table_1.docx

Introduction<p>Rituximab (RTX) is a monoclonal antibody targeting CD20, a transmembrane protein expressed on B cells, causing B cell depletion. RTX has shown great efficacy in studies of pemphigus vulgaris, but data of pemphigoid diseases are limited.</p>Objective<p>To assess the effectiveness and safety of RTX in pemphigoid diseases.</p>Methods<p>The medical records of 28 patients with pemphigoid diseases that were treated with RTX were reviewed retrospectively. Early and late endpoints, defined according to international consensus, were disease control (DC), partial remission (PR), complete remission (CR), and relapses. Safety was measured by reported adverse events.</p>Results<p>Patients with bullous pemphigoid (n = 8), mucous membrane pemphigoid (n = 14), epidermolysis bullosa acquisita (n = 5), and linear IgA disease (n = 1) were included. Treatment with 500 mg RTX (n = 6) or 1,000 mg RTX (n = 22) was administered on days 1 and 15. Eight patients received additional 500 mg RTX at months 6 and 12. Overall, DC was achieved in 67.9%, PR in 57.1%, and CR in 21.4% of the cases. During follow-up, 66.7% patients relapsed. Repeated treatment with RTX led to remission (PR or CR) in 85.7% of the retreated cases. No significant difference in response between pemphigoid subtypes was found. IgA-dominant cases (n = 5) achieved less DC (20 vs. 81.3%; p = 0.007), less PR (20 vs. 62.5%; p = 0.149), and less CR (0 vs. 18.8%; p = 0.549) compared to IgG-dominant cases (n = 16). Five severe adverse events and three deaths were reported. One death was possibly related to RTX and one death was disease related.</p>Conclusion<p>RTX can be effective in recalcitrant IgG-dominant pemphigoid diseases, however not in those where IgA is dominant.</p

Table_2_Keratinocyte Binding Assay Identifies Anti-Desmosomal Pemphigus Antibodies Where Other Tests Are Negative.docx

<p>The serological diagnosis of pemphigus relies on the detection of IgG autoantibodies directed against the epithelial cell surface by indirect immunofluorescence (IIF) on monkey esophagus and against desmoglein 1 (Dsg1) and Dsg3 by ELISA. Although being highly sensitive and specific tools, discrepancies can occur. It is not uncommon that sera testing positive by ELISA give a negative result by IIF and vice versa. This brings diagnostic challenges wherein pemphigus has to be ascertained or ruled out, especially when no biopsy is available. We utilized the ability of anti-Dsg3 and anti-Dsg1 IgG to bind in specific desmosomal patterns to living cells to investigate these discrepancies between IIF and ELISA. Living cultured primary normal human keratinocytes were grown under differentiating conditions to induce adequate expression of Dsg1 and Dsg3, incubated with patient serum for 1 h, and then stained to visualize bound IgG. We investigated two different groups; sera from patients with a positive direct immunofluorescence (DIF) and inconsistent serological findings (n = 43) and sera with positive ELISA or IIF but with negative DIF (n = 60). As positive controls we used 50 sera from patients who fulfilled all diagnostics criteria, and 10 sera from normal human subjects served as negative controls. In the DIF positive group, IgG from 39 of the 43 sera bound to the cells in a desmosomal pattern while in the DIF negative group none of the 60 sera bound to the cells. This shows that for pemphigus patients, ELISA and IIF can be negative while anti-desmosomal antibodies are present and vice versa that ELISA and IIF can be positive in non-pemphigus cases. In absence of a biopsy for DIF, such findings may lead to misdiagnosis.</p

table_1_Keratinocyte Binding Assay Identifies Anti-Desmosomal Pemphigus Antibodies Where Other Tests Are Negative.docx

<p>The serological diagnosis of pemphigus relies on the detection of IgG autoantibodies directed against the epithelial cell surface by indirect immunofluorescence (IIF) on monkey esophagus and against desmoglein 1 (Dsg1) and Dsg3 by ELISA. Although being highly sensitive and specific tools, discrepancies can occur. It is not uncommon that sera testing positive by ELISA give a negative result by IIF and vice versa. This brings diagnostic challenges wherein pemphigus has to be ascertained or ruled out, especially when no biopsy is available. We utilized the ability of anti-Dsg3 and anti-Dsg1 IgG to bind in specific desmosomal patterns to living cells to investigate these discrepancies between IIF and ELISA. Living cultured primary normal human keratinocytes were grown under differentiating conditions to induce adequate expression of Dsg1 and Dsg3, incubated with patient serum for 1 h, and then stained to visualize bound IgG. We investigated two different groups; sera from patients with a positive direct immunofluorescence (DIF) and inconsistent serological findings (n = 43) and sera with positive ELISA or IIF but with negative DIF (n = 60). As positive controls we used 50 sera from patients who fulfilled all diagnostics criteria, and 10 sera from normal human subjects served as negative controls. In the DIF positive group, IgG from 39 of the 43 sera bound to the cells in a desmosomal pattern while in the DIF negative group none of the 60 sera bound to the cells. This shows that for pemphigus patients, ELISA and IIF can be negative while anti-desmosomal antibodies are present and vice versa that ELISA and IIF can be positive in non-pemphigus cases. In absence of a biopsy for DIF, such findings may lead to misdiagnosis.</p

