Effect Of Imiquimod Research Articles

Spontaneous cytokine production by the cells from peripheral blood, lymph nodes, spleen, thymus and skin in a murine model of acute psoriasis-like dermatitis

Imiquimod-induced model of psoriasis in mice is used for the studies in psoriasis. Imiquimod administered as skin application triggers a complex multi-stage process simulating psoriatic inflammation of the skin, accompanied by changes in production of cytokines. The aim of this work was a comprehensive study of their contents in cell supernatants isolated from skin, blood, from central and peripheral lymphoid organs and the effect of imiquimod on these parameters. Forty-six C57BL/6 mice were divided into two groups: (1) control (n = 22), and (2) experimental (n = 24). Development of experimental pathology was evaluated by van de Fits et al. The mice from experimental group were treated with a cream containing 5% imiquimod (62.5 mg/ cm2/ day/mouse) for 7 days. On the 7th day of experiment, the animals were subject to blood sampling, extraction of spleen, lymph nodes, thymus, and skin biopsies were also made. To obtain cell suspensions, the tissues of spleen, lymph nodes, and thymus were crushed in a homogenizer, and then passed through cell filters (50 mcm pore size). The method of spontaneous migration was used to isolate skin cells. The cultures of blood, spleen, lymph nodes, thymus, and skin cells were incubated for 24 hours in RPMI-1640 followed by assessment of spontaneous cytokine production in supernatants. The cytokine level (IFNγ, IL-1β, IL-5, IL-6, IL-10, IL- 17, TNFα, IL-10) was determined using the murine Th1/Th2/Th17 Panel test system. Our study revealed that the cells isolated from blood and lymphoid organs synthesize increased amounts of pro-inflammatory cytokines: IL-1 and IL-17. The most pronounced changes in the cytokine profile are observed in cell supernatants from blood, skin and spleen cultures.

Abstract PO063: Transcriptomic analysis identifies changes in gene expression in Actinic Keratoses after treatment with imiquimod and differential gene expression

Abstract Background: The presence of actinic keratoses (AKs) increases a patient’s risk of developing squamous cell carcinoma (SCC) by greater than six-fold. It is difficult to predict which AKs will progress to become SCC, and topical treatments such as imiquimod are used to treat the entire affected field indiscriminately, effectively eliminating AKs in 50% of patients but sometimes resulting in severe inflammation and pain. Defining biomarkers to predict response to treatment would be helpful to guide therapy. Objectives: To evaluate the effect of imiquimod on the tumor microenvironment by measuring transcriptomic differences in AKs before and after treatment in patients treated with imiquimod. Methods: Biopsies were collected prospectively from 21 patients with presumptive AK and examined histologically. All patients were treated with imiquimod 3.75% on a short cyclical treatment regimen. 19 patients had available pre-treatment AK biopsies and 14 patients had paired pre- and post-treatment biopsies. RNA was extracted and transcriptomic analyses of 788 genes were performed using the nanoString assay. Results: Imiquimod significantly decreased total number of AKs by study endpoint at week 14 (p<0.0001). Post-imiquimod therapy, levels of CDK1, CXCL13, IL1B, GADPH, TTK, ILF3, EWSR1, BIRC5, PLAUR, ISG20, and C1QBP were significantly lower (p<0.05 after adjustment for multiple comparisons), with CXCL13 and IL1B showing a greater than 2-log2 fold decrease in expression post-treatment. Complete responders (CR) exhibited a distinct pattern of inflammatory gene expression pre-treatment relative to incomplete responders (IR), with significant alterations in 15 inflammatory pathways (p<0.05) reflecting differential expression of 103 genes, of which 95 were upregulated in CR (p<0.05). Presence of adverse effects was found to be related to an improved treatment response. Conclusions: NanoString analysis of AKs pre- and post-treatment with imiquimod shows downregulation of genes implicated in oncogenesis and immune suppression. Differences in gene expression were found between pre-treatment samples in CR versus IR, suggesting that higher levels of inflammation pre-treatment may play a part in regression of AKs. Further, presence of adverse effects in response to therapy was associated with an improved response to imiquimod. These findings show that the pre-treatment immune micro-environment impacts responsiveness of AKs to imiquimod and that clinical inflammation during treatment is associated with AK resolution. Further characterization of the immune micro-environment in AKs may help develop biomarkers predictive of response to topical immune modulators and may guide therapy in patients with high numbers of AKs at risk for SCC. Citation Format: Megan H. Trager, Emanuelle Rizk, Sharon Rose, Branden Lau, Ben Fullerton, Kuixi Zhu, Jaya Pradhan, Michael Moore, Ayush Chandra, Margaret Bogardus, Giselle Singer, Robyn Gartrell-Corrado, Rui Chang, Larisa Geskin, Yvonne Saenger, Gary Goldenbery. Transcriptomic analysis identifies changes in gene expression in Actinic Keratoses after treatment with imiquimod and differential gene expression [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO063.

