Topical resiquimod dosing regimens in patients with multiple actinic keratoses: a multicentre, partly placebo-controlled, double-blind clinical trial.

BACKGROUND: Topical immune response modifiers are established for actinic keratosis (AK) treatment and efforts are underway to make further improvements to their efficacy and safety. OBJECTIVES: To investigate the optimal dosing regimens of the Toll-like receptor 7/8 agonist resiquimod in terms of efficacy, safety and tolerability. METHODS: In a multicentre, partly placebo-controlled, double-blind clinical trial, we randomized 217 patients with AK lesions to 0·03% resiquimod gel once-daily application three times per week for 4 weeks or seven times within 2 weeks or five times for 1 week (arms 1/2/3) followed by a treatment-free interval of 8 weeks and one repetition of the cycle. In two additional arms (arms 4/5), patients applied either resiquimod gel 0·01% or 0·03% three times per week up to a biological end point defined by skin erosion or for a maximum duration of 8 weeks. Clearance was assessed clinically and histologically. RESULTS: Complete clinical clearance ranged from 56% to 85% with the highest rate observed in arm 2. Resiquimod 0·03% gel was more effective than 0·01% gel. Clearance rates in arms 1/2/3 were comparable and higher than with placebo and were reached with 24, 14 and 10 gel applications, respectively. Overall, 128 patients (59%) experienced treatment-related adverse reactions. CONCLUSIONS: Resiquimod 0·03% gel is more effective than 0·01% gel. From the perspectives of safety and tolerability, the lower concentration and shorter duration are preferable. The clinical response in arms 2/3 was reached with fewer gel applications. The dosing regimens that used the biological end point (arms 4/5) proved equally efficacious as predefined treatment durations and may therefore be suitable for personalized AK treatment.

Cytokine gene expression in epidermis with biological effects following injection of naked DNA.

The epidermis is readily accessible for genetic manipulation and is easily monitored. Using pig skin because it is very similar to human skin morphologically, we have developed a method to transiently express biologically active factors in epidermis. Following direct injection of naked plasmid DNA into skin, DNA is taken up and transiently expressed at high levels by epidermal keratinocytes. Injection of interleukin-8 plasmid DNA into skin results in the appropriate biological response of neutrophil recruitment, demonstrating functional utility. In addition to this model's therapeutic uses, the biological effects of structural gene products on the epidermis could also be studied in vivo.

[Off-label indications for topical tacrolimus]. / Off-label-Indikationen für topisches Tacrolimus.

The topical calcineurin antagonist tacrolimus plays an important role in the treatment of different forms of eczema because of its favorable risk profile. In addition, different off-label indications have been clinically tested where tacrolimus ointment has achieved clinical improvement. This article discusses off-label treatment of vitiligo, seborrheic dermatitis, steroid rosacea, perioral dermatitis, rosacea and lichen sclerosus.

[Important viral and bacterial infections of the skin and mucous membrane]. / Wichtige virale und bakterielle Infektionen der Haut und Schleimhäute.

Skin infections are more frequent in immunodeficient patients, seniors and infants than in healthy individuals with an intact immune system. The severity of infections ranges from localized, self-limiting cutaneous infections to widespread necrosis of the skin, muscle, and fascia. The most frequent infections of the skin and mucous membranes are caused by bacterial and viral infectious agents. Fungal infection are also important especially in immunocompromised patients. In particular, viruses play an important and increasing role in the development of several tumors including HPV-associated cervical, anal and penile cancer or HHV-8-induced Kaposi sarcoma.

[Trombiculiasis in humans]. / Trombidiose des Menschen.

Trombiculiasis is an epizoonosis that occurs worldwide and is caused by various types of chiggers. Clinical findings after accidental contact with the parasitic mite larvae include erythematous papules and urticarial plaques, to some extent with vesicular transformation, which can lead to excoriations due to the intense pruritus. Prophylaxis against exposure is recommended when visiting areas known to be colonized by mites. Reactions to bites can be treated by local application of alcohol compresses or zinc lotions and external application of agents containing polidocanol or glucocorticosteroid. Systemic antihistamines are indicated to control itching.

