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Investigating new Tx options for frontal fibrosing alopecia patients Alopecia

The Chronicle

of

& ALLERGY SKIN SKIN

Acne

P RACTICAL T HERAPEUTICS and C LINICAL N EWS from the W ORLD of D ERMATOLOGY

n

S E P T E M B E R 2018

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Prevention of recurrence key to improved management see page 4

Psoriasis Tx: Most important step is to match treatment to needs of patient n A biologic therapy should achieve at least PASI 75 in 90% of patients

N

by LOUISE GAGNON,

Correspondent, The Chronicle

umerous options are available to treat conditions like psoriasis and psoriatic arthritis, but the most important step is to match the therapy to the needs of the patient, according to Dr. Ron Vender, associate clinical professor in the Division of Dermatology, Department of Medicine at McMaster University in Hamilton. Speaking in Toronto at the annual conference of the Primary

Care Dermatology Society of Canada, Dr. Vender, founder and director of Dermatrials Research Inc. and Venderm Innovations in Psoriasis, noted that the evolution in biologics has brought forth options that are effective and highly safe. Safety first, efficacy later The tumour necrosis factors (TNF) inhibitors such as adalimumab, etanercept, and infliximab, are very safe, but they have been eclipsed in efficacy by newer biologics, such as secukinumab and ixekizumab, both of

Assessing factors that may increase potential for wound complications Wounds

n Recognition of wounds that would benefit from early interventions an important step

I

by LOUISE GAGNON, Correspondent, The Chronicle

dentifying patient and surgical factors that increase the risk of post-op wound complications, recognizing wounds that would benefit from interventions like negative pressure wound therapy (NPWT), and appreciating the roles of the healthcare team in caring for a surgical patient, are Please turn to Wounds page 8à

T

by JOHN EVANS,

Associate Editor, The Chronicle

Research into inflammatory process leads to new insights into

Clinical practice

n Increasing frequency, limited studies present therapeutic challenges in scarring condition

which are IL-17 inhibitors, and guselkumab, an IL-23 inhibitor. Dr. Vender, a dermatology consultant at St. Joseph’s Hospital in Hamilton, stressed that safety should be the first priority for a clinician. “I think safety first and efficacy later,” he said. Dr. Vender told conference attendees that Dr. Ronald their patients may Vender ask for a biologic by a brand name, because they have seen a television advertisement on a Please turn to Biologics page 10à

he incidence of frontal fibrosing alopecia [FFA] is on the rise, and while there is evidence that combination treatments can be effective in reducing symptoms and arresting hairline retreat, more research on therapies and the aetiology and clinical course of the condition is needed. These are the conclusions drawn from an update and review paper published in the Journal of Cutaneous Medicine and Surgery (Mar. 1, 2018; 22(2):182–189). “Frontal fibrosing alopecia is something we are seeing more commonly in clinic,” said the paper’s senior author Dr. Jennifer Beecker, staff dermatologist at The Ottawa Hospital and assistant professor in the division of dermatology, faculty of medicine at the Dr. Jennifer University of Beecker Ottawa. “A lot of dermatologists are reporting that 15 years ago they rarely saw it, and now we see it every day, it seems.” FFA data, research reviewed That increase in incidence and the absence of a consensus document to guide treatment led Dr. Beecker and her colleagues to review the data that existed regarding the etiopathogenesis, clinical features, and therapeutic options for FFA, she said. “We really wanted to do a comprehensive review for the everyday

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Vol. 24, No. 6

THE CHRONICLE of S K I N & A L L E R G Y

TOP of the MONTH

September 2018 · 3

A Message from the Medical Editor

Image courtesy David Welch, PhD

Special Report on Acne: Research into inflammatory process leads to new insights regarding treatment The toll like receptor 2 (TLR2) is implicated in inflammation in acne, and researchers may be able to look at molecules that can correct it in a more specific way . . . . . . . . . . 4 Surveying the dermatologic literature Minimizing light intensity can reduce pain during use of PDT for the treatment of AKs, even when covering large fields, according to a recent report in Photodermatology, Photoimmunology & Photomedicine (May 12, 2018), and more. . . .14

Far UV light treats skin infections

Chronicle Postgraduate Educational Supplement In this issue’s educational supplement, a team of researchers from the University of British Columbia and Germany’s University of Freiburg report that Granzyme B is elevated in autoimmune blistering diseases and cleaves key anchoring proteins of the dermal-epidermal junction, which may be an indication it could be amenable to a single targeted treatment . . . .19

Far-ultraviolet-C (UVC) light delivered through light-diffusing optical fibres is highly effective at killing drug-resistant bacteria and could be used to prevent epidermal infections around medical devices that penetrate the skin, such as catheters or mechanical heart pump drivelines (PLoS One 2018; 13(8):e0202275). Researchers used a laser to send 224 nm far-UVC light through a thin flexible optical fibre which diffused the light out along its length. The fibres were laid directly over tissue cultures containing MRSA, which was efficiently killed by the far-UVC light. “Our study suggests that far-UVC light, delivered by optical fibres that can be incorporated into skin-penetrating devices, could be used to prevent catheter-based and driveline infections,” said study author David J.

Sunscreens: Factors preventing proper application and use Speakers at the 4th International Conference on UV and Skin Cancer Prevention sessions in Toronto addressed a number of factors influencing the proper use of sunscreens, including lack of confidence in efficacy, cultural standards, and fear of vitamin D deficiency. . . . . . . . . . . 25

Optical fibres kill drug-resistant bacteria, and could be developed to treat epidermal infections.

Research

From the News Resources of The Chronicle

Brenner, PhD, professor and director of the Center for Radiological Research at Columbia University Irving Medical Center in New York, in a press release (https://goo.gl/pjcL9m). Previous research from the team has shown that narrow-spectrum, farUVC (207 to 224 nm) can kill MRSA bacteria without damaging human skin (PLoS One 2016; 11(6):e0138418). Conventional germicidal UV light at 254 nm effectively kills bacteria, but cannot be used in most healthcare settings since it can harm the skin and eyes. “This application would be used for catheters or drivelines that have to be kept in place for long periods of time, and it is hard to keep the area where they penetrate the skin sterile. Incorporating these thin, far-UVC-emitting fibres into the catheter or driveline may be the solution,” explained Dr. Brenner.

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n this issue of THE CHRONICLE we have multiple updates including acne, psoriasis, frontal fibrosing alopecia, wound care and sunscreen use. On the acne front, Drs. Hanna, Humphrey and Dutil not only update readers on new targets such as toll like receptor 2, they discuss innovative and novel approaches such as restoring the skin microbiome. On the practical side we get advice on the importance of the psychological impact of acne on our patients, as well as approaches to try and prevent post inflammatory hyperpigmentation in our acne patients (page 4). When it comes to treating psoriasis, Dr. Ron Vender stresses that we should think safety first and efficacy later, and reviews the significant impact that newer biologics are having on reaching previously unthinkable targets of PASI 90 and 100 in a significant number of patients. Targeting IL17 and 23 has certainly raised the bar of efficacy with a very acceptable safety profile (page 1). Dr. Jennifer Beecker contributes to our ability to treat a challenging and devastating form of scarring alopecia, i.e., frontal fibrosing alopecia (page 1). Although the evidence needs to be strengthened, combination therapy can Please turn to Message page 24à

Medical Editor

Wayne Gulliver,

MD, FRCPC

John P. Arlette, MD, FRCPC Benjamin Barankin, MD, FRCPC Marc Bourcier, MD, FRCPC Eric Goldstein, MD, FRCPC Peter Hull, MD, FRCPC Richard Langley, MD, FRCPC Danielle Marcoux, MD, FRCPC R.A.W. Miller, MD, FRCPC

Editor, Cosmetic Dermatology MD, FRCPC

Sheldon V. Pollack,

H. Eileen Murray, MD, FRCPC Kim Papp, MD, FRCPC Yves Poulin, MD, FRCPC Melanie D. Pratt, MD, FRCPC Denis Sasseville, MD, FRCPC Jerry Tan, MD, FRCPC Ronald B. Vender, MD, FRCPC

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Contacting The Chronicle

“We now know that toll like receptor 2 (TLR2) is significantly implicated in inflammation in acne. Once you know that, you might be able to look at molecules that can correct it in a more specific way.” Dr. Sam Hanna, a dermatologist at Dermatology on Bloor in Toronto (see page 4)

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4 · September 2018

Lead article

THE CHRONICLE of SKIN & A L L E R G Y

Clinical practice

Research into inflammatory process leading to new

A cne th e ra pie s I

n Patients aware of influence of microbiome on acne, and seeking topicals for microbiome balance by LOUISE GAGNON,

Correspondent, The Chronicle

mproved understanding of the inflammator y process in the development of acne is revealing new potential therapeutic targets, while evaluation of the role of the skin’s natural microbiome in the development of the chronic condition is also leading to potential new acne therapies.

“One of the things that has been really helpful with acne over the last few years is that we increasingly understand the markers and cytokines involved in the inflammation in acne,” explained Dr. Sam Hanna, a dermatologist at Dermatology on Bloor in Toronto. “We now know that toll like receptor 2 (TLR2) is significantly implicated in inflammation in acne. Once you know that, you might be able to look at molecules that can correct it in a more specific way.”

Role of microbiome in acne One of the therapies that is targeting the activity of TLR2 includes tetramethyl-hexadecenyl-cysteine-formylprolinate (SIG1459), which has been shown to have direct antibacterial activity and inhibit TLR2 inflammatory signalling, and was shown to be well-tolerated. Data from a vehicle-controlled clinical study published in Experimental

Dermatology showed that 1% SIG1459 outperformed 3% benzoyl peroxide in a head-to-head comparison (Exp Dermatol 2018 Dr. Sam Hanna May 24). Understanding the role of the microbiome in the management of dermatologic diseases has produced a surge in therapies. One therapy that is being studied for its ability to restore balance in the skin microbiome is Ammonia Oxidizing Bacteria (AOB), which is a topical formulation of Nitrosomonas eutropha and is currently in clinical trials. A Phase 2b study achieved the primary endpoint at week 12 of a statistically significant two-point reduction in an Investigator’s Global Assessment (IGA) of acne severity compared to vehicle control. The therapy was also well-tolerated in the study. Dr. Shannon Humphrey, medical director, Carruthers & Humphrey, clinical assistant professor, director of CME Department of Dermatology & Skin Science, University of British Columbia in Vancouver, says she is unfamiliar with AOB as a candidate therapy and its evidence for treatment, but notes there is abundant interest in therapies directed at microbiome correction. “I can say consumer demand and awareness of topical treatments that

target the microbiome is high,” pointed out Dr. Humphrey. “The evidence base has lagged significantly behind c o n s u m e r Dr. Shannon demand and Humphrey patient awareness, so what is promising is that the body of evidence is expanding to further explore the efficacy of this treatment.” Therapies are being developed to correct the perturbation of the skin microbiome, according to Dr. Hanna.

Sebum production still being studied “When you perturb it [skin microbiome] by cleansing [too much] or through the use of antibiotics that alter the balance [of the skin microbiome], it can contribute to the production of skin diseases,” says Dr. Hanna. “That is true for eczema, rosacea, acne, and other skin diseases.” Sebum production is the target for an established therapy like isotretinoin, and olumacostat glasaretil, formerly known as DRM01, presented a promising prospect as an acne therapy. “At present, [sebum production] can only be targeted with systemic isotretinoin,” says Dr. Humphrey. “We are already able to target inflammation, hyperkeratinisation, and P. acnes with topicals.”

Please turn to Acne page 6à


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At Week 12, TactuPump® FORTE demonstrated superior reductions in acne lesion counts vs. vehicle in patients with moderate and severe acne. • Mean reduction (and percent change—2° endpoint) in inflammatory lesions for TactuPump FORTE and vehicle: 27.8 (68.7%)* and 13.2 (39.2%), respectively. ®

• Mean reduction (and percent change—2° endpoint) in noninflammatory lesions for TactuPump FORTE and vehicle: 40.5 (68.3%)* and 19.7 (37.4%), respectively. ®

* p<0.001 vs. vehicle. Missing data was imputed using multiple imputation methodology, ITT population.

TactuPump® FORTE may also be considered for those patients who have moderate and severe acne vulgaris, who may have risk factors that worsen acne prognosis such as tendency toward cyclical relapses, pre-pubertal onset or long term history of acne, positive family/genetic history, those prone to or at risk for scarring, and those who may have intolerance or contraindication to systemic treatment. Clinical surveillance of these patients is recommended to ensure sufficient therapeutic response. Safety and effectiveness have not been established in geriatric patients (≥65 years). Contraindications: • Application to areas of skin affected by eczema or seborrheic dermatitis • Patients who are hypersensitive to adapalene, benzoyl peroxide or to any ingredient in the formulation or component of the container arnings and precautions: pr Most serious warnings • For external use only, not for ophthalmic use • Should not be used by pregnant women: use only after contraceptive counselling in women of childbearing age arnings and pr Other relevant warnings precautions: • Discontinue use if allergic/hypersensitivity reactions occur • Avoid contact with eyes, lips, angles of the nose, mucous membranes, abraded skin, open wounds, eczematous and sunburned skin • May bleach hair and coloured fabric; caution when applying near hairline

• Not recommended with concomitant topical acne therapy or potentially irritating topical products • Avoid electrolysis, “waxing” and chemical depilatories for hair removal on skin treated with TactuPump® FORTE • Patients should be advised to use non-comedogenic cosmetics • Certain cutaneous signs and symptoms can be expected with use • Avoid excessive sunlight, including sunlamps (avoid exposure or use protection, avoid sunburned skin); weather extremes, such as wind or cold, may be irritating • Caution in nursing women For more information: Please consult the TactuPump® FORTE Product Monograph at https://galderma.ca/Portals/4/pdf/2017/ TactuPump_TactuPump-Forte-PM-E.pdf for important information relating to adverse reactions, interactions and dosing information, which have not been discussed in this advertisement. The Product Monograph is also available by calling us at 1-800-467-2081. Study design: A multicentre, randomized, double-blind, parallel-group, active- and vehicle-controlled, 12-week study, comparing once-daily application of TactuPump® FORTE (n=217), TactuPump® (n=217) or vehicle gel (n=69) in moderate and severe acne patients 12 years of age and older. The co-primary endpoints were success rate defined as “Clear” or “Almost Clear” with at least 2-grade improvement in IGA (Investigator’s Global Assessment) score and mean absolute change from baseline at week 12 in both inflammatory and noninflammatory lesions. Reference: 1. TactuPump® FORTE Product Monograph, Galderma Canada Inc., May 19, 2017. ©2018 Galderma Canada Inc. TactuPump® FORTE is a registered trademark of Galderma Canada Inc.

