Santegoets, L.A.M.

An autoimmune phenotype in vulvar lichen sclerosus and lichen planus: A Th1 response and high levels of microRNA-155 (Article)

2012-03-01T00:00:00Z

Vulvar lichen sclerosus and lichen planus are T-cell-mediated chronic skin disorders. Although autoimmunity has been suggested, the exact pathogenesis of these disorders is still unknown. Therefore, the aim of the current study was to investigate the molecular and immunological mechanisms critical to the pathogenesis of vulvar lichen sclerosus and lichen planus. By using gene expression profiling and real-time RT-PCR experiments, we demonstrated a significantly increased expression of the pro-inflammatory cytokines (IFNγ, CXCR3, CXCL9, CXCL10, CXCL11, CCR5, CCL4, and CCL5) specific for a Th1 IFNγ-induced immune response. In addition, BIC/microRNA-155 (miR-155)a microRNA involved in regulation of the immune responsewas significantly upregulated in lichen sclerosus and lichen planus (9.5- and 17.7-fold change, respectively). Immunohistochemistry showed a significant T-cell response, with pronounced dermal infiltrates of CD4+, CD8+, and FOXP3+cells. In conclusion, these data demonstrate an autoimmune phenotype in vulvar lichen sclerosus and lichen planus, characterized by increased levels of Th1-specific cytokines, a dense T-cell infiltrate, and enhanced BIC/miR-155 expression.

Molecular and immunological mechanisms of epithelial disorders of the vulva (Doctoral Thesis)

2011-10-21T00:00:00Z

The vulva is the outer part of the female genital tract, bordered by the symphisis pubis, the labiocrural folds and the anus. It consists of the following structures: the mons pubis, the labia majora and minora, the clitoris, the vestibule of the vagina and the urethral orifice (Figure 1). Embryologically all three germ layers are present at the vulva; the cloacal endoderm, the urogenital ectoderm and the paramesonephric mesoderma. Different epithelia, from keratinized squamous epithelium on the labia majora and minora to squamous mucosa in the vestibule, cover the vulva. Dysplastic changes in the epithelium of the vulva are known as Vulvar Intraepithelial Neoplasia (VIN). In the past, various definitions have been used to describe these dysplastic vulvar lesions: morbus Bowen, Queyrat’s erythroplasia, carcinoma simplex, bowenoid papulosis, early vulvar cancer, vulvar atypia, hyperplastic dystrophy, carcinoma in situ and dysplasia (graded into mild dysplasia (VIN1), moderate dysplasia (VIN2) and severe dysplasia (VIN3)). To simplify this complexity of terms and to enhance diagnostic reproducibility, the International Society for the Study of Vulvovaginal Diseases (ISSVD) defined a new terminology in 2004.7 We nowadays classify two types of VIN: 1. Usual type VIN (uVIN), which is caused by a persistent Human Papilloma Virus (HPV) infection. 2. Differentiated type VIN (dVIN), which is not associated with HPV, but with chronic inflammatory and/or autoimmune processes, such as lichen sclerosus and lichen planus, involving vulvar mucosa and skin. Spontaneous regression of VIN lesions has been described, but malignant transformation into vulvar cancer has also been reported. Vulvar cancer is the fourth most common gynecological type of cancer.11 Worldwide the incidence rate is 1 to 2 per 100.000 women, and around 27.000 women are diagnosed each year.12 In the Netherlands about 200 new cases are identified each year.

Nonsteroidal anti-inflammatory drugs do not interfere with imiquimod treatment for usual type vulvar intraepithelial neoplasia (Article)

2011-05-01T00:00:00Z

Imiquimod has been shown to be an effective treatment for usual type vulvar intraepithelial neoplasia (uVIN). Since local inflammation and burning are common side effects, patients often use nonsteroidal anti-inflammatory drugs (NSAIDs). Our study investigated whether NSAID-use, which has been documented to inhibit the cell-mediated immune response, interferes with the outcome of imiquimod treatment. Monocyte-derived dendritic cells (moDCs) and Langerhans cells (moLCs) were cultured in the presence of NSAIDs. The expression of relevant surface markers (CD80, CD86, CD40, HLA-DR, CCR6 and CCR7), stimulatory function, and cytokine production were evaluated. Furthermore, we analyzed in uVIN patients whether frequent NSAID-use had an effect on the clinical response and on immunocompetent cell counts before and after imiquimod treatment. Although an effect was observed on the expression of moDC and moLC maturation markers, NSAIDs did not affect the ability of moDCs and moLCs to stimulate allogeneic T-cell proliferation, or the production of cytokines in an allogeneic T-cell stimulation assay. In agreement with this, in uVIN patients treated with imiquimod, no interference of frequent NSAID-use with clinical outcome was observed. However, we did notice that high CD1a+and CD207+cell counts in frequent NSAID-users before treatment seemed to predict a favourable response to imiquimod treatment. Our data indicate that NSAID-use does not seem to interfere with moDC and moLC function and does not interfere with immunomodulatory properties of imiquimod in uVIN patients. Therefore, NSAIDs can safely be used to reduce imiquimod side effects in uVIN patients during treatment. Copyright

