Position statement for a pragmatic approach to immunotherapeutics in patients with inflammatory skin diseases during the coronavirus disease 2019 pandemic and beyond.

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2, a novel RNA virus that was declared a global pandemic on 11 March 2020. The efficiency of infection with SARS-CoV-2 is reflected by its rapid global spread. The SARS-CoV-2 pandemic has implications for patients with inflammatory skin diseases on systemic immunotherapy who may be at increased risk of infection or more severe infection. This position paper is a focused examination of current evidence considering the mechanisms of action of immunotherapeutic drugs in relation to immune response to SARS-CoV-2. We aim to provide practical guidance for dermatologists managing patients with inflammatory skin conditions on systemic therapies during the current pandemic and beyond. Considering the limited and rapidly evolving evidence, mechanisms of action of therapies, and current knowledge of SARS-CoV-2 infection, we propose that systemic immunotherapy can be continued, with special considerations for at risk patients or those presenting with symptoms.

Developing classification criteria for skin-predominant dermatomyositis: the Delphi process.

BACKGROUND: The European League Against Rheumatism/American College of Rheumatology classification criteria for inflammatory myopathies are able to classify patients with skin-predominant dermatomyositis (DM). However, approximately 25% of patients with skin-predominant DM do not meet two of the three hallmark skin signs and fail to meet the criteria. OBJECTIVES: To develop a set of skin-focused classification criteria that will distinguish cutaneous DM from mimickers and allow a more inclusive definition of skin-predominant disease. METHODS: An extensive literature review was done to generate items for the Delphi process. Items were grouped into categories of distribution, morphology, symptoms, antibodies, histology and contextual factors. Using REDCap™, participants rated these items in terms of appropriateness and distinguishing ability from mimickers. The relevance score ranged from 1 to 100, and the median score determined a rank-ordered list. A prespecified median score cut-off was decided by the steering committee and the participants. There was a pre-Delphi and two rounds of actual Delphi. RESULTS: There were 50 participating dermatologists and rheumatologists from North America, South America, Europe and Asia. After a cut-off score of 70 during the first round, 37 of the initial 54 items were retained and carried over to the next round. The cut-off was raised to 80 during round two and a list of 25 items was generated. CONCLUSIONS: This project is a key step in the development of prospectively validated classification criteria that will create a more inclusive population of patients with DM for clinical research. What's already known about this topic? Proper classification of patients with skin-predominant dermatomyositis (DM) is indispensable in the appropriate conduct of clinical/translational research in the field. The only validated European League Against Rheumatism/American College of Rheumatology criteria for idiopathic inflammatory myopathies are able to classify skin-predominant DM. However, a quarter of amyopathic patients still fail the criteria and does not meet the disease classification. What does this study add? A list of 25 potential criteria divided into categories of distribution, morphology, symptomatology, pathology and contextual factors has been generated after several rounds of consensus exercise among experts in the field of DM. This Delphi project is a prerequisite to the development of a validated classification criteria set for skin-predominant DM.

Effect of biologics on depressive symptoms in patients with psoriasis: a systematic review.

BACKGROUND: Twenty to fifty percent of patients with psoriasis have depressive symptoms. OBJECTIVE: To describe the effects of biologics (tumour necrosis factor inhibitors [TNFi] or interleukin 12/23 inhibitors [IL-12/23i]) on depressive symptoms in patients with psoriasis. METHODS: Electronic databases were searched for randomized controlled trials (RCTs) examining the effects of biologics on depressive symptoms in adults with psoriasis. RESULTS: Of the 305 publications identified, three RCTs were included in a systematic review. In a trial evaluating ustekinumab, mean change in Hospital and Anxiety Depression Rating Scale at 24 weeks from baseline was 3.1 with ustekinumab (P < 0.001) vs. 0.21 with placebo (not significant). In a trial evaluating adalimumab, mean change in Zung Self-Rating Depression Scale at 12 weeks from baseline was -6.7 with adalimumab vs. -1.5 with placebo. In a trial evaluating etanercept, the between-group difference at 12 weeks in Beck Depression Inventory Scale was 1.8 (95% CI: 0.6, 2.90) in favour of etanercept over placebo. Limitations are that diagnostic criteria for depression were not used and scales and data from individual RCTs could not be combined. CONCLUSION: Adalimumab, etanercept and ustekinumab were associated with statistically significant reductions in depressive symptom scores using various scales in patients with moderate-to-severe psoriasis.