[Considerable variation in the range of time for complications to appear after tattoos]. / Erhebliche zeitliche Variationsbreite von Komplikationen nach Tätowierungen.

Side effects after tattoos are being observed with greater frequency in dermatological practice. The complications that occur can be classified into systemic and local reactions. The time course of cutaneous side effects ranges from direct complications during or following tattooing to reactions that first appear several years thereafter. The majority of allergic complications can be explained by the delayed degradation of the color pigment used for the tattoo and then release of potent allergens sometimes not until years later.

[Successful use of allogeneic stem cell transplantation for treatment-refractory mycosis fungoides]. / Erfolgreicher Einsatz einer allogenen Stammzelltransplantation bei therapierefraktärer Mycosis fungoides.

Mycosis fungoides is the most common type of cutaneous T-cell lymphoma and characterized by a chronic progressive course spanning decades. The choice of treatment options should be tailored to the stage depending on the extent and aggressiveness of the disease and taking the individual situation of the patient into consideration. Long-term complete remissions can only be achieved in the early phase of the disease, while there is no therapy that results in a cure or long-term remission in advanced stages. In young patients with a treatment-refractory course of mycosis fungoides, allogeneic stem cell transplantation represents an important alternative option to manage the disease since complete clinical remission can be obtained even in advanced stages.

Expression of naked DNA in human, pig, and mouse skin.

The insertion and expression of genes in the epidermis may have a variety of therapeutic uses, including the treatment of skin diseases. Here we show that when both human skin organ cultures and human skin grafts on immunocompromised mice are injected with naked DNA, the DNA is taken-up and genes are expressed in the epidermis in a manner similar to both pig skin injected in vivo and injected pig skin organ cultures. In contrast, DNA injected into mouse skin is expressed not just in the epidermis, but also in the dermis and underlying fat and muscle tissue, and is expressed at lower levels. These findings suggest that genes can be expressed in human skin, after injection of naked DNA, and indicate that pig skin is an appropriate model for the study of DNA uptake and gene expression in human skin. The organ cultures of human and pig skin may be useful in understanding how naked DNA is internalized and expressed after in vivo injections. Additionally, skin obtained from patients with skin disease may be studied as skin grafts and organ cultures to help optimize genetic approaches for the treatment of skin diseases prior to clinical trials, by determining if the injected gene can provide a therapeutic benefit.

Human immunodeficiency virus and other sexually transmitted diseases in Cuban women.

A cross-sectional study was performed in 60 Cuban women of child-bearing age who were seropositive for human immunodeficiency virus (HIV) and 60 controls. Human papillomavirus (HPV) was identified most frequently, with oncogenic HPV serotypes 16, 33 and 58 detected in HIV-positive patients, and serotypes 11, 33 and 51 in the controls (relative risk 4.41; 95% CI 2.21-8.29). Syphilis and hepatitis B and C viruses were detected exclusively in HIV-seropositive women (p<0.05). Sexually transmitted diseases (STDs) appeared to pose a substantial health problem, especially for HIV-positive women. Clinics should consider screening and treatment for STDs as part of their HIV prevention programmes.

Randomized, controlled phase II trial of subcutaneous interleukin-2 in combination with highly active antiretroviral therapy (HAART) in HIV patients.