Imiquimod-induced clearance of HPV is associated with normalization of immune cell counts in usual type vulvar intraepithelial neoplasia (Article)

2010-12-15T00:00:00Z

Recently, we reported on the efficacy of imiquimod for treatment of usual type vulvar intraepithelial neoplasia (uVIN). A histologic regression of uVIN to normal tissue was observed in 58% of patients. As success of treatment is related to clearance of high-risk human papilloma virus (HPV), the aim of our study was to assess differences in immune cell counts and in the expression of p16INK4a in VIN tissue before and after imiquimod treatment, in relation to HPV clearance and clinical response. Vulvar tissue samples taken prior to imiquimod treatment and 4 weeks after treatment were tested for the presence of HPV. Previously determined immune cell counts (CD1a, CD207, CD208, CD123/CD11c, CD94, CD4, CD8 and CD25/HLA-DR) in epidermis and dermis of 25 VIN patients and 19 healthy controls were completed with the counts for CD14 and CD68. The expression of p16INK4a was investigated by immunohistochemistry in 15 patients. Before imiquimod treatment, both HPV cleared and HPV noncleared patients showed mainly in the dermis significantly upregulated immune cell counts compared to healthy controls. However, in patients that cleared HPV and showed histologic regression already 4 weeks after imiquimod treatment, immune cell counts and p16INK4a expression were normalized. In conclusion, our data indicate that imiquimod-induced clearance of HPV results in normalization of counts for certain immune cells and is strongly correlated with histologic regression of the disease.

A retrospective study of 95 women with a clinical diagnosis of genital lichen planus (Article)

2010-10-01T00:00:00Z

Objective: The aim of this retrospective clinical study was to evaluate clinical features, histopathology, treatment regimen, and follow-up in 95 patients with genital lichen planus. Materials and Methods: We retrospectively analyzed data of 95 women diagnosed with genital lichen planus and visiting the vulvar clinic at Erasmus MC, Rotterdam, the Netherlands. Results: All patients were symptomatic, most often complaining of vulvar soreness and burning (31.6%). Of all women, 34% had persistent symptoms for more than 5 years. On physical examination, 81.1% showed sharply demarcated erythematous lesions, usually located at the vestibule, and 56.8% had oral lesions. Treatment usually consisted of potent topical corticosteroids. Seventeen women (17.9%) were referred to the gynecology department for additional surgical treatment. In two of them, a vulvar squamous cell carcinoma was detected, followed by radical surgery. Conclusions: In cases with vulvar soreness and burning, sharply demarcated erythematous vulvar lesions, and the concomitant presence of oral lesions, the diagnosis of lichen planus should be considered and treatment must be initiated accordingly, even when histopathology is discordant.

Genome-wide pathway analysis of folate-responsive genes to unravel the pathogenesis of orofacial clefting in man (Article)

2008-09-01T00:00:00Z

BACKGROUND: A cleft of the lip with or without the palate (CLP) is a frequent congenital malformation with a heterogeneous etiology, for which folic acid supplementation has a protective effect. To gain more insight into the molecular pathways affected by natural folate, we examined gene expression profiles of cultured B-lymphoblasts from CLP patients before and after the addition of 5-methyltetrahydrofolate (5-mTHF) to the cultures. METHODS: Immortalized B-lymphoblasts from five children with CLP were cultured in folate-deficient medium for 5 days. 5-mTHF was added to a concentration of 30 nM. Gene expression patterns were then evaluated before and after supplementation using Human Genome U133 Plus 2.0 arrays. Data analysis was performed with Omniviz and the GEPAS analysis suite. Differential genes were categorized into biological pathways with Ingenuity Pathway systems. Differential expression was validated by quantitative RT-PCR. RESULTS: Using supervised clustering, with a false discovery rate <1%, we identified 144 and 409 significantly up-regulated and down-regulated probesets, respectively, after 5-mTHF addition. The regulated genes were involved in a variety of biological pathways, including one carbon pool and cell cycle regulation, biosynthesis of amino acids and DNA/RNA nucleotides, protein processing, apoptosis, and DNA repair. CONCLUSIONS: The large variety of the identified folate responsive pathways fits with the modifying role of folate via the methylation pathway. From the present data we may conclude that folate deficiency deranges normal cell development, which might contribute to the development of CLP. The role of these folate responsive genes in CLP development is intriguing and needs further investigation.

