Skin autografts in epidermodysplasia verruciformis: human papillomavirus-associated cutaneous changes need over 20 years for malignant conversion.

Epidermodysplasia verruciformis (EV) is regarded as a model for cutaneous oncogenesis associated with specific human papillomaviruses (HPVs). Because genital HPV-associated carcinogenesis is a very long-lasting process requiring 20-30 years and epidemiological studies of this type for HPV-associated skin cancers are impossible in such a rare disease as EV, we observed for up to 20 years EV patients having surgery for carcinomas with consecutive autografts from uninvolved and non-sun-exposed skin. We noticed the appearance of premalignant and malignant changes around the grafts, whereas within the grafted skin, only benign macular lesions started to develop several years after transplantation. Thus, skin HPV-associated carcinogenesis appears to be a very slow process comparable to the genital carcinogenesis associated with high risk HPVs.

Cancer combination chemotherapy with retinoids: experimental rationale.

Retinoids, cytokines as well as 1,25-dihydroxyvitamin D3 and its analogs are all classes of compounds with pleiotropic actions. They inhibit proliferation in human transformed epithelial cell lines and induce differentiation in human transformed hemopoietic cell lines. In a murine model of tumor cell-induced angiogenesis all three classes of compounds inhibit the formation of new blood vessels, necessary for supplying the growing tumor with oxygen and nutrients. Combinations of compounds from the three different classes lead to higher efficacy than the compounds administered as single agents. The effects of combinations vary depending on the individual representatives of the three classes and on the particular test models used. Additive, synergistic and potentiating effects have been observed. The results obtained in experimental systems raise hope that combination therapy might be useful in the treatment of certain human neoplastic diseases.

Cancer combination chemotherapy with retinoids: experimental rationale.

Retinoids, cytokines as well as 1,25-dihydroxyvitamin D3 and its analogs are all classes of compounds with pleiotropic actions. They inhibit proliferation in human transformed epithelial cell lines and induce differentiation in human transformed hemopoietic cell lines. In a murine model of tumor cell-induced angiogenesis all three classes of compounds inhibit the formation of new blood vessels, necessary for supplying the growing tumor with oxygen and nutrients. Combinations of compounds from the three different classes lead to higher efficacy than the compounds administered as single agents. The effects of combinations vary depending on the individual representatives of the three classes and on the particular test models used. Additive, synergistic and potentiating effects have been observed. The results obtained in experimental systems raise hope that combination therapy might be useful in the treatment of certain human neoplastic diseases.

In vivo mobilization of polymorphonuclear leukocytes in psoriasis: relationship to clinical parameters and serum inhibitory factors.

Mean migration of polymorphonuclear leukocytes (PMNL) toward autologous and homologous control sera, evaluated by quantitative skin window chamber technique, was only slightly reduced in 60 patients with psoriasis as compared to 27 normal controls (p less than 0.1). A significant decrease in cell migration was found (1) in patients with actively spreading lesions, (2) in patients with extensive lesions involving more than 40-60% of the skin surface, (3) in the first 2 months of relapse, and (4) 5-6 months after onset of new lesions. However, PMNL migration was increased when psoriatic lesions lasted 3-4 months. Seventy-one percent of psoriatic sera exerted a suppressive effect on the psoriatic and normal PMNL migration. The inhibitors were found predominantly in patients with stationary and long-standing lesions. Some of the psoriatic sera had a stimulatory effect on the chemotaxis of psoriatic PMNL. These sera originated from those patients with active spreading lesions in the first 2 months of relapse. These data indicate that neutrophil migration is abnormal in the course of psoriasis and that it could be modified by different proportions of both inhibitors and stimulators of chemotaxis.

The activity of polymorphonuclear leukocyte neutral proteinases and their inhibitors in patients with psoriasis treated with a continuous peritoneal dialysis.

