Connexin Hemichannels: Methods for Dye Uptake and Leakage.

It is now clear that connexin-based, gap junction "hemichannels" in an undocked state are capable of opening and connecting cytoplasm to the extracellular milieu. Varied studies also suggest that such channel activity plays a vital role in diverse cell processes and abnormal hemichannel activity contributes to pathogenesis. To pursue fundamental questions in this area, investigators require methods for studying hemichannel permeability and dynamics that are quantitative, sensitive, versatile, and available to most cellular and molecular laboratories. Here we first provide a theoretical background for this work, including the role of cellular membrane potentials. We then describe in detail our computer-assisted methods for both dye uptake and leakage along with illustrative results from different cell systems. A key feature of our protocol is the inclusion of a mechanical stimulation step. We describe dye uptake, interpreted as connexin dependent, that is shown to be enhanced with reduced extracellular Ca2+, mechanically responsive, inhibited by TPA, inhibited by EL186 antibodies for Cx43 and sustained for more than 15 min following mechanical stimulation. We describe dye leakage that displays these same properties, with estimates of hemichannel numbers per cell being derived from leakage rates. We also describe dye uptake that is shown to be unaffected by a reduction in external Ca2+, insensitive to EL186 antibodies and relatively short-lived following mechanical stimulation; this uptake may occur via pannexin 1 channels expressed in the cells studied here. It is unlikely that cell damage plays a significant role in dye uptake following mechanical stimulation, given compelling results from various control experiments.

Reduced degree of irritation during a second cycle of ingenol mebutate gel 0.015% for the treatment of actinic keratosis.

Ingenol mebutate gel is a topical field treatment of actinic keratosis (AK). One of several proposed mechanisms of action for ingenol mebutate is induction of cell death in proliferating keratinocytes, suggesting a preferential action on AKs rather than healthy skin. Local skin reactions (LSRs) during 2 sequential 4-week cycles of AK treatment with ingenol mebutate gel 0.015% on the face or scalp were evaluated to test the hypothesis that reapplication of the study product would produce lower LSR scores than during the first treatment cycle. In this unblinded study, 20 participants with AKs on the face or scalp were treated with ingenol mebutate gel 0.015% once daily for 3 days in 2 sequential 4-week cycles. Composite LSR scores were evaluated during both cycles. The composite LSR score during the second cycle was found to be significantly lower than the first cycle (P=.0002). The proportion of participants who experienced LSRs in the second treatment cycle was less than the first cycle. Ingenol mebutate gel 0.015% may cumulatively reduce the burden of sun-damaged skin over 2 treatment cycles by targeting and removing transformed keratinocytes.

Afamelanotide and narrowband UV-B phototherapy for the treatment of vitiligo: a randomized multicenter trial.

IMPORTANCE: Narrowband UV-B (NB-UV-B) phototherapy is used extensively to treat vitiligo. Afamelanotide, an analogue of α-melanocyte-stimulating hormone, is known to induce tanning of the skin. OBJECTIVE: To evaluate the efficacy and safety of combination therapy for generalized vitiligo consisting of afamelanotide implant and NB-UV-B phototherapy. DESIGN, SETTING, AND PARTICIPANTS: This study was performed in 2 academic outpatient dermatology centers and 1 private dermatology practice. We enrolled men and women 18 years or older with Fitzpatrick skin phototypes (SPTs) III to VI and a confirmed diagnosis of nonsegmental vitiligo that involved 15% to 50% of total body surface area. Vitiligo was stable or slowly progressive for 3 months. Patients were randomized to combination therapy (n = 28) vs NB-UV-B monotherapy (n = 27). After 1 month of NB-UV-B phototherapy, 16 mg of afamelanotide was administered subcutaneously to the combination therapy group monthly for 4 months while NB-UV-B phototherapy continued; the other group continued to receive NB-UV-B monotherapy. INTERVENTIONS: Narrowband UV-B monotherapy vs combined NB-UV-B phototherapy and afamelanotide. MAIN OUTCOMES AND MEASURES: Response on the Vitiligo Area Scoring Index and Vitiligo European Task Force scoring system. RESULTS: Response in the combination therapy group was superior to that in the NB-UV-B monotherapy group (P < .05) at day 56. For the face and upper extremities, a significantly higher percentage of patients in the combination therapy group achieved repigmentation, and at earlier times (face, 41.0 vs 61.0 days [P = .001]; upper extremities, 46.0 vs 69.0 days [P = .003]). In the combination therapy group, repigmentation was 48.64% (95% CI, 39.49%-57.80%) at day 168 vs 33.26% (95% CI, 24.18%-42.33%) in the NB-UV-B monotherapy group. Notable adverse events included erythema in both groups and minor infections and nausea in the combination therapy group. Comparison between Fitzpatrick SPTs showed patients with SPTs IV to VI in the combination therapy group had improvement in the Vitiligo Area Scoring Index at days 56 and 84 (P < .05); no significant difference was noted in patients with SPT III. CONCLUSIONS AND RELEVANCE: A combination of afamelanotide implant and NB-UV-B phototherapy resulted in clinically apparent, statistically significant superior and faster repigmentation compared with NB-UV-B monotherapy. The response was more noticeable in patients with SPTs IV to VI. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01430195.