2214-0518E

Indication and clinical use: TactuPump® FORTE (adapalene 0.3%/benzoyl peroxide 2.5%) Topical Gel is indicated for the treatment of moderate and severe acne vulgaris, characterized by comedones, inflammatory papules/pustules with or without occasional nodules in patients 12 years of age and older.


ng ancers

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Relevant warnings andand Relevant warnings precautions: precautions: â&#x20AC;˘ May risk of Â&#x2022; increase 7(,"0$ the 1$2'$0(1)-% infection and should be be infection and should used with caution in patients 31$#5(2'" 32(-,(,. 2($,21 with5(2'"*(,(" clinically important **7(+.-02 ,2 chronic or active infection. "'0-,("-0 "2(4$(,%$"2(-, â&#x20AC;˘ Tuberculosis (TB): Should 93!$0"3*-1(1'-3*# not,-2!$&(4$,2-. be given to patients with 2($,215(2' active TB. Evaluate for TB "2(4$4 *3 2$%-0 infection prior to initiating2(,& (,%$"2(-,.0(-02-(,(2( treatment. Initiate treatment 20$ 2+$,2,(2( 2$20$ 2+$,2 of latent TB infection prior -%* 2$,2(,%$"2(-,.0(-0 to administering Taltz. *28 2- #+(,(12$0(,& Consider anti-TB therapy .7 -,1(#$0 ,2(2'$0 prior to initiating2(,& Taltz in .0(-02-(,(2( *28(, patients with a history of . 2($,215(2' '(12-07-% latent or active TB and * 2$,2-0 "2(4$ ,# in whom an adequate (,5'-+ , #$/3 2$ course of treatment cannot course of treatment cannot be !$"-,<0+$#-,(2-0 confirmed. Monitor patients closely for signs . 2($,21"*-1$*7%-01(&,1 and ,#17+.2-+1-% symptoms of active TB "2(4$ during andand after treatment during after treatment with5(2' Taltz. *28 â&#x20AC;˘ Serious hypersensitivity 9$0(-31'7.$01$,1(2(4(27 reactions, including 0$ "2(-,1(,"*3#(,& anaphylaxis, angioedema, , .'7* 6(1 ,&(-$#$+  and ,#302(" urticaria,0(have ' been 4$!$$, reported in Taltz-treated 0$.-02$#(, *2820$ 2$# patients in clinical trials. . 2($,21(,"*(,(" *20( *1 â&#x20AC;˘ Caution should be be Â&#x2022; Caution should exercised in patients with $6$0"(1$#(,. 2($,215(2' inflammatory bowel disease, (,= ++ 2-07!-5$*#(1$ 1$ including Crohnâ&#x20AC;&#x2122;s disease including CrohnÂ&#x2019;s disease andand ulcerative colitis; ulcerative colitis; monitor patients who have 4$ +-,(2-0. 2($,215'-' inflammatory bowel disease. 1$ (,= ++ 2-07!-5$*#(1$ â&#x20AC;˘ Prior to initiating therapy, 90(-02-(,(2( 2(,&2'$0 .7 consider completion "-,1(#$0"-+.*$2(-, of all age -% ** appropriate &$ ..0-.0( 2$ immunizations; patients (++3,(8 2(-,1. 2($,21 treated with Taltz should notnot treated with Taltz should receive live vaccines. 0$"$(4$*(4$4 ""(,$1 â&#x20AC;˘ No clinical studies have Â&#x2022; No clinical studies have been conducted in pregnant ,2 !$$,"-,#3"2$#(,.0$&, women to establish safety%$27 5-+$,2-$12 !*(1'1 during pregnancy.,"7 #30(,&.0$&, â&#x20AC;˘ Caution should be exercised 9 32(-,1'-3*#!$$6$0"(1$# when administered to to when administered nursing women. ,301(,&5-+$, data are available â&#x20AC;˘ No Â&#x2022; No data are available on on thethe effect of Taltz on on effect of Taltz human fertility. '3+ ,%$02(*(27 â&#x20AC;˘ Safety and ,#$%%$"2(4$,$11(, effectiveness in 9 %$27 patients <18 years of01-% age &$ . 2($,21 7$ have not been evaluated. ' 4$,-2!$$,$4 *3 2$# â&#x20AC;˘ There is insufficient data 2  9'$0$(1(,13%<"($,2# to determine whether to determine whether patients â&#x2030;Ľ65 years age &$ . 2($,21; 7$ of01-% respond differently from 0$1.-,##(%%$0$,2*7%0-+ younger patients. 7-3,&$0. 2($,21 ForFor more information: more information: Please the product *$ consult 1$"-,13*22'$.0-#3"2 monograph at www.lilly.ca/ +-,-&0 .' 2555*(**7"  taltzpm/en for important ,2 2 *28.+$,%-0(+.-02 information relating to adverse information relating to adverse reactions, drug interactions, 0$ "2(-,1#03&(,2$0 "2(-,1 andand dosing information which dosing information which have not been discussed have not been discussed in this piece. The product (,2'(1.($"$'$.0-#3"2 monograph is also *1- available by +-,-&0 .'(1 4 (* !*$!7 calling us at 1-888-545-5972. " **(,&31 2     Reference: 1. Taltz Product Monograph.  *280-#3"2-,-&0 .' Lilly Reference: Canada Inc, 1. March 29, 2018. (**7 , # ," 0"'    TALTZ is a registered trademark owned by (1 #$+ 0)-5,$#!7 or licensed to 0$&(12$0$#20 Eli Lilly and Company, its -0*("$,1$#2-*((**7 subsidiaries or affiliates. ,#-+. ,7(21 13!1(#( 0($1-0 %<*( 2$1 Š 2018, Eli Lilly and Company. :  *((**7 All rights reserved. ,#-+. ,7 **0(&'210$1$04$# PP-IX-CA-0096 

6 ¡ September 2018

Acne: TLR2 significantly implicated in acne inflammation, may lead to new Tx targets Continued from page 4 Unfortunately, data released earlier this year revealed olumacostat glasaretil did not meet its co-primary endpoints in two double-blind, international Phase III trials to treat moderate-to-severe acne vulgaris, and a decision followed to discontinue development of the therapy. Antibiotic stewardship top of mind One of the factors that influences the choice of a therapy is antibiotic stewardship, said Dr. Maha Dutil, assistant professor of medicine at the University of Toronto. â&#x20AC;&#x153;I think one message has sunk in well: that neither oral nor topical antibiotics should be used as monotherapy for acne treatment in order to avoid increasing antimicrobial resistance,â&#x20AC;? Dr. Dutil told T HE C HRONICLE . â&#x20AC;&#x153;The challenge in some cases is in limiting the use of systemic antibiotics to three to four months in moderate-to-severe acne.â&#x20AC;? she said. â&#x20AC;&#x153;Another challenge is in increasing the use of topical retinoids in every case and early in treatment.â&#x20AC;? Pregnancy is another consideration for women who are looking for acne treatment. Dr. Humphrey suggests that women look to resources such as the Motherisk Program at Torontoâ&#x20AC;&#x2122;s Hospital for Sick Children, a program that supplies information about the risk and safety of medications during preg-

nancy and breastfeeding. â&#x20AC;&#x153;They have an FAQ/blog on topical treatments in pregnant women that can be used to guide therapeutic options in these patients,â&#x20AC;? said Dr. Humphrey. Skin type is another factor to consider in choosing an acne therapy for Canadian clinicians. â&#x20AC;&#x153;Patients with higher Fitzpatrick skin types will be prone to post-inflammatory hyperpigmentation, so through the course of treatment, that may be a consideration,â&#x20AC;? said Dr. Humphrey. â&#x20AC;&#x153;Azelaic acid may be used earlier in the algorithm for these patients to target acne and pigmentation. Patients with lighter phototypes will often have post-inflammatory erythema after treatment, and as such, may be candidates for vascular laser or IPL [intense pulse light] to speed the improvement of the erythema,â&#x20AC;? she said. Psychological issues common Dr. Dutil agreed that azelaic acid is a good option for patients who are prone to post-inflammatory hyperpigmentation and notes topical retinoids are another wise choice for such patients. â&#x20AC;&#x153;Topical retinoids manage the acne and can also help with the hyperpigmentation,â&#x20AC;? said Dr. Dutil. â&#x20AC;&#x153;It is crucial to give clear instructions to the patient on how to use the retinoid in order to avoid the irritation, which may itself lead to the hyperpigmentation you were trying to prevent.â&#x20AC;?

It is not uncommon for patients with acne to experience psychological conditions, noted Dr. Dutil. â&#x20AC;&#x153;Psychological comorbidities include anxiety, depression, attention-deficit disorder, attention deficit hyperactivity disorder, and insomnia,â&#x20AC;? she said. â&#x20AC;&#x153;I ask these patients to fill out the PHQ [Patient Health Questionnaire]-9, and that guides me as to whether they need referral to address these issues.â&#x20AC;? There are elevated rates of suicidal ideation among young women with acne compared to their peers without acne, independent of acne severity, Dr. Hanna said. â&#x20AC;&#x153;Even with mild acne, there is a commensurate rise in mood change,â&#x20AC;? he says Effective treatment of acne can result in improvement of psychosocial impacts, added Dr. Hanna â&#x20AC;&#x153;There are data to suggest that there is a reduction in mood change when you treat acne,â&#x20AC;? he said. Non-proprietary and brand names of therapies: tetramethyl-hexadecenylcysteine-formylprolinate (SIG1459) (not licensed in Canada); Ammonia Oxidizing Bacteria (AOB) (topical formulation of Nitrosomonas eutropha) (not licensed in Canada); isotretinoin (Accutane, Roche; Epuris, Cipher); olumacostat glasaretil (DRM01) (not licensed in Canada); azelaic acid (Finacea 15% gel, Bayer).

Research

Differences in AD severity seen in patients of African descent: Study report From the News Resources of The Chronicle via derm.city Atopic dermatitis (AD) appears to be more difficult to treat in patients of African descent, and a more personalized approach to medication dosing may be required. This is a conclusion of a paper published online ahead of print in Annals of Allergy, Asthma, & Immunology (Sept. 14, 2018). â&#x20AC;&#x153;Research shows about 19 per cent of African Americans and 16 per cent of European Americans are diagnosed with AD,â&#x20AC;? said senior author Emma GuttmanYassky, in a press release. â&#x20AC;&#x153;Our study found there are significant differences in the skin of people with AD than in those without the condition. Furthermore, we found African Americans with AD have more inflammation than European Americans with the condition.â&#x20AC;? Dr. Guttman-Yassky is a professor of dermatology, medicine, and clinical immunology, and vice chair of the department of dermatology at the Icahn School of Medicine at Mount Sinai Medical, New York. Investigators conducted genetic and immunohistochemistry studies in lesional and non-lesional skin of psoriasis patients of both African and European backgrounds, and in healthy controls. They found the AD lesions in patients of African descent showed a greater infiltration of dendritic cells compared to the lesions of patients with a European background. As

well, the lesions in patients with an African background featured decreased expression of innate immune markers, and T-helper (Th)1- and Th17-cell-related markers. AD clinical severity significantly correlated with Th2 and Th22-related products, as well as serum IgE, in the patients of African background. Fillagrin was only downregulated in AD patients with a European background. While loricrin was downregulated in both AD cohorts, it negatively correlated with clinical severity in the African-background patients. According to the release, molecular profiling of skin is being used to develop newer, more effective treatments for people with AD, yet to date only AD patients with a European background have been included in the research to develop this profiling technique. â&#x20AC;&#x153;This study looked for differences in the molecular profile of the skin of African Americans with AD compared to the skin of European Americans with AD to determine if there are differences that might improve treatment options for African Americans,â&#x20AC;? said Dr. Guttman-Yassky. â&#x20AC;&#x153;The results indicated that the immune profile was more unbalanced in African Americans with AD compared to European Americans.â&#x20AC;? The findings from this study could explain variances in the severity of AD in patients of African descent and differences in response to similar treatments.


A selective IL-17A inhibitor with binding affinity of <3 pM1*

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Taltz is indicated for the treatment of: â&#x20AC;˘ Adult patients with moderate-to-severe plaque psoriasis who are candidates for systemic therapy or phototherapy. â&#x20AC;˘ Adult patients with active psoriatic arthritis who have responded inadequately to, or are intolerant to one or more disease-modifying antirheumatic drugs (DMARD). Taltz can be used alone or in combination with a conventional disease-modifying antirheumatic drug (cDMARD) (e.g., methotrexate).

* Clinical significance has not been established.

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Relevant warnings g and p precautions: • Do not initiate treatment in patients with any clinically important active infections until infection resolves or is adequately treated • Discontinue treatment if patient develops a serious infection or is not responding to standard therapy for infection • Evaluate patients for tuberculosis infection prior to therapy and monitor for active tuberculosis during and after treatment • Consider completion of all immunizations prior to treatment • Concurrent use with live vaccines is not recommended • Women of childbearing potential should use adequate contraception • Use during pregnancy only if clearly needed • The benefits of breastfeeding should be considered along with the mother’s clinical need • Effect on human fertility has not been evaluated • Safety and efficacy in pediatric patients have not been evaluated • Data in patients ≥65 years of age are limited For more information: Please consult the Product Monograph at http://www.janssen.com/ canada/products#prod-966 for important information relating to adverse reactions, drug interactions, and dosing that has not been discussed in this piece. The Product Monograph is also available by calling 1-800-567-3331. DLQI=Dermatology Life Quality Index; IL=interleukin; NRI=non-responder imputation; PASI=Psoriasis Area Severity Index. † VOYAGE 1: A multicentre, randomized, double-blind, placebo- and active comparator–controlled phase 3 study in 837 adult patients with moderate to severe plaque psoriasis (body surface area involvement ≥10%, PASI score ≥12, Investigator’s Global Assessment ≥3) with or without psoriatic arthritis who were candidates for systemic therapy or phototherapy. Patients were randomized to receive subcutaneous injections of TREMFYA™ 100 mg at Weeks 0 and 4, then every 8 weeks (n=329); adalimumab 80 mg at Week 0, 40 mg at Week 1, then 40 mg every 2 weeks (n=334); or placebo at Weeks 0, 4 and 12 (n=174). At Week 16, patients receiving placebo crossed over to TREMFYA™ 100 mg at Weeks 16 and 20, then every 8 weeks.