Topical Imiquimod as a Treatment Option for Nodular Basal Cell Carcinoma: A Systematic Review.

Surgical excision is considered standard treatment for nodular basal cell carcinoma (nBCC). However, patients who reject or are unsuited for surgery may benefit from imiquimod (IMQ) 5% cream as an alternative treatment. The objective of this study was to conduct a systematic review on the efficacy and safety of IMQ for the treatment of nBCC. The terms basal cell carcinoma AND imiquimod OR Aldara were searched on Ovid-MEDLINE, EMBASE, and Cochrane Library databases. Articles were included if they reported the efficacy or side effects of IMQ for nBCC. Primary outcomes included clearance (clinical and histological), recurrence rates, and adverse events. Number of lesions/subjects, treatment regimens, length of treatment, and time to recurrence were secondary outcomes. Thirty-nine publications, totaling 738 lesions, revealed a 77.4% (335/433 lesions) clinical and 72.9% (390/535 lesions) histological clearance rate. Regimens ranged from once daily 2 days a week to twice daily 7 days a week. Average treatment duration was 8.81 (±3.49) weeks. There was a 1.80% recurrence rate after an average follow-up period of 13.03 (±15.09) months. Common adverse effects included erythema (77.2%), crusting (50.5%), pruritus (34.1%), tenderness/irritation (27.3%), ulceration (25.4%), burning (22.1%), and erosion (21.7%). Unforeseen side effects included conjunctivitis, keratitis, depigmentation, comedone formation, and ruptured epidermoid cysts. Imiquimod showed clinical and histological clearance rates of over 70% for nBCC, with a recurrence rate of 1.80%. Although clearance rates are lower than surgery, IMQ can be considered as a treatment option for nBCC in those who decline or are unfit for surgical intervention.

Efficacy of imiquimod in the management of lentigo maligna.

10074 Background: Lentigo maligna (LM), a melanocytic proliferation occurring on photoexposed skin, might progress to LM melanoma. Surgery is recommended as first-line treatment. However, the main challenge is the size of the excision inducing often-aesthetic injuries on the face and thus often refused by patients. The excision margins of 5 to 10 mm remain without international consensus. Several studies have shown that imiquimod induced LM regression, acting by enhancing IFN-γ production and effector function of T cells. The main goal of this study is to investigate the effect of imiquimod versus placebo in neoadjuvant setting to decrease the excision size as from the first surgical procedure. Methods: We performed a prospective, randomized, open, multicenter, phase III clinical study (NCT01720407). The health authority and ethics committee approvals were obtained and all subjects signed an informed consent. The primary endpoint was to demonstrate that in neoadjuvant situation, imiquimod could reduce the surgical excision size of LM with a healthy tissue margin of 5 mm. The main inclusion criteria were: Patients > 18 years fit for surgery. LM of the head histologically confirmed and not previously treated. Surface lesion ≥ to 1cm² and ≤ to 20cm². The two treatment arms were imiquimod or placebo followed by LM excision. Imiquimod or placebo were applied once daily, 5 days/week for 4 weeks followed by 5 mm margin surgery performed four weeks after the last treatment application. For sample size, 268 patients were expected to demonstrate a difference of 15% between the two arms in a bilateral situation with an alpha risk of 5% and a beta risk of 20%. Results: The trial involved 273 patients, 238 (105 men (44%) and 133 women (56%), mean age of 71 ± 10.2 years, were analyzed in modified ITT. Statistical analysis was performed on 122 patients in the imiquimod arm and 116 patients in the placebo arm. For the primary endpoint, the first extralesionnal excision has been achieved for 112 (91.8%) patients in the imiquimod arm and for 98 (84.5%) placebo patients group. There was no significant difference (p value = 0.1067) between the two arms. However, regarding the surface of LM, imiquimod allowed a highly significant reduction (4.2 cm² ± 4.6 to 2.3 cm² ± 3.3) compared to LM treated by placebo (4.0 cm² ± 3.5 to 4.0 cm² ±3.3; p < 0.0001). Conclusions: This randomized prospective study shows that imiquimod reduces the LM area (-50%) after one month of treatment. Reducing the surface of LM with imiquimod is not associated with a higher risk of intralesional excision (marge 5mm), with a significant esthetic result (less excised surface). Clinical trial information: NCT01720407.

