypersensitivity, hypersensitivity was limited to group B corticosteroids and it was in fact a connubial contact dermatitis.

Immunological aspects of psoriasis: V. T. cell subsets and suppressor cell functions regulating immune responses in peripheral blood.

T cell subsets bearing Fc-receptors for either IgG (TG) or IgM (TM), and suppressor cell activity of peripheral blood mononuclear cells on in vitro lymphoproliferative responses were studied in patients with untreated psoriasis. The proportions of TG and TM cells were unmodified in 15 patients compared to 15 control subjects studied in parallel experiments. The concanavalin A-induced suppressor cell activity, as well as the spontaneous suppressor cell function of in vitro short-lived adherent cells, were in the normal range for 7 out of 8 psoriatic patients investigated. The data argue against the possibility that a generalized suppressor cell defect occurs in psoriasis.

Mutation of the tumor suppressor gene TP53 is not detected in psoriatic skin.

We investigated the tumor-suppressor gene, TP53, in psoriatic skin at gene (GP53) and protein (p53) levels. No mutation was detected in the seven exons analyzed using a combination of polymerase chain reaction and single-strand conformation polymorphism techniques. In addition, by immunohistochemistry using three different anti-p53 antibodies, no positive staining, reported to be associated with mutations and/or abnormal expression of the TP53 gene, was observed in psoriatic skin biopsies, in contrast to recent reports. These results suggest that the proliferative status of psoriatic keratinocytes does not implicate the TP53 gene.

Characterization and subcellular localization of calcium-dependent phospholipid binding proteins (annexins) in normal human skin and reconstituted epidermis.

Annexins represent a widespread family of Ca(++)-dependent phospholipid binding proteins. Although their precise functions are still unknown, they probably play an important role in cell regulation because they are major substrates for various growth factor receptor kinases. We characterized annexins in human skin using three different antisera raised against annexin II, annexin V, and a synthetic peptide that resembles the consensus sequence of all annexins. In normal human skin, using SDS-PAGE, two-dimensional gel electrophoresis and immunoblot analysis, we identified two major 34-kDa proteins and one 36-kDa protein, with respective isoelectric points of 6.5, 5.2, and 7.2-7.9. According to these criteria they were identified as annexins, I, V, and II, respectively. Minor 45-51 kDa and 68-kDa proteins with 6.1-6.7 and 6.8-7.1 isoelectric points were also present, and likely corresponded to annexins VII and VI, respectively. We investigated the ability of these proteins to bind phospholipids in the presence of calcium using liposomes formed from a mixture of phosphatidylserine and phosphatidylcholine. The cellular distribution of annexins in normal human skin was determined by immunofluorescence with antiannexin II and anti-annexin V antibodies. Labeling with both antibodies was observed predominantly at the cell membrane with some cytoplasmic staining also being apparent. Specificity was confirmed by the absence of staining using pre-immune sera or after the absorption of the antibodies with their corresponding antigens. These proteins were also characterized in vitro in a reconstituted human skin model. All were present in this system except annexin VI and VII, which were lost after phospholipid purification. Further experiments should now be carried out using this system to clarify the role and regulation of these proteins within the epidermis.

C-fos and c-jun proto-oncogene expression is decreased in psoriasis: an in situ quantitative analysis.

