Double-Stranded Structure of the Polyinosinic-Polycytidylic Acid Molecule to Elicit TLR3 Signaling and Adjuvant Activity in Murine Intranasal A(H1N1)pdm09 Influenza Vaccination.

Polyinosinic-polycytidylic acid (PIC) is a potent double-stranded RNA (dsRNA) adjuvant useful in intranasal influenza vaccination. In mice, the intensity and duration of immune responses to PIC correlated with the double-stranded chain length. A rational method to avoid PIC chain extension in PIC production is to use multiple short poly(I) molecules and one long poly(C) molecule for PIC assembly. In this study, we elucidate that a newly developed uPIC100-400 molecule comprising multiple 0.1 kb poly(I) molecules and one 0.4 kb poly(C) molecule effectively enhanced the immune responses in mice, by preventing the challenged viral propagation and inducing hemagglutinin-specific IgA, after intranasal A(H1N1)pdm09 influenza vaccination. Reduced intraperitoneal toxicity of PIC prepared with multiple short poly(I) molecules in mice indicates the widened effective range of uPIC100-400 as an adjuvant. In contrast to uPIC100-400, the PIC molecule comprising multiple 0.05 kb poly(I) molecules failed to elicit mouse mucosal immunity. These results were consistent with TLR3 response but not retinoic acid inducible gene I (RIG-I)-like receptor response in the cell assays, which suggests that the adjuvant effect of PIC in mouse intranasal immunization depends on TLR3 signaling. In conclusion, the double-stranded PIC with reduced toxicity developed in this study would contribute to the development of PIC-adjuvanted vaccines.

A 12-Day Course of Imiquimod 5% for the Treatment of Actinic Keratosis: Effectiveness and Local Reactions. / Tratamiento con imiquimod al 5% durante 12 días para las queratosis actínicas: estudio de la eficacia y la reacción local.

INTRODUCTION AND OBJECTIVES: Imiquimod is an excellent option for patients with actinic keratosis, although its use may be limited by the long course of treatment required (4 weeks) and the likelihood of local skin reactions. The objectives of the present study were to demonstrate the effectiveness of a 12-day course of imiquimod 5% for the treatment of actinic keratosis and to examine the association between treatment effectiveness and severity of local reactions. PATIENTS AND METHODS: We included patients with at least 8 actinic keratoses treated with imiquimod 5% cream for 12 consecutive days. Local reactions were classified as mild, moderate, or severe. The statistical analysis of the association between local reactions and clinical response was based on the Pearson χ2 test and the Spearman rank correlation test. RESULTS: Sixty-five patients completed the study. Complete response was recorded in 52.3% and partial response in 75.4%. We found a statistically significant association between severity of the local reaction and response to treatment in both the Pearson χ2 test and the Spearman rank correlation test. CONCLUSIONS: A 12-day course of imiquimod 5% proved effective for the treatment of actinic keratosis. Severity of local reactions during treatment was correlated with clinical response.

Immune responses and outcome after vaccination with glioma-associated antigen peptides and poly-ICLC in a pilot study for pediatric recurrent low-grade gliomas.