Retapamulin 1% Ointment and Clobetasol Propionate 0.05% Foam is More Efficacious than Vehicle Ointment and Clobetasol 0.05% Propionate Foam in the Treatment of Hand/Foot Dermatitis: A Single Center, Randomized, Double-blind Study.

BACKGROUND: Staphylococcus aureus has been implicated in the pathogenesis of adult hand/foot dermatitis. OBJECTIVE: The authors hypothesized that retapamulin 1% ointment and clobetasol propionate 0.05% foam would decrease disease severity in subjects with hand/foot dermatitis and provide a higher clearance of Staphylococcus aureus colonization, when compared to vehicle (placebo) ointment and clobetasol propionate 0.05% foam. METHODS: Adult subjects with moderate to very severe hand/foot dermatitis had twice-daily topical application of clobetasol propionate 0.05% foam to hands/feet for 14 days and were randomized to apply either retapamulin 1% ointment or vehicle ointment twice daily to hands/feet and nares for five days. RESULTS: Seventy-three percent of subjects in the retapamulin/clobetasol group were clear/almost clear at Day 15 compared to 47 percent of subjects in the vehicle/clobetasol group (p-value of 0.04). The percentage of subjects who had both negative skin and nares cultures and were clear/almost clear was also statistically significant in favor of the retapamulin/clobetasol group at Day 15 (p-value of 0.05). LIMITATIONS: Sample size, study population. CONCLUSION: At Day 15, retapamulin 1% ointment with clobetasol propionate 0.05% foam was more efficacious than vehicle ointment and clobetasol propionate 0.05% foam for disease improvement and Staphylococcus aureus clearance in adult subjects with hand/foot dermatitis.

Evaluating the Efficacy of Photodynamic Therapy with 20% Aminolevulinic Acid and Microdermabrasion as a Combination Treatment Regimen for Acne Scarring: A Split-face, Randomized, Double-blind Pilot Study.

Acne scarring is a consequence of abnormal resolution of wound healing after damage that occurs in the sebaceous follicle during acne inflammation. No trial to date has evaluated the efficacy of the combination of microdermabrasion and photodynamic therapy for acne scarring. This single-center, double-blinded pilot study enrolled subjects with moderate-to-severe acne scarring who were randomly assigned in a blinded fashion to use aminolevulinic acid and vehicle in a split-face fashion after full-face treatment with microdermabrasion. On average, 80 percent of the patients displayed more improvement in scarring on the aminolevulinic acid split face versus the vehicle split face after five treatments. Using two different noninvasive mechanisms of targeting acne scarring provided for a safe treatment regimen characterized by more efficacious results with respect to higher rates of scarring improvement.

A single-blinded randomized controlled study to assess the efficacy of twice daily application of sinecatechins 15% ointment when used sequentially with cryotherapy in the treatment of external genital warts.

OBJECTIVE: To evaluate the efficacy of sequential therapy of cryotherapy and sinecatechins 15% ointment BID versus cryotherapy alone in treatment of external genital warts (EGW). METHODS: Forty-two subjects with at least two EGW lesions underwent cryotherapy to all lesions. One week following cryotherapy, subjects were randomized 1:1 to receive either no additional treatment or treatment with sinecatechins 15% ointment BID up to 16 weeks or until complete clearance. The total number of visible baseline and new EGW were recorded at each visit. Subjects were followed for a total of 65 weeks post-treatment. RESULTS: There was a significant reduction in mean number of lesions from baseline after 16 weeks of treatment in the cryotherapy-sinecatechins ointment group compared to cryotherapy alone (-5.0 lesions vs -2.1 lesions respectively, P=0.07). CONCLUSION: Cryotherapy plus sinecatechins 15% ointment BID resulted in a significant improvement in the reduction of EGW compared to cryotherapy alone. Clinicaltrials.gov registration identifier: NCT02147353.