Imiquimod to prevent keloid recurrence postexcision: A systematic review and meta-analysis.

Imiquimod 5% cream, an immune response modifier capable of inducing IFN-α, TNF-α, and interleukins 1, 6, and 8. It was approved for use in the management of genital and perianal warts and soon embraced as a method to diminish the recurrence of keloids postexcision. A previous meta-analysis included four studies. This meta-analysis is part of a larger systematic review project on the effect of moisturizers on scars. It was conducted following an a priori protocol and the guidance of the Joanna Briggs Institute. Databases searched included PubMed, CINAHL, Embase, and Web of Science. After screening and critical appraisal, subgroup meta-analysis on excision method and location of the keloid was conducted using the Miller approach for proportional meta-analysis and a random effects model. Seven studies, including 77 participants and 82 keloids were included. Meta-analysis revealed a recurrence rate of 39% (95% CI = 8.474.4%; I2 = 87.5%) following application of Imiquimod postexcision. The use of primary excision or tangential excision did not alter the outcome. For analysis based on the location of the keloid scar, earlobe keloids had a recurrence rate of 5.4% (95% CI = 0-21.7%; I2 = 52.9%). For keloids excised from other areas (predominantly on the trunk) recurrence rate was higher, at 76.8% (95% CI = 36.1-100%). For keloids, Imiquimod application postexcision results in highly variable recurrence rates. There is very low certainty in the effect of Imiquimod and it therefore is not recommended as a treatment option.

Evaluation of Morphine with Imiquimod as Opioid Growth Factor Receptor or Nalmefene as Opioid Blocking Drug on Leishmaniasis Caused by Leishmania major in Vitro

In this research, the effect of morphine on promastigotes and amastigotes of Leishmania major has been investigated in the presence of nalmefene as a blocking opioid drug and imiquimod as an opioid growth factor receptor. This study was conducted at Tarbiat Modares University, Tehran, Iran in 2015-2018. Morphine with different concentration (0.1, 1, 10 and 100 1μg/ml) alone and with imiquimod (0.01, 0.1 and 1μg/ml) and nalmefene (0.1, 1 and 10 μg/ml) on promastigotes and amastigotes in macrophages and also the percentage of infected macrophages was investigated. For evaluation of the apoptosis, we used flow cytometry method. The effect of imiquimod and nalmefene on glucantime and amphotericin B as current drugs for treatment of leishmaniasis was evaluated too. The effect of morphine on promastigotes and amastigotes has a reverse relationship with its concentration. The results of flow cytometry for drug-treated promastigotes revealed that apoptosis and necrosis did not increase markedly relative to the control group. A combination of morphine and imiquimod in concentrations of 0.05, 5 and 5 μg/ml had a pronounced effect on reduction and prevention of macrophage infection with amastigotes. Morphine at a concentration of 0.1 μg/ml plays the role of adjunctive treatment. In amastigote assay we found the better results in group that get glucantime 25 μg/ml+ imiquimod 0.5 μg/ml. This effect is strengthened with imiquimod and weakened with nalmefene. Using high dose morphine and nalmefene had reverse effects. They suppress immune system and had no controlling effect in macrophages amastigote infection and reduction of promastigotes.

