Tannic acid induces in vitro acantholysis of keratinocytes via IL-1alpha and TNF-alpha.

The mechanism of acantholysis in pemphigus vulgaris (PV) is an intriguing argument since several chemical mediators are implicated. We previously reported a central role for IL-1alpha and TNF- alpha, both able to regulate complement activation and plasminogen activators. Very little is known about what triggers the disease (drugs, viruses or food). In this study, we evaluate the molecular role of tannins in acantholysis. By HPLC chromatography we measured tannic acid (TA) and gallic acid (GA) in blister fluid of 4 groups of patients divided according to their dietary habits, including a regular diet, a diet rich in tannins, a diet free of tannins, and a group of pemphigus patients. Blister fluid was obtained from patients using a suction blister apparatus. We show that people with a diet rich in tannins have increased tannin metabolites (TA and GA) in the skin in respect to controls (tannin-rich diet: GA = 194.52+/-2.39 nmol/ml; TA = 348.28+/-1.4 nmol/ml versus tannin-Mediterranean diet: GA = 15.28+/-1.63 nmol/ml; TA = 22.81+/-1.68 nmol/ml). PV patients showed similar values to the Mediterranean diet population (PV patients: GA = 95.8+/-1.97 nmol/ml; TA = 199.09+/-4.15 nmol/ml versus Mediterranean diet: GA = 83.53+/-2.35 nmol/ml; TA = 195.1+/-2.50 nmol/ml). In an in vitro acantholysis system using TA and PV-IgG we show that TA 0.1 mM in NHEK culture is able to induce acantholysis. This effect was able to amplify the acantholytic action of PV-IgG in vitro. A blocking study using anti IL-1 alpha and anti TNF-alpha antibodies showed a reduction in TA-induced acantholysis. Taken together, these results suggest that a diet rich in tannins could be a trigger in genetically predisposed patients. If these data are confirmed, a complementary diet poor in tannins may be useful in patients affected by PV.

Elevated 8-isoprostane levels in basal cell carcinoma and in UVA irradiated skin.

Isoprostanes are prostaglandin isomers produced from the peroxidation of polyunsaturated fatty acids from the cellular membrane. They have been used as a specific index of cellular lipoperoxidation and as an indirect measure of oxidative stress. However, these molecules also present several biological activities. An oxidative environment measured as the presence of other indirect measurements of reactive oxygen species lipoperoxidation has recently been described in basal cell carcinoma, the most frequent type of non-melanoma skin cancer. This study aims to measure the levels of 8-isoprostaglandin F2alpha, an isoprostane widely studied in other models as a by-product of ROS-induced lipid peroxidation, in basal cell carcinoma and in UVA irradiated healthy skin. We found that 8-iso-PGF2 alpha is present in higher levels in BCC specimens compared to healthy non sun-exposed skin, confirming previous studies on the production of lipoperoxidation in this tumor. Moreover, we demonstrated that topical pre-treatment with a compound containing vitamin E is capable of reducing 8-iso-PGF2 alpha formation in UV irradiated skin suggesting a role for isoprostanes in UV induced inflammation and eventually carcinogenesis and confirming the function of vitamin E as an antioxidant in this model.

Samvisterin, a new natural antiplasmodial betulin derivative from Uapaca paludosa (Euphorbiaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Uapaca paludosa is used in African traditional medicine for the treatment of malaria. MATERIALS AND METHODS: A bioguided fractionation of U. paludosa trunk bark extracts was performed on the basis of their antiplasmodial activity against Plasmodium falciparum. RESULTS: A new natural betulin derivative named samvisterin (2) was isolated. In addition, 12 already known compounds were isolated from U. paludosa and tested against P. falciparum: squalene (1); lupeol (3), betulonic acid methyl ester (4), ß-sitosterol (5), stigmasterol (6), betulin (7), betulinic acid (8), pentadecanoic acid (9), palmitic acid (10), margaric acid (11), stearic acid (12), methyl palmitate (13). With the exception of betulinic acid, all were isolated for the first time from U. paludosa. Their chemical structures were established on the basis of spectroscopic analysis. The antiplasmodial activity of compounds 1-8 was confirmed on the chloroquine-resistant strain of P. falciparum, FcM29-Cameroon, with IC50 values ranging from 0.7µg/ml (for 1) to 30µg/mL (for 3). The cytotoxicity of the fractions and isolated compounds was also determined on KB and Vero cell lines in order to determine the cytotoxicity/activity ratio of each one. CONCLUSIONS: The results obtained with samvisterin (2) show that this new compound is the most promising of the series, with a weak cytotoxicity leading to the best selectivity index values.