3 of 16 patients with extensive psoriasis have been completely cleared of skin lesions within 2-3 weeks of continuous peritoneal dialysis, and 2 of them up to 2 mo after termination of therapy. In 5 cases there was a great improvement of psoriatic lesions and in 6 remaining cases only a slight improvement was found. The remission of psoriasis was correlated with extremely high polymorphonuclear leukocyte depletion through the peritoneal cavity in a short time. Neutral serine proteinases were extracted from polymorphonuclear leukocytes and quantitated. The quantity of enzymes in the cells recovered from peritoneal dialysates was found to decrease with duration of treatment, and it was 2-5 times lower than amounts of neutral proteinases extracted from peripheral blood polymorphonuclear leukocytes of psoriatics and normals. The enzyme content per polymorphonuclear leukocyte of patients with active psoriasis was significantly higher (2-fold) than that in inactive psoriasis and in normal controls. Proteinase activity was also found in the sera of psoriatics and normals, as well as in the peritoneal dialysates. However, this activity appeared to be about 30-50 times lower than serum inhibitory activity against neutral proteinases. The concentration of neutral proteinase inhibitors in 5 of 17 sera of patients with psoriasis was significantly lower than that in normal sera. These data indicate that the depletion of activated PMNL with increased amounts of neutral proteinases may account for the beneficial effect of peritoneal dialysis in the clearing of psoriatic lesions.

Enhanced angiogenic capability of monocyte-enriched mononuclear cell suspensions from patients with systemic scleroderma.

Different subsets of peripheral blood mononuclear cells (MNC) from 15 patients with systemic scleroderma were tested for their ability to evoke angiogenesis in a xenogenic system. The angiogenic capability of total MNC from patients with systemic scleroderma was lower than that of normal human cells, irrespective of the form of the disease. However, the capability of a monocyte-enriched subset of MNC from patients with scleroderma was found to be increased, as compared with their total MNC and with that of the corresponding subset from healthy individuals. This might be due to the activation of monocytes in the disease.

Interleukin-12 inhibits angiogenesis induced by human tumor cell lines in vivo.

Tumor cell-induced angiogenesis, i.e., new blood vessel formation within tumor tissue, is an essential requirement for the growth of solid neoplasms. Interleukin-12 (IL-12) inhibits growth of a variety of experimental tumors in vivo. We tested whether antitumor activity of IL-12 is related to the inhibition of angiogenesis induced by tumor cell lines. Angiogenesis was induced in x-ray immunosuppressed Balb/c mice by intradermal injection of the following human tumor cells: T47D, originating from mammary carcinoma; A431, derived from vulval carcinoma; and Skv, established from bowenoid papulosis, Systemic treatment of the mice with murine IL-12 significantly decreased angiogenesis induced by human tumor cells in a time-and dose-dependent manner. Preincubation of human cells in vitro with IL-12 did not inhibit tumor cell-induced angiogenesis, suggesting that the antiangiogenic capacity of IL-12 is restricted to in vivo conditions. Treatment of the mice with rat antibody against murine interferon-gamma (IFN-gamma) resulted in counteracting the antiangiogenic effect of murine IL-12. Furthermore, human IFN-gamma inhibited the angiogenic activity of human tumor cell lines. This indicates that IFN-gamma is a mediator of the antiangiogenic effect of IL-12. The results show that the mechanism of antitumor action of IL-12 may depend not only on the immunostimulatory activity of this cytokine but also on its effect on tumor cell-induced angiogenesis. IL-12 should be considered as a potential candidate for the treatment of angiogenesis-dependent malignancies.

Defective function of T lymphocytes in psoriasis.

The distribution of thymus-derived (T) and bone marrow-derived (B) lymphocytes in 100 patients with psoriasis were studied by the rosetting techniques. Depression of the number of T lymphocytes forming spontaneous rosettes with sheep erythrocytes (E rosettes) occurred in 66% of patients, whereas no difference in B lymphocytes bearing C3 receptor (EAC rosettes) was observed between psoriatics and normals. The decrease in E rosettes was associated with the active phase of the disease. This disappeared 4-6 wk after onset of remission, which suggested that the abnormality in T-cell marker distribution is transitional. Lymphocytes forming neither E nor EAC rosettes, which were found to be significantly increased in active psoriasis, were identified as T lymphocytes since they reacquired normal E rosette function during short-term preincubation with concanavalin A (Con A). A serum factor was also demonstrated which inhibited E rosette formation by normal peripheral blood lymphocytes. Its activity increased linearly within 2 mo from the onset of skin lesions. The data suggest that in active psoriasis serum factors may be coated on the lymphocyte surface membrane which may be responsible for blocking of specific receptor for sheep erythrocytes and/or interfere with T lymphocyte function.

Human papillomavirus 8-L1 immune serum: a new diagnostic possibility for Epidermodysplasia verruciformis-specific HPVs.