Treatment of HPV-associated Anal Lesions in HIV-positive Patients - Comparison of Surgical Treatment and Topical Therapy with Imiquimod

In HIV+-patients, routine proctological assessment is warranted due to the high incidence of human papilloma virus (HPV) infection-related anogenital lesions, such as Condylomata acuminata (C. ac.), anal intraepithelial dysplasia (AIN) and anal cancer. For C. ac. and AIN, surgical resection and topical therapy with imiquimod have been discussed as treatment options. In this study, we contrasted surgical resection and topical imiquimod therapy of HPV-associated anal lesions in HIV+-patients, with a focus on healing rates and clinical outcome. We also analysed whether a synergistic treatment effect was detectable. This was a retrospective analysis of 97 HIV+ patients who underwent proctological evaluation and treatment over a 10-year period (11/2004 - 11/2015) at our centre. Initial success of surgical treatment, topical imiquimod therapy and the combination of the two strategies were compared. In 53/97 patients (54%), HPV-associated anal disease was diagnosed upon the first visit. In approx. 50% of the patients, the HIV infection was adequately controlled (52 patients with viral load < 40 copies [53.6%]) under cART. The mean age was 41.0 ± 11.6 years. In 7/53 patients with macroscopic C. ac., low-grade and in 18/53 patients high-grade AIN were additionally confirmed. Success rates of surgical resection, imiquimod treatment and the combination of the two were compared. Complete remission of C. ac. and AIN four weeks after treatment was considered a therapeutic success. For C. ac., success rates with imiquimod were 5/25 (20.0%) vs. surgery* 30/57 (52.6%, Mann-Whitney U test p < 0.05) vs. surgery+imiquimod 7/15 (46.7%). For AIN, success rates with imiquimod were 4/24 (16.7%) vs. surgery* 47/83 (56.7%, Mann-Whitney U test p < 0.05) vs. surgery+imiquimod 9/21 (42.8%). In 7/92 (13%) of surgical treatments, complications were reported: four minor and two significant bleeding episodes and one perianal thrombosis. No side effects of imiquimod were documented besides skin irritation. Surgery is more effective than topical imiquimod as initial therapy of HPV-related anogenital disease in HIV+-patients. A synergistic effect could not be demonstrated. On this basis, we recommend surgical treatment of C. ac. and AIN in HIV+-patients as first line treatment.

Effects of Imiquimod 5% Cream in the Prevention of Excised Keloid Recurrence

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IRF-2 haploinsufficiency causes enhanced imiquimod-induced psoriasis-like skin inflammation

BackgroundsIFN regulatory factor (IRF)-2 is one of the potential susceptibility genes for psoriasis, but how this gene influences psoriasis pathogenesis is unclear. Topical application of imiquimod (IMQ), a TLR7 ligand, induces psoriasis-like skin lesions in mice. ObjectiveThe aim of this study was to investigate whether IRF-2 gene status would influence severity of skin disease in IMQ-treated mice. MethodsImiquimod-induced psoriasis-like skin inflammation was assessed by clinical findings, histology, and cytokine expression. The effects of imiquimod or IFN on peritoneal macrophages were analyzed in vitro. ResultsIMQ-induced skin inflammation assessed by clinical findings and histology was more severe in IRF-2+/− mice than in wild-type mice. In inflamed skin, mRNA expression levels of tumor necrosis factor (TNF)-α, IL-12/23p40, IL-17A, and IL-22 were significantly elevated in IRF-2+/− mice compared to wild-type mice. Stimulation of peritoneal macrophages by IMQ significantly increased mRNA levels of TNF-α, IL-12/23p40, IL-23p19, IL-12p35, and IL-36. Interestingly, macrophages from IRF-2+/− mice expressed higher levels of TNF-α, IL-12/23p40, and IL-36 compared to those from wild-type mice 24 h after stimulation, while they expressed similar levels of IL-12p35 and IL-23p19. Moreover, elevated mRNA expression of inducible nitric oxide synthase was observed only in IMQ-stimulated macrophages derived from IRF-2+/− mice, which correlated with angiogenesis in IMQ-treated ears of IRF-2+/− mice. ConclusionsThese results suggest that IRF-2 haploinsufficiency creates heightened biologic responses to IFN-α that phenotypically lead to enhanced angiogenesis and psoriasis-like inflammation within skin.