Reduced local immunity in HPV-related VIN: Expression of chemokines and involvement of immunocompetent cells (Article)

2008-08-01T00:00:00Z

Usual type VIN is a premalignant disorder caused by persistent HPV infection. High prevalence of VIN in immuno-suppressed women suggests that a good innate and adaptive immune response is important for defense against HPV. Here, we explored expression levels of chemokines and related these to the presence or absence of immuno-competent cells (dendritic and T-cells) in affected (HPV-positive VIN) and non-affected (HPV-negative) vulvar tissues from the same patients. Combining microarray data with quantitative real-time RT-PCR, it was observed that several important chemokines were differentially expressed between VIN and control samples (up-regulation of IL8, CXCL10, CCL20 and CCL22 and down-regulation of CXCL12, CCL21 and CCL14). Furthermore, an increased number of mature dendritic cells (CD208+) seemed to be bottled up in the dermis, and although a T-cell response (increased CD4+and CD8+cells) was observed in VIN, a much larger response is required to clear the infection. In summary, it seems that most mature dendritic cells do not receive the proper chemokine signal for migration and will stay in the dermis, not able to present viral antigen to naive T-cells in the lymph node. Consequently the adaptive immune response diminishes, resulting in a persistent HPV infection with increased risk for neoplasia.

Signaling by estrogens and tamoxifen in the human endometrium (Article)

2008-04-01T00:00:00Z

Tamoxifen is used as adjuvant treatment for postmenopausal breast cancer patients. The mechanism of action of tamoxifen in breast cancer patients is that tamoxifen inhibits growth of cancer cells by competitive antagonism for estrogens at the estrogen receptor (ER). In the endometrium, tamoxifen has an effect that varies with the ambient concentration of estrogen: in premenopausal women (high estrogen levels), tamoxifen displays an estrogen-antagonistic effect, while in postmenopausal women (low estrogen levels), tamoxifen displays an estrogen-agonistic mode of action. Here, using microarray technology we have compared estrogen signaling with tamoxifen signaling in the human endometrium. It was observed that on the one hand tamoxifen-treatment results in modulation of expression of specific genes (370 genes) and on the other hand tamoxifen-treatment results in modulation of a set of genes which are also regulated by estrogen treatment (142 genes). Upon focusing on regulation of proliferation, we found that tamoxifen-induced endometrial proliferation is largely accomplished by using the same set of genes as are regulated by estradiol. So, as far as regulation of proliferation goes, tamoxifen seems to act as estrogen agonist. Furthermore, tamoxifen-specific gene regulation may explain why tamoxifen-induced endometrial tumors behave more aggressively than sporadic endometrial tumors.

Genomic and nongenomic effects of estrogen signaling in human endometrial cells: Involvement of the growth factor receptor signaling downstream AKT pathway (Article)

2007-10-01T00:00:00Z

For the endometrium, estradiol and tamoxifen induce proliferation, and consequently, tamoxifen treatment of breast cancer results in a 2-fold to 7-fold increased risk for endometrial cancer. Here, the role of activation of growth factor receptor signaling in mediating the e fects of estrogen and tamoxifen is determined. Microarray analysis of ECC-1 cells treated with estradiol or tamoxifen indicate that rapid responses to treatment (1 hour) are very distinct from long-term responses (>24 hours). Furthermore, estradiol and tamoxifen are observed to induce AKT activation. Comparing long-term estrogen- and tamoxifen-regulated genes with genes regulated by insulin-like growth factor 1 and amphiregulin reveals that the late e fects of estrogen and tamoxifen signaling may partly be mediated via activation of growth factor receptor signaling pathways. It is hypothesized that both early and late e fects of estrogen and tamoxifen signaling in the endometrium are partly mediated via the activation of growth factor receptor signaling, putatively at the level of AKT activation.

HPV related VIN: Highly proliferative and diminished responsiveness to extracellular signals (Article)

2007-08-15T00:00:00Z

Vulvar intraepithelial neoplasia (VIN) is a premalignant disorder caused by human papillomaviruses. Basic knowledge about the molecular pathogenesis of VIN is sparse. Therefore, we have analyzed the gene expression profile of 9 VIN samples in comparison to 10 control samples by using genome wide Affymetrix Human U133A plus2 GeneChips. Results were validated by quantitative real-time RT-PCR analysis and immunostaining of a few representative genes (TACSTD1, CCNE2, AR and ESR1). Significance analysis of microarrays (SAM) showed that 1,497 genes were differentially expressed in VIN compared to controls. By analyzing the biological processes affected by the observed differences, we found that VIN appears to be a highly proliferative disease; many cyclins (CCNA, CCNB and CCNE) and almost all prereplication complex proteins are upregulated. Thereby, VIN does not seem to depend for its proliferation on paracrine or endocrine signals. Many receptors (for example ESR1 and AR) and ligands are downregulated. Furthermore, although VIN is not an invasive disease, the inhibition of expression of a marked number of cell-cell adhesion molecules seems to indicate development towards invasion. Upon reviewing apoptosis and angiogenesis, it was observed that these processes have not become significantly disregulated in VIN. In conclusion: although VIN is still a premalignant disease, it already displays several hallmarks of cancer.