Human papillomavirus (HPV) 8 induces flat macular skin lesions with a high risk of malignant conversion. A 600-bp fragment from the center of the major structural protein gene L1 was cloned into pEX2 to produce a beta-galactosidase-L1 fusion protein. The expressed part of L1 is masked in intact virions and not detected by sera of infected patients. Immunization of guinea pigs with purified fusion protein raised high-titer antisera, which reacted with capsid proteins of HPV8 and closely related viruses in Western blot and indirect immunofluorescence tests. Structural proteins of HPV1, 2, and 3 were not detected, indicating specificity for the subgroup of ev-specific HPVs. The sera present themselves as convenient diagnostic reagents for the demonstration of infections with potentially oncogenic HPVs using routine immunofluorescence procedures.

Theophylline-resistant and theophylline-sensitive "active" and "total" E rosette-forming lymphocytes in patients with systemic scleroderma.

Using the E rosette test and its modification with theophylline, we have studied T regulatory lymphocytes in various forms of systemic scleroderma. Mean percentages of active rosette-forming cells (ARFC) as well as the fraction resistant to theophylline incubation (ARFC-res) were significantly decreased, irrespective of the variety of the disease, compared to the age-matched controls. Late ("cold") rosette-forming fractions were unimpaired. The theophylline-sensitive fraction of total rosette-forming cells (TRFC-sens), which contains mainly cells from the suppressor circuit, was found to be lowered in all patients groups studied, whereas the ARFC-sens fraction was significantly decreased only in patients with diffuse scleroderma over 50 years of age, in whom there was a tendency to a more severe course, as manifested by pronounced systemic organ involvement. The lowered values of E rosette tests were found in a majority of SSc patients and were correlated with the appearance in the sera of factors capable of inhibiting ARFC formation by normal human peripheral blood T lymphocytes. Normal values of the E rosette test were related to the presence in the patients' sera of factors stimulating ARFC formation by normal lymphocytes. We surmise from the results that in SSc patients the T-cell defect is not only restricted to T suppressor cells but also refers to the active theophylline-resistant fraction containing mainly T inducer and T cytotoxic cells.

Premalignant lesions and cancers of the skin in the general population: evaluation of the role of human papillomaviruses.

To evaluate the role of human papillomaviruses (HPV) in the development of premalignant lesions and cancers of the skin in the general population, 314 biopsies obtained from 227 patients with benign neoplasms, premalignant lesions, and cancers of the skin and from 25 patients with squamous cell carcinoma of the lip were analyzed by Southern blot hybridization. DNA probes specific for various cutaneous and genital HPV types were used in hybridizations conducted under nonstringent or stringent conditions. HPV DNA sequences were only detected in eight specimens obtained from six patients: HPV 34 in one case of periungual Bowen's disease, HPV 36 and an as yet uncharacterized HPV in two cases of actinic keratosis, HPV 20 in one case of basal cell carcinoma, an as yet unrecognized HPV in one case of squamous cell carcinoma, and HPV 16 in one case of squamous cell carcinoma of the lip. None of the specimens of cutaneous horn and keratoacanthoma contained detectable HPV DNA. In contrast, HPV DNA sequences, mostly HPV 16, were detected in 13 of 23 cases of anogenital Bowen's disease and invasive Bowen's carcinoma. HPV DNA sequences were not detected in 90 cutaneous samples further analyzed by the polymerase chain-reaction technique, using amplification primers that contain conserved sequences among the genomes of HPV. These results strongly suggest that the known HPV types play only a minor role, if any, in skin carcinogenesis in the general population.

Lowered angiogeneic capability of peripheral blood lymphocytes in progressive systemic sclerosis (scleroderma).

Peripheral blood lymphocytes isolated from 19 patients with progressive systemic sclerosis (7 with diffuse scleroderma and 12 with CREST syndrome) and from 19 healthy control individuals were tested in a lymphocyte-induced angiogenesis assay. The cells were injected intradermally into x-ray-immunosuppressed mice and their capability to induce new blood vessel formation was assessed by morphologic criteria. The lymphocytes derived from patients with systemic scleroderma showed a significant decrease in angiogeneic capability compared with controls. No significant difference in this capability was found between patients with diffuse scleroderma and those with CREST syndrome. The decrease in the angiogeneic capability of lymphocytes reflects a depression in cell-mediated immunity and might be relevant to the capillary loss observed in systemic scleroderma.

TGF beta-1 and TNF alpha expression in the epidermis of patients with epidermodysplasia verruciformis.

