Con el objeto de estudiar anticuerpos de virus oral, genital, citomegálico y Epstein-Barr en el suero de pacientes con carcinomas de cuello uterino, condiloma acuminado y en controles de Cali y Estados Unidos, se practicaron pruebas de inmunoflorescencia indirecta. No se vio diferencia significativa en la prevalencia de anticuerpos tardíos de virus oral y citomegálico entres los grupos estudiados. Se encontraron títulos más altos contra virus genital en los 3 grupos de Cali que en el grupo control de Estados Unidos. También hubo títulos altos de antígenos tardíos y tempranos contra Epstein-Barr en los mismos 3 grupos. Se discute el significado de estos hallazgos.

Prostate stem cells are thought to be responsible for generation of all prostate epithelial cells and for tissue maintenance. The lineage relationship between basal and luminal cells in the prostate is not well clarified. We developed a mouse model to trace cell fate and a mouse model with a slowly cycling cell label to provide insight into this question. The results obtained indicate that putative mouse prostate stem cells are likely to reside in the basal layer.

Abstract Caspase recruitment domain-containing protein 14 mutations are the first monogenic defect causing psoriasis and pityriasisi rubra pilaris (PRP), linking psoriasis and PRP into one disease spectrum. Interestingly the disease responds well to the treatment with anti-p40 antibody (Ustekinumab) blocking IL-12 and IL-23. The mutant protein is mainly expressed in keratinocytes, leading us to explore whether keratinocyte dysfunction triggers the inflammatory phenotype through the recruitment of hematopoietic cells to the skin. CAR14 mutant keratinocytes produced high level of IL-8 to Toll-like receptor 3, 5, 9, IL-1a/b, IL-36, and TNF stimulation than control cells. IL-17A synergizes with Poly IC, Flagellin, and TNF, but not with IL-12/23, suggesting that Ustekinumab may not directly block keratinocytes but indirectly through inhibit IL-17A produced from likely hematopoietic cells. Using siRNA knock down assay of CMB (CARD-MALT1-BCL10) complex, we show that IL-17A induced IL-8 production is MALT1-dependent; thus raising the question what is the role of CARD14 in IL-17 mediated signal. Our data suggests that while IL-12/23 does not activate keratinocytes, IL-17A is a potent activator and even synergistically with other cytokine stimulant from hematopoietic cells during skin inflammation.

The isolation and characterization of living human epithelial stem cells is difficult because distinguishing cell surface markers have not been identified with certainty. Side population keratinocytes (SP-KCs) that efflux Hoechst 33342 fluorescent dye, analogous to bone marrow-derived side population (SP) hematopoietic stem cells, have been identified in human skin, but their potential to function as keratinocyte stem cells (KSCs) in vivo is not known. On the other hand, human keratinocyte populations that express elevated levels of beta1 and alpha6 integrins and are distinct from SP-KCs, which express low levels of integrins, may be enriched for KSCs based on reported results of in vitro cell culture assays. When in vitro assays were used to measure total cell output of human SP-KCs and integrin-bright keratinocytes, we could not document their superior long-term proliferative activity versus unfractionated keratinocytes. To further assess the KSC characteristics in SP-KCs and integrin-bright keratinocytes, we used an in vivo competitive repopulation assay in which bioengineered human epidermis containing competing keratinocyte populations with different human major histocompatibility (MHC) class I antigens were grafted onto immunocompromised mice, and the intrinsic MHC class I antigens are used to quantify expansion of competing populations. In these in vivo studies, human SP-KCs showed little competitive expansion in vivo and were not enriched for KSCs. In contrast, keratinocytes expressing elevated levels of alpha6 integrin and low levels of CD71 (alpha6-bright/CD71-dim) expanded over 200-fold during the 33-week in vivo study. These results definitively demonstrate that human alpha6-bright/CD71-dim keratinocytes are enriched with KSCs, whereas SP-KCs are not.

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 > or = 1,280), durable (present > or = 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.