References: 1. TREMFYA™ Product Monograph. Janssen Inc. November 10, 2017. 2. Blauvelt A, Papp KA, Griffiths CE, et al. J Am Acad Dermatol 2017;76(3):405-17.

Toronto, Ontario 19 Green Belt Drive | Toronto, Janssen Inc. | | M3C 11L9 L 9 | www www.janssen.com .janssen.com /canada | © 2018 Janssen www.janssen.com/canada All tr ademarks used under license. license. | TFBR170602E trademarks

8 · September 2018

Wounds and complications Continued from page 1

Factors affecting complications Factors that are surgical in nature that increase the likelihood of a wound complication include whether the surgery is emergency surgery (which may elevate the risk of contamination because of time pressures), whether implants are placed in the body (which may result in a reaction such as infection), extended length of surgery (which leaves the body vulnerable to contamination) or an error in technique (which can also result in increased blood loss), said Lewis. A patient’s characteristics can also contribute to increasing the potential for surgical wound complications. Those characteristics include the presence of comorbidities such

as diabetes, advanced age, elevated body mass index (BMI), the use of nicotine, and immunosuppression. “If a patient has an elevated BMI, there is increased tension on suture lines,” she said. “If a patient is immunocompromised, that patient will have a harder time healing.” Not only do oncology therapies cause a patient to be immunocompromised but other medications can also influence the immune system and how a patient heals after surgery, said Lewis. With the majority of surgeries performed on an outpatient basis, it is vital that members of the healthcare team work in a highly collaborative manner, for patients will be healing in their homes rather than in healthcare facilities, said Lewis. Part of that collaboration involves identifying the most effective form of communication to alert other members of the healthcare team, such as the surgeon, when something is amiss during the course of healing, she said. “The [healthcare] team needs to operate without walls,” said Lewis. “If we see that something is going south [with the patient], it is better to communicate sooner rather than later. We have to identify how the surgeon likes to receive information, whether it’s by phone or email. In this way, we are supporting the patient.” NPWT is a valuable therapeutic option over closed surgical incesions for patients who are at high risk for complications. This therapy bolsters and stabilizes the incision line and removes peri-wound fluid; this in turn can prevent dehiscence. NWPT is an intervention that can also address dehiscence and wound exudate, but NPWT cannot be employed in all surgical cases, stressed Lewis, citing cases such as unexplored fistulas, untreated imfection including osteomyelitis, as well as cases where there is malignancy. In addition, NPWT involves a financial cost and also requires a commitment on the part of the patient to adhere to using the therapy.

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three factors that should be top of mind for clinicians caring for patients with surgical wounds. That is according to Kerstin Lewis, RN, BSN, IIWCC, a wound care nurse at Island Health in Victoria, B.C., speaking at the Wounds Canada conference in Mississauga, Ont. “The two complications that we most often see are infection and dehiscence,” said Lewis, noting infections usually occur within 30 days post-operatively and dehiscence usually occurs anywhere from Kirsten Lewis four to 14 days following surgery. “There are other complications that include hematomas, seromas, and graft failures,” she said. A surgical wound is considered an intentional opening into the skin or any part of the integumentary system. Following surgery, a wound develops a healing ridge in the first few days that further extends to the entire incision line, gradually softening and flattening until the ridge resolves, typically a few weeks after surgery.

THERAPEUTIC INDEX

Celgene Otezla . . . . . . . . . . . . . . . . 17

Galderma TactuPump Forte . . . . . . . . . . . . 5

Janssen Tremfya . . . . . . . . . . . . . . . . . .9, 8 Lilly Taltz . . . . . . . . . . . . . . .7, 6

Pfizer Canada Inc. Eucrisa . . . . . . . . . . . .2

Procter & Gamble Head & Shoulders. . . . . . . . . . . 15 Olay . . . . . . . . . . . . . . . . . .27

Wounds Canada Meeting announcement . . . . . . . 10


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TREMFYA is the first and only fully human p19-subunit selective IL-23 inhibitor for the treatment of plaque psoriasis* TM

73%

POWERFUL LASTING EFFICACY DEMONSTRATED in moderate to severe plaque psoriasis

of patients achieved PASI 90 at Week 16 with TREMFYA vs. 3% with placebo (co-primary endpoint) and 50% with adalimumab (secondary endpoint) TM

(TREMFYA™ 241/329, 100 mg at Weeks 0 and 4, then every 8 weeks; placebo 5/174; adalimumab 166/334, 80 mg at Week 0, 40 mg at Week 1, then 40 mg every 2 weeks; p<0.001, NRI) 1,2†

76% of patients achieved PASI 90 at Week 48 with TREMFYA vs. 48% with adalimumab TM

(TREMFYA™ 251/329, 100 mg at Weeks 0 and 4, then every 8 weeks; adalimumab 160/334, 80 mg at Week 0, 40 mg at Week 1, then 40 mg every 2 weeks; p<0.001; secondary endpoint, NRI) 1,2†

-11.2 -11.2 mean change from baseline in DLQI at Week 16 for TREMFYA vs. -0.6 with placebo TM

(TREMFYA™ n=322, 100 mg at Weeks 0 and 4, then every 8 weeks; placebo n=170; p<0.001; secondary endpoint, NRI)

1,2†

At Week 16, patients receiving placebo crossed over to TREMFYA 100 mg at Weeks 16 and 20, then every 8 weeks. TM

TREMFYA (guselkumab) is indicated for the treatment of adult patients with moderate to severe plaque psoriasis who are candidates for systemic therapy or phototherapy. TM

* Comparative clinical significance unknown.

Toronto, Ontario | M3C 1L9 1L 9 | www.janssen.com/canada www.janssen.com /canada 19 Green Belt Drive | Toronto, Janssen Inc. | All trademarks trademarks used under license © 2018 Janssen license.. | TFBR170602E

Program Pr ogram


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THE CHRONICLE of S K I N & A L L E R G Y

10 · September 2018

Biologics must achieve at least PASI 75 in 90% of patients

Continued from page 1 U.S.-based TV channel. Clinicians should consider that a patient may be more adherent to a therapy if they have asked for it by name, since the patient may then have a greater sense of being part of the decision-making process in the selection of therapy, noted Dr. Vender. There is no one biologic that should be automatically considered over others, and factor such as frequency of dosing, whether monthly,

weekly, or every other week, to name a few regimens, are taken into consideration in making a therapeutic decision about the right biologic for the right patient, he said. “The best biologic is the one that works for the patient,” said Dr. Vender. “We are trying to pick the biologic that we think will work for the patient.” The goals for psoriasis clearance are being continually revised, with clinicians using

Psoriasis Area Severity Index (PASI) 75, 90, and even 100, to measure how effective a therapy is in getting patients “clear or almost clear,” according to Dr. Vender.

Response targets higher Any emerging therapy that does not meet PASI 75 in about 90% of patients is failing to produce impressive efficacy, said Dr. Vender. New therapies are expected to achieve PASI 90 in 60% of patients, and achieve PASI 100 in 40%, he added.

The American College of Rheumatology (ACR) has developed similar targets, ACR 20/50/70, for improvement in psoriatic arthritis, Dr. Vender noted. Some newer biologic agents, such as ixekizumab and guselkumab, are hitting those targets. When TNF inhibitors were studied in clinical trials, PASI 100 did not exist as a target endpoint to reach, said Dr. Vender. Another variable that

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influences the decision about therapy is how sustained the impact of any given therapy is, he said. “If you take a drug like secukinumab, we [dermatologists involved in clinical trials] noticed that you get drop off [in efficacy] over time,” explained Dr. Vender. “It means that you may have to dose optimize over time.” Considerations such as pregnancy influence the choice of a biologic therapy, explained Dr. Vender. A biologic like etanercept offers less transplacental transport than adalimumab or infliximab, making it a wise option for women contemplating pregnancy. “Etanercept is a drug that has good recapture and is a good choice during pregnancy,” said Dr. Vender, but noted etanercept is not very impressive in terms of clearance of psoriasis. Still another good option to consider for women who are contemplating pregnancy or who want to continue biologic treatment while they are pregnant is certolizumab pegol, a biologic that is pegylated and offers minimal placental transport. New Tx approved for psoriasis “It [certolizumab pegol] is approved for psoriasis,” said Dr. Vender, noting certolizumab pegol was approved for psoriatic arthritis previously. “It is a very good option for a patient with psoriasis who wants to get pregnant. It is not excreted into breastmilk.” In his own practice, Dr. Vender recommends that women stop taking a biologic therapy if they discover they are pregnant, with the exception of the biologics that are deemed safer to take during pregnancy. But he underlined all biologics, without exception, should be discontinued in the third trimester of pregnancy. Traditional therapies for psoriasis such as methotrexate, acitretin, and cyclosporine are still prescribed to treat psoriasis, and the argument for prescribing such therapies is largely an economic one, according to Dr. Vender.

Please turn to Biologics page 25à


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Vol. 20, No. 5

14 · September 2018

Surveying the current

Dermatologic literature Intralesional diprospan/topical timolol 0.5% for superficial infantile hemangiomas

Minimizing light intensity can reduce pain during PDT for AK

Suction blister grafts for vitiligo involving the angles of the lip

A

THE CHRONICLE of S K I N & A L L E R G Y

Recently come across something from the peer-review literature that you consider to be interesting or impactful? Share it with your colleagues. E-mail your clippings, along with your comments, to: health@chronicle.org

COMBINATION TREATMENT INVOLVING INTRALESIONAL INJECTION OF DIPROSPAN ALONG WITH A TOPICAL

application of timolol 0.5% cream, appears to be a suitable and safe strategy for thick superficial infantile hemangiomas (IHs), researchers report online ahead of print in Dermatologic Therapy (Apr. 30, 2018).

The authors note that topical application of timolol cream is known to be an effective and convenient option for the treatment of superficial IHs, and that intralesional injections of corticosteroids—such as diprospan—can be a useful way of treating superficial IHs while avoiding systemic side effects. To investigate whether combining intralesional diprospan injection and topical timolol 0.5% cream would be more efficient than timolol cream alone in treating thick superficial IHs, investigators recruited 38 patients with 39 thick superficial IHs. Each of the lesions was randomly divided into two equal parts, and then one part was treated with topical timolol 0.5% cream (timolol cream group), while the other part was treated with the combination treatment (combined treatment group). The infants were then followed every four weeks to determine whether the diprospan injections should be continued. Timolol cream was applied four times daily for five months. During the five-month treatment period, three specialist physicians were invited to evaluate the therapeutic effects. Overall, the combined treatment group had better lesion involution than the timolol cream group with regard to lesion thickness and colour. —For more information visit https://goo.gl/9nk2fJ

(PDT), APPEARS TO reduce pain while still allowing for a high clearance rate of actinic keratoses (AKs), investigators report online ahead of print in Photodermatology, Photoimmunology & Photomedicine (May 12, 2018).

M

INIMIZING LIGHT INTENSITY DURING THE ILLUMINATION PHASE OF PHOTODYNAMIC THERAPY

Noting that while PDT can be used to treat large fields of AK with high clearance rates, the authors say that one of the challenges with PDT for AK is the pain experienced by patients during illumination. In an attempt to address this, researchers set out to optimize the illumination protocol during conventional PDT in order to reduce pain without compromising treatment effectiveness. Investigators from the University of Gothenburg in Gothenburg, Sweden conducted a prospective, randomized study in which 29 patients with a total of 399 symmetrically-distributed AKs were included. In the split-face study, all patients were treated using an ALA 78 mg/g gel. One side of each subject’s face was illuminated with the Aktilite CL‐128 lamp and the other side with the RhodoLED lamp in which the light intensity gradually increased to a maxi2 mum of 60%. Both sides of the face received a total light dose of 37 J/cm . Investigators measured pain during the treatment using a visual analogue scale (VAS), and the clinical effectiveness of the two treated sides was assessed after 12 weeks. The authors report that the study showed that illumination with the gradually increasing light intensity resulted in a decrease of the median VAS score by 1.1 points. Clearance rates were similar between the two lamps, the researchers noted. —For more information visit https://goo.gl/gbypRZ

S

(SBG) IS A COST-EFFECTIVE APPROACH FOR REPIGMENTATION IN VITILIGO involving the angles of the lip, with long-lasting results, researchers report in the Journal of Cutaneous and Aesthetic Surgery (Jan.-Mar. 2018; 11(1):13-19). UCTION BLISTER EPIDERMAL GRAFTING

The authors note that lip vitiligo is frequently resistant to medical modalities of treatment due to the lack of hair follicles and the associated pigment cell reservoir for recolonization. In this area, surgery is an alternative with some evidence. In particular, SBG has been used for many years with a high rate of success. However, the authors explain there have been no long-term follow-up studies of patients with SBG at a difficult-to-treat site such as the angles of the lip. To assess the pigmentation rate and patient satisfaction with SBG on vitiligo involving the angles of the lip, investigators conducted a prospective study that recruited 112 patients with stable vitiligo involving the angles of the lip recruited from a single outpatient department of a tertiary care centre between Aug. 2010 and July 2013. SBG was carried out in all the patients using the standard procedure. Patients were advised to apply topical psoralen followed by sun exposure (PUVASOL) for eight to 12 weeks after their operation. The patients were followed up at 3, 6, 12, 18, and 24 months post-treatment for assessment of pigmentation and overall satisfaction. The authors observed a pigmentation success rate of 83.7%, 84.9%, 85.7%, 78.3%, and 77.8% in the patients who were followed up at 3, 6, 12, 18, and 24 months, respectively. A total of 77.8% of patients who came for follow-up at the end of 24 months were reported to be very happy with the treatment. —For more information visit https://goo.gl/hyC81B


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THE CHRONICLE of S K I N & A L L E R G Y