Toll-like receptor 7 stimulation promotes autoimmune diabetes in the NOD mouse.

AIMS/HYPOTHESIS: The role of Toll-like receptor 7 (TLR7), a sensor of viral and self RNA, in promoting autoimmune diabetes remains unclear. Our goal was to determine the effect of TLR7 stimulation on the priming and activation of diabetogenic CD8(+) T cells. METHODS: We explored the effects of CL097 (TLR7/8 agonist) and immunoregulatory sequence 661 (IRS661, TLR7 inhibitor) on bone marrow-derived dendritic cells (BMDCs), diabetogenic CD8(+) T cell function and autoimmune diabetes onset in NOD and 8.3 NOD T cell receptor transgenic mice (8.3 NOD mice). RESULTS: TLR7 stimulation of NOD BMDCs increased activation and production of proinflammatory cytokines. In vivo administration of CL097 activated T cells and dendritic cells and increased levels of proinflammatory cytokines and type 1/2 IFNs in NOD mice. In vivo antigen-specific cytotoxicity studies revealed enhanced cytotoxicity against islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP, an islet autoantigen) peptide pulsed targets in NOD mice treated with CL097 plus CD40 agonist. This combination treatment accelerated the onset of autoimmune diabetes in 8.3 NOD mice. Likewise, topical treatment of NOD mice with a TLR7 agonist accelerated diabetes onset. Spontaneous disease in 8.3 NOD mice and accelerated disease in CL097+CD40 agonist-treated 8.3 NOD mice were delayed by IRS661 treatment, which is associated with inhibition of the endogenous upregulation of IFN-α levels within the pancreatic lymph nodes. CONCLUSIONS/INTERPRETATION: TLR7 stimulation accelerates the spontaneous onset of autoimmune diabetes in 8.3 NOD and NOD mice. Conversely, TLR7 inhibition prevents the early events associated with diabetogenesis.

CXCR3 ligands promote expression of functional indoleamine 2,3-dioxygenase in basal cell carcinoma keratinocytes.

BACKGROUND: Basal cell carcinoma (BCC) is the most common malignancy in humans worldwide. Studies suggest that BCCs exhibit immunoprotection, similar to other keratinocyte carcinomas, although the mechanisms of defence have not been defined. OBJECTIVES: To examine if indoleamine 2,3-dioxygenase (IDO), an immune privilege-associated enzyme, would be expressed in BCC, regulated in part by CXCR3. METHODS: We analysed the expression and function of IDO in human BCC (hBCC) tissues using nonlesional skin epithelial (NL) tissues as a control. RESULTS: Quantitative real-time reverse transcription-polymerase chain reaction (qPCR) revealed significant upregulation of IDO1 and IDO2 (12·5- and 19·14-fold change, respectively) in nodular hBCCs as compared with NL tissues. Immunohistochemistry showed that IDO colocalized with keratin 17, a BCC keratinocyte marker, in hBCC tissues. Western blot identified a full-length IDO (42 kDa) product and a splice variant (∼30 kDa) in BCC tissues. Kynurenine assays and qPCR were conducted to determine IDO enzymatic activity in hBCCs in vitro with CXCL11 supplementation, which has previously been shown to be required for the tumour cell growth. Addition of CXCL11 upregulated IDO2 and increased l-kynurenine concentration in a dose-dependent manner in hBCCs while normal primary keratinocytes exhibited no response. CONCLUSIONS: The expression of IDO at both mRNA and protein levels in hBCC tissues, the upregulation of IDO2 and the IDO-mediated l-kynurenine production in hBCCs with CXCL11 treatment suggest that functional IDO is synthesized by hBCC tumours and may be used as a method of immunoprotection during tumorigenesis. Also, IDO enzymatic activity may be modulated by CXCR3/CXCL11 signalling in BCCs.

Gut barrier disruption by an enteric bacterial pathogen accelerates insulitis in NOD mice.