BACKGROUND: Low-grade gliomas (LGGs) are the most common brain tumors of childhood. Although surgical resection is curative for well-circumscribed superficial lesions, tumors that are infiltrative or arise from deep structures are therapeutically challenging, and new treatment approaches are needed. Having identified a panel of glioma-associated antigens (GAAs) overexpressed in these tumors, we initiated a pilot trial of vaccinations with peptides for GAA epitopes in human leukocyte antigen-A2+ children with recurrent LGG that had progressed after at least 2 prior regimens. METHODS: Peptide epitopes for 3 GAAs (EphA2, IL-13Rα2, and survivin) were emulsified in Montanide-ISA-51 and administered subcutaneously adjacent to intramuscular injections of polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose every 3 weeks for 8 courses, followed by booster vaccines every 6 weeks. Primary endpoints were safety and T-lymphocyte responses against GAA epitopes. Treatment response was evaluated clinically and by MRI. RESULTS: Fourteen children were enrolled. Other than grade 3 urticaria in one child, no regimen-limiting toxicity was encountered. Vaccination induced immunoreactivity to at least one vaccine-targeted GAA in all 12 evaluable patients: to IL-13Rα2 in 3, EphA2 in 11, and survivin in 3. One child with a metastatic LGG had asymptomatic pseudoprogression noted 6 weeks after starting vaccination, followed by dramatic disease regression with >75% shrinkage of primary tumor and regression of metastatic disease, persisting >57 months. Three other children had sustained partial responses, lasting >10, >31, and >45 months, and one had a transient response. CONCLUSIONS: GAA peptide vaccination in children with recurrent LGGs is generally well tolerated, with preliminary evidence of immunological and clinical activity.

Imiquimod for anogenital warts in non-immunocompromised adults.

BACKGROUND: 30% of people with anogenital warts (AGW) have spontaneous regression of lesions but there is no way to determine whether a specific lesion will remain. There are a wide range of options available for treating people with AGW and selection is based on clinician's experience, patient preferences and adverse effects. The imiquimod could offer the advantages of patient-applied therapies without incurring the limitations of provider-administered treatments. OBJECTIVES: To assess the effectiveness and safety of imiquimod for the treatment of AGW in non-immunocompromised adults. SEARCH METHODS: We searched the Cochrane Sexually Transmitted Infections Group Specialized Register (15 April 2014), CENTRAL (1991 to 15 April 2014), MEDLINE (1946 to 15 April 2014), EMBASE (1947 to 15 April 2014), LILACS (1982 to 15 April 2014), World Health Organization International Clinical Trials Registry (ICTRP) (15 April 2014), ClinicalTrials.gov (15 April 2014), Web of Science (2001 to 15 April 2014) and OpenGrey (15 April 2014). We also handsearched conference proceedings, contacted trial authors and reviewed the reference lists of retrieved studies. SELECTION CRITERIA: Randomized controlled trials (RCTs) comparing the use of imiquimod with placebo, any other patient-applied or any other provider-administered treatment (excluding interferon and 5-fluorouracil which are assessed in other Cochrane Reviews) for the treatment of AGW in non-immunocompromised adults. DATA COLLECTION AND ANALYSIS: Three review authors independently assessed trials for inclusion, extracted data and assessed risk of bias. We resolved any disagreements through consensus. The quality of the evidence was assessed using the GRADE approach. MAIN RESULTS: Ten RCTs (1734 participants) met our inclusion criteria of which six were funded by industry. We judged the risk of bias of the included trials as high. Six trials (1294 participants) compared the use of imiquimod versus placebo. There was very low quality evidence that imiquimod was superior to placebo in achieving complete and partial regression (RR 4.03, 95% CI 2.03 to 7.99; RR 2.56, 95% CI 2.05 to 3.20, respectively). When compared with placebo, the effects of imiquimod on recurrence (RR 2.76, 95% CI 0.70 to 10.91), appearance of new warts (RR 0.76, 95% CI 0.58 to 1.00) and frequency of systemic adverse reactions (RR 0.91, 95% CI 0.63 to 1.32) were imprecise. We downgraded the quality of evidence to low or very low. There was low quality evidence that imiquimod led to more local adverse reactions (RR 1.73, 95% CI 1.18 to 2.53) and pain (RR 11.84, 95% CI 3.36 to 41.63).Two trials (105 participants) compared the use of imiquimod versus any other patient-applied treatment (podophyllotoxin and podophyllin). The estimated effects of imiquimod on complete regression (RR 1.09, 95% CI 0.80 to 1.48), partial regression (RR 0.77, 95% CI 0.40 to 1.47), recurrence (RR 0.49, 95% CI 0.21 to 1.11) or the presence of local adverse reactions (RR 1.24, 95% CI 1.00 to 1.54) were imprecise (very low quality evidence). There was low quality evidence that systemic adverse reactions were less frequent with imiquimod (RR 0.30, 95% CI 0.09 to 0.98).Finally, two trials (335 participants) compared imiquimod with any other provider-administered treatment (ablative methods and cryotherapy). There was very low quality of evidence that imiquimod did not have a lower frequency of complete regression (RR 0.84, 95% CI 0.56 to 1.28). There was very low quality evidence that imiquimod led to a lower rate of recurrence during six-month follow-up (RR 0.24, 95% CI 0.10 to 0.56) but this did not translate in to a lower recurrence from six to 12 months (RR 0.71, 95% CI 0.40 to 1.25; very low quality evidence). There was very low quality evidence that imiquimod was associated with less pain (RR 0.30, 95% CI 0.17 to 0.54) and fewer local reactions (RR 0.55, 95% CI 0.40 to 0.74). AUTHORS' CONCLUSIONS: The benefits and harms of imiquimod compared with placebo should be regarded with caution due to the risk of bias, imprecision and inconsistency for many of the outcomes we assessed in this Cochrane Review. The evidence for many of the outcomes that show imiquimod and patient-applied treatment (podophyllotoxin or podophyllin) confer similar benefits but fewer systematic reactions with the Imiquimod, is of low or very low quality. The quality of evidence for the outcomes assessing imiquimod and other provider-administered treatment were of very low quality.