16 · September 2018

Vender on psoriasis Research

 Assessing clinical advances in Pso care and research FASTEST ONSET OF ACTION SEEN WITH BRODALUMAB FOR MODERATE TO SEVERE PSO

and legs. As well, both ethnic and regional differences in response have been reported. With all of the biologics evaluated, clinically meaningful outcomes were achieved within two to 11 weeks. Broken down by medication, the Brodalumab may have the fastest onset of accalculated times for 25% of patients to achieve PASI 75 were 2.1 [95% contion of any current biological therapy for psofidence interval (CI) 2.0–2.3] riaiss, researchers report in Journal of Drugs weeks with brodalumab, 2.4 in Dermatology (Mar. 2018; 17(3):247–250). Comment: This is an important analysis, however its true clinical with ixekizumab, 3.0 with high Drs. Kim A. Papp and Mark Lebwohl relevance is minimal. A more meaningful analysis is the number dose secukinumab, 3.5 with inconducted a review of the published and of weeks for 50% of the patients to achieve a 50% response—in fliximab, 4.6 with adalimumab presented efficacy data on adalimumab, other words a mean change in baseline PASI scores. and high-dose ustekinumab, 5.1 infliximab, ustekinumab, etanercept, broThis study reveals that brodalumab and ixekizumab with low-dose ustekinumab, 6.6 dalumab, ixekizumab, and secukinumab are virtually the same. This allows the clinician to explain with high-dose etanercept, and in order to estimate how long each took to to their patients that on average they could expect half of 9.5 with low-dose etanercept. achieve clinically meaningful outcomes— their psoriasis to be gone in two weeks with brodalumab The calculated times for a 50% which they defined as the time for 25% of and ixekizumab. Secukinumab is simply one week later. All reduction in baseline PASI patients to achieve either Psoriasis Area of these values are a great improvement over the 1st genscores were 1.8 [95% CI 1.7–1.9] and Severity Index (PASI) 75 or a 50% reeration subcutaneous biologics. weeks with brodalumab, 1.9 duction in mean baseline PASI. The auwith ixekizumab, 3.0 [95% CI thors note that onset of action has not been used as a primary or secondary end2.8–3.2] with secukinumab, 3.5 point in any clinical trial. They also point out with adalimumab, 3.7 with inflixthat different body regions may provide difimab, 5.1 with low-dose ustekfering responses. The head/neck and trunk inumab, 6.5 with high-dose have been shown to respond more quickly than psoriasis of the arms etanercept, and 10.9 with low-dose etanercept. We invite your comments and questions about this feature at www.derm.city. Dr. Ron Vender is a certified dermatologist with 27 years of clinical practice experience and over 75 clinical trials in psoriasis. He is founder and director of Venderm Innovations in Psoriasis, a center of excellence for Psoriasis offering a comprehensive management solution for individuals with psoriasis. www.psoriasis.vip

Have a comment about any of the articles in this issue of The Chronicle?

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Share your knowledge and opinions with worldwide peers. Visit www.

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have been treated with OTEZLA globally to date, combined across both psoriasis and psoriatic arthritis indications2


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THE CHRONICLE of S K I N & A L L E R G Y

18 ¡ September 2018

FFA incidence on rise, but evidence for therapeutics unclear

Continued from page 1 clinician to say: â&#x20AC;&#x2DC;here are all the treatment options, this is the level of evidence or the strength of the evidence, and here are the response rates,â&#x20AC;&#x2122; and help people make more informed choices for treatment.â&#x20AC;? FFA is very resistant to treatment, she said. The researchers conducted a literature search of both PubMed and Cochrane databases, the former starting from 1946, the latter from

1991. All English-language retrospective and prospective studies were included, though only those case studies with a reported treatment regimen and outcome were included. No randomized, controlled trials were discovered in the search. They found there was an increasing incidence of FFA, mostly in postmenopausal women. This larger number of cases had contributed to the clinical and histological under-

standing of the condition.

Ranking treatment agents However, there were reported results from different treatment methods, including intralesional or oral corticosteroids, anti-androgens, antimalarials, antibiotics, and surgery. At the time of publication, no randomized controlled trials for FFA treatment had been conducted. â&#x20AC;&#x153;Despite the fact that we do not have great quality evidence, we tried to rank [treat-

ment agents],â&#x20AC;? she said. In the paper, therapeutic options were clustered by treatment type and overall response. â&#x20AC;&#x153;What people can take away overall is it seems that intralesional triamcinolone injections seem to have the most data for high efficacy,â&#x20AC;? said Dr. Beecker. â&#x20AC;&#x153;There are several different trials that show good results, and the overall response rate seems to be at least 50 per cent, and in some of the studies it is even

   

      

  

                   

      

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higher. There have been studies that showed 97 per cent reduction in symptoms and arrested hairline, but on average it was around 56 per cent.â&#x20AC;? Though not as solid as randomized clinical trial data would be, these observations are helpful for making treatment decisions from both a patient and clinicianâ&#x20AC;&#x2122;s perspective, Dr. Beecker said. Patients will be more likely to take the time for the multiple injection visits needed if they have some confidence the therapy will help their hair loss. From a clinicianâ&#x20AC;&#x2122;s point of view, the observations that arrest of hairline recession typically occurs in three to six months provides a timeline for trialling the medication in a patient, she said.

Evidence for Tx assessed Anti-androgens are another promising option, said Dr. Beecker, and one many practitioners are becoming familiar with. â&#x20AC;&#x153;I think it is good to see that although the numbers in the studies were small, the efficacy was quite high againâ&#x20AC;&#x201D; roughly around 50 per cent.â&#x20AC;? Other treatment approaches commonly used in other forms of hair loss, such as antimalarials and doxycycline, have less evidence and seem to have less efficacy in FFA, she said. While there is, overall, poor evidence for any treatment for FFA, the data that is available can be used to create an order of response rateâ&#x20AC;&#x201D;an algorithm for which treatments to try, said Dr. Beecker. â&#x20AC;&#x153;Maybe starting with intralesional, perhaps combining with an anti-androgen, and then going on to other therapies from there, based on the evidence. I still think it gives a framework for clinicians to at least start with.â&#x20AC;? Dr. Beecker emphasizes that one thing that is reasonably clear from the data is that patients with FFA achieve better outcomes on combined therapies, rather than on monotherapy. A second take-away from the study that deserves emphasis, she said, â&#x20AC;&#x153;would be to just not discount other options including camouflageâ&#x20AC;&#x201D;such as eyebrow tattooing, hairpieces or wigs. Hair transplants seemed less successful, but [it is helpful] to think of those other options.â&#x20AC;?


POSTGRADUATE EDUCATIONAL SUPPLEMENT Chronicle

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Granzyme B is elevated in autoimmune blistering diseases and cleaves key anchoring proteins of the dermal-epidermal junction

Valerio Russo, Theo Klein, Darielle J. Lim, Nestor Solis,4,5 Yoan Machado,4,5 Sho Hiroyasu,1,2,3 Layla Nabai,,3 Yue Shen,1,2,3 Matthew R. Zeglinski,1,2,3 Hongyan Zhao,1,2,3 Cameron P. Oram,1,2,3 Peter A. Lennox,6 Nancy Van Laeken,6 Nick J. Carr,6 Richard I. Crawford,2,7 Claus-Werner Franzke,8 Christopher M. Overall,4,5 and David J. Granville1,2,3* 1,2,3

4,5

3

from 1International Collaboration On Repair Discoveries (ICORD) Research Centre, Vancouver, Canada; 2Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver; 3BC Professional Firefighters’ Burn and Wound Healing Research Laboratory, Vancouver; 4Centre for Blood Research, University of British Columbia, Vancouver; 5Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver; 6Department of Surgery, University of British Columbia, Vancouver; 7Department of Dermatology and Skin Science, University of British Columbia, Vancouver; 8Department of Dermatology, Medical Center and Faculty of Medicine—University of Freiburg, Germany; *Corresponding Author

ABSTRACT

In healthy skin, epidermis and dermis are anchored together at the dermal-epidermal junction (DEJ), a specialized basement membrane pivotal for skin integrity and function. However, increased inflammation in the DEJ is associated with the disruption and separation of this junction and sub-epidermal blistering. Granzyme B (GzmB) is a serine protease secreted by immune cells. Dysregulated inflammation may lead to increased GzmB accumulation and proteolysis in the extracellular milieu. Although elevated GzmB is observed at the level of the DEJ in inflammatory and blistering skin conditions, the present study is the first to explore GzmB in the context of DEJ degradation in autoimmune sub-epidermal blistering. In the present study, GzmB induced separation of the DEJ in healthy human skin. Subsequently, α6/β4 integrin, collagen VII, and collagen XVII were identified as extracellular substrates for GzmB through western blot, and specific cleavage sites were identified by mass spectrometry. In human bullous pemphigoid, dermatitis herpetiformis, and epidermolysis bullosa acquisita, GzmB was elevated at the DEJ when compared to healthy samples, while α6/β4 integrin, collagen VII, and collagen XVII were reduced or absent in the area of blistering. In summary, our results suggest that regardless of the initial causation of sub-epidermal blistering, GzmB activity is a common final pathway that could be amenable to a single targeted treatment approach.

B

INTRODUCTION

listering is a hallmark of many dermatological conditions, and can manifest itself with varying degrees of severity, but is typically characterized by erosions or fluid-filled elevations from the skin surface caused by disruption of the cell to

cell attachment in different layers of the epidermis, or detachment of the epidermis from dermis. Due to the critical role that skin plays as a barrier in regulating fluid/electrolyte retention, thermoregulation, and protection against infection, depending on the size and severity of blistering, such functions can be compromised and potentially fatal.1 Based

Reprinted with permission from: Scientific Reports June 26, 2018; 8:9690 © 2018 the authors. Published by Nature. Distributed under the terms of the Creative Commons Attribution License https://www.nature.com/articles/s41598-018-28070-0/ Content may have been edited to conform with the Canadian Press Publication Style Guide.

on the etiology, these dermatoses are generally classified in four major groups: (a) antibody-mediated, (b) cutaneous adverse drug reactions, (c) congenital conditions, and (d) blistering caused by external insults such as burns, friction, sunlight, insect bites, and chemical weapons. With respect to autoimmune skin blistering diseases, auto-antibodies are produced against structural or adhesive molecules of the skin and based on the location of the specific auto-antigens and level of the blister formation, these diseases are further classified into intraepidermal and sub-epidermal blistering diseases. In sub-epidermal blistering dermatoses such as bullous pemphigoid, dermatitis herpetiformis and epidermolysis bullosa acquisita (EBA), auto-antibodies targeting components of the dermal-epidermal junction (DEJ) lead to the disruption of this basement membrane and consequent detachment of the epidermis.2 Granzyme B (GzmB) is a serine protease widely known for its proapoptotic role in cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-mediated killing of target cells whereby the pore-forming protein perforin is secreted along with GzmB and facilitates its entry into target cells. 3 However, in recent years it has become clear that GzmB accumulation in the extracellular space can contribute to other pathological processes. Indeed, the directed secretion of GzmB from the effector cell toward the target cell is not efficient, resulting in leakage into the extracellular milieu.4 Furthermore, it is now recognized that other immune and nonimmune cell types, that do not express perforin and/or form immunological synapses such as plasmacytoid dendritic cells, B cells, mast cells, and keratinocytes may also express and secrete GzmB under certain conditions (reviewed in5). As such, the extracellular function of GzmB has received much attention in recent years as its role in the onset of several inflammatory conditions continues to be revealed. GzmB is capable of cleaving cell receptors, cellular adhesion proteins, cytokines and important extracellular matrix (ECM) proteins, thus affecting tissue structure and function.6,7,8 Of particular relevance to autoimmune skin blistering, GzmB accumulation at the DEJ has been reported by previous studies in bullous diseases and cutaneous adverse drug reactions.9,10,11 However, with respect to mechanism of action in skin diseases, GzmB has been viewed almost exclusively in the context of CTL/NK-mediated keratinocyte apoptosis12,13,14,15 while the recently recognized role of extracellular GzmB proteolysis 5,8,16 has not been considered. As GzmB accumulates on both sides of the DEJ, including the dermis which is devoid of keratinocytes, and given the established potential of this enzyme to cleave multiple ECM proteins, we hypothesized that GzmB

compromises DEJ integrity and function through cleavage of key basement membrane components α6/β4 integrin, collagen VII, and collagen XVII, thus directly contributing to epidermal detachment and blistering through extracellular mechanisms. Using in vitro cleavage assays and amino-terminal oriented mass spectrometry (ATOMS)17,18 on purified proteins and intact human skin, we provide evidence that GzmB cleaves important proteins of the DEJ and induces epidermal detachment of healthy skin. This work demonstrates for the first time a role for extracellular GzmB in the blistering process that goes beyond a cytotoxic effect on basal keratinocytes.

