Diagnostic Techniques in Autoimmune Blistering Diseases.

Autoimmune blistering diseases (AIBD) comprise a heterogeneous group of uncommon disorders of the skin and mucous membranes, characterised by antibodies targeting structural proteins within epithelial tissue and the underlying basement membrane. There can be significant overlap in clinical presentation of these diseases and accurate diagnosis relies on the detection and characterisation of relevant autoantibodies. Immunofluorescence provides the gold-standard diagnostic tool for these diseases, identifying both tissue-bound autoantibodies in biopsy material using direct immunofluorescence and circulating antibodies in serum through indirect immunofluorescence. Following advances in the identification and subsequent characterisation of numerous antigenic targets in these diseases, the development of antigen-specific tests, in particular, enzyme-linked immunosorbent assays on serum specimens, has provided a third key tool to not only identify, but also quantify AIBD autoantibodies. This quantification has proven particularly useful in monitoring disease activity and informing clinical management decisions. Accurate diagnosis of these diseases is important since optimal treatment strategies differ between them and, prognostically, some diagnoses are associated with an increased risk of malignancy. This review outlines the molecular pathology underlying the major AIBD and describes how the three principal techniques can be used in combination, to provide best practice for diagnosis and treatment monitoring.

Epidermolysis bullosa acquisita: a case series of three paediatric patients.

Epidermolysis bullosa acquisita is a highly uncommon condition in the paediatric population. This article describes three children with this disease, different clinical presentation and management. It also reviews the most relevant articles on this topic.

Autoantibody Detection for Diagnosis in Direct Immunofluorescence-Negative Mucous Membrane Pemphigoid: Ocular and Other Sites Compared.

PURPOSE: To assess whether a panel of serum pemphigoid autoantibody tests could be used to confirm an immunopathologic diagnosis of mucous membrane pemphigoid (MMP) in direct immunofluorescent negative (DIF-) MMP patients. DESIGN: Prospective cross-sectional study. PARTICIPANTS: Seventy-six patients with multisite MMP with 45 matched control participants. METHODS: Enzyme-linked immunosorbent assays (ELISAs) for BP180 and BP230 (MBL International), immunoglobulin A (IgA) A and immunoglobulin G indirect immunofluorescence (IIF) on human salt-split skin and the keratinocyte footprint assay for anti-laminin 332 antibodies. MAIN OUTCOME MEASURES: Sensitivity and specificity of autoantibody detection and significant differences for individual tests and test combinations for MMP involving different sites. RESULTS: All DIF- patients (24/73 [31.8%]) had either ocular-only disease or ocular involvement in multisite disease. Serum pemphigoid autoantibodies were detected in 29 of 76 MMP patients (38.2%) compared with 3 of 45 control participants (6.7%). Autoantibody reactivity detected by any 1 or more of the tests was present in 6 of 24 DIF- patients (25%) compared with 22 of 49 DIF positive (DIF+) patients (44.9%). Ocular-only MMP serum reactivity was not significantly different for any test or test combination compared with control participants, whereas DIF- multisite ocular MMP differed for 1 ELISA and 3 of 7 test combinations. By contrast, for DIF+ nonocular MMP patients, all the individual tests, apart from IgA IIF, and all test combinations were significantly different compared with those for control participants. For the entire MMP cohort, the sensitivity of all individual tests was low, having a maximum of 21.05% for BP180 reactivity but increasing to 38.16% for an optimal test combination. Disease activity was associated strongly with positive serologic findings. CONCLUSIONS: Pemphigoid serum autoantibody tests did not provide immunopathologic evidence of MMP in ocular-only MMP patients but showed limited value in DIF- multisite ocular MMP patients. The requirement for immunopathologic confirmation of MMP by autoantibody detection is inappropriate for DIF- ocular-only MMP patients, resulting in missed diagnoses, delayed therapy, and poor outcomes. Alternative diagnostic criteria for ocular-only MMP are required to exclude the other causes of scarring conjunctivitis until more sensitive and specific immunopathologic tests become available.

A homozygous nonsense mutation within the dystonin gene coding for the coiled-coil domain of the epithelial isoform of BPAG1 underlies a new subtype of autosomal recessive epidermolysis bullosa simplex.