AIMS/HYPOTHESIS: Increased exposure to enteric microbes as a result of intestinal barrier disruption is thought to contribute to the development of several intestinal inflammatory diseases; however, it less clear whether such exposure modulates the development of extra-intestinal inflammatory and autoimmune diseases. The goal of this study was to examine the potential role of pathogenic enteric microbes and intestinal barrier dysfunction in the pathogenesis of type 1 diabetes. METHODS: Using NOD mice, we assessed: (1) intrinsic barrier function in mice at different ages by measuring serum levels of FITC-labelled dextran; and (2) the impact on insulitis development of infection by strains of an enteric bacterial pathogen (Citrobacter rodentium) either capable (wild-type) or incapable (lacking Escherichia coli secreted protein F virulence factor owing to deletion of the gene [DeltaespF]) of causing intestinal epithelial barrier disruption. RESULTS: Here we demonstrate that prediabetic (12-week-old) NOD mice display increased intestinal permeability compared with non-obese diabetes-resistant and C57BL/6 mice. We also found that young (4-week-old) NOD mice infected with wild-type C. rodentium exhibited accelerated development of insulitis in concert with infection-induced barrier disruption. In contrast, insulitis development was not altered in NOD mice infected with the non-barrier-disrupting DeltaespF strain. Moreover, C. rodentium-infected NOD mice demonstrated increased activation and proliferation of pancreatic-draining lymph node T cells, including diabetogenic CD8(+) T cells, compared with uninfected NOD mice. CONCLUSIONS/INTERPRETATION: This is the first demonstration that a loss of intestinal barrier integrity caused by an enteric bacterial pathogen results in the activation of diabetogenic CD8(+) T cells and modulates insulitis.

Type 1 interferon signature in the scalp lesions of alopecia areata.

BACKGROUND: Autoimmune attack of the bulbar region of anagen phase hair follicles by CD8+ T cells and Th1 cytokines has been proposed to result in hair loss in alopecia areata (AA). The initiating stimuli are unknown. As interferon-alpha therapy may trigger AA, we propose that type 1 interferons are involved in the induction of disease. OBJECTIVES: To compare lesional scalp from patients with AA with scalp lesions of cutaneous diseases associated with local type 1 interferon-related protein expression. METHODS: Lesional scalp of patients with AA, discoid lupus erythematosus, lichen planopilaris and androgenetic alopecia was examined by immunohistochemistry for expression of the type 1 interferon-inducible myxovirus protein A (MxA), the chemokine receptor CXCR3, and the cytotoxic proteins granzyme B (GrB) and T-cell intracytoplasmic antigen 1 (TiA-1). RESULTS: MxA was expressed in the intradermal and subcutaneous compartments of the hair follicle including sebaceous glands in inflammatory AA similar to lesions of cicatricial alopecia (discoid lupus erythematosus, lichen planopilaris) but not in the epidermal compartment of AA, and not at all in noninflammatory AA or androgenetic alopecia. The location of CXCR3-expressing cells correlated with MxA expression. The inflammatory cells around the hair follicle in AA included a lower number of GrB+ and TiA-1+ cells compared with cicatricial alopecia and demonstrated predominant TiA-1+ expression. CONCLUSIONS: We demonstrate the expression of type 1 interferon-related proteins in the inflammatory lesions of AA. The distribution pattern of the interferon signature and cytotoxicity-associated proteins in AA differs from cicatricial alopecia.

Cutaneous lupus erythematosus: recent lessons from animal models.

Cutaneous lupus erythematosus (CLE) may present as a clinically heterogeneous group of lupus-specific skin lesions that have common histopathological findings. Determination of the immunopathological sequence of events in this group of disorders has been challenging for dermatologists and immunologists but is vital for therapeutic targeting. We review animal models in which different aspects of immune alteration in CLE have been addressed. The MRL/lpr mouse develops spontaneous skin disease with some features of CLE. Study of this strain and related gene-manipulated strains has revealed roles for multiple cytokines, including interleukin (IL)-6, IL-18, and IL-21, in disease pathogenesis. A role for the growth factor colony stimulating factor 1 and the inflammatory protein high-mobility group box 1 has also been suggested. We discuss potential novel treatment options suggested by these models.

Neonatal beta-cell apoptosis: a trigger for autoimmune diabetes?