OBJECTIVE: To determine the immunological, virological and clinical effects of subcutaneous IL-2 in 44 HIV-patients in conjunction with pre-existing tri-therapy (zidovudine, 3TC, saquinavir). DESIGN: Partially randomized, controlled, prospective trial. SETTING: Single center study at tertiary care center. PATIENTS: Sixty four patients (CD4 count 200-500 x 10(6)/l). INTERVENTION: Fourty four patients were randomized to receive 5-day cycles of IL-2 (9 Mio IU/d) every 6 weeks (Group A) or whenever the CD4 cell count dropped below the 1.25-fold of baseline (Group B), whereas 20 control patients received the same HAART without IL-2. OUTCOME MEASURES: The optimal individual treatment interval and the immunological and virological effects of subcutaneously administered IL-2 were analysed. Importantly, the level of cellular in vivo immunity and the frequency of dermatological marker diseases and infectious complications were assessed. RESULTS: IL-2 was well tolerated although fever, influenza-like symptoms and indurated injection sites were commonly encountered. After 1 year of IL-2, there was a median increase of more than 100 x 10(6)/l CD4 cells in both IL-2 groups in contrast to the controls (P < 0.01, 0.01 and not significant). The median HIV load did not increase either in plasma or in lymph nodes. Lymphocyte activation decreased as assessed by MHC class II (P < 0.001), CD25 (P < 0.001) and CD38 expression (P < 0.005). Although delayed type hypersensitivity against common recall antigens increased in both IL-2 groups, it did not reach statistical significance. However, it is of note, that in 7 of 11 (63.6%) patients delayed type hypersensitivity against recombinant HIV antigens improved significantly. Whereas there was no opportunistic infection in either IL-2 group, three cases of Kaposi's sarcoma occurred in the controls. Dermatological indicator diseases (thrush, condyloma, herpes simplex) were found to occur more frequently in the control group. CONCLUSIONS: Subcutaneous IL-2 in addition to HAART was safe and led to sustained qualitative and quantitative immunological improvements in the majority of patients. Individualisation of therapy intervals further improved the efficacy and tolerance of IL-2.

Viral interference during simultaneous transduction with two independent helper-free retroviral vectors.

The ability to stably transduce a single cell with two independent retroviral vectors would have distinct advantages for gene therapy. We determined that cells can be transduced with two distinct retroviral vectors and have quantitated transduction efficiencies in cells infected sequentially and simultaneously. Two amphotropic, helper virus-free, retroviral vectors, a murine Moloney sarcoma virus-based vector containing the nuclear beta-galactosidase and neomycin resistance genes (MMSVn beta-gal/neoR) and a Harvey virus-derived vector containing the human multidrug resistance gene (HaMDR) were introduced into NIH-3T3 cells, pig keratinocytes, and primary pig fibroblasts simultaneously and sequentially. Analytical flow cytometry was utilized to determine retroviral transduction efficiency by assessing the percentage of cells transduced by either one or both retroviruses, in the absence of selection. Simultaneous retroviral transductions were infrequent events. In addition, transduction of previously infected cells (sequential transductions) occurred at lower than expected frequencies. Our data suggest that there is quantifiable viral interference in sequential retroviral transductions. This interference occurs by a mechanism that appears to be independent of the amphotropic retroviral receptor. Thus, such dual transductions will likely require in vitro selection or the use of a single retrovirus which contains both desired genes on the same genome.

Selection of keratinocytes transduced with the multidrug resistance gene in an in vitro skin model presents a strategy for enhancing gene expression in vivo.

In gene therapy studies, achieving prolonged, high-level gene expression in a significant percentage of cells has been difficult. One solution to enhance expression would be to select for cells expressing both the desired gene and a linked selectable marker gene in a bicistronic vector. As a potential target tissue, the skin is easily accessible for safe topical application of a selecting agent that could lead to significant gene expression in a high percentage of keratinocytes. To test the feasibility of such an approach, a skin raft culture model was developed. Human keratinocytes were transduced with the multidrug resistance (MDR) gene, which confers resistance to a variety of cytostatic and antimitotic compounds, such as colchicine. While growing on acellular dermis, transduced keratinocytes were treated with various doses of colchicine (10-50 ng/ml). Colchicine treatment increased the percentage of keratinocytes expressing MDR to almost 100% in raft cultures, Significantly, keratinocytes in colchicine-treated, MDR-transduced raft cultures were able to proliferate normally and form a stratified, differentiated epidermis. This model suggests that topical selection for MDR-expressing keratinocytes in vivo should be feasible without hampering the biologic integrity of skin. Thus, topical selection leading to enhanced expression of a desired gene, linked to a resistance gene, holds future promise for skin gene therapy.