The effects of imiquimod on the level of BALF IL-12, IL-13 and sera IgE in asthmatic murine

Objective To observe the effects of aerosol imiquimod on the level of Interleukin-12(IL-12) and interleukin-13(IL-13) in bronchial alveolar lavage fluid(BALF) and serum immunoglobulin E( IgE ) level in mouse asthma models. Methods Thirty BALB/c mice were divided into 3 groups, control group, asthma group and imiquimod group, ten in each group.The asthma group and imiquimod group were established by sensitization and challenge with ovalbumin (OVA), imiquimod group was interfered with aerosol imiquimod 30 min before challenge.Serum in each group was collected by excising eyeball 24 h after the last challenge.The level of IgE in serum was measured by ELISA.Bronchoalveolar lavage fluid (BALF) was performed, and total cell counts, cell differential were examined.The level of IL-12 and IL-13 in BALF was measured by ELISA and total cell counts, cell differential were examined in BALF.The inflammation changes of lung was observed by HE staining. Results (1)By hematoxylin-eosin(HE) staining, the result of asthma mice were small airway wall thickening, secretion increased, epithelial cells fall off, arranging disorderly, and submucosal had plenty of inflammatory cells infiltration, mainly with lymphocytes.But imquimod group attenuated the airway inflammation of asthmatic mice.(2)Total cell counts and all kinds of inflammatory cells of asthma group were increased greatly compared with normal group; Eosinophil and lymphocyte cells were much lower in imquimod group than those in asthma group (P<0.05). (3) The level of IL-13 in BALF and the level of IgE in serum in imiquimod group was obviously higher than that in control group, IL-13 level were positively correlated with IgE level(r=0.774, P<0.05), but imiquimod group was greatly decreased compared with control group and increased in comparison with that in group A; The level of IL-12 in asthma group was reduced and the level of IgE was increased compared with normal group.IL-12 level in imiquimod group was evidently higher and IgE level was lower than that in asthma group.IL-12 level negatively correlated with IgE level(r=-0.802, P<0.05). Conclusion Imiquimod aerosol inhalation may up-regulate the level of Th1 chemokine IL-12, down-regulates the level of Th2 chemokine IL-13 and decrease the level of IgE in serum so that it may suppress antigen-induced airway inflammation. Key words: Asthma; IgE; Imiquimod

Imiquimod inhibits the growth of SGC‑7901 cells in vitro through induction of autophagy and apoptosis.

Imiquimod, the most prominent Toll‑like receptor 7 agonist, has direct anti‑tumor activity and can induce autophagy and apoptosis in various types of human cancer. The aim of the present study was to examine the anti‑tumor effects of imiquimod and their underlying mechanisms in SGC‑7901 cells. Imiquimod exerted an inhibitory effect on cell proliferation in a dose‑ and time‑dependent manner as indicated by an MTT assay. Imiquimod induced autophagy as well as apoptosis, while simultaneous treatment with 3‑methyladenine (3-MA), an autophagy inhibitor, decreased the toxicity of imiquimod. Furthermore, blocking of autophagy by 3‑MA exerted an inhibitory effect on imiquimod-induced apoptosis, which indicated that autophagy can function as a mechanism which, upon activation, directly leads to apoptosis and cell death of SGC‑7901 cells. The results of the present study suggested that imiquimod has potent direct activity against gastric cancer cells by inducing autophagy and apoptosis.

P0137 Imiquimod induces HIF-1α expression and aerobic glycolysis to protect imiquimod-induced apoptosis in cancer cells

Background Tumour cells increase glucose uptake and predominantly utilise glycolysis instead of mitochondrial respiration, even in the presence of abundant oxygen (aerobic glycolysis, the Warburg effect). HIF-1 α is a major determinant for aerobic glycolysis and cancer cell homeostasis under cytotoxic stress. Targeting HIF-1 α may be a specific strategy for chemotherapy. Methods and Findings In this study, we demonstrated imiquimod, a synthetic Toll-like receptor (TLR) 7/8 ligand which contains anti-tumour effects, can promote aerobic glycolysis that was not required for expression of TLR7 or TLR8. This effect depends on up-regulation of HIF-1 α expression at the transcriptional and translational levels via ROS mediated STAT3-dependent and Akt-dependent pathways. The genetic silencing of HIF-1 α not only repressed imiquimod-induced aerobic glycolysis but also sensitised imiquimod-induced apoptosis due to faster ATP and Mcl-1 depletion. Moreover, the glucose analogue 2-DG and the HIF-1 α inhibitor 17-AAG synergised with imiquimod to induce tumour cell apoptosis in vitro and significantly inhibited tumour growth in vivo. Interpretation We suggest that imiquimod-induced aerobic glycolysis and HIF-1 α expression is a protective effect in response to imiquimod treatment, and therefore, co-treatment with inhibitors of HIF-1 α and/or glycolysis may be a useful therapeutic strategy to enhance the anti-tumour effects of imiquimod in clinical settings.