Efficacy of combination therapy of oral zinc sulfate with imiquimod, podophyllin or cryotherapy in the treatment of vulvar warts.

AIM: Zinc sulfate is beneficial in the treatment of epithelial warts. We conducted this study to compare the efficacy of combination therapy of oral zinc sulfate with conventional treatments in the treatment of vulvar warts. MATERIAL AND METHODS: This study was a randomized controlled trial. The sample size was 42 in each group. Women aged 20-50 years were placed by the block randomized method into six groups: the podophyllin-, imiquimod- and cryotherapy-treated groups, and another three groups receiving 8-week combination therapy of 400 mg oral zinc sulfate with one of the above-mentioned treatments. Data were analyzed using anova and Fischer's exact test with spss16. RESULTS: A total of 228 patients were recruited and completed the study in six treatment groups. No significant difference was observed in the response to treatment among these groups. Relapse after 6 months was significantly higher in the podophyllin-, imiquimod- and cryotherapy-treated patients compared to patients receiving these treatments in combination with oral zinc sulfate (P<0.05). CONCLUSIONS: Combined therapy of oral zinc sulfate with conventional treatments of vulvar warts appears to reduce the relapse rate.

Prevention of airway allograft tolerance by polyinosinic:polycytidylic acid requires type I interferon responsiveness for mouse airway obliteration.

BACKGROUND: Respiratory RNA viruses are associated with bronchiolitis obliterans syndrome (BOS) in lung transplant recipients (LTRS); however, the immune mechanisms that regulate airway obliteration remain incompletely understood. METHODS: Using the mouse heterotopic tracheal transplant model of obliterative airway disease (OAD), we studied the role of double-stranded (ds)RNA using polyinosinic:polycytidylic acid (poly[I:C]), a synthetic analog of viral dsRNA, in abrogating airway allograft tolerance established with donor-specific transfusion (DST) and anti-CD154 monoclonal antibody therapy. RESULTS: Wild-type (WT) B6 recipients of accepted BALB/c airway grafts demonstrated significantly reduced intragraft CD8+ T cells, with markedly impaired allospecific interferon (INF)-γ and tumor necrosis factor-α secretion, uncoupled from an activated phenotype, and evidence of proliferation. Administration of poly(I:C) to DST/anti-CD154-treated recipients restored OAD pathology and CD8+ alloeffector responses to levels observed in untreated mice. However, B6 type I IFN receptor-deficient (IFN-αßR(-/-)) recipients were resistant to the abrogation of tolerance mediated by poly(I:C) and did not develop CD8+ alloeffector responses or OAD. Further, adoptive transfers of WT CD8+ T cells or CD11c+ dendritic cells alone into B6 IFNαßR(-/-) recipients treated with poly(I:C) and DST/anti-CD154 were incapable of abrogating airway graft tolerance. CONCLUSIONS: Together, these data indicate abrogation of DST/anti-CD154-induced airway allograft tolerance via dsRNA requires type-I IFN responsiveness for mouse airway obliteration.