Epidermolysis bullosa (EB) is a group of autosomal dominant and recessive blistering skin diseases in which pathogenic mutations have been reported in 13 different genes encoding structural proteins involved in keratinocyte integrity, as well as cell-matrix or cell-cell adhesion. We now report an inherited skin fragility disorder with a homozygous nonsense mutation in the dystonin gene (DST) that encodes the coiled-coil domain of the epithelial isoform of bullous pemphigoid antigen 1, BPAG1-e (also known as BP230). The mutation, p.Gln1124X, leads to the loss of hemidesmosomal inner plaques and a complete absence of skin immunostaining for BPAG1-e, as well as reduced labeling for plectin, the beta4 integrin subunit, and for type XVII collagen. The 38-year-old affected individual has lifelong generalized trauma-induced spontaneous blisters and erosions, particularly around the ankles. In addition, he experiences episodic numbness in his limbs, which started at the age of 37 years. These neurological symptoms may also be due to DST gene mutation, although he has a concomitant diagnosis of CADASIL (cerebral arteriopathy, autosomal dominant, with subcortical infarcts and leukoencephalopathy), a cerebral small-vessel arteriopathy, which thus complicates the genotype-phenotype interpretation. With regard to skin blistering, the clinicopathological findings expand the molecular basis of EB by identifying BPAG1-e pathology in a new form of autosomal recessive EB simplex.

Loss-of-function FERMT1 mutations in kindler syndrome implicate a role for fermitin family homolog-1 in integrin activation.

Kindler syndrome is an autosomal recessive disorder characterized by skin atrophy and blistering. It results from loss-of-function mutations in the FERMT1 gene encoding the focal adhesion protein, fermitin family homolog-1. How and why deficiency of fermitin family homolog-1 results in skin atrophy and blistering are unclear. In this study, we investigated the epidermal basement membrane and keratinocyte biology abnormalities in Kindler syndrome. We identified altered distribution of several basement membrane proteins, including types IV, VII, and XVII collagens and laminin-332 in Kindler syndrome skin. In addition, reduced immunolabeling intensity of epidermal cell markers such as beta1 and alpha6 integrins and cytokeratin 15 was noted. At the cellular level, there was loss of beta4 integrin immunolocalization and random distribution of laminin-332 in Kindler syndrome keratinocytes. Of note, active beta1 integrin was reduced but overexpression of fermitin family homolog-1 restored integrin activation and partially rescued the Kindler syndrome cellular phenotype. This study provides evidence that fermitin family homolog-1 is implicated in integrin activation and demonstrates that lack of this protein leads to pathological changes beyond focal adhesions, with disruption of several hemidesmosomal components and reduced expression of keratinocyte stem cell markers. These findings collectively provide novel data on the role of fermitin family homolog-1 in skin and further insight into the pathophysiology of Kindler syndrome.

IL-18 is a key proximal mediator of contact hypersensitivity and allergen-induced Langerhans cell migration in murine epidermis.

Langerhans cells (LC) migrate rapidly from epidermis to lymph node following epicutaneous application of antigen. In this study, we have explored the role of IL-18, a cytokine with structural similarities to IL-1 beta, in murine LC migration and contact hypersensitivity (CHS), which to oxazolone (OX) and 2-4,dinitrofluorobenzene (DNFB) was suppressed significantly in IL-18 knockout (IL-18-/-) mice and could be rescued by local intradermal administration of IL-18 prior to sensitization, suggesting that the defect in these mice was in the afferent phase of CHS. To determine the effect of IL-18 on LC migration, mice were treated topically with OX or DNFB, and remaining LC numbers were assessed. A significant decline in remaining epidermal LC occurred in wild-type (WT) mice but did not occur in IL-18-/- mice. Sodium lauryl sulfate, a nonantigenic LC migratory stimulus, induced equivalent LC migration in IL-18-/- and WT mice. In IL-18-/- mice, IL-1 beta and TNF-alpha were equally able to mobilize LC from epidermis, indicating that migration in response to these cytokines is not dependent on IL-18 and suggesting that IL-18 acts upstream of these cytokines in the initiation of antigen-induced LC migration. Moreover, IL-1 beta but not IL-18 was able to rescue the defective CHS response observed in caspase-1-/- mice, which have no functional IL-1 beta or IL-18. These data indicate that IL-18 is a key proximal mediator of LC migration and CHS, acting upstream of IL-1 beta and TNF-alpha, and may play a central role in regulation of cutaneous immune responses.