RESULTS

GzmB accumulates at the level of the DEJ in bullous pemphigoid, dermatitis herpetiformis, and EBA Immunohistochemistry of patient skin samples from bullous pemphigoid, dermatitis herpetiformis, and EBA indicated that GzmB accumulated at the DEJ. Specifically, H&E staining of bullous pemphigoid revealed sub-epidermal blistering with dense inflammatory infiltrate consisting predominantly of eosinophils and neutrophils (Fig. 1a, b). Intense GzmB staining was observed at the level of the DEJ in most neutrophils but not in eosinophils as expected, both within the blister and immediately below the detached epidermal layer (Fig. 1b). Dermatitis herpetiformis was characterized by pathognomonic subepidermal clefts and papillary abscesses, consisting mostly of neutrophils and a few eosinophils, at the tips of dermal papillae (Fig. 1a,b). These papillary abscesses were heavily stained for GzmB suggesting neutrophil and lymphocyte involvement in its secretion (Fig. 1b). GzmB presence was predominantly observed in the upper papillary dermis adjacent to the DEJ, but positive cells could also be detected embedded within the epidermal layer. EBA skin displayed epidermal detachment with abundant immune infiltrate, mostly composed of neutrophils and lymphocytes, in the interstitial space between the separated epidermis and the dermis (Fig. 1a, b). Similar to what was observed in bullous pemphigoid and dermatitis herpetiformis, GzmB was found predominantly in neutrophils (Fig. 1b). GzmB cleaves α6 and β4 integrins in vitro in domains pivotal for their function Once we ascertained GzmB presence at the level of the DEJ in diseased skin, we hypothesized that GzmB mediates cleavage of α6/β4 integrin, collagen VII, and collagen XVII, which are important components of the basement membrane critical for DEJ function. Both α6 and β4 integrin sub-units were cleaved by GzmB (Fig. 2a, b); α6 integrin was


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POSTGRADUATE EDUCATIONAL SUPPLEMENT

Figure 1: GzmB levels are elevated in the DEJ of sub-epidermal blistering diseases. (a) In the upper row, representative images of H&E staining of healthy skin, bullous pemphigoid (BP), dermatitis herpetiformis (DH), and epidermolysis bullosa acquisita (EBA). Dotted lines delineate blisters. In the lower row, GzmB immunostaining of healthy, bullous pemphigoid (BP), dermatitis herpetiformis (DH), and epidermolysis bullosa acquisita (EBA) biopsies. Abundant GzmB is observed at the level of the dermal-epidermal junction in diseased skin particularly in areas of epidermal separation (black arrowheads). Dotted lines indicate separation between the epidermis and the dermis. Scale bars represent 200 μm. (b) In the upper row, representative images of H&E staining of, bullous pemphigoid (BP), dermatitis herpetiformis (DH), and epidermolysis bullosa acquisita (EBA). In lower row, GzmB staining for the same tissue sections. In all conditions studied GzmB co-localizes with neutrophils (red circles). Scale bars represent 40 μm. detected at 150 kDa with fragments at ~20, 25, and 37 kDa, whereas cleavage of β4 integrin ectodomain (100 kDa) yielded an evident fragment at ~65 kDa and a weaker band at 50 kDa. To confirm that cleavage of these DEJ components was indeed mediated by GzmB, compound 20, a GzmB-specific competitive inhibitor, and serpin A3N, an irreversible serine protease inhibitor, were included in the cleavage assay. Both inhibitors prevented the cleavage of α6 and β4 integrin subunits at 100 μM and 600 nM respectively (Fig. 2a, b). Furthermore, mass spectrometry by ATOMS was used to identify cleavage sites on these proteins to assess whether GzmB-mediated cleavage of α6 and β4 integrins could impair DEJ function. We focussed on the extracellular domains of α6 and β4 integrins as this protein region is more likely to be exposed to GzmB. α6 integrin was cleaved by GzmB at Asp100, Asp166, Asp199, Asp302, Asp311, Asp358, Asp482, and Asp488 in the FGGAP repeats 2, 3, 4, 5, and 7 within the extracellular β-propeller domain (Fig. 3a and Supplemental Fig. S1), a cleavage site at Glu856 was also detected. Cleavage of the β4 integrin sub-unit fell within the Von Willebrand factor A domain at Glu223, Asp237 and Asp272, as well as within the Cysteine Rich Region 1 at Asp611, and within the linker region between these two domains at Asp351, Asp442 and Asp447 (Fig. 3b and Supplemental Fig. S2). GzmB cleaves collagen VII in ligand binding regions Once we ascertained GzmB presence at the level of the DEJ in diseased skin, we hypothesized that GzmB mediates

cleavage of α6/β4 integrin, collagen VII, and collagen XVII, which are important components of the basement membrane critical for DEJ function. Both α6 and β4 integrin sub-units were cleaved by GzmB (Fig. 2a, b); α6 integrin was detected at 150 kDa with fragments at ~20, 25, and 37 kDa, whereas cleavage of β4 integrin ectodomain (100 kDa) yielded an evident fragment at ~65 kDa and a weaker band at 50 kDa. To confirm that cleavage of these DEJ components was indeed mediated by GzmB, compound 20, a GzmB-specific competitive inhibitor, and serpin A3N, an irreversible serine protease inhibitor, were included in the cleavage assay. Both inhibitors prevented the cleavage of α6 and β4 integrin sub-units at 100 μM and 600 nM respectively (Fig. 2a, b). Furthermore, mass spectrometry by ATOMS was used to identify cleavage sites on these proteins to assess whether GzmB-mediated cleavage of α6 and β4 integrins could impair DEJ function. We focussed on the extracellular domains of α6 and β4 integrins as this protein region is more likely to be exposed to GzmB. α6 integrin was cleaved by GzmB at Asp100, Asp166, Asp199, Asp302, Asp311, Asp358, Asp482, and Asp488 in the FG-GAP repeats 2, 3, 4, 5, and 7 within the extracellular β-propeller domain (Fig. 3a and Supplemental Fig. S1), a cleavage site at Glu856 was also detected. Cleavage of the β4 integrin sub-unit fell within the Von Willebrand factor A domain at Glu223, Asp237 and Asp272, as well as within the Cysteine Rich Region 1 at Asp611, and within the linker region between these two domains at Asp351, Asp442 and Asp447 (Fig. 3b and Supplemental Fig. S2).

Figure 2: GzmB Cleavage assays on dermal-epidermal junction proteins. (a) 4– 20% SDS-PAGE western blot of GzmB-mediated cleavage of α6 integrin (α6 int) sub-unit with and without inhibitors serpin A3N (SA3N) and compound 20 (Com20). Black arrows indicate cleavage fragments and * indicates full-length proteins. At a concentration of 200 nM GzmB produces cleavage bands, and this cleavage is prevented by GzmB inhibitors. Full-length blot is presented in Supplemental Fig. S5. (b) 4–20% SDS-PAGE western blot of GzmB-mediated cleavage of β4 integrin sub-unit (β4 int) with and without inhibitors SA3N and Com20. Black arrows indicate cleavage fragments and * indicates full-length proteins. At a concentration of 200 nM GzmB produces cleavage bands, but its inhibition prevents the appearance of these bands. Full-length blot is presented in Supplemental Fig. S6. (c) 10% SDS-PAGE western blot of GzmB-mediated cleavage of collagen VII (coll VII) with and without inhibitors SA3N and Com20. Black arrows indicate cleavage fragments and * indicates full-length proteins. GzmB produces cleavage bands, and this cleavage is reduced by the addition of Com20 and abolished by SA3N. Full-length blot is presented in Supplemental Fig. S7. (d) 8% SDS-PAGE western blot of GzmB-mediated cleavage of collagen XVII (coll XVII) with and without inhibitors SA3N and Com20. Black arrow indicates cleavage fragment and * indicates full-length protein. GzmB produces a cleavage band, and this cleavage is abolished by the addition of Com20 or SA3N. Full-length blot is presented in Supplemental Fig. S8. GzmB cleaves collagen VII in ligand binding regions

Collagen XVII is a substrate for GzmB cleavage

Western blot was used to assess cleavage of collagen VII domain a.a 199–482 by GzmB. This fragment is part of the non collagenous region 1 (NC1), which is pivotal for collagen VII interactions with other proteins of the ECM19,20. Untreated collagen VII fragment was detected at ~30 kDa, and its cleavage by GzmB yielded bands at ~20 and 25 kDa (Fig. 2c). On the other hand, collagen I, the most common collagen in the human body, was not cleaved by GzmB (Supplemental Fig. S3). Inhibition of collagen VII cleavage with serpin A3N prevented the appearance of both ~20 and 25 kDa bands, whereas compound 20 inhibition was incomplete and a cleavage band could still be detected at ~25 kDa (Fig. 2c). As for the location of cleavage, the NC1 fragment of collagen VII we tested was cleaved by GzmB in the Von Willebrand factor A domain at Asp193, in the fibronectin-like domain III-2 at Glu332 and Asp390, and within fibronectin-like domain III-3 at Asp414 (Fig. 3c and Supplemental Fig. S4).

As a crucial component of the hemidesmosomes, collagen XVII plays a critical role in bridging the intracellular and the extracellular structural elements involved in epidermal adhesion21. Treatment of collagen XVII ectodomain (a.a 490–1497) with GzmB resulted in cleavage of this region, and in the appearance of a cleavage band at ~95 kDa (full length protein ~120 kDa). Preincubation of GzmB with both compound 20 and serpin A3N prevented NC16 cleavage (Fig. 2d). ATOMS was attempted on collagen XVII using 1 or 2 µg of protein. GzmB-digested and undigested forms of collagen XVII were compared by labelling exposed N-termini with heavy (13CD2O formaldehyde) and light (12CH2O formaldehyde) dimethylated tags respectively. However, detection of N-terminal peptides was confounded due to ion suppression by more intense ions from other contaminating proteins in the sample. Furthermore, agglutination of collagen XVII was observed after dimethylation, which likely decreased the efficiency of trypsin digestion. For these reasons, we were unable to obtain cleavage site data for this protein.


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Figure 3: Mass spectrometry analysis on GzmB-cleaved DEJ substrates. (a) Extracellular domain schematics for α6 integrin. GzmB mediated cleavage sites identified proteomically by ATOMs (red arrows) fall within the ligand-binding domains in the β-propeller region. GzmB, granzyme B; TM, transmembrane helix. (b) Extracellular domain schematics for β4 integrin. GzmB mediated cleavage sites identified proteomically by ATOMS (red arrows) falls within ligand-binding domains in the specificity-determining loop. (c) Extracellular domain schematics for collagen VII. GzmB mediated cleavage sites identified proteomically by ATOMS (red arrows) falls in the von Willebrand factor A and fibronectin type III-2 domains, which mediate collagen VII attachment to other dermal-epidermal junction components, such as laminins and collagen IV. TM, transmembrane helix; PSI, plexin-semaphorin-integrin; VWFA, von Willebrand factor A; CRR, cysteine-rich region; FNIII, fibronectin-like III domain; NC, non collagenous region; CMP, cartilage matrix protein; VWFA1/2, von Willebrand factor A 1/2; Pi, protein inhibitor. α6/β4 integrin, collagen VII and collagen XVII lining at the DEJ are disrupted in diseased skin Following Western blot and ATOMS analyses indicating that both α6 and β4 integrin sub-units, collagen VII and collagen XVII are substrates for GzmB, we sought to assess their integrity in bullous pemphigoid, dermatitis herpetiformis, and EBA skin samples. Staining for α6 integrin in normal skin was mainly localized perivascularly in the dermis and as a continuous line at the DEJ. When diseased skin samples were investigated, the pattern of α6 integrin localization was similar for all conditions, exhibiting scattered staining throughout the entire sections, which could be indicative of protein fragmentation (Fig. 4). At the DEJ, staining was absent, weak, or disorganized both in areas of epidermal detachment and in sections where the epidermis was still attached to the dermis (Fig. 4). As for integrin β4, a strong well localized staining delineated the DEJ in healthy skin, as well as in bullous pemphigoid and dermatitis herpetiformis in areas where the epidermis was anchored to the dermis (Fig. 4). However, integrin β4 staining was completely absent or faint in areas of epidermal separation in all conditions studied (Fig. 4). Mooney, et al have previously shown fragmented collagen VII in the DEJ of patients with discoid lupus erythematosus.22 While normal skin displayed a continuous, strong staining for

collagen VII lining the interface between epidermis and dermis (Fig. 4), in bullous pemphigoid and dermatitis herpetiformis collagen VII staining was weak or absent. Weak staining in both diseases was localized on the dermal side of a blister or papillary abscess, consistent with a separation of the fibrillar zone of the DEJ from the lamina densa above it due to cleavage of collagen VII in the NC1 (Fig. 4). In dermatitis herpetiformis, collagen VII staining presented a peculiar pattern, with short stained sections perpendicular to the epidermis rather than parallel to it. Collagen VII staining of EBA samples showed an intermittent pattern, with DEJ segments presenting weak staining alternated by areas with a stronger staining (Fig. 4). Finally, collagen XVII staining pattern for all conditions was similar to what was observed for the other substrates. A clear, uninterrupted line of staining was detected in healthy skin, whereas in diseased skin weak staining was observed in areas of reduced DEJ integrity and was mostly absent in the sections of skin where the epidermis had detached (Fig. 4). Incubation with GzmB results in epidermal separation in healthy human skin and is inhibited by Compound 20 As GzmB is elevated at the DEJ of bullous pemphigoid, dermatitis herpetiformis, and EBA, and capable of cleaving key junctional proteins, the direct impact of GzmB proteolysis on DEJ

Figure 4: Immunohistochemistry of DEJ components in healthy and diseased skin biopsies. α6 and β4 integrin, collagen VII, and collagen XVII immunostaining of healthy, bullous pemphigoid (BP), dermatitis herpetiformis (DH), and epidermolysis bullosa acquisita (EBA) biopsies. Blue arrowheads indicate intact protein in areas of dermo-epidermal adhesion, black arrowheads indicate weak or absent staining. Dotted lines indicate separation between the epidermis and the dermis. Scale bars represent 200 μm. α6 integrin is fragmented and scattered throughout the dermis in diseased skin, whereas in healthy biopsies this protein lines the DEJ. β4 integrin appears to be crucial for adhesion: in the bullous pemphigoid sample, a flap of dermis in the lower right corner is attached to the epidermis and shows strong β4 staining; this area is flanked by separated epidermis with faint β4 integrin staining. Scale bars represent 200 μm. Collagen VII lining is intact (blue arrowheads) in healthy skin, but weak or absent immunoreactivity was observed in diseased samples (black arrowheads). Collagen XVII lining is intact (blue arrowheads) in healthy skin, but weak or absent immunoreactivity was observed in diseased samples (black arrowheads).

Figure 5: GzmB induces DEJ separation. H&E staining of healthy skin incubated for 12 h at 37 °C in PBS, 200 nM GzmB, or 200 nM GzmB previously inactivated with 100 μM Compound 20 (Com20). Clefts between the epidermis and the dermis (black arrowheads) were observed in GzmB-treated samples but were absent in PBS control and in the sample where GzmB was inhibited by Com20. Scale bars represent 200 μm. integrity in freshly-isolated human skin was assessed. A ~1 mm × 4 mm strip of healthy skin comprising epidermis and dermis was immersed and incubated for 12 h at 37 °C in a 200 nM solution of GzmB. Upon incubation, the skin was viable but H&E staining revealed the appearance of clefts between the epidermis and the dermis in the GzmBtreated sample that were mostly absent in the PBS control (Fig. 5 and Supplemental Fig. S9). Inactivation of GzmB with the GzmB-specific inhibitor Compound 20 prevented the formation of DEJ clefts, revealing a tissue morphology similar to PBS control (Fig. 5).