Effect of Imiquimod as Compared With Surgery on the Cancerization Field in Basal Cell carcinoma

BackgroundPatients with basal cell carcinoma (BCC) have an increased risk of subsequent BCCs. It is possible that imiquimod might reduce this risk by acting on the cancerization field. ObjectiveTo examine the ability of imiquimod to reduce subsequent BCCs. MethodsRetrospective cohort study of patients with BCC treated at our hospital between 2003 and 2011. The patients were divided into 2 groups depending on whether they had been treated with surgery or with imiquimod. Comparing the 2 groups, we analyzed the development of new BCCs, the time that elapsed between first and subsequent tumors, and the site of occurrence of the second BCC with respect to the first one (local, same lymphatic drainage basin or anatomic region, or other). Survival methods were used to analyze the data. ResultsWe reviewed the charts of 623 patients. Of these, 550 had been treated with surgery (88.3%) and 71 with imiquimod (11.4%). Overall, a second BCC occurred in 36.4% of patients (n=227). The rate of occurrence was 38.2% in the surgery group and 23.9% in the imiquimod group (P=.02). The hazard ratio for the occurrence of a subsequent BCC was 2.13 (95% CI, 1.28-3.53) for patients treated with surgery compared with those treated with imiquimod. Imiquimod reduced the risk of a second BCC locally, regionally, and in the lymphatic drainage area. Our findings are limited by the retrospective nature of our study and the small number of patients treated with imiquimod. ConclusionsImiquimod may reduce the risk of subsequent BCC in patients treated for BCC and its effect could last for up to 2 years in local, regional and lymphatic cancerization fields. We believe that the cancerization field concept should be expanded to include not only the local area, but also the pertinent anatomic region and the regional lymphatic drainage area.

Imiquimod: the biochemical mechanisms of immunomodulatory and anti-inflammatory activity

Imidazoquinolins represent a new group of compounds that recently entered into clinical practice as anti-tumor and anti-viral immune modulators. They are low molecular weight synthetic guanosine-like molecules. Although imiquimod, the most widely used imidazoquinolin, is recommended for the treatment of several forms of skin cancer and papillomas, the molecular mechanisms of its action are not fully understood. In particular, imiquimod has been characterized as a specific agonist of Toll-like receptor 7 (TLR7) and is widely used in this capacity in a large number of experimental studies and clinical trials. However, detailed analysis of the published data with the use of imiquimod, suggests that its biological activity can not be explained only by interaction with TLR7. There are indications of a direct interaction of imiquimod with adenosine receptors and other molecules that regulate the synthesis of cyclic adenosine monophosphate. A detailed understanding of the biochemical basis of imiquimod immunomodulating and antitumor effect will increase its clinical effectiveness and accelerate the development of new drugs with similar but improved medical properties. This review summarizes the published data concerning the effects of imiquimod on a variety of intracellular biochemical processes and signaling pathways.

Effects of imiquimod and low-intensity laser (λ660nm) in chemically induced oral carcinomas in hamster buccal pouch mucosa

Squamous cell carcinoma (SCC) is the most common neoplasm of the oral cavity. It is aggressive, highly proliferative, and metastatic. This study aimed to evaluate the effect of LLLT and imiquimod on DMBA chemically induced lesions on the oral mucosa of hamsters. SCCs were induced on 25 hamsters. Animals of G1 (control 1) were killed and the presence of tumors confirmed; G2 (control 2) suffered no interventions for additional 4 weeks; animals of G3 (laser treatment) were irradiated (λ660 nm, 50 mW, CW, Ø=3 mm, 0.07 cm(2), 714.2 mW/cm(2), 133 s, 95 J/cm(2), 6.65 J) at every other day for 4 weeks; animals of G4 (imiquimod treatment) received 5 % imiquimod three times a week for 4 weeks; and animals of G5 (imiquimod and laser treatment) received both treatments for the same period. Samples were taken and underwent histological analysis by light microscopy and were investigated using immunohistochemistry for S-100(+) dendritic cells. In G1, G2, and G3, the evaluations showed malignant tumors and the absence of S-100(+) dendritic cells in the tumor stroma. In G4, 60 % of the animals had no malignant tumors, and S-100(+) dendritic cells were present in the stroma of the tumors as well as dysplasia. In G5, 40 % of the animals presented SCC, with scarce or no S-100(+) dendritic cells. The imiquimod treatment played a direct effect on SCC, demonstrated by the increased number of S-100(+) dendritic cells, which could suggest an important role of immune surveillance against neoplastic proliferation. Furthermore, its association with laser needs to be further investigated.