Psoriasis is a common, sometimes severe, non-malignant skin disease characterized by hyperproliferation and abnormal differentiation of keratinocytes. Because proto-oncogenes are implicated in both cell proliferation and differentiation, their expression could be modified in skin diseases such as psoriasis. The c-fos and c-jun proto-oncogenes, whose products associate to form a heterodimeric transcription factor, are among the first genes to be expressed when certain cells are stimulated to either proliferate or differentiate. Recent studies in our laboratory have shown that the c-fos proto-oncogene is highly expressed in normal human adult skin. In the present study, we used in situ hybridization with RNA to compare the expression and localization of c-fos and c-jun transcripts in 15 lesional and non-lesional psoriatic skin samples. Two clinical variants of psoriasis were studied: the most severe and chronic form or plaque-type psoriasis (N = 10) and rapidly resolutive guttate-type psoriasis (N = 5). Quantitative analysis was performed using a semi-automatic image analyzer and the "Starwise grain" software program. Our control samples included 10 normal skins and eight specimens from other benign hyperproliferative non-psoriatic skin diseases, consisting of three with inflammation (seborrheic dermatitis and atopic dermatitis), and 5 without inflammation (seborrheic keratoses). Control genes we used for in situ hybridization and RNA integrity were keratin 14, which is expressed in the epidermis and was normally expressed in all tissue analyzed, and ribosomal RNA. Our data showed that c-fos and c-jun were expressed to an equivalent extent, both spatially and quantitatively, in all specimens tested. Expression was significantly decreased in plaque-type but not in guttate-type psoriasis. It was also decreased in the three other benign inflammatory cutaneous hyperproliferative disorders, but not in the five non-inflammatory cases. These results were surprising because hyperproliferation was here associated with a decrease in proto-oncogene expression, thus suggesting that c-fos and c-jun do not play a crucial role in the control of keratinocyte proliferation in vivo. However, their reduced expression in some abnormally differentiated skins indicates that both c-fos and c-jun proto-oncogenes may play a key role in keratinocyte differentiation. Their altered expression correlated with severity of the disease and the presence of an inflammatory infiltrate. These data offer a new insight into the role and regulation of these proto-oncogenes in vivo in humans.

TP53 tumor suppressor gene and skin carcinogenesis.

The tumor suppressor gene TP53 encodes for a nuclear phosphoprotein involved in the control of cell proliferation, particularly in stressed cells. TP53 gene mutations are the most frequent genetic event found in human cancers. Most mutations locate in the highly conserved domains of the gene. Their localizations vary according to the tissue and tumor type, but define some hot spot regions that may have a certain degree of tissue specificity. In certain cases, the type of nucleotide substitutions observed can help to find the carcinogenic agent. In recent years, TP53 gene mutations have been frequently observed in human skin tumors. In epithelial carcinomas, they involve mainly exons 5, 7, and 8. Interestingly, many are C to T transitions at dipyrimidine sites; particularly, one can find CC to TT double-base changes that are known to be specific to ultraviolet radiation. These data confirm at the molecular level the role of ultraviolet radiation as an important etiologic factor in the genesis of these lesions. The high incidence of TP53 mutations suggest that they play a role in keratinocyte transformation. Nevertheless, this event has not yet been defined as an early or late event. In melanomas, most studies have shown the detection of the p53 protein by immunohistochemistry, suggestive of the presence of a mutation in the gene prolonging the protein half-life. Anti-p53 reactivity is frequent in these tumors and seems to correlate with tumor aggressiveness. Confirmation and characterization of TP53 gene mutation at the DNA level would help to precisely define the role of this gene in the development of these tumors.

C3d,g deposits in inflammatory skin diseases: use of psoriatic skin as a model of cutaneous inflammation.

Recent studies in our laboratories have shown that human keratinocytes synthesize and secrete complement components including C3. Moreover, human keratinocyte-derived C3 is regarded as a potential source of C3d,g, a recently described constituent of the sublamina densa region of normal epidermal basement membrane. Additionally, human keratinocyte-derived C3 may also contribute to epidermal basement membrane deposits of C3 in autoimmune or inflammatory skin disorders. To further our understanding of the specificity and origin of epidermal basement membrane C3 deposits in normal and diseased skin, we have characterized in situ deposits of C3 and C3 cleavage fragments in various inflammatory skin diseases and utilized a skin equivalent model to assess the deposition of C3 cleavage fragments in neo-basement membrane of epidermal outgrowths from normal or diseased human skin. C3d,g reactivity was found to be greater in all samples of inflamed skin, and typically associated with C3c reactivity at these sites. No immunoglobulins or other complement components were detected. When lesional psoriatic skin rich in epidermal basement membrane C3c was used in our organ culture system, C3 incorporation within neo-basement membrane was observed. These results show that human keratinocyte-derived C3 may contribute to inflammatory reactions in skin as well as account for deposits of C3d,g in normal epidermal basement membrane.

High levels of c-fos proto-oncogene expression in normal human adult skin.