The psoriatic transcriptome closely resembles that induced by interleukin-1 in cultured keratinocytes: dominance of innate immune responses in psoriasis.

Psoriasis has been considered an autoimmune, T cell-mediated disorder in which adaptive immune responses predominate over those of non-antigen-specific innate immunity. To test this hypothesis, we profiled the transcriptome of psoriatic tissue and compared the data with that from cultured human keratinocytes exposed to the proinflammatory cytokine interleukin (IL)-1alpha and the Th1 cytokine interferon-gamma. When compared with patient-matched, nonlesional skin biopsies, psoriatic samples exhibited regulation of 90 transcripts including several members of the epidermal differentiation complex, molecules with antimicrobial activity, and hyperproliferation-associated keratins. Stimulation of keratinocytes with interferon-gamma resulted in regulation of 252 transcripts, with particularly strong expression of the CXCR3-binding ligands CXCL9, -10, and -11 and class II major histocompatibility complex genes, primarily those of the HLA-DR and -DP families. In contrast, the transcriptome resulting from exposure of keratinocytes to IL-1alpha elicited differences in just 19 transcripts, particularly genes within the epidermal differentiation complex and antimicrobial molecules, including PI3 and DEFB4. Major differences between the two keratinocyte transcriptomes were exhibited with only five induced IL-1alpha transcripts also regulated in the interferon-gamma set. Unexpectedly, there was a high correlation between psoriatic lesional tissue and the IL-1alpha transcriptome. These findings suggest that the inflammatory milieu in the epidermal microenvironment in psoriasis is more likely dependent on evolutionarily ancient cytokines such as IL-1, rather than those of the adaptive immune response.

Interleukin-1: a key inflammatory mediator in psoriasis?

The pro-inflammatory cytokine interleukin-1 (IL-1) is constitutively expressed by keratinocytes in vivo and has been shown to be expressed in psoriatic lesional skin. To determine what role the IL-1 system might contribute to the inflammatory process in psoriasis, semi-quantitative RT-PCR and cRNA microarray studies were performed on biopsies excised from lesional and non-lesional skin. Whilst IL-1alpha mRNA levels showed a reduction in lesional skin in a subset of patients, steady state IL-1beta mRNA was increased markedly. Neither of the two IL-1 receptor transcripts nor total IL-1 receptor antagonist exhibited major changes within the lesion. Expression of the IL-1-induced chemokine IL-8 was only observed in lesional epidermis. Functional genomic experiments comparing transcriptome profiles derived from psoriatic lesional skin and IL-1 stimulated keratinocytes demonstrated a striking level of overlap. Taken together, these data suggest that IL-1 is likely to be an important mediator in the initiation and maintenance of psoriatic plaques and may represent an attractive therapeutic target.

Counter-regulation of interleukin-1alpha (IL-1alpha) and IL-1 receptor antagonist in murine keratinocytes.

Interleukin-1alpha (IL-1alpha) is a potent proinflammatory cytokine constitutively expressed by keratinocytes, which also synthesize a specific inhibitor of IL-1 activity, intracellular IL-1 receptor antagonist (IL-1ra). Although homeostatic regulation of the IL-1 system in keratinocytes has long been suspected, there is currently little evidence for this. To explore this issue, the PAM212 murine keratinocyte cell line was exposed to increasing concentrations of either IL-1alpha or IL-1ra and the opposing ligand was assessed by ELISA. Release of IL-1ra was induced following stimulation by murine IL-1alpha in a concentration-dependent manner and, conversely, IL-1ra stimulation increased IL-1alpha release. To determine whether a similar homeostatic circuit operates in vivo, epidermis from transgenic mice in which overexpression of IL-1alpha or IL-1ra was targeted to keratinocytes was analyzed. Epidermal sheets derived from IL-1alpha transgenic mice released eight times more IL-1ra than those from wild-type mice following ex vivo culture and similarly, IL-1alpha release was increased 3-4-fold in epidermal sheets derived from IL-1ra transgenic epidermis, Use of specific neutralizing antibodies against type I and type II IL-1 receptors indicated that the counter-regulation mechanism is mediated extracellularly through the type I IL-1 receptor alone. Taken together, these observations provide the first demonstration of mutual counter-regulation of IL-1 receptor ligands in keratinocytes.