In neonatal rodents, the beta-cell mass undergoes a phase of remodeling that includes a wave of apoptosis. Using both mathematical modeling and histochemical detection methods, we have demonstrated that beta-cell apoptosis is significantly increased in neonates as compared with adult rats, peaking at approximately 2 weeks of age. Other tissues, including the kidney and nervous system, also exhibit neonatal waves of apoptosis, suggesting that this is a normal developmental phenomenon. We have demonstrated that increased neonatal beta-cell apoptosis is also present in animal models of autoimmune diabetes, including both the BB rat and NOD mouse. Traditionally, apoptosis has been considered a process that does not induce an immune response. However, recent studies indicate that apoptotic cells can do the following: 1) display autoreactive antigen in their surface blebs; 2) preferentially activate dendritic cells capable of priming tissue-specific cytotoxic T-cells; and 3) induce the formation of autoantibodies. These findings suggest that in some circumstances physiological apoptosis may, in fact, initiate autoimmunity. Initiation of beta-cell-directed autoimmunity in murine models appears to be fixed at approximately 15 days of age, even when diabetes onset is dramatically accelerated. Taken together, these observations have led us to hypothesize that the neonatal wave of beta-cell apoptosis is a trigger for beta-cell-directed autoimmunity.

A cytotoxic T lymphocyte clone can recognize the same naturally occurring self peptide in association with a self and nonself class I MHC protein.

The alloreactive CD8+ cytotoxic T lymphocyte (CTL) clone 2C was previously shown to recognize complexes made up of the class I MHC (MHC-I) molecule Ld and an octapeptide (LSPFPFDL, termed p2Ca) isolated from tissues of H-2d mice. Because peptide p2Ca has also been found in BALB.B (H-2b) mice, the strain from which clone 2C originated, the question arises as to whether these T cells can recognize peptide p2Ca in association with a self MHC protein of the H-2b haplotype. Here we show that 2C CTL do indeed recognize peptide p2Ca in association with Kb on the surface of H-2b cells or on transfected cells expressing Kb, but that an approximately 1000-fold higher concentration of this peptide is required to sensitize Kb+ than Ld+ target cells for lysis by 2C cells. However, the peptide's binding to Kb was not much weaker than to Ld, with only an approximately 10-fold difference in the respective equilibrium constants. These results predict that the T cell receptor (TcR) of clone 2C has a much lower intrinsic affinity for p2Ca-Kb complexes than for p2Ca-Ld complexes, and they provide some quantitative limits on the requirements for triggering T cell-mediated autoimmune reactivity.

Redefining cutaneous lupus erythematosus: a proposed international consensus approach and results of a preliminary questionnaire.

There is currently no uniform definition of cutaneous lupus erythematosus (CLE) upon which to base a study population for observational and interventional trials. A preliminary questionnaire was derived from and sent to a panel of CLE experts which demonstrated consensus agreement that (1) there is a need for new definitions for CLE (2) CLE is distinct from systemic lupus erythematosus and that a CLE grouping scheme should remain apart from current systemic lupus erythematosus schema (3) current CLE grouping schemes are inadequate around communication, prognostic information and to meet the needs of researchers, clinicians, patients and payers.

Immunosuppressive agents in dermatology. An update.

Azathioprine, cyclophosphamide, methotrexate, and cyclosporine are the immunosuppressive agents most commonly used by dermatologists. Azathioprine has a relatively good safety profile and is therefore often preferred for the treatment of chronic eczematous dermatitides and bullous disorders. Awareness of the role of genetic polymorphisms in its metabolism can increase the efficacy and safety of this drug. Cyclophosphamide is an antimetabolite that has a more rapid onset of immunosuppressive effect than azathioprine, but has significant short-term and long-term toxicity. It is of use in fulminant, life-threatening cutaneous disease. Methotrexate is an antimetabolite that has significant anti-inflammatory activity. Despite its hepatotoxicity, its role in inflammatory dermatoses is broadening. Likewise, the role of cyclosporine is being expanded. This drug has potent T-cell inhibitory effects secondary to interference with intracellular signal transduction. Given the evidence for cumulative renal toxicity, it currently has a role in the short-term treatment of refractory psoriasis and atopic dermatitis, as well as in select inflammatory dermatoses. Familiarity with disease-specific clinical efficacy, side-effect profile, and dosage allows the successful and judicious use of these drugs in dermatologic disorders.

Treatment options for localized scleroderma.