In vitro and in vivo expression of interleukin-1alpha and tumor necrosis factor-alpha mRNA in pemphigus vulgaris: interleukin-1alpha and tumor necrosis factor-alpha are involved in acantholysis.

Keratinocyte-derived cytokines have been implicated in the pathogenesis of a number of skin diseases. In this study we examined the possible role of keratinocyte-derived cytokines in the development of acantholysis in pemphigus vulgaris. Nineteen patients with pemphigus vulgaris, demonstrating the characteristic clinical, pathologic, and immunopathologic findings were studied. In situ immunolabeling demonstrated the presence of two cytokines interleukin-1alpha and tumor necrosis factor-alpha, in lesional and perilesional areas. Results were confirmed by reverse transcriptase-polymerase chain reaction, demonstrating overexpression of both cytokines in vivo. To study the role of these cytokines in the pathogenesis of pemphigus vulgaris both in vitro and in vivo studies were performed. The results of the in vitro study demonstrated that pemphigus vulgaris IgG induced interleukin-1alpha and tumor necrosis factor-alpha mRNA in the skin. The potential pathogenic role of these mediators was demonstrated by a blocking study using antibodies against human interleukin-1alpha and tumor necrosis factor-alpha in keratinocytes cultures. A combination of anti-interleukin-1alpha and anti-tumor necrosis factor-alpha antibodies inhibited in vitro pemphigus vulgaris IgG induced acantholysis. To confirm the role of interleukin-1 and tumor necrosis factor-alpha in pemphigus, we utilized passive transfer studies using interleukin-1 deficient mice (ICE-/-, interleukin-1beta-/-) and tumor necrosis factor-alpha receptor deficient mice (TNFR1R2-/-). Both groups demonstrated a decreased susceptibility to the passive transfer of pemphigus. Our data support the role of cytokines interleukin-1 and tumor necrosis factor-alpha in the pathogenesis of pemphigus vulgaris.

Imiquimod, a topical immune response modifier, induces migration of Langerhans cells.

Langerhans cells are bone marrow derived dendritic cells that represent the major antigen-presenting cells in the skin. Langerhans cells take up and process antigen within the epidermis and present processed antigen to T lymphocyte in the regional lymph nodes and thus form an integral part of the cutaneous immune response. The cutaneous immune response can be modified by a number of pharmacologic agents, including corticosteroids, cyclosporine, and retinoids as well as physical agents, such as ultraviolet light. For the most part these agents act by suppressing immune function. A topical immune response modifier, imiquimod has been shown to enhance the cutaneous immune response. Imiquimod has anti-viral and anti-tumor effects in animal models and has been approved for the topical treatment of external genital and perianal warts in humans. The biologic activity of imiquimod in part is due to its effect as a cytokine inducer. Preliminary data suggested that imiquimod could have an effect on Langerhans cells. In order to clarify this effect on Langerhans cells, we examined Langerhans cell morphology and migration in imiquimod-treated skin. The density of Ia + cells decreased 2 d after treatment, falling to approximately 43% by day 10. The Ia positive in cells remaining in the skin appeared larger and more dendritic suggesting an activated state. ATPase staining of epidermal sheet confirmed the decreased number of Langerhans cells. To clarify status of Langerhans cells, the activation of B7 was examined. Activation of B7-1 or B7-2 was not detected. Imiquimod, however, did enhance Langerhans cell migration from skin to draining lymph nodes. This enhanced Langerhans cell migration was also associated with an enhanced allergic contact hypersensitivity. These results suggest that the mechanism of modulation of immune response by imiquimod is in part due to effects on Langerhans cells.

Impaired ultraviolet-B-induced cytokine induction in xeroderma pigmentosum fibroblasts.