DISCUSSION

It is now widely acknowledged in the literature that extracellular GzmB exerts a pathogenic, perforin-independent, role in conditions associated with dysregulated and/or chronic inflammation and impaired tissue repair due to ECM cleavage.7,16,23,24,25 In the present study, we used western blot and ATOMS to show for the first time that α6/β4 integrin and collagen VII, which are key components of the

DEJ, are cleaved by GzmB in key regions for their anchoring functions. We also demonstrated that in diseased human skin biopsies of bullous pemphigoid, dermatitis herpetiformis and EBA, sub-epidermal blisters display elevated levels of GzmB at the DEJ accompanied by degradation of the newly discovered GzmB substrates α6/β4 integrin, collagen VII, and collagen XVII. Supporting the pathological relevance of GzmB activity, we reported partial detachment of the epidermis from the dermis in healthy human skin exposed to a physiologically relevant concentration of GzmB,26,27 and showed that this separation can be prevented by a GzmBspecific inhibitor. These data suggest that, in addition to previously studied proteases,28 extracellular GzmB could also contribute to epidermal separation in autoimmune sub-epidermal blistering pathologies. Evidence for the importance of α6/β4 integrin comes from both animal models29 and from severe, often lethal, human blistering diseases. Mutations and deficiencies of the integrin complex


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POSTGRADUATE EDUCATIONAL SUPPLEMENT involving α6/β4, as well as production of anti-α6/β4 integrin autoantibodies,30,31 contribute to lethal phenotypes characterized by widespread muco-cutaneous blistering. Several studies have identified mutations in the genes coding for α6/β4 integrin32,33,34 or absence at the protein level of one of its sub-units,35 in numerous subtypes of epidermolysis bullosa. In particular, Phillips, et al speculated that in situ proteolytic cleavage of the epitopes by an as yet unidentified protease, might be responsible for the loss of β4 subunit immunoreactivity in patients with junctional epidermolysis bullosa.36 In this study we show that GzmB cleaves both α6 and β4 integrin sub-units at several sites in their ligand-binding extracellular domains,37,38,39,40 possibly compromising the adhesive properties of this molecule. The importance of these extracellular regions is particularly evident for integrin β4 as collectively, most of the missense mutations and the amino acid deletions described in lethal junctional epidermolysis bullosa were located in its extracellular domain, 32,41,42,43 while missense or splice mutations associated with the non-lethal form were frequently located in the cytoplasmic portion.41,44,45 Interestingly, Nakano et al. identified the lethal mutation p.D131Y/p.G273D, which may abolish important ligand binding sites of integrin β4 as it falls within a highly conserved region. 41 Since one of the GzmB cleavage sites we have identified is at Asp272, this is suggestive that GzmB-mediated cleavage of α6/β4 integrin could likewise severely affect the adhesive properties of this molecule. As the main component of the papillary dermis, another protein fundamental for the integrity of the DEJ is collagen VII. Among collagen VII domains, fibronectin-like regions are pivotal for the interaction with several ECM components. Studies with a recombinant version of the NC1 region revealed strong binding affinity of fibronectin-like domains with collagen I, collagen IV, laminin 332 and fibronectin,19,20 which allows anchorage of the papillary dermis to the lamina densa. Mutations of collagen VII or production of auto-antibodies against this region result in severe disruption of the DEJ, causing dystrophic epidermolysis bullosa and EBA respectively.46,47 These conditions are characterized by extensive epidermal detachment and formation of blisters. We show herein GzmB-mediated cleavage of collagen VII in the fibronectin-like III-2 domain, as well as in the von Willebrand factor A domain, and inhibition of this cleavage by the GzmB inhibitors serpin A3N and compound 2048. Moreover, GzmB-mediated cleavage of collagen XVII was also observed. Collagen XVII is another important component of the hemidesmosome, whose interaction with α6/β4 integrin is required for the assembly of protein complexes that anchor basal keratinocytes to the lamina lucida44. Taken together, these

observations suggest that GzmB can disrupt the basal keratinocyte/lamina lucida connection through cleavage of α6/β4 integrin and collagen XVII, and lamina densa/fibrillar zone adhesion through cleavage of collagen VII. GzmB accumulation at the DEJ is observed in bullous dermatoses such bullous pemphigoid9 and dermatitis herpetiformis,10 as well as in cutaneous adverse drug reactions including lichenoid drug eruption, 49 SJS/TEN and generalized bullous fixed drug eruption.11 However, as the newly discovered, non-apoptotic roles for GzmB were not established at the time, none of the aforementioned studies considered extracellular GzmB-mediated proteolysis as a mechanism for DEJ disruption. Rather, GzmB was proposed to contribute to CD8+ T-cell- and NKmediated, keratinocyte and melanocyte50 apoptosis in a perforindependent manner. While mostly absent in normal skin, GzmB is present at the DEJ in the sub-epidermal blistering conditions bullous pemphigoid and dermatitis herpetiformis in agreement with previous studies,9,10,11 and we have shown for the first time accumulation of this protease at the DEJ in EBA. It has long been hypothesized that anti-DEJ auto-antibody-triggered sub-epidermal blister formation is mediated by proteases secreted by infiltrating inflammatory cells. 51 Indeed, auto-antibody-mediated immune cell recruitment to the DEJ is a mandatory condition for dermo-epidermal separation as it dictates the localized and concentrated degranulation of proteinases.52 Confirming this mechanism, auto-antibodies contained in the serum of patients with bullous pemphigoid,53 EBA,54 and pemphigoid gestationis 55 promote leukocyte recruitment to the DEJ resulting in its separation. We suggest that GzmB could be an important contributor to this DEJ separation in autoimmune conditions as the DEJ substrates we have identified are degraded proximal to the area in which blistering is occurring in these diseases. Although in vivo studies will be necessary to fully elucidate GzmB contribution to autoimmune skin blistering, further evidence for a role of GzmB in this process derives from our observation that incubation of healthy human skin with a physiologically relevant concentration of this protease results in clefts formation at the DEJ. One limitation of this model is that, unlike in diseased skin, staining for α6/β4, collagen VII, and collagen XVII remained detectable in areas of epidermal separation (data not shown). There are several explanations for this result. Firstly, in this model the entire skin section is bathed in a solution containing cationic GzmB, which is attracted to, and sequestered by negatively charged proteoglycans rather than concentrating at the DEJ, whereas in sub-epidermal blistering conditions, GzmB is directed specifically to the basement membrane by auto-antibodies. Thus, it is possible that in areas of mild epidermal separation observed in

our ex vivo model, the antibodies we used for IHC could still recognize their epitopes, while the lack of staining in diseased human samples characterized by complete epidermal detachment is a consequence of more severe protein degradation due to a long-term and DEJ-localized exposure to GzmB. Another explanation is that given this is a static environment, cleavage fragments likely remain in the DEJ and are subsequently detected by immunohistochemistry. Despite these limitations, this model shows a causative relation between GzmB exposure and epidermal separation, although not as widespread and dramatic as observed in vivo, providing proof of concept that GzmB alone can dramatically affect DEJ anchoring function. The pathological role of extracellular GzmB in autoimmune diseases might not be limited to the physical disruption of important substrates. Growing evidence suggest that antigenic, GzmB-generated peptide fragments are part of a feed-forward loop that sustains the propagation of several autoimmune diseases (reviewed in 56 ). Studies suggest that GzmB is instrumental in auto-antigen generation in certain autoimmune conditions. 57,58,59 In our study, GzmB cleaves α6/β4 integrin and collagen VII in epitope regions recognized by autoantibodies present in the sera of patients with certain pemphigoid diseases, and EBA respectively. In oral pemphigoid, one of the identified auto-epitopes is represented by the peptide a.a. 292–305 of α6 integrin.60 In vitro, GzmB cleaves α6 integrin at residues Asp199 and Asp302, thus we speculate that GzmB could potentially generate this antigenic fragment in vivo, establishing a cycle of sustained immune response and further generation of antigenic fragments. As mentioned above, we also observed GzmB-mediated cleavage of the ectodomain of collagen XVII. The production of collagen XVII auto-antibodies results in bullous pemphigoid61,62 whereas mutations in the ectodomain of this protein have been associated with certain forms of junctional epidermolysis bullosa. 63,64 Although we were not able to identify the specific GzmB cleavage sites in the NC16 region of collagen XVII, this domain is the immunodominant region in bullous pemphigoid and its recombinant forms are used for detecting specific auto-antibodies in approximately 85% of patients affected by this condition.65,66 Finally, several groups have demonstrated that in EBA, T and B cells target identical regions of the NC1 domain of collagen VII. 67,68 In particular, Lapiere, et al incubated different fragments of collagen VII with sera from 19 EBA patients, and observed that 16 sera strongly reacted with the fusion protein composed of the fibronectin-like III domains 1 to 469. The NC1 domain of collagen VII also mediates Fc-dependent neutrophil activation and induction of

dermo-epidermal separation.54 Given the presence of GzmB cleavage sites in the immunodominant regions of α6/β4 integrin, collagen XVII, and collagen VII, this protease may contribute to the generation of antigenic peptides either directly, or by revealing a cryptic epitope due to a change in substrate structure. If confirmed, this hypothesis would have substantial therapeutic relevance as the molecular target of GzmB includes, but is not limited to the antigenic target of the auto-antibodies. Thus, even though the disease-defining antigenic targets are all different, they result in a common final pathway that may be amenable to the single targeted treatment approach of inhibiting GzmB. In summary, the present study suggests for the first time that GzmB mighty directly contribute to sub-epidermal autoimmune blistering via extracellular proteolysis-mediated DEJ impairment, thus playing a role in this pathological process that goes beyond cytotoxicity. Inhibition of GzmB represents a novel therapeutic approach for the treatment and prevention of autoimmune sub-epidermal blistering.

MATERIALS AND METHODS

All human studies were approved by the University of British Columbia Review Ethic Board (H15-03345). Waiver of consent was obtained as per Tri-Council Policy Statement article 12.3B. Immunohistochemistry and Histology on Diseased Skin One hundred sixty-four (66%) subjects from the CSU group and 31 (24%) from the control group reported at least one reaction with some food (p<0.01) (Figure 1) and 92% of them make a dietary restriction. In the group with urticaria, the patients self-reported an exacerbation of urticaria with these foods, while in the control group the symptoms that were self-reported were mainly cutaneous type pruritus, but 50% also reported erythema and hives. According to the reports of patients and control subjects, the primary food or food products suspected of causing reactions were pork and sauces for both groups (Figure 2). At least two foods were suspected of causing reaction in 40% of the CSU and 12 of the control group. Self-reported exacerbations were always higher than sensitization or positive challenge test except for shrimp (Figure 3). Paraffin-embedded skin samples from patients affected by bullous pemphigoid (n=3), dermatitis herpetiformis (n=3), and inflammatory EBA (n=3) were sectioned (5 μm) for immunohistochemical analysis of GzmB (Cat # ab4059, ABCAM) and collagen VII (Cat # ab93350, ABCAM, Toronto) using 3,3′-Diaminobenzidine for visualization, as well as α6 (Cat # ab181551, ABCAM) and β4 (Cat # ab182120, ABCAM) integrins, and collagen XVII (NC16a-370) visualized through Novared. Healthy skin obtained from patients undergoing


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elective abdominoplasty was used as a control. In order to observe cellular infiltrate and tissue architecture, H&E staining was also performed using established methods. Following image acquisition, these H&E stained sections were de-stained by serial incubation in xylene (2 min), 100% EtOH (2 min), 95% EtOH (2 min), water (10 min), and acidic alcohol solution (10 min). In order to detect GzmB-producing cells, destained H&E sections were probed for GzmB (Cat # ab4059, ABCAM). All slides were scanned using a Aperio CS2 slide scanner (Leica, Concord, Ont.). DEJ Proteins Cleavage Assay The recombinant human integrin α6/β4 (α6 a.a. 24–878, β4 a.a. 28–710, Cat # 5497-A6-050, R&D Systems, Minneapolis, Minn.), collagen VII (199– 482 a.a., Cat # 2018615, MyBiosource, San Diego), collagen XVII ectodomain (490–1497 a.a., generous gift from Dr. Claus-Werner Franzke), and collagen I (Cat # NBP1-97266, Novus Biologicals, Littleton, CO) were incubated for 24 h at 37 °C in 200 nM purified human GzmB (EmeraldBio, Bainbridge Island, Wash.). For inhibition studies, prior to the addition of substrates to the reaction, GzmB was incubated in the presence of 600 nm serpin A3N (generous gift from Dr. Chris R. Bleackley, University of Alberta, Edmonton) or 100 μM of the small molecule inhibitor compound 20 (courtesy of the Centre for Drug Research and Development, Vancouver) for 1 h at 37 °C. After the 24 h incubation, proteins were denatured, separated on a 4 to 20% (for integrin α6/β4), 10% (for collagen VII), 8–10% (for collagen XVII), or 7.5% (for collagen I) SDS-polyacrylamide gel. Cleavage was detected by western blot using anti-human integrin α6 sub-unit (Cat # ab181551, ABCAM), anti-human integrin β4 sub-unit (Cat # MAB4060, R&D Systems, Minneapolis, Minn.), anti-collagen VII (Cat # ab93350, ABCAM), NC16A antibody directed against the NC16A domain of human collagen XVII71 and imaged using a LiCor Odissey FC (Li-Cor, Lincoln, Neb.). Coomassie staining was used for the assessment of collagen I cleavage following manufacturer instructions. LC-MS/MS analysis GzmB cleavage sites were identified by ATOMS as described earlier.17,18 Briefly, GzmB-digested or control substrates were denatured, cysteines reduced with dithiothreitol and alkylated with iodoacetamide. Primary amine groups were dimethylated with formaldehyde and sodium cyanoborohydride before acetone precipitation. Pellets were redissolved in digestion buffer with 1 μg/mL MS-grade trypsin (Thermo Fisher Scientific, Waltham, Mass.) and desalted using StageTips.72 Samples were analyzed on an Impact II Q-TOF (Bruker, Billerica, Mass.) on ReproSilPur 120 C18AQ 1.9 μ m particles (Dr. Maisch) columns and resolved by a gradient of acetonitrile (0.1% v/v formic acid) in water delivered by an easy-nLC system (Thermo Fisher Scientific) preceding

data-dependent precursor selection of the top 18 peaks. Spectra were extracted using DataAnalysis 4.3 (Bruker) and searched against a Uniprot human proteome database (downloaded 2016-0224; 70,472 sequences) using semi-specific ArgC as enzyme specificity, carbamidomethylation (C) and dimethylation (K) as fixed modifications, and deamidation (N,Q), dimethylation (Nterm), pyroglutamation (Q) and oxidation (M) as variable modifications. Potential GzmB cleavage sites were defined as semi-specific, N-terminally dimethylated peptides with an acidic residue N-terminal to the identified sequence identified at a peptide expect value <0.01. Skin Cleavage Assay Fresh healthy human skin obtained from patients undergoing elective plastic surgery was transported to the lab and cut into ≈1 mm × 4 mm strips (n=3). The adipose tissue layer was removed to obtain a strip of dermis and epidermis. Skin strips were then incubated at 37 °C for 12 h in 300 μL of either PBS, 200 nM GzmB, or 200 nM GzmB previously inactivated through 1 h incubation at 37 °C with 100 μM Compound 20. Following incubation, samples were fixed in 10% buffered formalin overnight, paraffin embedded and sectioned for H&E staining using established methods, and TUNEL (cell death) analysis following manufacturer’s instructions (Cat # 11684795910, Sigma). Outermost sections of the samples were used for staining, as GzmB might not penetrate deep into the tissue.