Localized scleroderma, or morphea, is a chronic disease that causes a thickening and induration of the skin. For plaque type morphea, the treatments of choice include super-potent corticosteroids and calcipotriol. For the more generalized forms, as well as the linear forms, UVA is currently the best therapeutic modality. Patients with localized scleroderma are managed by both rheumatologists and dermatologists. There is still much therapeutic uncertainty in this disease.

Treatment options for the cutaneous manifestations of systemic sclerosis.

Systemic Sclerosis is a multisystem disorder with vascular instability as a clinical hallmark. Treatment currently consists of recognition and management of end-organ damage. Dermatologists can assist in the management of these patients by facilitating early diagnosis, and treating cutaneous manifestations such as Raynaud's phenomenon, cutaneous calcinosis, and digital ulceration. New potentially disease-modifying therapies are now undergoing clinical trials.

PP065. dNK and dNK-CM mediated alterations of DNA methylation in extravillous cytotrophoblasts (EVTS).

INTRODUCTION: Placental DNA methylation is thought to be influenced by environmental exposure. Decidual natural killer cells (dNKs) directly contact with cytotrophoblasts in the early stage of pregnancy. dNKs may affect DNA methylation of extravillous cytotrophoblasts (EVTs) directly or indirectly through their secreted soluble factors. OBJECTIVES: Previously, we showed that EVT outgrowth and migration on collagen gel were restricted by exposure to dNK or dNK-derived conditioned medium (dNK-CM) (ref [1]). The aim of this study was to determine if EVT DNA methylation was altered by treating with dNK or dNK-CM. METHODS: Placental explants collected from 6 first-trimester healthy pregnancy terminations were cultured on a rat collagen gel model. Outgrowth EVTs from each subject were treated with medium or concordant dNK (trapped inside of hollow fibers) or dNK-CM. EVTs were harvested after 96-hour co-culturing and underwent DNA extraction. DNA methylation was quantified using the Infinium Human Methylation 450 BeadChip, which targets over 450,000 CpG sites in the human genome. Differential methylation was defined by having p<0.05 (Student's t-test) and average DNA methylation change >10% before and after treatments. Functional enrichment was assessed by gene ontology analysis with False Discovery Rate <10% defined as significantly enriched. RESULTS: Increased DNA methylation was observed for 360 loci and 572 loci by dNK or dNK-CM respectively, and decreased DNA methylation was shown for 62 loci and 188 loci by dNK or dNK-CM respectively. DNA methylation at 44 loci was altered by both dNK and dNK-CM. The common loci were overrepresented for associations with EVT differentiation, adhesion and migration. Examples of the relevant overlapped loci with increased DNA methylations were MYO15A and PRDM16 (PR domain zinc finger protein 16); and the overlapped loci with reduced DNA methylation were CDH9 and USP29 (ubiquitin specific protein 29). dNK but not dNK-CM reduced IL18 methylation and increased methylation on ITGAL (integrin, alpha L) and ITGB7. dNK-CM but not dNK reduced methylation of ITGAD and PCDH8 (protocadherin 8) and increased methylation of CDH4 and CDH6. CONCLUSION: DNA methylation of EVT was altered by exposure to surrounding dNK and their secreted soluble molecules. These results serve as a basis for further investigations on whether DNA methylation can mediate the changes in protein expression that influence EVT differentiation, adhesion and migration.

Decidual NK cell-derived conditioned medium enhances capillary tube and network organization in an extravillous cytotrophoblast cell line.