Xeroderma pigmentosum is a rare, autosomal recessive disease in which patients develop excessive solar damage at an early age and have a 1000-fold increased risk of developing cutaneous neoplasms. Xeroderma pigmentosum can be classified into seven complementation groups (A-G) with defects in different DNA nucleotide excision repair genes. Xeroderma pigmentosum patients also have impaired immune function including reduced natural killer cell activity and impaired induction of interferon-gamma. We hypothesized that altered cytokine induction may contribute to the immune defect in xeroderma pigmentosum patients. We examined cytokine mRNA expression after ultraviolet B irradiation using reverse transcriptase polymerase chain reaction in fibroblasts derived from five xeroderma pigmentosum patients in complementation groups A, C, and D and in complemented XP-A and XP-D cells. Cytokines interleukin-1beta and interleukin-6 displayed impaired ultraviolet B induction whereas interleukin-8 had normal induction in the xeroderma pigmentosum fibroblasts. Stable complementation of XP-A and XP-D cell lines increased ultraviolet-B-induced interleukin-1beta and interleukin-6 expression. These results demonstrate a deficient response of xeroderma pigmentosum fibroblasts to ultraviolet B in terms of cytokine interleukin-1beta and interleukin-6 induction but normal interleukin-8 induction and exhibit a role for DNA repair in cytokine induction.

Delayed hypersensitivity in the hamster cheek pouch.

A method is described for eliciting a delayed hypersensitive cell mediated immune response in the Syrian hamster cheek pouch. Using a sensitizing dose of 0.1% DNCB and a challenge dose of 0.01% DNCB 14 days later resulted in a histologically positive lymphocyte infiltrate in the challenge area. These results indicate that the hamster cheek pouch may not be an immunologically inert site and may be used for cell mediated immunity studies.

Lectin binding to normal, dysplastic, and neoplastic cervical epithelium.

Avidin-biotin-peroxidase labeling technic was used to localize the binding sites of Concanavalin agglutinin (Con A), Ricinus communis (RCA-I), Ulex europaeus (UEA-I), and Limus flafus (LFA) in the cervical epithelia afflicted with condyloma (2 cases), moderate dysplasia (6), severe dysplasia (3), carcinoma in situ (9), squamous cell carcinoma (18), adenosquamous carcinoma (2), adenocarcinoma (1), and glassy cell carcinoma (1). Normal squamous epithelium displayed binding sites predominantly located on the cellular membranes for all the tested lectins except UEA. Normal glandular epithelium showed cytoplasmic localization of the lectins. Neoplastic transformation of squamous epithelium was associated with an increased intensity of the reaction and the appearance of the binding sites in the cytoplasm. UEA binding has changed from negative in normal epithelium to moderately positive in dysplasia and strongly positive in carcinoma. Invasive squamous carcinomas demonstrated an extremely variable pattern of lectin binding, some with very high intensity, allowing easy recognition of malignant cells even in minute metastatic foci.

Immunotherapy of chemically-induced tumors in the hamster cheek pouch with dinitrochlorobenzene.

Immune stimulation with an agent such as dinitrochlorobenzene (DNCB) may delay chemical carcinogenesis. Dimethylbenzanthracene (DMBA) was used to chemically induce tumors in the hamster buccal pouch. Hamsters were studied for the effect of DNCB sensitization in the buccal pouch prior to or after DMBA tumor induction. At appropriate time intervals the hamsters were sacrificed and each cheek pouch was examined histologically for the development of DMBA-induced tumors and for the presence of lymphoid cells infiltrating the tumor site. The results show that DNCB immunotherapy or immunoprophylaxis prior to or following DMBA tumor induction can alter the type of tumor produced and stimulate an infiltration of lymphoid cells into the tumor area probably invoking immune defense mechanisms.

Hamster cheek pouch carcinoma: effect of incision and cortisone on growth, invasion, and metastasis.

After cheek pouch carcinomas were induced in hamsters by the application of dimethylbenzanthracene (DMBA) to the right pouch for 13 weeks, the animals were divided into four groups and observed for seven more weeks. The control group received no further treatment, two experimental groups had incisional biopsies performed on tumors in their pouches, one of these also received injections of cortisone throughout the 20-week experimental period, and a fourth group received cortisone only. The wet weights of the cancerous cheek pouches were determined, and the submandibular and parotid salivary glands with associated cervical lymph nodes, the lungs, and the liver were examined with light microscopy. The cancerous pouches of the animals that received cortisone weighed significantly less than those of animals that received no cortisone but had incisional biopsies of the tumors. There was no significant difference in the degree of histodifferentiation of the tumors among the four groups. The animals in the two groups that received cortisone had significantly more tumors that were invasive than did the animals that did not receive cortisone. Cervical lymph node metastasis occurred in 21% to 38% of the animals but was not significantly different in the four groups. Distant metastases to the lungs or the liver were not found. Incisional biopsy of the tumors stimulated local growth of the cheek pouch tumors, and systemic cortisone administration produced more invasive cheek pouch tumors.