Local and systemic adverse effects of imiquimod therapy for external anogenital warts in men: report of three cases.

Imiquimod is a topical immune response modifier used to treat anogenital warts. Although considered a safe drug, mild to moderate local and systemic side-effects may occasionally occur. We report three cases of local and systemic adverse effects related to imiquimod, including one case that mimicked meningitis, which promptly resolved with drug cessation.

[Herpetic infection in patients with psoriasis: the improvement of therapy].

The aim of the study was to estimate the efficacy of liniment cycloferon included in combined therapy of herpetic infection in 30 patients with psoriasis divided into 2 groups. Combined treatment of patients with recurrent herpetic infection promoted elimination of general infection syndrome, shortened duration of eruption and local inflammation, accelerated epithelization of herpetic erosion, and decreased the frequency of relapses during the follow-up.

[Efficacy of antiviral agents in the treatment of Crimean hemorrhagic fever].

The study aimed at estimating the pharmacotherapeutic efficacy of medications possessed of antiviral activity, such as ribavirin, cycloferon solution and tablets, in 410 patients with Crimean hemorrhagic fever: The early beginning of therapy (days 1-4 after the onset of the disease) with these drugs reduced the number of severe cases and manifestations of hemorrhagic syndrome. The duration of the disease decreased, the occurrence of intoxication syndrome reduced to a minimum, hemorrhagic rash rapidly disappeared, and the frequency of complications decreased. Adverse events were documented in 5.2% of the patients (n = 15); most of them developed on day 2 after using the drugs, their duration did not exceed 2.2 days. There were no cases of drug withdrawal.

[Evaluation of safety and pharmacotherapeutic efficacy of cycloferon in treatment of Astrakhan rickettsial fever].

Clinicopathogenetic impact of cycloferon, an endogenous interferon inductor, on the process of Astrakhan rikettsial fever, its complications and outcomes was analysed. The treatment scheme with addition of cycloferon to the complex therapy was optimized. The specificity of the disease clinical process and the level of the interferon status in the patients treated with cycloferon alone or with combination of the standard therapy and cycloferon was shown. It was observed that in the patients with moderate severity of the disease the combined use of the standard therapy and cycloferon was in favour of arresting the disease clinical signs (fever, headache, weakness, eruption, hepatomegaly, arthralgia and myalgia, lymphatic gland inflammation, primary affect) and lowered the hospitalization term vs. the standard therapy alone. In the patients with moderate severity of the disease the levels of the serous interferon-alpha before the treatment were found lower, while those of interferon-gamma were higher. The use of cycloferon in the treatment scheme resulted in increase of the interferon-alpha levels and decrease of the higher levels of interferon-gamma. The standard therapy in combination with cycloferon in the patients with moderate severity of the disease provided changes in the immune status: increase of the relative content of T- and B-lymphocytes and normalization of their absolute number. Normalization of the phagocytic activity and the coefficient of the active phagocytes, as well as increase of the phagocytic index, the levels of immunoglobulins G, A and M and the number of the circulating immune cells were stated. The standard therapy with addition of cycloferon resulted in normalization of the levels not only of succinic denydrogenase, lactate dehydrogenase and glucose-6-dehydrogenase but also of alpha-naphthylacetate esterase and alpha-naphthylbutirate esterase in the neutrophils, as well as of the whole spectrum of the monocyte enzymes, except NAD-diaphorase. The adverse reactions were observed in 2.5% of the cases (9 subjects). All of them were mild and did not require discontinuation of the drugs use.