Reduced burning and stinging associated with topical application of lactic acid 10% with strontium versus ammonium lactate 12%.

Burning and/or stinging is one of the most common concerns expressed by patients using topical therapies for treatment of dermatologic disorders. Topical lactic acid preparations often are used to treat dry scaly skin. In this study, we compared the level of burning/stinging reported by participants with application of lactic acid cream 10% containing strontium versus ammonium lactate lotion 12% and cetearyl alcohol lotion. The mean rating of burning/stinging reported for lactic acid cream 10% with strontium and cetearyl alcohol lotion was lower than ammonium lactate lotion 12% (P<.0001). Based on the study results, lactic acid cream 10% with strontium causes less burning/stinging than ammonium lactate lotion 12%.

Efficacy and safety of tretinoin 0.025%/clindamycin phosphate 1.2% gel in combination with benzoyl peroxide 6% cleansing cloths for the treatment of facial acne vulgaris.

Combination therapy using medications with complementary mechanisms of action is the standard of care in treating acne. We report results of a clinical trial evaluating the use of a fixed-dose tretinoin 0.025%/clindamycin phosphate 1.2% (T/CP) gel in combination with a benzoyl peroxide 6% foaming cloth compared with T/CP alone for facial acne. At week 12, the combination therapy group showed a trend toward greater efficacy compared with T/CP alone. There was a high success rate observed in the study, which may be attributable to the large percentage of adult female acne patients enrolled. Cutaneous adverse events were not statistically different in using combination therapy compared with T/CP alone.

Efficacy and safety of a ceramide containing moisturizer followed by fixed-dose clindamycin phosphate 1.2%/benzoyl peroxide 2.5% gel in the morning in combination with a ceramide containing moisturizer followed by tretinoin 0.05% gel in the evening for the treatment of facial acne vulgaris.

Combination therapy addressing multiple pathogenic factors should be used to achieve optimal outcomes in treating acne. The following study demonstrated both safety and efficacy of fixed-dose clindamycin phosphate 1.2%/benzoyl peroxide 2.5% in the morning with micronized tretinoin 0.05% gel in the evening. Both products were applied to the skin following the use of a ceramide containing moisturizing lotion.

Why are there significant differences in published narrowband ultraviolet B dosimetry recommendations? The need for national standardization of phototherapy treatment.

Narrowband ultraviolet B phototherapy is an important treatment option for psoriasis and other skin diseases. When narrowband ultraviolet B phototherapy is initiated, one method involves determining the minimal erythema dose for each patient with the starting dose at 50% to 70% of the minimal erythema dose. An alternative method involves using the recommended narrowband ultraviolet B exposure dose based on a patient's Fitzpatrick skin type. When the recommended narrowband ultraviolet B exposure doses of separate publications are compared, alarming differences are found. These discrepancies not only create confusion but also suggest the risk of phototoxicity, or its opposite, namely the risk of suboptimal dosimetry. For these reasons, this article discusses possible explanations for the wide variation in dosimetry recommendations. To remedy the current situation, the authors advocate a national standard for the practice of phototherapy treatment with the guidelines of the United Kingdom as a possible model for emulation.

Is tuberculin skin testing reliable during anti-tumor necrosis factor-alfa therapy? A case report and review of the literature.

The increased risk to patients on anti-tumor necrosis factor-alfa therapy of developing active tuberculosis supports screening of these patients for latent tuberculosis infection. The current practice of determining the presence of latent tuberculosis infection primarily, and often entirely, depends on the use of tuberculin skin testing (TST). We report a patient with psoriasis on long-term etanercept therapy who had a negative TST result and a positive interferon-gamma release assay result. Similar cases have also been found through a review of the literature. These findings suggest that TST might be unreliable during long-term anti-tumor necrosis factor-alfa therapy. QuantiFeron-TB Gold testing may be a more appropriate primary test in patients with risk factors for false-negative TST results.