ADDITIONAL INFORMATION

AUTHOR INFORMATION Affiliations International Collaboration On Repair Discoveries (ICORD) Research Centre, Vancouver, B.C., V5Z 1M9, Canada Valerio Russo, Darielle J. Lim, Sho Hiroyasu, Layla Nabai, Yue Shen, Matthew R. Zeglinski, Hongyan Zhao, Cameron P. Oram, David J. Granville Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, B.C., V6T 2B5, Canada Valerio Russo, Sho Hiroyasu, Yue Shen, Matthew R. Zeglinski, Hongyan Zhao, Cameron P. Oram, Richard I. Crawford, David J. Granville BC Professional Firefighters’ Burn and Wound Healing Research Laboratory, Vancouver, B.C., V5Z 1M9, Canada Valerio Russo, Sho Hiroyasu, Layla Nabai, Yue Shen, Matthew R. Zeglinski, Hongyan Zhao, Cameron P. Oram, David J. Granville Centre for Blood Research, University of British Columbia, Vancouver, B.C., V6T 1Z3, Canada Theo Klein, Nestor Solis, Yoan Machado, Christopher M. Overall Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, B.C., V6T 1Z3, Canada Theo Klein, Nestor Solis, Yoan Machado, Christopher M. Overall Department of Surgery, University of British Columbia, Vancouver, B.C., V5Z 1M9, Canada

Original Publisher’s Note

Peter A. Lennox, Nancy Van Laeken, Nick J. Carr

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Department of Dermatology and Skin Science, University of British Columbia, Vancouver, B.C., V5Z 4E8, Canada

ACKNOWLEDGEMENTS

The authors are thankful to Dr. Leena Bruckner-Tuderman and Dr. Alexander Nyström, University of Freiburg, for generously providing collagen VII and recessive dystrophic epidermolysis bullosa samples for analysis. We also thank Dr. Jenny Chik, ICORD, for her help editing the manuscript, Amrit Samra, Centre for Heart Lung Innovation, for embedding human skin, and Dr. Chris Bleackley, University of Alberta, for donating serpin A3N inhibitor. Finally we would like to thank Tammy Rathbone, Toni Trevisan, Kelle Mobley, and Michelle Hawser for their assistance in coordinating sample collection from the plastic surgery clinics. Funding for this project was provided by grants-in-aid from the Canadian Institutes of Health Research (CIHR), Michael Smith Foundation for Health Research (DJG, CMO) and Rick Hansen Institute (DJG), a Canada Research Chair in Protease Proteomics and Systems Biology (CMO), and the Canada Foundation for Innovation (CMO).

Richard I. Crawford Department of Dermatology, Medical Center and Faculty of Medicine University of Freiburg, 79104, Freiburg, Germany Claus-Werner Franzke Contributions V.R. contributed by designing the study, performing experiments, acquiring and analyzing data, preparing images, and writing the manuscript. T.K. contributed by performing experiments, acquiring and analyzing mass spectrometry data, preparing images, and writing the manuscript. D.J.L. contributed by performing experiments, and preparing images. N.S. and Y.N. contributed by performing mass spectrometry experiments. S.H. contributed by performing cleavage assays and IHC experiments. L.N., Y.S. and M.R.Z. contributed to experimental design. H.Z. and C.P.O. contributed by performing IHC experiments. P.A.L., N.V.L., and N.J.C. contributed by providing healthy human skin samples for analysis. R.I.C. contributed to experimental design, and

provided diseased human skin samples for analysis. C.W.F. contributed by providing collagen XVII and anticollagen XVII antibodies. C.M.O. contributed to experimental design and wrote the manuscript. D.J.G. contributed to experimental design, data analysis, and wrote the manuscript. Competing Interests DJG is a co-founder and serves as the Chief Scientific Officer and member of the scientific advisory board to viDA Therapeutics. No viDA-derived products, reagents, or inhibitors were used in this study. Corresponding Author Correspondence to David J. Granville.

ELECTRONIC SUPPLEMENTARY MATERIAL

https://goo.gl/a2g6A7

RIGHTS AND PERMISSIONS

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Message from the Medical Editor Continued from page 3

be effective with significant improvement seen in about half of patients. In the wound care arena Kerstin Lewis emphasizes the role of the wound care team in managing com-

plications (page 1). She notes that negative pressure wound therapy may be an approach, especially in complications such as dehiscence or wounds with excessive exudate. Another article I would also like to

mutations in the beta 4 integrin gene (ITGB4) and genotype/phenotype correlations. Pediatr Res 2001; 49:618–626. 42. Pulkkinen L, Bruckner-Tuderman L, August C, and Uitto J: Compound heterozygosity for missense (L156P) and nonsense (R554X) mutations in the beta4 integrin gene (ITGB4) underlies mild, nonlethal phenotype of epidermolysis bullosa with pyloric atresia. AJPA 1998; 152:935–941. 43. Pulkkinen L, Kim DU, Uitto J: Epidermolysis bullosa with pyloric atresia: novel mutations in the beta4 integrin gene (ITGB4). AJPA 1998; 152:157–166. 44. Koster J, Geerts D, Favre B, Borradori L, Sonnenberg A: Analysis of the interactions between BP180, BP230, plectin and the integrin alpha6beta4 important for hemidesmosome assembly. J Cell Sci 2003; 116:387–399. 45. Kambham N, et al: Congenital focal segmental glomerulosclerosis associated with beta4 integrin mutation and epidermolysis bullosa. Am J Kidney Dis 2000; 36:190–196. 46. Kim JH, Kim SC: Epidermolysis bullosa acquisita. J Eur Acad Dermatol Venereol 2013; 27:1204–1213. 47. Dang N, Murrell DF: Mutation analysis and characterization of COL7A1 mutations in dystrophic epidermolysis bullosa. Exp Dermatol 2008; 17:553–568. 48. Willoughby CA, et al: Discovery of potent, selective human granzyme B inhibitors that inhibit CTL mediated apoptosis. Bioorg Med Chem Lett 2002; 12:2197–2200. 49. Lage D, Juliano PB, Metze K, de Souza EM, Cintra ML: Lichen planus and lichenoid drug-induced eruption: a histological and immunohistochemical study. Int J Dermatol 2012; 51:1199–1205. 50. van den Wijngaard R, et al: Local immune response in skin of generalized vitiligo patients. Destruction of melanocytes is associated with the prominent presence of CLA+ T cells at the perilesional site. Lab Invest 2000; 80:1299–1309. 51. Jordon RE, Kawana S, Fritz KA: Immunopathologic mechanisms in pemphigus and bullous pemphigoid. J Invest Dermatol 1985; 85:72s–78s. 52. Sitaru C, Zillikens D: Mechanisms of blister induction by autoantibodies. Exp Dermatol 2005; 14:861–875. 53. Mihai S, et al: IgG4 autoantibodies induce dermal–epidermal separation. J Cell Mol Med 2007; 11:1117–1128. 54. Sitaru C, Kromminga A, Hashimoto T, Bröcker E-B, Zillikens D: Autoantibodies to type VII collagen mediate Fcgamma-dependent neutrophil activation and induce dermalepidermal separation in cryosections of human skin. AJPA 2002; 161:301–311. 55. Herrero-González JE, et a:. Immunoadsorption against two distinct epitopes on human type XVII collagen abolishes dermal-epidermal separation induced in vitro by autoantibodies from pemphigoid gestationis patients. Eur J Immunol 2006; 36:1039–1048. 56. Darrah E, Rosen A: Granzyme B cleavage of autoantigens in autoimmunity. Cell Death Differ 2010; 17:624–632. 57. Darrah E, et al: Proteolysis by Granzyme B enhances presentation of autoantigenic peptidylarginine deiminase 4 epitopes in rheumatoid arthritis. J Proteome Res 2017; 16:355–365. 58. Niland B, et al: Cleavage of transaldolase by granzyme B causes the loss of enzymatic activity with retention of anti-

bring to your attention is “Sun Screen Use: Factors Preventing Proper Application” in which Craig Sinclair does a brilliant job tackling this important topic (page 25). He also leaves readers with some very practical advice on sun screen use to pass along to our patients, i.e., use at least SPF30,

genicity for multiple sclerosis patients. J Immunol 2010; 184:4025–4032. 59. Nagaraju K, Cox A, Casciola-Rosen L, Rosen A: Novel fragments of the Sjögren’s syndrome autoantigens αfodrin and type 3 muscarinic acetylcholine receptor generated during cytotoxic lymphocyte granuleinduced cell death. Arthritis Rheum 2001; 44:2376–2386. 60. Rashid KA, Stern JNH, Ahmed AR: Identification of an epitope within human integrin alpha 6 subunit for the binding of autoantibody and its role in basement membrane separation in oral pemphigoid. J Immunol 2006; 176:1968–1977. 61. Zimina EP, et al: Bullous pemphigoid autoantibodies preferentially recognize phosphoepitopes in collagen XVII. J Invest Dermatol 2008; 128:2736–2739. 62. Nishie W: Update on the pathogenesis of bullous pemphigoid: an autoantibody-mediated blistering disease targeting collagen XVII. J Dermatol Sci 2014; 73:179–186. 63. McGrath JA, et al: Compound heterozygosity for a dominant glycine substitution and a recessive internal duplication mutation in the type XVII collagen gene results in junctional epidermolysis bullosa and abnormal dentition. AJPA 1996; 148:1787–1796. 64. Schumann H, et al: Three novel homozygous point mutations and a new polymorphism in the COL17A1 gene: relation to biological and clinical phenotypes of junctional epidermolysis bullosa. Am J Hum Genet 1997; 60:1344–1353. 65. Giudice GJ, et al: Bullous pemphigoid and herpes gestationis autoantibodies recognize a common non-collagenous site on the BP180 ectodomain. J Immunol 1993; 151:5742–5750. 66. Murakami H, et al: Analysis of antigens recognized by autoantibodies in herpes gestationis. Usefulness of immunoblotting using a fusion protein representing an extracellular domain of the 180 kD bullous pemphigoid antigen. J Dermatol Sci 1996; 13:112–117. 67. Müller R, et al: T and B cells target identical regions of the non-collagenous domain 1 of type VII collagen in epidermolysis bullosa acquisita. Clin Immunol 2010; 135:99–107. 68. Jones DA, Hunt SW, Prisayanh PS, Briggaman RA, Gammon WR:. Immunodominant autoepitopes of type VII collagen are short, paired peptide sequences within the fibronectin type III homology region of the noncollagenous (NC1) domain. J Invest Dermatol 1995; 104:231–235. 69. Lapiere JC, et al: Epitope mapping of type VII collagen. Identification of discrete peptide sequences recognized by sera from patients with acquired epidermolysis bullosa. J Clin Invest 1993; 92:1831–1839. 70. Hofmann SC, et al: Plasmin plays a role in the in vitro generation of the linear IgA dermatosis antigen LADB97. J Invest Dermatol 2009; 129:1730–1739. 71. Franzke CW, et al: Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs. EMBO J 2002; 21:5026– 5035. 72. Rappsilber J, Mann M, Ishiham Y: Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2007; 2:1896–1906.

any brand you like and using it as often as possible. It is certainly an informing read, since we have seen and heard all the excuses over the years.. As always, we invite your comments at www.derm.city. —Wayne P. Gulliver, MD, FRCPC, Medical Editor


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THE CHRONICLE of S K I N & A L L E R G Y

September 2018 · 25

Sunscreens: Factors preventing proper application Research

n Speakers at 4th International Conference on UV and Skin Cancer Prevention address urban myths

L

by BIANCA QUIJANO,

Assistant Editor, The Chronicle

ack of confidence in the efficacy of sunscreen formulations, fear of vitamin D deficiency, and cultural standards regarding the characteristics of beautiful skin are some of the factors preventing people from properly using sunscreen. This issue was discussed during presentations at the 4th International Conference on UV and Skin Cancer Prevention in Toronto. Stories of children getting adverse reactions from sunscreens have contributed to the growing distrust in sunscreen use, causing people to focus more on the potential dangers of sun protection and less on its benefits said Craig Sinclair during his lecture. In Canada, for example, the Banana Boat brand was linked to burns. In Australia, a Peppa Pig brand of sunscreen was reported to cause skin reactions among children, and pressure was placed on Cancer Council Australia to withdraw the product from the Craig Sinclair market. However, adverse reactions to sunscreens are rare, and occur in less than 1% of users, according to Sinclair. In order to combat this growing negative perception in the public, he says that skin protection advocates must develop clearer campaign messaging. “Many times, our messaging gets caught up in the debate of, for example, which SPF strength is best or should consumers use sunscreens that contain more UV blockers than absorbers,” said Sinclair, director of the World Health Organization’s (WHO) Collaborative Centre for UV Radiation, associate professor at Queensland University of Technology, and Head of Cancer Prevention at the Cancer Council Victoria, Australia. “Instead we should be telling people to simply choose at least SPF30 sunscreen they like and apply it as much as they possibly can. The most important thing is to get people to choose a product that they actually like applying, because the more they apply, the better protected they will be.” Confusion regarding when it is necessary to apply sunscreen also contributes to improper usage. Sinclair pointed out that public health agencies all over the world have different recommendations. In Australia, it is recommended to apply sun-

screen when the UV index is greater than three. In contrast, health agencies in the U.S. recommend using sunscreen everyday, regardless of UV index levels. In the U.K., instead of the UV index, sunburns are used as a marker for sunscreen use, with health organizations advising that sunscreens can be used to prevent burns. In an effort to simplify their messaging, Sinclair said that Cancer Council Australia is moving toward recommending daily sunscreen use. “That is not our current recommendation. There is an ongoing debate as to whether we should be getting people to use sunscreen every day, no matter what, in the same way that it is recommended for people to brush their teeth on a daily basis,” he said.