Skin reactions due to anti-epileptic drugs: several case-reports with long-term follow-up.

In this study, the clinical findings and management of allergic skin reactions induced by the most used antiepileptic drugs, Lamotrigine (LMT) and Carbamazepine (CBZ), were evaluated. Lamotrigine is an antiepileptic drug recently released in several countries; it is effective for a variety of seizure types in adults and children, both as an add-on agent and in monotherapy, and it is generally well tolerated. Clinical and epidemiologic evidence suggest serious cutaneous reactions to antiepileptic drugs are more likely to occur during the first 8 weeks and they appear to increase when drugs are administered with other anticonvulsants, such as Valproate (VPA). We selected 10 patients who presented an idiosyncratic skin rash when treated with carbamazepine (8 patients) and lamotrigine (2 patients) administered as monotherapy, and we followed up on these patients for several years. Seven reactions were mild/severe cutaneous eruptions; one Toxic Epidermal Necrolysis, a case of Stevens-Johnson and a case of Hypersensitivity Syndrome. All severe skin drug reactions were induced by Carbamazepine. In five patients the AEDs were ceased abruptly (sometimes with the administration of a different molecule), tapered in four and continued unchanged in one. We conclude that the discontinuation of the drug with substitution with another is the most effective treatment and that corticosteroids are helpful in mild cutaneous reactions, while in severe skin reactions, such as Toxic Epidermal Necrolysis, corticosteroids are only a complementary therapy since intravenous immunoglobulins are the first choice treatment.

Osseous coagulum: a histologic evaluation.

Chronic periodontitis can be successfully simulated in primates by the method employed in this study. Osseous defects can be created with a marked degree of similarity to one another and subsequently rendered into chronic lesions for healing-repair studies. The chronic periodontal ossious defects corrected by the osseous coagulum technique and by curettage in the rhesus monkey in this study were repaired by the regeneration of the architecture of the lost tissue. The use of the osseous coagulum in two- and three-walled periodontal osseous defects led to a more rapid osteogenesis in such defects as compared to correction by curettage alone. This rapid filling of the osseous defects may serve to inhibit the apical migration of the epithelial attachment during the early stages of repair, and thereby inhibit a subsequent recurrence of the defect. Clinically and histologically, no readily apparent distinction could be made in the healing process between the two- and three-walled lesions.

Activated macrophages in human periodontitis.

Fifteen patients, eight males and seven females, ranging from 30 to 88 years of age with advanced periodontal disease were selected for this study. Biopsies and blood samples were taken of both normal and inflamed gingival tissues, and processed for detection of nonspecific esterase and acid phosphatase activity in monocytes and macrophages. Activated macrophages, as indicated by their intense reaction to acid phosphatase and nonspecific esterase, were found in the gingival epithelium, lamina propria, perivascular tissues and in the blood vessels in human chronic periodontitis. Blood smears of monocytes showed variability of stain intensity suggesting that their activation occurred in blood vessels where they marginate and emigrate into the perivascular tissues in chronic periodontitis. They then appear as macrophages that migrate through the connective tissue, penetrate the basement membrane and continue through the epithelium. The nonspecific esterase stain identified T-cells, by a singular dot-like granule, and plasma cells by multiple granules in the cytoplasm. Lymphocytes containing multiple cytoplasmic nonspecific esterase positive granules commonly were found only in the perivascular connective tissue and may represent B-cell differentiation to plasma cells. The plasma cell predominance, the presence of T-cells and activated macrophages indicated both humoral and cell-mediated responses are operative in human chronic periodontitis.

The effects of hypophysectomy upon DNA synthesis in rat oral epithelium.