In epidermodysplasia verruciformis (EV), the infection with specific human papillomaviruses (HPV) might be under control of the local immunosurveillance mechanisms related to cytokines produced by epidermal cells. We have investigated by in situ hybridization the expression of mRNA coding for TGF beta-1 and TNF alpha in the skin of patients with EV (n = 4) as compared to the skin lesions of patients with other premalignant (actinic keratosis; n = 5) or malignant (squamous cell carcinoma; n = 4) skin lesions, and to the skin of healthy individuals (n = 5). The expression of TGF beta-1 and TNF alpha mRNA was higher in the epidermis of EV patients as compared to the control skin from healthy individuals. The increased expression of mRNA for both cytokines was confirmed by northern blot analysis of RNA isolated from the skin lesions of the patient with EV. No specific signals for TGF beta-1 and TNF alpha were detected in actinic keratosis, and in cases of squamous cell carcinomas only single neoplastic cells were positive for TGF beta-1. It is conceivable that in EV TGF beta-1 and TNF alpha can be involved in the regulation of the growth and differentiation of HPV-infected keratinocytes and in the persistence of HPV-induced skin lesions.

Juvenile dermatitis herpetiformis versus "benign chronic bullous dermatosis of childhood." Are these immunologic diseases?

Seven cases of juvenile dermatitis herpetiformis have been investigated. Immunofluorescence and histologic studies were made in all and jejunal biopsies in three. Immunopathologic results were positive in all cases including one that had previously been reported to be negative. Two groups could be distinguished according to clinical and histologic criteria, response to sulfapyridine, and character of the immunoglobulin deposits. The first corresponded to dermatitis herpetiformis (DH) of adults, with characteristic lesions of the jejunal mucosa; the second corresponded either to bullous pemphigoid (BP), although in the majority of the cases without circulating antibasement-membrane antibodies, or to a mixed type with the combined features of DH and BP. Repeated biopsies with serial sections are essential for demonstrating immune deposits. The question arises whether any immunologically negative cases of "benign chronic bullous dermatosis of childhood" actually exist.

Identification of papillomaviruses in butchers' warts.

We have studied the papillomaviruses found in the hand warts of 60 butchers, most of them from 2 distant slaughterhouses. Warts differing in morphology and location were studied separately. The viruses were identified by molecular hybridization, restriction enzyme analysis and immunofluorescence. Four known human papillomaviruses (HPV-1, HPV-2, HPV-3, HPV-4) were detected and one hitherto unknown papillomavirus was identified in 9 butchers. The DNA of the latter virus did not anneal with any of the RNAs complementary to either HPV-1 to HPV-5 or bovine papillomavirus type 1 (BPV-1) DNAs, and showed a Hind II + III restriction enzyme cleavage pattern distinct from those of known HPVs and BPVs. This virus showed distinct antigenic properties, as shown by immunofluorescence, using HPV-1, -2, -3, -5, and BPV-1 antisera. It may represent a new type of human papillomavirus (HPV-7) or a yet unidentified animal papillomavirus. In addition, 6 butchers were found to be infected with a papillomavirus, distinct from the known skin HPVs and from BPV-1, which could not be characterized by restriction enzyme analysis. Eleven butchers were found to be infected by 2 viruses. A characteristic histological pattern was found to be associated with the different papillomaviruses.

The ultrastructural localization of IgA deposits in chronic bullous disease of childhood (CBDC).

A case of bullous disease in a child with linear IgA immune deposits at the basement membrane zone and with some clinical, histological, and electron microscopic characteristics both of dermatitis herpetiformis and bullous pemphigoid, is described. The bulla formed between the basal lamina and basal cell membranes as in bullous pemphigoid, but at the same time there were numerous inflammatory cells in the dermis just below the partly destroyed basal lamina and also abundant fibrin deposits in very recent bulla and in the skin, all of which is rather characteristic of dermatitis herpetiformis. Ultrastructurally, the IgA deposits were located chiefly below the lamina basalis (the dermal type) but also, though less abundantly, in the lamina lucida, very much as we have seen them to be in adult cases with linear IgA immune deposits at the basement membrane zone. The investigations have supplied further evidence showing the chronic bullous disease of childhood to be actually a counterpart of the form in adults with the same linear localization of IgA deposits.

Detection of DNA-psoralen photoadducts in mammalian skin.

