Human Bullous Pemphigoid Antigen 2 Transgenic Skin Elicits Specific IgG in Wild-Type Mice

Bullous pemphigoid antigen 2 (BPAG2) is targeted by autoantibodies in patients with bullous pemphigoid (BP), and absent in patients with one type of epidermolysis bullosa (OMIM #226650). A keratin 14 promoter construct was used to produce transgenic (Tg) mice appropriately expressing human BPAG2 (hBPAG2) in murine epidermal basement membrane (BM). Grafts of Tg skin placed on gender-matched, syngeneic wild type (Wt) or major histocompatibility complex I (MHC I)−/− mice elicited IgG that bound human epidermal BM and BPAG2. Production of such IgG in grafted mice was prompt (detectable within 16±2 days), robust (titer ≥1,280), durable (present ≥380 days), and correlated with the involution and loss of Tg skin grafts. MHC II−/− mice grafted with Tg skin did not develop anti-hBPAG2 IgG or graft loss indicating that MHC II:CD4+ T cell interactions were crucial for these responses. Tg skin grafts on Wt mice developed neutrophil-rich infiltrates, dermal edema, subepidermal blisters, and deposits of immunoreactants in epidermal BM. This model shows fidelity to alterations seen in patients with BP, has relevance to immune responses that may arise in patients with epidermolysis bullosa following BPAG2 gene replacement, and can be used to identify interventions that may block production of IgG against proteins in epidermal BM

MicroRNA-135b Regulates Leucine Zipper Tumor Suppressor 1 in Cutaneous Squamous Cell Carcinoma

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin malignancy and it presents a therapeutic challenge in organ transplant recipient patients. Despite the need, there are only a few targeted drug treatment options. Recent studies have revealed a pivotal role played by microRNAs (miRNAs) in multiple cancers, but only a few studies tested their function in cSCC. Here, we analyzed differential expression of 88 cancer related miRNAs in 43 study participants with cSCC; 32 immunocompetent, 11 OTR patients, and 15 non-lesional skin samples by microarray analysis. Of the examined miRNAs, miR-135b was the most upregulated (13.3-fold, 21.5-fold; p=0.0001) in both patient groups. Similarly, the miR-135b expression was also upregulated in three cSCC cell lines when evaluated by quantitative real-time PCR. In functional studies, inhibition of miR-135b by specific anti-miR oligonucleotides resulted in upregulation of its target gene LZTS1 mRNA and protein levels and led to decreased cell motility and invasion of both primary and metastatic cSCC cell lines. In contrast, miR-135b overexpression by synthetic miR-135b mimic induced further down-regulation of LZTS1 mRNA in vitro and increased cancer cell motility and invasiveness. Immunohistochemical evaluation of 67 cSCC tumor tissues demonstrated that miR-135b expression inversely correlated with LZTS1 staining intensity and the tumor grade. These results indicate that miR-135b functions as an oncogene in cSCC and provide new understanding into its pathological role in cSCC progression and invasiveness

Sequential Intramolecular Epitope Spreading of Humoral Responses to Human BPAG2 in a Transgenic Model

Bullous pemphigoid (BP) is a subepidermal autoimmune disease characterized by a humoral response to an epidermal basement membrane (BM) component, BP antigen 2 (BPAG2). BP patients have IgG autoantibodies against an immunodominant BPAG2 extracellular domain termed NC16A as well as additional epitopes located both in the intracellular and extracellular domains (ICD and ECD, respectively) of this autoantigen. To study the evolution of humoral responses to BPAG2, sequential serum samples obtained from C57BL/6Ncr mice grafted with otherwise syngeneic skin from transgenic mice expressing human BPAG2 (hBPAG2) in epidermal BM were studied for IgG reactivity to seven ECD and ICD hBPAG2 epitopes. All grafted mice developed specific IgG against hBPAG2 ECD and ICD epitopes. In seven of eight mice, anti-hBPAG2 IgG was initially directed against ECD epitopes; in six mice, humoral responses subsequently targeted additional ECD and ICD BPAG2 epitopes. In contrast to IgG specific for ECD epitopes, IgG against ICD epitopes was present at lower levels, detectable for shorter periods, and non-complement fixing. Interestingly, the appearance of IgG directed against ICD epitopes correlated with the development of graft loss in this experimental model. These studies provide a comprehensive and prospective characterization of the evolution of humoral immune responses to hBPAG2 in vivo

miR-135b overexpression increases cell migration and invasion in MET1, PM1 and MET4 cSCC cell lines.

<p>A, B) miR-135b mimic significantly increases cSCC cell migration in the wound scratch assay within 24 hours post-transfection. C, D) miR-135b mimic significantly increases cSCC cell invasion in the Matrigel transwell invasion assay in PM1 cell line. * indicate p values less than 0.01. A) <i>Bar = 100um</i>; B) <i>Bar = 200um</i>.</p

Immunohistochemical analysis of LZTS1 protein levels in cells transfected with miR-135b inhibitor.

<p>A) cSCC cell lines transfected with miR-135b inhibitor showed increased LZTS1staining intensity 48 hours post-transfection when compared to negative control. B) Mean fluorescent intensity per cell was quantified using NIH image J program in cell transfected with miR-135b inhibitor and negative control oligonucleotiides. * indicates p values less than 0.001. <i>Bar = 1000um</i>.</p