Safety and pharmacokinetics of naked plasmid DNA in the skin: studies on dissemination and ectopic expression.

Gene therapy using naked DNA injected into muscle and skin is increasingly being used for vaccination and treatment purposes. Favorably, naked plasmid DNA does not exhibit the various limitations inherent to viral vectors, such as the elicitation of adverse immune responses and the risk of insertional mutagenesis. In order to assess the distribution and safety of naked plasmid DNA in a relevant animal model, we analyzed if intracutaneously injected plasmid DNA was transported to other organs and if ectopic expression occurred. When a "superdose" of a marker plasmid was injected intradermally, most organs were found transiently to contain the plasmid DNA for several days, whereas integration into the host genome was not detected. With the exception of ovary, however, mRNA expression only occurred in the skin, regional lymph nodes, and muscular tissues. From a safety standpoint, skin gene therapy with naked plasmid DNA can be considered safe due to the rapid biodegradation of plasmid DNA and the exclusive and transient expression of foreign genes in tissues known to take up DNA.

Efficient expression of naked plasmid DNA in mucosal epithelium: prospective for the treatment of skin lesions.

Mucocutaneous gene therapy offers exciting new treatment modalities for skin lesions. Transient expression of naked plasmid DNA could be used as a local treatment of various skin lesions where the corresponding gene product (protein) has therapeutic or immunization potential. We analyzed the time course, magnitude, and histologic expression of the indicator plasmid DNA (pCMV:beta-Gal) in mucosal epithelium and papilloma lesions. Upon direct injection of naked plasmid DNA (20 microg) into oral mucosa, expression occurred at high local concentrations, up to 35-fold higher than in comparable injections into the epidermis. Due to the accelerated turnover of mucosal epithelium beta-galactosidase positive epithelial cells were detected in the basal and suprabasal layers as early as 3 h after injection, whereas only the most superficial mucosal layers demonstrated beta-galactosidase staining at 24 h post-injection. These biologic characteristics need to be taken into consideration when clinical applications of expressing naked plasmid DNA in epithelial tissues are considered.

The derivation and characterization of pig keratinocyte cell lines that retain the ability to differentiate.

Pig skin may be a very useful model for studying human skin biology, since its morphology closely resembles that of human skin. To manipulate pig keratinocytes in vitro, we have analyzed different culture conditions for optimal pig keratinocyte growth and describe here a simple method for culture and extended passage of primary pig keratinocytes on collagen substrates. The colony-forming efficiency and proliferative capacity of primary pig keratinocytes were readily supported by Type I collagen and a final calcium concentration of 0.075 microM. These culture conditions permitted efficient gene transfer into keratinocytes using various cationic lipids at a 4:1 ratio (lipid: DNA). In addition, immortalized pig keratinocyte cell lines, which maintained a normal phenotype, were derived using these optimized culture conditions. By karyotype analysis, two independently derived cell lines had the same chromosomal abnormalities, suggesting a causal role in their immortalization. The keratinocyte cell lines exhibited a differentiated phenotype in response to elevated calcium concentration and were nontumorigenic in in vivo tumor assays. Immortalized pig keratinocyte cell lines that maintain the ability to differentiation may become a valuable tool in the study of epidermal differentiation both in vitro and in vivo. In addition, methods using keratinocytes to deliver genes to pigs in vivo could also be enhanced with these pig keratinocyte cell lines.

Topical immunomodulators--progress towards treating inflammation, infection, and cancer.