Fear of vitamin D deficiency Concerns about vitamin D deficiency caused by sunscreen use is another notion undermining sun protection initiatives, according to Rachel Neale, PhD, associate professor at QIMR Berghofer Medical Research Institute in Brisbane, Australia. She also Nynne Johanne holds adjunct Sahl Freeriksen appointments at Queensland University of Technology and the University of Queensland in St. Lucia, Australia. Sunscreens that do not block vitamin D are now being marketed and there is an ongoing discussion as to whether they should be endorsed by sun protection societies. Dr. Neale conducted a systematic review of studies on how sunscreen usage affects vitamin D levels. Her report showed that study results are too varied, at this point, to conclude whether or not sunscreen use can cause harmful vitamin D depletion (Photochem Photobiol Sci June 15, 2018). “The observational studies mostly suggest that vitamin D deficiency from sunscreen use does not occur in practice, but they do have considerable limitations,” Dr. Neale said. “The trials show that moderate SPF sunscreen application does not seem to affect circulating vitamin D levels, but there are no trials in the general population of the much higher SPF sunscreens that are now frequently used, so this is a gap that we need to fill.”

Influence of societal norms An ethnographic study of sunscreen usage among Danes on sunny holidays abroad showed that the desire to have tanned skin, particularly dur-

ing vacations, also prevents people from practicing proper sun protection. Nynne Johanne Sahl Frederiksen presented these findings during her lecture at the UV and Skin Cancer Prevention summit. Denmark has a temperate climate with short summers and long winters, but skin cancer is still the most common form of cancer in Denmark. “About 38 per cent of adult Danes travel to sunny destinations each year and among these one out of three report having been sunburned during their vacation,” said Frederiksen, anthropologist and senior project manager of the sun safety campaign at the Danish Cancer Society in Copenhagen. The study focussed on Danish families during their holidays to a Spanish island called Mallorca. Sixteen respondents travelling with their children were interviewed before, during, and after their vacations. Participant observations were also conducted (Frederiksen NS, Krüger-Jensen C: Sunscreen: An Ambiguous Tool—-Findings from an Ethnographic Study Among Danes On Holiday, UV and Skin Cancer Prevention, Toronto). First, the researchers observed that the way hotels are designed encouraged vacationers to spend most of their time directly exposed to the sun. “The hotel rooms were quite small and they did not have the facilities to entertain a whole family inside. The hotels were also built around swimming pools, the beach, and surrounding trails,” said Frederiksen. The respondents knew that spending many hours outside warranted sunscreen use, but their motivation was

not primarily focussed on reducing their risk of skin cancer. Instead, they used sunscreen to prolong the time they could spend exposed to the sun and to avoid the physical discomfort of being sunburned. “We found that sunscreen was used to mediate between two contrasting logics during their vacation. First, respondents acted upon a logic of protection. Second was a logic based on a wish to get a tan, which signalized to others that they had a successful sunny vacation upon returning home,” said Frederiksen. Notably, the researchers found that this logic of protection was not applied equally to adults and children. Participants reported that children must always wear sunscreen, whereas for adults, the notion is negotiable. The investigators also observed that participants applied just enough sunscreen so that they would not get sunburned, but not so much that they would not get a tan. Participants applied smaller and smaller doses of sunscreen than recommended, guided by their individual body sensations, the temperatures of the day, cloud coverage, and their planned activities. “They did not apply sunscreen according to the UV index. The perception was that if they were using sunscreen, then they would be protected no matter what,” explained Frederiksen. “This often led to sunburns, but the respondents did not blame themselves for the sunburn. The perception was that if they were using sunscreen and still got sunburned, then it was unavoidable and a natural part of being on holiday in a sunny destination.”

Biologics must achieve at least PASI 75 in 90% of patients Continued from page 10

“We use them because they are cheap,” he said. “They are old therapies.” A disadvantage of a therapy like methotrexate is that it requires ongoing monitoring of patients for liver function and other parameters, said Dr. Vender. Clinicians may have to prescribe traditional therapies to patients who have financial constraints, he said. Because of that fact, clinicians need to conduct thorough baseline examinations that include a complete blood count, chest X-ray and ultrasound to ensure that patients do not have a condition like fatty liver disease which would rule out methotrexate as a potential therapy. Non-proprietary and brand names of therapies: adalimumab (Humira, Abbvie); etanercept (Enbrel, Amgen); infliximab (Remicade, Janssen); secukinumab (Cosentyx, Novartis); ixekizumab (Taltz, Lilly); guselkumab (Tremfya, Janssen): certolizumab pegol (Cimzia, UCB Canada Inc.); acitretin (Soriatane, Nuvo Pharmaceuticals).


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CERAMIDE LEVELS IN SKIN OF ACNE PATIENTS CHANGE WITH THE SEASONS

esearchers analyzed ceramides in the stratum corneum of healthy and acne skin, with a focus on seasonal variation over the course of a year. They identified 283 ceramides in the skin. Overall, the acne-affected skin showed lower ceramide levels, particularly CER[NH] and CER[AH], as well as lower levels of the acylceramides CER[EOS] and CER[EOH]. These differences were more apparent during the winter. Lower ceramide levels were associated with an increase in trans-epidermal water loss (TEWL) in acne, compared to healthy skin, which partly resolves in the summer. Finally, individual cermaide species with 18-carbon 6-hydroxysphingosine (H) bases—including CER[N(24)H(18)], CER[N(26)H(18)], CER[A(24)H(18)], and CER[A(26)H(18)]—were significantly reduced in acne skin, which the authors say suggests CER[NH] and CER[AH] ceramide species may be particularly important for a healthy skin barrier. A. Pappas, A.C. Kendall, L.C. Brownbridge, N. Batchvarova, A. Nicolaou: Seasonal changes in epidermal ceramides are linked to impaired barrier function in acne patients, in Experimental Dermatology (Aug. 2018; 27(8):833-836).

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TOPICAL SIROLIMUS 0.1% FOR THE TX OF VASCULAR ANOMALIES IN CHILDREN

o evaluate the efficacy, tolerance, and absorption of topical sirolimus 0.1% with different types of vascular anomalies in children, investigators applied the medication to anomalies in six children aged two to 17 years. These anomalies included three extratruncular micro- and macrocystic lymphatic malformations, a verrucous venous malformation, a truncular lymphatic malformation with angiokeratomas, and an infantile hemangioma. The children’s sirolimus blood levels were then measured after one week, one month, and three months. There was a rapid decrease in the size of superficial lymphatic malformations in three of six patients and a significant decrease in discharge from oozing lesions. Treatment response occurred in less than three months. The truncular lymphatic malformation, verrucous venous malformation, and infantile hemangioma did not respond to treatment. Blood sirolimus levels were undetectable, and adverse effects were limited to local irritation. Researchers conclude that topical sirolimus 0.1% is a useful treatment for cutaneous manifestations of extratruncular lymphatic malformations, with no systemic effects expected, due to clinically insignificant blood levels. S. Le Sage, M. David, J. Dubois, J. Powell, C.C. McCuaig, Y. Théorêt, and N. Kleiber: Efficacy and absorption of topical sirolimus for the treatment of vascular anomalies in children: A case series, in Pediatric Dermatology (July/Aug. 2018; 35(4):472-477).

Diagnostic Quiz

A. Impetigo B. Shingles C. Dermatitis herpetiformis D. Candidiasis

THE EDITORS invite your participation in this regular feature of the journal. Please send all images and correspondence to: Medical Editor, The Chronicle of Skin & Allergy 555 Burnhamthorpe Road, Suite 306, Toronto, Ont. M9C 2Y3. Telephone: (416) 916-2476 E-mail: health@chronicle.org

What THE LAY PRESS is saying about . . .

FREQUENT BCC A RISK FACTOR FOR OTHER CANCERS Frequent recurrence of basal cell carcinoma raises the risk of bowel, prostate, and colon cancer, reports The Daily Mail (Aug. 9, 2018). The news outlet reports that a study from the Stanford University School of Medicine in Stanford, Calif., found that patients with abnormally frequent cases of basal cell carcinoma appear to be at a three-fold greater risk of developing other cancers. This increased susceptibility may be caused by mutations in a panel of proteins responsible for repairing DNA damage. The study authors suggest that the skin could serve as a sort of early warning system to reveal an individual's overall cancer susceptibility. Assistant professor of dermatology and study co-author Dr. Kavita Sarin was quoted as saying: “We discovered that people who develop six or more basal cell carcinomas during a 10-year period are about three times more likely than the general population to develop other, unrelated cancers.” Dr. Sarin said these findings raise the prospects of identifying individuals at risk of developing lifethreatening malignancies before there are clinical signs.

ACNE POSITIVITY MOVEMENT GROWING ON SOCIAL MEDIA A growing acne positivity movement is encouraging people to be more open about their skin challenges and to feel less shame for having acne, reports The Guardian (Sept. 18, 2018). The trend may trace back to British blogger Em Ford, who in 2015 posted a YouTube video titled You Look Disgusting. This video showed Ford both in full makeup and barefaced with her acne visible, alongside negative comments she had received about her skin. That video had more than 10 million views in its first week and Ford's channel My Pale Skin has more than one million subscribers. Not only is the movement growing among the general public— Colorado-based Hailey Wait on Instagram and the British beauty blogger Kadeeja Khan are examples—but celebrities are speaking up as well. Examples include U.S. actress and model Kylie Jenner who has promoted her favourite spot treatment; Canadian musician Justin Bieber posted on Instagram that “Pimples are in”; and New Zealandborn singer and songwriter Lorde’s has used her social media to push back at individuals giving her unsolicited skincare advice.

Image by: Fisle, via Wikimedia Commons https://goo.gl/TBFt8T

R

Research of Note

THE CHRONICLE of S K I N & A L L E R G Y

EXPERIMENTAL MEDICATIONS REVERSES DIET-RELATED HAIR LOSS IN A MOUSE MODEL Researchers from Johns Hopkins University School of Medicine in Baltimore have developed an experimental medication that reversed dietrelated hair greying and hair loss in mice, reports Men's Health (Aug. 1, 2018). In the study, the mice were given a diet high in fat and cholesterol to model the Western diet. Mice on the test diet had their hair change to grey or white from black, or they experienced hair loss. Some mice on the diet also experienced skin inflammation. When investigators gave the mice the experimental compound, which researchers believe stops the production of a group of fats known as glycosphingolipids (GSLs), the animals' hair was restored while their skin cleared up. “Our findings show that a Western diet causes hair loss, hair whitening and skin inflammation in mice, and we believe a similar process occurs in men who lose hair and experience hair whitening when they eat a diet high in fat and cholesterol,” the magazine quoted senior author Dr. Suboroto Chatterjee as saying. Dr. Chatterjee is a professor of paediatrics and medicine at the John Hopkins University School of Medicine.

Correct answer: B. Shingles

Journal Club

26 · September 2018

MYTHS ABOUT DIET AND ACNE PERSIST ON ONLINE FORUMS IN SPITE OF LACK OF EVIDENCE A belief that diet can control acne remains widespread and common, with everything from fat elimination to high-fat ketogenic diets being suggested online as an easy solution to breakouts. Yet the science supporting dietary acne control is still not strong, reports The Atlantic (Aug. 7, 2018). The news article quotes several users of the reddit.com community SkincareAddiction presenting anecdotes of how giving up one type or category of food resolved their acne completely—often contradicting each other. It also cites a small survey published in Dermatology Practical & Conceptual (Apr. 2016; 6(2):21-27) in which 46 of 49 patients said they believed diet, particularly greasy food, could impact acne. However, the article notes that the American Academy of Dermatology does not currently recommend diet changes to manage acne, citing lack of sufficient data. The most promising correlation among the limited data is with dietary sugar. “Multiple studies have now found that diets with a high glycemic load can trigger acne in certain persons,” Dr. Rajani Katta, a clinical professor of dermatology at the University of Texas at Houston, says in the article. Department Editor: John Evans


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See what HUMIRA can do for your patients • Treatment of active moderate to severe hidradenitis suppurativa (HS) in adult patients, who have not responded to conventional therapy (including systemic antibiotics). Please consult the Product Monograph at abbvie.ca/content/dam/abbviecorp/ ca/en/docs/HUMIRA_PM_EN.pdf for contraindications, warnings, precautions, adverse reactions, interactions, dosing and conditions of clinical use. The Product Monograph is also available by calling 1-888-704-8271. Reference: 1. AbbVie Corporation. Data on file.

PsA

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DATES OF INTRODUCTION IN CANADIAN PRACTICE:1

2006

THE ABBVIE DERMATOLOGY PORTFOLIO

ESTABLISHED EXPERIENCE*

2015

HUMIRA is indicated for: • Treatment of adult patients with chronic moderate to severe plaque psoriasis (Ps) who are candidates for systemic therapy. For patients with chronic moderate plaque Ps, HUMIRA should be used after phototherapy has been shown to be ineffective or inappropriate. • Reducing the signs and symptoms of active arthritis and inhibiting the progression of structural damage and improving the physical function in adult psoriatic arthritis (PsA) patients. HUMIRA can be used in combination with methotrexate (MTX) in patients who do not respond adequately to MTX alone.

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The Chronicle of Skin & Allergy September 2018  

The Chronicle of Skin & Allergy September 2018 Dr. Wayne Gulliver, Medical Editor Dr. Sheldon V. Pollack, Editor, Cosmetic Dermatology Featu...

The Chronicle of Skin & Allergy September 2018  

The Chronicle of Skin & Allergy September 2018 Dr. Wayne Gulliver, Medical Editor Dr. Sheldon V. Pollack, Editor, Cosmetic Dermatology Featu...

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