Alex Kleinjan

Treatment of Vulvar Intraepithelial Neoplasia with Topical Imiquimod

BACKGROUNDnAlternatives to surgery are needed for the treatment of vulvar intraepithelial neoplasia. We investigated the effectiveness of imiquimod 5% cream, a topical immune-response modulator, for the treatment of this condition.nnnMETHODSnFifty-two patients with grade 2 or 3 vulvar intraepithelial neoplasia were randomly assigned to receive either imiquimod or placebo, applied twice weekly for 16 weeks. The primary outcome was a reduction of more than 25% in lesion size at 20 weeks. Secondary outcomes were histologic regression, clearance of human papillomavirus (HPV) from the lesion, changes in immune cells in the epidermis and dermis of the vulva, relief of symptoms, improvement of quality of life, and durability of response. Reduction in lesion size was classified as complete response (elimination), strong partial response (76 to 99% reduction), weak partial response (26 to 75% reduction), or no response (< or =25% reduction). The follow-up period was 12 months.nnnRESULTSnLesion size was reduced by more than 25% at 20 weeks in 21 of the 26 patients (81%) treated with imiquimod and in none of those treated with placebo (P<0.001). Histologic regression was significantly greater in the imiquimod group than in the placebo group (P<0.001). At baseline, 50 patients (96%) tested positive for HPV DNA. HPV cleared from the lesion in 15 patients in the imiquimod group (58%), as compared with 2 in the placebo group (8%) (P<0.001). The number of immune epidermal cells increased significantly and the number of immune dermal cells decreased significantly with imiquimod as compared with placebo. Imiquimod reduced pruritus and pain at 20 weeks (P=0.008 and P=0.004, respectively) and at 12 months (P=0.04 and P=0.02, respectively). The lesion progressed to invasion (to a depth of <1 mm) in 3 of 49 patients (6%) followed for 12 months (2 in the placebo group and 1 in the imiquimod group). Nine patients, all treated with imiquimod, had a complete response at 20 weeks and remained free from disease at 12 months.nnnCONCLUSIONSnImiquimod is effective in the treatment of vulvar intraepithelial neoplasia. (Current Controlled Trials number, ISRCTN11290871 [controlled-trials.com].).

Mucosal and systemic inflammatory changes in allergic rhinitis and asthma: a comparison between upper and lower airways

Background Local airway inflammation and airway remodelling are considered important in the clinical expression of allergic asthma.

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

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.

Enforced Expression of Gata3 in T Cells and Group 2 Innate Lymphoid Cells Increases Susceptibility to Allergic Airway Inflammation in Mice

Airway inflammation in allergic asthma reflects a threshold response of the innate immune system, including group 2 innate lymphoid cells (ILC2), followed by an adaptive Th2 cell–mediated response. Transcription factor Gata3 is essential for differentiation of both Th2 cells and ILC2. We investigated the effects of enforced Gata3 expression in T cells and ILC2 on the susceptibility of mice to allergic airway inflammation (AAI). We used CD2-Gata3 transgenic (Tg) mice with enforced Gata3 expression driven by the CD2 promoter, which is active both in T cells and during ILC2 development. CD2-Gata3 Tg mice and wild-type (WT) littermates were analyzed in mild models of AAI without adjuvants. Whereas OVA allergen exposure did not induce inflammation in WT controls, CD2-Gata3 Tg mice showed clear AAI and enhanced levels of IL-5 and IL-13 in bronchoalveolar lavage. Likewise, in house dust mite–driven asthma, CD2-Gata3 Tg mice were significantly more susceptible to AAI than WT littermates, whereby both ILC2 and Th2 cells were important cellular sources of IL-5 and IL-13 in bronchoalveolar lavage and lung tissue. Compared with WT littermates, CD2-Gata3 Tg mice contained increased numbers of ILC2, which expressed high levels of IL-33R and contributed significantly to early production of IL-4, IL-5, and IL-13. CD2-Gata3 Tg mice also had a unique population of IL-33–responsive non-B/non-T lymphoid cells expressing IFN-γ. Enforced Gata3 expression is therefore sufficient to enhance Th2 and ILC2 activity, and leads to increased susceptibility to AAI after mild exposure to inhaled harmless Ags that otherwise induce Ag tolerance.

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

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.

Disturbed Patterns of Immunocompetent Cells in Usual-Type Vulvar Intraepithelial Neoplasia

Genital infection with human papillomavirus (HPV) is usually transient, as the immune system is capable of eliminating the virus. When immunity fails and the infection persists, vulvar intraepithelial neoplasia (VIN) may develop. In this study, we examined the distribution of inflammatory cells in 51 patients with HPV-associated usual-type VIN and in 19 healthy controls. Frozen vulvar tissue samples were tested for the presence of HPV-DNA, and immunohistochemical staining for the markers CD1a, CD207, CD208, CD123/CD11c, CD94, CD4, CD8, and CD25/HLA-DR was performed. Cells were counted in both the epidermis and dermis over at least 2 mm of basal membrane length. In the epidermis of VIN patients, CD1a(+) and CD207(+) (Langerin) dendritic cells (DC) and CD8(+) T cells were significantly lower than in controls, whereas the number of CD123(+)/CD11c(-) plasmacytoid DCs (pDC) was significantly increased. No significant changes were observed for CD208(+) DCs, CD94(+) natural killer (NK) cells, CD4(+) T cells, and CD25(+)/HLA-DR(+) regulatory T cells. In the dermis of VIN patients, elevated numbers of CD208(+), CD123(+)/CD11c(-), CD94(+), CD4(+), CD8(+), and CD25(+)/HLA-DR(+) cells were observed when compared with healthy controls. The numbers of CD1a(+) and CD207(+) DCs were not different between groups. In summary, high-risk HPV-related usual-type VIN lesions are characterized by an immunosuppressive state in the epidermis, showing a reduction of immature myeloid DCs (mDC) and CD8(+) T cells. In the dermis, inflammatory activation is reflected by the influx of mature mDCs and pDCs, NK cells, and T cells, suggesting that the cellular immune response on viral HPV infection occurs in the dermis of VIN patients.

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

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.