Potent immune activation in chronic hepatitis C patients upon administration of an oral inducer of endogenous interferons that acts via Toll-like receptor 7.

BACKGROUND: ANA773, an oral prodrug of a small-molecule Toll-like receptor (TLR)7 agonist, induces a dose-related decrease in serum HCV RNA levels in chronic hepatitis C patients. METHODS: The prodrug ANA773 was administered to healthy individuals and chronic hepatitis C patients. At different time points during the course of treatment, modulation of the phenotype and function of peripheral leukocytes were evaluated to determine the role of distinct immune cells on the clinical outcome of therapy. RESULTS: Early after administration of the TLR7 agonist, a mild transient reduction of the number of lymphocytes was observed in both healthy individuals and chronic hepatitis C patients. Moreover, repeated administration of ANA773 resulted in transiently reduced numbers of myeloid and plasmacytoid dendritic cells (DC) in blood. Interestingly, reduced plasmacytoid DC numbers as well as increased serum interferon (IFN)-α and IFN-γ inducible protein (IP)-10 levels were observed only in virological responders (≥1 log(10) IU/ml reduction of HCV RNA levels upon ANA773 treatment), but were absent in virological non-responders. In vitro stimulation of peripheral blood mononuclear cells from virological responders showed a high frequency of IFN-α-producing plasmacytoid DC upon stimulation in vitro with ANA773, whereas no IFN-α was induced in non-responders. CONCLUSIONS: These findings indicate that the viral load decline in chronic hepatitis C patients treated with the TLR7 agonist ANA773 is likely due to intrinsic differences in the induction of endogenous IFNs and IFN-stimulated gene products (IFN-α and IP-10) upon TLR7 ligation.

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.

[Frequently ill child syndrome].

The efficacy of cycloferon use in the treatment of frequently ill children (FIC) was estimated by the dynamics of the blood plasma proteomic profile. A group of FIC at the age of 4 to 10 years were observed. Cycloferon was administered according to the standard schemes. The results were analysed by the computer programme complex, including the anamnesis, clinical symptoms, infection index, findings of the electrophoretic and mass-spectrometric analyses of the blood plasma before and after the drug use. The intensity indices of the Rho and Ras proteins, the signal pathways, in the blood proteomic profile proved to be sensitive and specific parameters for estimating the regimens of the therapy and prophylaxis of respiratory tract infection in FIC. The epidemiologic efficacy of cycloferon, as an agent of nonspecific immunoprophylaxis for FIC during seasonal prevalence of respiratory tract infection and influenza cases was shown.

[Monitoring of side effects and estimation of cycloferon efficacy in treatment of children with frequent and prolonged diseases].

Two hundred fifty patients, including 100 children with frequent and prolonged diseases at the age of 4 to 7 years, 76 children at the age of 7 to 18 years and 74 subjects at the age of 22 to 57 years were observed. The patients were treated with cycloferon in two courses with a 2-week interval according to the standard scheme. The tonsil surface microflora and its susceptibility to antibiotics were determined. Cycloferon lowered the Staphylococcus aureus titre and increased the culture susceptibility to benzylpenicillin, oxacillin, rifampicin, and erythromycin, reducing the variety of the fauces nonpathogenic microflora. The use of cycloferon induced no adverse (pathologic) reactions in 94.8% of the cases. In 4.4% of the children under school age the adverse reactions were transitory and did not require discontinuation of the drug use. Unforeseen reactions were recorded in 0.8% of the children and the use of the drug in them was discontinued. The use of cycloferon in two courses with a 2-week interval according to the standard scheme is recommended for prophylaxis of acute respiratory diseases in the group of children with frequent and prolonged diseases during epidemiologically unfavourable periods and for complex therapy of rhinopharinx infections as an agent increasing efficacy of other antibacterials.

Sequential use of photodynamic therapy and imiquimod 5% cream for the treatment of actinic cheilitis: a 12-month follow-up study.