Cytokine Dermatitis and Febrile Seizure From Imiquimod

Cytokine dermatitis is a well-known and common clinical adverse effect of imiquimod 5% cream (Aldara, 3M). Data from initial Phase III clinical trials reveal a minority of study drug patients experience systemic adverse effects, including fever, arthralgia, headache, myalgia, and lymphadenopathy. These adverse effects are caused, presumably, from increased absorption of study drug over the area of dermatitis, leading to systemic cytokine release. Furthermore, the incidence of systemic reactions was rarely statistically increased above control patients. We describe herein a case of severe cytokine dermatitis in a 2-year-old female patient treated with daily imiquimod for molluscum contagiosum who subsequently developed febrile seizure. We believe this to be the first reported case of seizure associated with imiquimod 5% cream (Aldara, 3M) in a pediatric setting.

Virostatische und immunmodulatorische Lokaltherapie bei rezidivierenden HPV-assoziierten hochgradigen vulvären intraepithelialen Neoplasien: Eine Pilotstudie

OBJECTIVES: Aim of the study was to test the efficacy of a local virostatic and immunomodulatory therapy for recurrent HPV-associated vulvar intraepithelial neoplasias (VIN) and to evaluate the effect of imiquimod on dendritic cells. METHODS: Three women with recurrent VIN treated previously with surgical excision and laser destruction developed new VIN lesions. They received local treatment with 5 % imiquimod cream for 2 weeks alternating with 1 % cidofovir gel for 2 weeks for a total period of 12 weeks. Expression surface markers on antigen-presenting dendritic cells after treatment with imiquimod were analyzed. RESULTS: All biopsies revealed HPV 16 DNA. At 1 and 5 months after completion of local therapy, all 3 patients had clinical and histological remission without detectable HPV 16 DNA. One patient was disease-free for 5 years before developing a recurrent VIN lesion again. She was treated locally with imiquimod and cidovir for a second time, which resulted in complete remission. Both other patients remained free of recurrence during the whole period. After incubation with imiquimod, dendritic cells revealed an increased expression of surface markers CD80/B7.1, CD 86/B7.2 and CD40, CD54/ICAM-1 and CD83. CONCLUSION: Virostatic and immunomodulatory local therapy is an effective noninvasive treatment alternative for recurrent HPV-associated high grade VIN. The effect of imiquimod might be based on an enhanced presentation of HPV-antigens to lymphocytes with subsequent destruction. This might be the result of an increased expression of co-stimulatory molecules and surface markers on the cell membrane of antigen presenting dendritic cells.

Abstract 5623: Synergy of the immunomodulators GM-CSF and imiquimod for peptide-based therapeutic vaccines

Abstract All approved immunomodulators currently in use with therapeutic peptide-based cancer vaccines in are of only moderate efficacy in terms of direct activation of antigen-presenting cells which is a key prerequisite for efficient priming of cytotoxic T cells (CTLs). Moreover, induction of immune responses against tumor-associated antigens (TAAs) is further hampered by an in most cases immunosuppressive environment in cancer patients and by effective tolerization of the immune system against self-antigens, as the majority of TAAs is not exclusively expressed in tumors but also to some extent present on normal tissues. Because the reliable induction of tumor-directed immune responses is crucial for the efficacy of therapeutic cancer vaccines, novel immunomodulator regimens have to be introduced into clinical testing to achieve a better clinical outcome. Therefore, we analyzed safety and efficacy of several approved immunomodulatory substances and combinations thereof in a dedicated pre-clinical screening program. The combination of subcutaneously (s.c.) applied granulocyte-macrophage colony-stimulating factor (GM-CSF) with topical application of the TLR7 ligand imiquimod resulted in synergistic enhancement of peptide-vaccine induced CTL responses without any signs of toxicity or cumulated adverse effects. GM-CSF applied s.c. combined with imiquimod cream applied topically to the shaved skin of C57BL/6 mice at the vaccination site significantly increased the number of vaccine-specific CTLs induced by s.c. vaccinations with model MHC class I-binding peptides compared with either substance alone. The induced immune response was comparable to the effect of CpG deoxyoligonucleotides or poly-IC, two potent immunomodulators currently not approved and therefore not widely available for clinical trials in humans. However, the timing of imiquimod application in relation to the vaccination schedule is crucial, as the synergistic effect of imiquimod and GM-CSF was only observed, if imiquimod was applied at the day of vaccination and/or 24 h later, but not if imiquimod was applied the day before peptide vaccination. In total, 45 mice were immunized with the combination of GM-CSF and imiquimod without any signs of local or systemic toxicities. Further results from in vitro activation assays with human PBMCs and isolated antigen-presenting cells and the distinct molecular mechanisms of immunostimulation employed by both substances provide further support for the anticipated safety of this regimen in humans. GM-CSF and imiquimod is therefore considered as a promising and safe alternative immunomodulator regimen for future clinical trials. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5623.