Immunomodulators include both immunostimulatory and immunosuppressive agents. Only recently have the basic mechanisms of topical immunotherapy been elucidated. Besides topical contact sensitisers (eg, diphencyprone or dinitrochlorobenzene), newer agents of the imidazoquinoline family such as imiquimod and resiquimod act by inducing cytokine secretion from monocytes or macrophages (interferon-alpha, interleukin-12, tumour-necrosis factor-alpha). The locally generated immune milieu leads to a Th1-dominance and cell-mediated immunity that have been used clinically to treat viral infections such as human papillomavirus (HPV), herpes simplex virus (HSV), mollusca, and cancerous lesions including initial squamous cell and basal cell carcinoma in immunocompetent and immunosuppressed patients. While these agents improve antigen-presentation by dendritic cells, they also act on B cells and lead to the synthesis of antibodies such as IgG2a much like the recently discovered immunostimulatory CpG-sequences that stimulate innate immunity. These sequences act as "danger signals" since they occur in bacterial and viral DNA, but are selectively methylated and inactivated in the mammalian genome. They share the induction of the same cytokines as imidazoquinolines but they show different magnitudes and kinetics of response. Topical immunotherapy with immunostimulatory agents shows potential for effective and patient-friendly treatment of inflammatory, infectious, and cancerous skin diseases. Immunoenhancers such as imdazoquinolines and CpG-sequences also have adjuvant properties that could improve conventional (protein) and DNA vaccination against cancer, atopy, and allergies.

Long-term chemotherapy of HIV-associated Kaposi's sarcoma with liposomal doxorubicin.

The aim of this study was to examine the outcome, adverse events and clinical complications of long-term chemotherapy with pegylated liposomal doxorubicin (PegLiposomal DOX) for human immunodeficiency virus (HIV)-associated Kaposi's sarcoma (KS) in the pre-highly active antiretroviral therapy (HAART) era. A phase II study over a 4-year period in a tertiary care university hospital was carried out. 52 acquired immunodeficiency syndrome (AIDS)-patients with advanced KS received long-term chemotherapy (71+/-51 weeks) with a mean of 22.8+/-18.2 cycles and a mean cumulative liposomal doxorubicin dose of 456+/-364 mg/m(2) (120-1040 mg/m(2)). Tumour burden, duration and dosage of PegLiposomal DOX, adverse events, opportunistic infections, immunological parameters and HIV load were measured. A complete (10%) or partial response (56%) was achieved while on chemotherapy. 10 patients (19%) showed stable disease. Tumour progression was observed in 8 patients (15%). Importantly, chemotherapy with PegLiposomal DOX was also successful after previous cytostatic therapy with bleomycin and vincristine. The most common adverse events included leucopenia, neutropenia, anaemia, and increased liver function tests. 34 patients (65%) developed new opportunistic infections and 29 patients (56%) died during the study period. To conclude, pegylated liposomal doxorubicin is a safe and effective drug for long-term chemotherapy of advanced (AIDS) KS without adverse effects on CD4 cell counts and HIV viral load.

Human papillomavirus genotype distribution and risk factors for infection in women from a small municipality in north east Brazil.

In order to assess the prevalence of human papillomavirus (HPV) infection, the HPV genotypes and factors associated with infection, we conducted a population-based survey in a small municipality in north east Brazil among women aged between 12 and 49 years. A questionnaire regarding socioeconomic variables, reproductive life and sexual behaviour was used, and women were examined gynaecologically, followed by collection of vaginal lavage with saline solution for HPV DNA determination. HPV DNA was detected by the Digene(®) SHARP Signal(TM)-System, and further genotyped by INNO-LiPA Genotyping System(®). Of 579 women, HPV infection was present in 68 (prevalence: 11.7%; 95% CI: 9.3-14.7). The most common HPV types were 16, 31 and 74, each accounting for 14.7% of infections. Of all HPV-positive women, 35.3% showed multiple HPV genotypes. Variables independently associated with HPV infection were: ≥3 partners in life (adjusted OR [aOR]: 3.06; 95% CI: 1.68-5.60) and the use of oral contraception in the last 12 months (aOR: 2.39; 95% CI: 1.33-4.30). Previous participation in a cervical cancer screening programme was protective (aOR: 0.28; 95% CI: 0.13-0.60). HPV infection is common among women from rural Brazil, and HPV genotypes identified indicate that immunization could be an important preventive measure in this population.