BACKGROUND: Actinic cheilitis (AC) may progress into fully developed squamous cell carcinoma with a relatively high risk of metastasizing and therefore requires early identification and treatment. OBJECTIVES: To assess the clinical and histological long-term outcome as well as the safety and tolerability of sequential use of photodynamic therapy (PDT) and imiquimod in AC. METHODS: Patients with histologically confirmed grade 1 and 2 AC received two methyl aminolaevulinate-PDT sessions 2weeks apart. After a 2-week rest period the patients started applying imiquimod 5% cream 3days per week for 4weeks. At the first follow-up visits at 3 and 6months, noncomplete clinical responders were biopsied and excluded from the study if histological alterations were indicative for AC. At the last 12-month follow-up all remaining patients were biopsied. Adverse events were noted at weeks 2, 4, 6 and 8 of the treatment phase and at every visit of the follow-up period. RESULTS: Of the 34 enrolled patients, 30 completed the study. Complete clinical response was achieved by 27 patients in 3months. At 6months, clinical and histological recurrence occurred in two patients, while at 12months the complete clinical cure rate obtained was 80% and the histological complete cure rate was 73%. Treatment was well tolerated and adverse events were as expected and transient. CONCLUSION: Sequential use of PDT and imiquimod cream is of significant benefit for the treatment of AC. Further studies are needed in order to confirm the improved outcome using the combination treatment, to clarify the involved mechanisms and to optimize the therapeutic protocol.

Randomised clinical trial: anti-viral activity of ANA773, an oral inducer of endogenous interferons acting via TLR7, in chronic HCV.

BACKGROUND: The ANA773 is an oral prodrug of a small-molecule toll-like receptor (TLR)7 agonist. Preclinical and healthy volunteer clinical studies with ANA773 have demonstrated induction of endogenous interferon-α (IFN-α) of multiple subtypes, which supports the potential utility in the treatment of chronic hepatitis C virus (HCV) infection. AIM: To examine safety, tolerability, pharmacodynamics, pharmacokinetics and anti-viral activity of ANA773. METHODS: The ANA773 was investigated in a double-blind, placebo-controlled study in 34 patients chronically infected with HCV of any genotype. Patients were treatment-naïve or had relapsed following previous interferon-based treatment. This dose escalation study was composed of four dose groups (800, 1200, 1600 and 2000mg). In each group, six to eight patients received ANA773 and two received placebo. Patients were dosed with ANA773 every-other-day for either 28 days (800, 1200 or 1600mg) or 10days (2000mg). RESULTS: Mild to moderate adverse events were reported, with an increase in frequency and intensity with increasing dose. No serious AEs were reported and there were no early discontinuations. There were dose-related increases in various markers of IFN-α response. The mean maximum change in serum HCV RNA level from baseline was -0.34, -0.29, -0.40, -0.97 and -1.26log(10) in the placebo, 800, 1200, 1600 and 2000mg cohorts, respectively. At the 2000mg dose, ANA773 significantly (P=0.037) reduced serum HCV RNA levels (range: 0.14 to -3.10log(10) ). CONCLUSION: The ANA773 was generally well tolerated and resulted in a dose-related IFN-dependent response leading to a significant decrease in serum HCV RNA levels in the 2000mg dose group.

Acute renal failure caused by imiquimod 5% cream in a renal transplant patient: review of the literature on side effects of imiquimod.

Imiquimod is an immunomodulator of the imidazoquinoline group which possesses antiviral and antitumour activities. Although its mechanism of action has not been entirely elucidated yet, imiquimod 5% cream has been shown to be an efficient, long-lasting and safe therapy for multiple actinic keratoses in non-immunosuppressed patients and in transplant recipients. We report the case of a 44-year-old patient with a third renal transplant who developed an acute tubular necrosis confirmed by renal biopsy after the use of imiquimod 5% cream. The result of a literature search revealed a wide variety of side effects attributable to the use of imiquimod.