Abstract C88: Imiquimod induces reactive oxygen species- and caspase-independent cell cycle arrest and apoptosis in MDA-MB-468 breast cancer cells

Abstract Imiquimod is an immune modulator currently used as a topical treatment for superficial basal cell carcinoma. Recently, apoptosis-inducing activity in neoplastic cells has been attributed to imiquimod at doses over a 1000-fold lower than those used in topical applications. The present study examined the direct effect of imiquimod on MDA-MB-468 breast cancer cells. Oregon Green 488 staining of imiquimod-treated MDA-MB-468 cells indicated that imiquimod had a dose-dependent, anti-proliferative effect; cell cycle analysis indicated that arrest occurred in S phase after 72 h exposure to imiquimod. In addition, imiquimod had a time- and dose-dependent cytotoxic effect on a panel of breast cancer cells (MDA-MB-468, MDA-MB-231, MCF-7, and T47D). Double staining of imiquimod-treated MDA-MB-468 cells using Annexin-V-FLOUS and propidium iodide indicated that imiquimod caused apoptosis, while the lack of lactate dehydrogenase release confirmed the absence of necrosis. Unlike previously reported data for other neoplastic cells, imiquimod-induced apoptosis was not caspase-dependent in MDA-MB-468 cells; however, DiOC6 staining revealed destabilization of the mitochondrial membrane following imiquimod treatment. Pretreatment with reduced glutathione did not rescue MDA-MB-468 cells from imiquimod-induced apoptosis, indicating that reactive oxygen species were not required for imiquimod-induced cytotoxicity. Western blotting showed that imiquimod-treated MDA-MB-468 cells had increased cytosolic cytochrome c and PARP cleavage compared to controls, which was consistent with induction of the mitochondrial pathway of apoptosis. These data suggest that imiquimod exerts a significant caspase-independent apoptosis-promoting effect on breast cancer cells. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C88.

Mutations in Classical Swine Fever Virus NS4B Affect Virulence in Swine

NS4B is one of the nonstructural proteins of classical swine fever virus (CSFV), the etiological agent of a severe, highly lethal disease of swine. Protein domain analysis of the predicted amino acid sequence of the NS4B protein of highly pathogenic CSFV strain Brescia (BICv) identified a putative Toll/interleukin-1 receptor (TIR)-like domain. This TIR-like motif harbors two conserved domains, box 1 and box 2, also observed in other members of the TIR superfamily, including Toll-like receptors (TLRs). Mutations within the BICv NS4B box 2 domain (V2566A, G2567A, I2568A) produced recombinant virus NS4B.VGIv, with an altered phenotype displaying enhanced transcriptional activation of TLR-7-induced genes in swine macrophages, including a significant sustained accumulation of interleukin-6 (IL-6) mRNA. Transfection of swine macrophages with the wild-type NS4B gene partially blocked the TLR-7-activating effect of imiquimod (R837), while transfection with the NS4B gene harboring mutations in either of the putative boxes displayed decreased blocking activity. NS4B.VGIv showed an attenuated phenotype in swine, displaying reduced replication in the oronasal cavity and limited spread from the inoculation site to secondary target organs. Furthermore, the level and duration of IL-6 production in the tonsils of pigs intranasally inoculated with NS4B.VGIv were significantly higher than those for animals infected with BICv. The peak of IL-6 production in infected animals paralleled the ability of animals infected with NS4B.VGIv to resist challenge with virulent BICv. Interestingly, treatment of peripheral blood mononuclear cell cultures with recombinant porcine IL-6 results in a significant decrease in BICv replication.