The proto-oncogene c-fos is thought to play an important role in the modulation of cell growth and differentiation. In normal tissues that have been studied to date, c-fos expression has been found to be regulated in a tissue-specific manner. Actually, little is known about its expression in normal human adult skin (NHAS). Moreover, the epidermis is a useful tissue to study the role of cellular oncogenes because keratinocytes can be observed simultaneously in their proliferative as well as differentiated state. We studied c-fos expression in NHAS using different molecular approaches which permit us to characterize and localize c-fos products within the epidermis, specifically, at the RNA level by Northern blot and in situ hybridization, and at the protein level by immunofluorescence and Western blotting. Here, we show that both c-fos mRNA and protein are present at high levels in NHAS. These results contrast with the low level of c-fos expression reported for most human adult tissues. Furthermore, c-fos expression is visible throughout the epidermal layers indicating that it is not restricted to proliferating basal cells. The epidermis, therefore, represents the first human adult tissue where c-fos is expressed at high levels in vivo and provides an interesting model to further elucidate the role of this proto-oncogene in normal and pathologic conditions.

Dapsone as initial treatment in superficial pemphigus. Report of nine cases.

Nine cases of superficial pemphigus that were treated by dapsone as the initial mode of therapy are reported. Five patients, all having low or negative serum anti-intercellular cement substance antibody titers, responded dramatically, while the other four patients, showing high titers of anti-intercellular cement substance antibodies, did not respond to this treatment. Complications of therapy were observed in three cases.

Immunofluorescence in psoriasis: studies of immunoglobulins, complement deposits, and three membrane markers.

Sixteen psoriatic patients and 11 control subjects were investigated by immunofluorescence for skin immunoglobulins (IG) and complement (c) deposits and for keratinocyte membrane markers with anti beta 2 microglobulin (beta 2 m) antibodies (Ab), Con A, and pemphigus Ab. IG and/or c deposits were almost constant in involved epidermis. Three patterns were associated: (1) parakeratotic nuclear (dots-dashes), (2) stratum corneum (SC) intercellular (lamellar pattern), (3) vascular (granular or linear deposits in papillary dermal vessels). In uninvolved epidermis nuclear or vascular deposits could occasionally be present but intercellular pattern was never found in SC. Control specimens were always negative. The three surface markers investigated (beta 2 m, Con A receptor, Pemphigus antigen) could be demonstrated on psoriatic keratinocytes with only slight differences in distribution when compared with controls. Thus, by this methodology no important abnormality could be found in psoriatic keratinocyte membrane antigens.

Neutrophil chemotaxis in psoriasis before and after PUVA therapy.

Chemokinesis and chemotaxis of neutrophil leucocytes were studied by migration under agarose in 35 patients with psoriasis and compared with 35 healthy controls. Eschericheae coli filtrate and lipopolysaccharide were used as chemo-attractants. Of these patients, 14 were tested before and after photochemotherapy. The chemotactic capacities of psoriatic serum before and after PUVA therapy were also investigated. The increase in the chemotactic activity of psoriatic polymorphonuclear leucocytes (PMNs) was not significant and remained unchanged after PUVA therapy. The activated psoriatic serum showed a slight increase in chemotactic activity when compared with normal activated serum. These results do not support an intrinsic abnormality of psoriatic PMNs, and their migration into psoriatic lesions could be chiefly due to the presence of chemotactic factors in involved epidermis.

Studies of polymorphonuclear migration into psoriatic skin using a new in vivo method.

A new method, employing a skin-implanted cell trap already used to study chemotaxis in cancer patients, was applied to 35 healthy volunteers and 12 psoriatic patients. A dacron disk impregnated with 10 microliters of 4-6.10(6) live BCG suspension was implanted in the deep dermis. After 24 h the disk was removed, and five sections of each disk were counted for polymorphonuclear leukocytes (PMNs) and monocytes. Involved and uninvolved psoriatic skin showed a decrease of PMN migration into the disk as compared with controls. No difference could be demonstrated between involved and uninvolved skin. Mononuclear cell chemotaxis was the same in psoriasis as in controls. These results are in agreement with other in vivo data using mainly the skin chamber technique indicating a decrease of PMN chemotaxis in psoriatic skin at 24 h.