Membrane proteins are responsible for many critical cellular functions and identifying cell surface proteins on different keratinocyte populations by proteomic approaches would improve our understanding of their biological function. The ability to characterize membrane proteins, however, has lagged behind that of soluble proteins both in terms of throughput and protein coverage. In this study, a membrane proteomic investigation of keratinocytes using a two-dimensional liquid chromatography (LC) tandem-mass spectrometry (MS/MS) approach that relies on a buffered methanol-based solubilization, and tryptic digestion of purified plasma membrane is described. A highly enriched plasma membrane fraction was prepared from newborn foreskins using sucrose gradient centrifugation, followed by a single-tube solubilization and tryptic digestion of membrane proteins. This digestate was fractionated by strong cation-exchange chromatography and analyzed using microcapillary reversed-phase LC-MS/MS. In a set of 1306 identified proteins, 866 had a gene ontology (GO) annotation for cellular component, and 496 of these annotated proteins (57.3%) were assigned as known integral membrane proteins or membrane-associated proteins. Included in the identification of a large number of aqueous insoluble integral membrane proteins were many known intercellular adhesion proteins and gap junction proteins. Furthermore, 121 proteins from cholesterol-rich plasma membrane domains (caveolar and lipid rafts) were identified.

We report the clinical and immunopathologic findings in a cohort of 35 patients with anti-epiligrin cicatricial pemphigoid (AECP). These patients have a mucosal predominant subepithelial blistering disease that is clinically indistinguishable from other forms of cicatricial pemphigoid. The mucosal surfaces of the mouth and eye are most commonly involved. The skin is also involved in most patients, but usually this is less severe than mucosal involvement. AECP is characterized by the binding of circulating IgG autoantibodies to the dermal side of 1M NaCl split human skin on indirect immunofluorescence microscopy. These IgG antibasement membrane autoantibodies target laminin 5, a heterotrimeric protein consisting of alpha3, beta3, and gamma2 subunits. IgG autoantibodies predominantly target the G domain within the alpha subunit. The presence of circulating IgG autoantibodies are specific for the diagnosis of AECP and are not seen in patients with other autoimmune blistering diseases or normal volunteers. Furthermore, we expand on data previously reported on the finding of an increased relative risk for solid cancer in patients with AECP, especially in the first year after blister onset. The majority of cancers documented in a cohort of 35 patients assembled over 12 years of study were adenocarcinomas that were at an advanced stage at their time of detection. This circumstance is thought to account for a high incidence of mortality among AECP patients who develop an associated cancer. AECP patients also demonstrate a significant risk for mortality as a consequence of treatment with systemic immunosuppressives. The current longitudinal study suggests that only a minority of AECP patients go into remission.

Abstract: Gene therapy of large genes (e.g. plectin and collagen genes) is hampered by size limitations for insertions of the currently used viral vectors. To reduce the size of these insertions spliceosome‐mediated RNA trans‐splicing (SMaRT™), which provides intron‐specific gene‐correction at the pre‐RNA level, can be an alternative approach. To test its applicability in skin gene therapy, SMaRT™ was used in the context of the 4003delTC mutation in the collagen XVII gene (COL17A1) causing generalized atrophic benign junctional epidermolysis bullosa. A β‐galactosidase (β‐gal) trans‐splicing assay system was established using intron 51 of COL17A1 as the target for trans‐splicing. In this system, intron 51 is flanked by the 5′exon and the 3′exon of the β‐gal gene, the latter containing two in‐frame stop codons. Cotransfection of a pre‐trans‐splicing molecule consisting of the binding domain of intron 51 and the 3′exon of β‐gal without the stop codons resulted in a 300‐fold increase of β‐gal activity compared to controls. A 2–3‐fold increase in efficiency was obtained through an elongation of the binding domains. Replacement of the complete 3′end of the COL17A1 gene was shown using a collagen XVII mini‐gene construct. The β‐gal assay was used in human keratinocytes to evaluate the influence of a keratinocyte‐specific spliceosome background. Reverse transcription polymerase chain reaction and β‐gal activity assay showed functional correction of the stop‐codons in cultured human keratinocytes and in an immortalized GABEB cell line harbouring the 4003delTC mutation. These results demonstrate that SMaRT™ is feasible in a keratinocyte‐specific context and therefore may be applied in skin gene therapy.