Extravillous cytotrophoblast (EVT) migration, invasion and endovascular differentiation are regulated by a variety of growth factors, cytokines and adhesion molecules. Decidual natural killer cells (dNK) and their secreted cytokines probably modulate these processes. In this study, we used dNK-derived conditioned medium (dNK-CM) to investigate whether or not (i) dNK-CM was able to enhance capillary tube and network formation of an EVT cell line, HTR8/SVneo, on Matrigel, (ii) PI3K/AKT pathway and p38 MAPK pathway activation were involved, and (iii) HTR8/SVneo surface ICAM-1 played a role in the process of HTR8/SVneo endovascular differentiation. The results demonstrated that HTR8/SVneo constitutively form 'vascular' tubes and networks after culture on Matrigel. dNK-CM enhanced and maintained tube and network formation, acquiring an endothelium-like angiogenic morphology followed by increased VEGF-C production. HTR8/SVneo cell expression level of VE-cadherin, PECAM-1, VCAM-1 and alphavbeta3 was unaltered by dNK-CM, whereas ICAM-1 expression level was increased. Anti-human ICAM-1 blocking antibody inhibited HTR8/SVneo migration and partially reversed dNK-CM-mediated enhancement of HTR8/SVneo tube and network formation. PI3K/AKT and p38 MAPK pathways were activated in dNK-CM-mediated enhancement of HTR8/SVneo tube and network formation. The PI3K/AKT and p38 MAPK pathway inhibitors (LY294002 and SB202190, respectively) decreased dNK-CM-stimulated ICAM-1 induction, HTR8/SVneo migration, and reversed tube and network formation. The results suggest that dNK cell-secreted growth factors and cytokines participate in the regulation of HTR8/SVneo endothelium-like tube formation. Adhesion molecules, particularly ICAM-1, expressed on EVT may participate in the process. To our knowledge, this is the first report of a role for ICAM-1 in EVT angiogenesis, as previously reported for endothelial cells.

A comparative study of calcipotriol and anthralin for chronic plaque psoriasis in a day care treatment center.

Eighteen patients with symmetric plaque-type psoriasis were recruited for an open, controlled, bilateral half-body comparison study to evaluate the efficacy of calcipotriol/tar/UVB vs. anthralin/tar/UVB in a day care treatment setting. No patient had been on systemic antipsoriatic agents for at least 3 months prior to enrolment. One half-body was arbitrarily assigned to treatment with gradually increasing concentrations of anthralin as tolerated. The other half-body received calcipotriol ointment twice daily. Both sides received UVB and additional coal tar distillate in accordance with our standard day care regimen. Patients who were admitted to the day care program attended the clinic for UVB, anthralin, and calcipotriol on weekdays for two consecutive weeks. Anthralin was applied to psoriatic plaques on one side in the following fashion: anthralin 0.1% with salicylic acid 3% in zinc oxide paste on days 1 and 2; anthralin 0.2% with salicylic acid 3% in zinc oxide paste on days 3-5; anthralin 1% with salicylic acid 3% in hydrophilic petrolatum for 60 min on days 8-10 to thicker lesions; and anthralin 2% with salicylic acid 3% in hydrophilic petrolatum for 60 min on day 11 to thicker lesions. On the contralateral side, calcipotriol ointment 0.05 microgram/mL (Leo Pharmaceuticals, Ajax, Ontario) was applied to lesions twice daily. No anthralin or calcipotriol was applied on weekends. All patients applied coal tar oil 50% (Doak Oil Forte, Trans CanaDerm, St-Laurent, Québec, equivalent to 5% coal tar distillate) with salicylic acid 5% in hydrophilic petrolatum to their lesions at home in the evenings and on weekends. UVB (FSX72T12 lamps, National Biologic Corporation, Twinsburg, Ohio) was administered twice daily on weekdays in increasing doses as tolerated (to erythema) prior to the application of the topical medications. No trial medications were applied to the face, scalp, or genital regions. For clinical evaluation, the standard Psoriasis Activity and Severity Index (PASI) score was modified by splitting the score for area under 10%; the modified score (mPASI) for an area of coverage of 1%-4% was 0.5 and for an area of 5%-9% was 1. The head and neck area was excluded from the analysis since neither anthralin nor calcipotriol was used at these sites. Each half-body was considered to represent 100% in the area score determination. The maximum modified score for each side was 64.8 (vs. 72 in the standard PASI scoring system). Clinical evaluations were completed at days 0 (baseline), 3, 7, 10, and 42. The primary end-point was day 10. On day 10, patients were asked to compare the calcipotriol ointment to the anthralin on a five-point scale in terms of efficacy and irritancy and to state their future preferred treatment modality. Following discharge from day care, patients were continued on outpatient UVB and tar treatments three times weekly and asked to return for a repeat clinical assessment after 4 weeks (day 42). Blood samples taken prior to treatment and at day 10 were analyzed for serum calcium. Comparisons of treatment efficacy were based on changes in the mPASI scores from onset of treatment to day 10, as well as on the corresponding percentage changes. Analyses were carried out using the Wilcoxon test. Subjective patient comparisons of effectiveness and irritancy, as well as patient preference, were tested for equiprobability using the chi-square goodness-of-fit test with an examination of the adjusted residuals.