Table_3_Keratinocyte Binding Assay Identifies Anti-Desmosomal Pemphigus Antibodies Where Other Tests Are Negative.docx

<p>The serological diagnosis of pemphigus relies on the detection of IgG autoantibodies directed against the epithelial cell surface by indirect immunofluorescence (IIF) on monkey esophagus and against desmoglein 1 (Dsg1) and Dsg3 by ELISA. Although being highly sensitive and specific tools, discrepancies can occur. It is not uncommon that sera testing positive by ELISA give a negative result by IIF and vice versa. This brings diagnostic challenges wherein pemphigus has to be ascertained or ruled out, especially when no biopsy is available. We utilized the ability of anti-Dsg3 and anti-Dsg1 IgG to bind in specific desmosomal patterns to living cells to investigate these discrepancies between IIF and ELISA. Living cultured primary normal human keratinocytes were grown under differentiating conditions to induce adequate expression of Dsg1 and Dsg3, incubated with patient serum for 1 h, and then stained to visualize bound IgG. We investigated two different groups; sera from patients with a positive direct immunofluorescence (DIF) and inconsistent serological findings (n = 43) and sera with positive ELISA or IIF but with negative DIF (n = 60). As positive controls we used 50 sera from patients who fulfilled all diagnostics criteria, and 10 sera from normal human subjects served as negative controls. In the DIF positive group, IgG from 39 of the 43 sera bound to the cells in a desmosomal pattern while in the DIF negative group none of the 60 sera bound to the cells. This shows that for pemphigus patients, ELISA and IIF can be negative while anti-desmosomal antibodies are present and vice versa that ELISA and IIF can be positive in non-pemphigus cases. In absence of a biopsy for DIF, such findings may lead to misdiagnosis.</p

Quantitative estimates and calculated values in our developmental model of the skin.

<p>Quantitative estimates and calculated values in our developmental model of the skin.</p

Revertant mosaicism in genetic diseases other than epidermolysis bullosa.

<p>Revertant mosaicism in genetic diseases other than epidermolysis bullosa.</p

A selective growth advantage of revertant cells is supported by the patterns of the revertant skin patches.

<p>(A) Dorsal forearm of an 8-year old boy (EB134-01) with generalized intermediate junctional EB due to the compound heterozygous <i>COL17A1</i> mutations c.[1260del];[3496_3497del], p.[Thr421Leufs*72];[Ser1166Leufs*6] showing a revertant patch (indicated by blue line). (B) The lines of Blaschko on the dorsal forearm as deduced by Blaschko and Happle [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192994#pone.0192994.ref049" target="_blank">49</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192994#pone.0192994.ref050" target="_blank">50</a>]. (C) The lines of Blaschko projected on the revertant patch on the dorsal forearm of patient EB134-01. The revertant patch has clearly exceeded the boundaries of the Blaschko lines and has grown into adjacent Blaschko line segments. The parents of patient EB134-01 have given written informed consent (as outlined in PLOS consent form) to publish this photograph.</p

Revertant mosaicism in epidermolysis bullosa.

<p>Revertant mosaicism in epidermolysis bullosa.</p

The smallest revertant patch observed in any of our patients.

<p>Photograph of the revertant patch on the dorsal middle finger of patient EB093-01 (deceased) who had generalized intermediate junctional EB due to compound heterozygous <i>COL17A1</i> mutations c.[3676C>T];[4319dup], p.[Arg1226*];[Gly1441Trpfs*14] [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192994#pone.0192994.ref009" target="_blank">9</a>]. Note stitched biopsy site for confirmation of type XVII collagen re-expression. The size of the patch was approximately 2 cm² and it allowed him to wear a wedding ring. Based on this, we concluded revertant patches should be ~1 cm² minimum in order to be clinically recognizable.</p

Relative numbers of MLVF types detected in the chronic wounds of five EB patients.

<p>A, The percentages of different <i>S. aureus</i> MLVF types detected in individual chronic wounds from patients 1, 62, 63 and 64 at one particular time point are indicated by bar diagrams. Different MLVF types are marked in black, white or grey shading. Note that, for matters of simplicity, the MLVF type numbers were arbitrarily attributed to different <i>S. aureus</i> types isolated from individual patients. Hence, distinct <i>S. aureus</i> types isolated from different patients can have the same type number. B, percentages of different <i>S. aureus</i> MLVF types detected in chronic foot and breast wounds from patient 14. Replica plating of used bandages was performed thrice at two-weekly intervals. Black bars mark the first, white bars the second and grey bars the third time point of sampling.</p