An immunofluorescence (IF) method for the detection of 8-methoxypsoralen (8-MOP) photoadducts to DNA has been developed to assess nuclear damage in keratinocytes and melanocytes after psoralen plus UVA (PUVA) treatment, both under in vitro and in vivo conditions. Cryostat sections of the albino and pigmented guinea pig and human skin were used for in vitro studies to establish minimal and maximal drug concentration and UVA dosimetry for the detection of DNA-8-MOP photoadducts. Limits of detection were as low as 10 ng/cm2 8-MOP and 1 J/cm2 UVA for skin sections and sodium bromide-split epidermal sheets. Guinea pigs treated with topical PUVA revealed positive IF stain in epidermal cell nuclei at a threshold dose of 100 micrograms/cm2 8-MOP and 13 J/cm2 UVA. Pretreatments of cryostat cuts with ethanol and alkali before IF test enhanced the sensitivity of detection in vivo about 10-fold and enabled us to follow the repair of DNA damage after treating normal guinea pig skin with a dose of 50 micrograms/cm2 8-MOP plus 6 J/cm2 UVA. The most interesting findings were as follows: A sensitive method to detect PUVA-induced nuclear damage in epidermal and dermal cells was developed. PUVA treatment induced nuclear DNA damage to melanocytes as well as to adjacent keratinocytes, and melanocytes appeared to be 10 times less vulnerable to photo-damage than keratinocytes. There was a greater propensity for the proliferative cells to be damaged by PUVA. PUVA induced nuclear damage up to 700 micron depth in the dermis. The usefulness of the IF test in detecting DNA damage in microgram and ng amounts in vivo and in following the repair of damaged DNA induced by PUVA.

Human papillomavirus (HPV) types specific of epidermodysplasia verruciformis detected in warts induced by HPV3 or HPV3-related types in immunosuppressed patients.

Epidermodysplasia verruciformis (EV) is characterized by an abnormal genetic predisposition to infection with specific types of human papillomavirus (HPV). Specific defects of the cell-mediated immunity and/or of the control of HPV infection in keratinocytes are assumed to be involved in the development of the disease. As a model to test this hypothesis, we have studied the prevalence of EV-specific HPV in skin warts of 56 immunosuppressed patients. All main types of cutaneous HPV (HPV1, 2, 3, 4, 10, and 28) responsible for skin warts in the general population were detected by blot hybridization. EV-specific HPV (HPV5, 20, and 23) were detected in three patients. Four additional patients were found infected with HPV49, first characterized in the course of this study, and found to be related to EV HPV. A most important finding was that HPV5, 20, 23, and 49 were always codetected with HPV3 or the related types HPV10 and 28. None of the specimens showed the typical clinical morphology of EV lesions. In none of these specimens was the specific cytopathic effect of EV recognized; instead that of HPV3 and related types was seen. No evidence for productive EV HPV DNA replication was obtained for the three specimens that could be further analyzed by in situ hybridization. Our data suggest that HPV3 infection favors infection with EV HPV in immunosuppressed patients but that the full expression of EV HPV is usually restricted as in the general population.

Psoriasis: A possible reservoir for human papillomavirus type 5, the virus associated with skin carcinomas of epidermodysplasia verruciformis.

Recent polymerase chain reaction data have shown that most human papillomavirus (HPV) genotypes associated with epidermodysplasia verruciformis (EV) are widespread; however, HPV5 associated with EV skin carcinomas has only rarely been detected in non-EV patients. To identify the reservoir of this virus, we examined 335 sera from different groups of patients for the presence of HPV5 antibodies by an enzyme-linked immunosorbent assay test based on HPV5 virus-like particles. The prevalence of antibodies reacting with HPV5 virus-like particles was found to be significantly higher in psoriatic patients (24.5%) than in other groups (2-5%), including patients with atopic dermatitis and renal transplant recipients. Analysis of scrapings of lesional and uninvolved skin by a nested polymerase chain reaction method, using degenerate EV HPV primers, disclosed HPV DNA in 91.7% of 48 psoriatic skin samples and 35.5% of 31 atopic dermatitis specimens. Eleven EV HPV genotypes, most frequently HPV5 and HPV36, and a putative novel genotype (PsoX1) were identified in psoriasis. Five EV HPV genotypes and two putative novel genotypes (ADX1 and ADX2) were detected in atopic dermatitis patients. HPV5 was not found in atopic dermatitis patients. Using type specific primers, HPV5, HPV36, and HPV1 were found in 89.4%, 84.2%, and 42.1% of specimens from psoriatic patients, whereas HPV36 was detected in 22.5% of specimens from atopic dermatitis patients. HPV16 was never detected. On the whole, 27 HPV5 and 13 HPV36 DNA variants were disclosed after sequencing amplification products. Our data confirm that EV HPV are widespread and point to psoriasis as a reservoir for HPV5. Whether HPV5 is involved in the pathogenesis of psoriasis remains to be determined.