Ten hypophysectomized and 10 normal female albino rats, 50-days-old, were kept for 5 days and treated with tritiated thymidine 1 hour before sacrifice of the animals. The animals were weighed and the histomorphology of the palate epithelium was studied including the thickness, cell density, and DNA labeling index. The hypophysectomized rats failed to gain weight after 5 days. The palatal epithelium showed a normal morphology indicating the hypophysectomy allowed for differentiation of squamous epithelium. There was a significant reduction in the thickness of the epithelium and a reduced cell density. This was attributed to a significant decrease in DNA synthesis. The epithelial cells were lost from the surface without adequate replacement due to an expected depression in mitotic activity. DNA synthesis may be depressed due to reduced ATP synthesis resulting from suboptimal glucose metabolism and depression in protein synthesis.

Insulin I125 distribution within oral tissues.

The localization of insulin I125 in oral tissues of the rat was investigated. Eight rats were made chemically diabetic in order to study the tissue distribution of insulin I125. Two nondiabetic control animals were also injected with the tracer. Another animal served as a complete control (i.e., not injected with either the diabetic inducer or insulin I125). Additional controls were sections of thyroid and liver. Experimental rats were sacrificed at preselected time intervals and autoradiographic procedures were performed subsequent to conventional histologic studies. Qualitative grain counts were made of each slide in order to compare with the background count. All oral tissues studied except dentin showed insulin I125 uptake.

Levels of immunoglobulins IgG, IgA, and IgM in the human inflamed gingiva.

Resected inflamed gingival tissue obtained from 16 periodontal patients and a pooled sample of noninflamed gingiva from five edentulous patients were assayed for immunoglobulins IgG, IgA, and IgM using low-level immunodiffusion plates. Findings based on the gingival assays include:(1) IgA and IgG are present in both inflamed and normal gingiva and although their levels are substanitally higher in the inflamed gingiva, their ratio, one to another, remains the same; (2) IgM can not be consistently demonstrated in inflamed gingiva with the assay technique employed: (3) local immune response exists in the inflamed gingiva of humans with chronic periodontal disease; and (4) there is an unknown homeostatic mechanism which regulates the globulin levels in states of health and inflammation. Further study is needed to correlate clinical impressions with histological and quantitative measure of immunoglobulin levels in the inflamed gingiva and to investigate the role of delayed hypersensitivity in the disease process. Work must also be directed toward the detection of specific antigens responsible for elicting the immune response in the gingival tissue of the periodontal patient.

Xenogeneic implants in primates. Collagen and chondroitin sulfate.

This study was undertaken to examine histologically, in monkeys, the sequential healing phenomena of created two-walled osseous defects, which have been corrected by purified collagen and a mixed isomer of chondroitin sulfate. Four adult rhesus monkeys were used as experimental models and provided 23 specimens, from 0 to 56 days postoperatively. Sixteen of these served as implant specimens, and seven served as control specimens in which defects were corrected by curettage only. Two types of implant materials were utilized. The implants were compatible with osteogenesis. The two implant materials themselves did not differ in the healing events.

Immunoglobulins and complement in human periodontitis.

The gingiva in human periodontitis shows IgG, IgM, and C3 suggesting an antigen-antibody response binding and activating complement. The irregular distribution of the fluorescence on the basement membrane suggests binding of antigen-antibody complexes.

Identification of Langerhans cells in human gingival epithelium.

The purpose of this study was to qualitatively compare three recent techniques of Langerhans cells detection in oral epithelium and to quantitatively compare Langerhans cells in clinically normal and clinically inflamed human gingival biopsies. Eleven subjects were selected who displayed chronic periodontitis and moderate gingival inflammation. A quadrant associated with clinically inflamed tissues was not treated, while the remaining teeth were scaled and root-planed. Two gingival biopsies were taken: clinically normal, treated tissue; and clinically inflamed, untreated tissue. Langerhans cells were stained using HLD-DR, S-100 and OKT6. They were quantitated using a standard grid for OKT6-stained sections only. Approximately 5 times as many Langerhans cells were identified in the biopsy specimens of clinically inflamed human gingiva as in clinically normal gingiva of the same patient. Of the methods studied, OKT6 was qualitatively determined to be the best for visualization of these cells. An immunologic role in the host response to chronic periodontal disease is postulated for Langerhans cells.

Large cell, multilobated, B-cell lymphoma of the palate. A case report.

A case of B-large cell non-Hodgkin's lymphoma (NHL) with multilobated nuclei arising in the palatal mucosa is described. Immunologic typing of tumor cells was crucial to determine the exact cell of origin of this lesion.