Somatostatin treatment of psoriasis.

Somatostatin treatment was administered to 20 psoriatic patients according to the following protocol: Continuous infusion (250 micrograms/h) for at least 2 days followed either by short infusions (1 h) at 8 A.M. and 8 P.M. (12 cases) or by repeating the initial 2-day infusion (eight patients). Before treatment (day 0) and on day 6, biopsy specimens were taken for routine examination (12 patients) and for ultrastructure (seven patients). In vitro immunological studies were carried out on peripheral blood lymphocytes (six patients) on day 0 and day 8. In two patients, somatostatin was stopped because of serious side effects. Thus, clinical results were evaluated in 18 patients, on day 30. In ten of them no improvement whatsoever occurred, two had a partial clearing and an almost complete remission was achieved in six others. Ultrastructural studies showed, on day 6, enlargement of the intercellular spaces with deposits of granular material of glucidic composition, associated with features of cellular damage. Percentages of T and B cells were unmodified but a significant depression of mitogenic stimulation by PHA and ConA was clearly observed on day 8. Even if somatostatin treatment may have a beneficial effect in some patients it seems much less valid than other well-known therapies for psoriasis.

Unlikely role of Epstein-Barr virus in the pathogenesis of primary cutaneous CD30+ anaplastic large cell lymphoma.

BACKGROUND: Primary cutaneous CD30+ anaplastic large cell lymphoma (ALCL) is a rare subset of cutaneous lymphoma, with a much better prognosis than its nodal counterpart. The pathogenesis of both nodal and primary cutaneous CD30+ ALCL is largely unknown but experimental data support the hypothesis that the Epstein-Barr virus could play a role in the nodal subset. OBJECTIVE: To evaluate the involvement of Epstein-Barr Virus (EBV) in primary cutaneous CD30+ ALCL by searching for both nucleic acids and EBV proteins in cutaneous lesions. SETTING: Two University Hospitals in Southern France (secondary referral hospitals). PATIENTS: Eight consecutive patients with typical primary cutaneous CD30+ anaplastic large cell lymphoma were studied. METHODS: Search for the presence of DNA, RNA and EBV proteins in cutaneous lesions by PCR, in situ hybridization and immunohistochemistry. RESULTS: EBV DNA and RNA was identified in only one lesion of primary cutaneous CD30+ ALCL and in none of the normal adjacent skin samples. In situ hybridization and immunohistological studies were consistently negative in all samples. CONCLUSION: These results do not support an early role of EBV in the oncogenetic pathogenesis of primary cutaneous CD30+ ALCL.

Skin perfusion pressure in leg ulcers assessed by photoplethysmography.

The aetiology of lower limb ulcers is often the result of intricate vascular pathology and the quality of the peri-ulcer microcirculation is a major factor in the prognosis of the disease. We measured the skin perfusion pressure (S.P.P.) using an infra-red photoplethysmograph of the PPG type (ESM, Mauguio, France). The study included 30 healthy subjects, 25 patients with leg ulcers of varied etiology (venous: 14, arterial: 11), and 15 patients with uncomplicated varicose veins. Measurements were undertaken in dorsal decubitus position after a 15 minute rest. The S.P.P. was expressed as the percentage of humeral blood pressure. The results showed an important decrease of S.P.P. in patients with arterial ulcers when compared to patients with healthy legs (39.9 +/- 16.4% and 82.8 +/- 10.0% respectively). The S.P.P. values were intermediate (54.4 +/- 19.3%) in patients with venous ulcers while they were normal in the patients with uncomplicated varicose veins (87.3 +/- 12.3%). Among this population, 18 patients (11 with uncomplicated varicose veins and 7 with venous ulcers) were investigated before and after a one month treatment with a flavonoid (Daflon 500 mg, 2 tablets per day). An increase of S.P.P. to normal values after treatment was found in patients with venous ulcers though not in patients with varicose veins. This non invasive procedure appreciating the microcirculation status in peri-ulcerous skin could be of predictive value for the healing potential. It could be a good method to assess the efficacy of vasculotropic agents.