Anti-p200 pemphigoid is a recently defined subepidermal immunobullous disease. It is characterized by the binding of circulating IgG autoantibodies to the dermal side of 1 M NaCl split skin and by reactivity of these autoantibodies to a unique 200-kd antigen on immunoblot of dermal extract. On immunoelectron microscopic examination, these autoantibodies deposit at the lamina lucida-lamina densa interface. We describe the clinical, histologic, and immunopathologic features in a patient with anti-p200 pemphigoid, as well as his favorable response to treatment with systemic glucocorticosteroids and dapsone. (J Am Acad Dermatol 2002;46:786-9.)

Abstract: Generalized atrophic benign epidermolysis bullosa [GABEB (OMIM no. 226650)] is an inherited subepidermal blistering disease typically caused by null mutations in COL17A1, the gene encoding type XVII collagen. Studies of GABEB keratinocytes homozygous for 4003delTC showed that this 2 bp deletion results in markedly reduced COL17A1 transcripts due to nonsense mediated‐mRNA decay. To explore consequences of this null mutation in COL17A1 on the expression of other genes, RNA samples from reference GABEB and normal keratinocytes were profiled in comparative screens of microarrays of known cDNAs (n = 6180) and expressed sequence tags (ESTs) (n = 15 144). All comparative hybridization experiments were performed ≥ twice; data were quantitated by densitometry and analyzed using peak quantification statistical comparative analysis (P‐SCAN) software to identify differentially expressed genes. Representative genes found to be differentially expressed were verified using real‐time reverse transcription‐polymerase chain reaction (RT‐PCR). These experiments determined that expression of nonsense‐mediated mRNA decay trans‐acting factor (NMD‐F), the regulator of nonsense transcripts (i.e. the human homolog of the yeast Upf1 protein), was upregulated in GABEB keratinocytes. NMD‐F was subsequently found to be upregulated in cultured keratinocytes from other GABEB patients homozygous for 4003delTC. These findings indicate that the gene responsible for nonsense‐mediated mRNA decay is upregulated in keratinocytes known to eliminate mutant COL17A1 transcripts via this highly conserved mechanism.

It is not known whether patients with anti-epiligrin cicatricial pemphigoid (AECP) have an increased risk of malignancy. We calculated the expected numbers of cancers in a c hort of 35 such patients based on respective incidence rates for all cancers in the National Cancer Institute's Surveillance, Epidemiology, and End Results (NCI SEER) Registry. Ten patients in this cohort had solitary solid cancers; eight patients developed cancer after onset of AECP (seven within 14 months). The relative risk (RR) for cancer in this cohort was 6·8 (95% confidence intervals [CI]: 3·3-12·5). AECP seems to be associated with an increased relative risk for cancer.

Anti-epiligrin cicatricial pemphigoid (AECP) is a mucosal-predominant, subepithelial blistering disease characterized by IgG anti-basement membrane autoantibodies to laminin 5 (alpha3beta3gamma2). This and prior studies found that autoantibodies from most patients recognize the alpha-subunit of this laminin isoform. Accordingly, sera from 10 representative patients were tested against prokaryotic recombinants of this polypeptide in epitope mapping studies. cDNAs spanning the full length of the alpha-subunit were generated by PCR, directionally cloned into the pGEX-4T-3 vector, and expressed as glutathione-S-transferase fusion proteins of appropriate size and immunoreactivity. Sera from 9 of 10 AECP patients immunoblotted fusion proteins corresponding to subdomains G2, G3, G4, and G5 at the carboxyl terminus of the laminin 5 alpha-subunit. Serum from 1 patient (and that from normal volunteers) showed no reactivity to any fusion proteins; no sera bound recombinant glutathione-S-transferase alone. Immunoadsorption of patient sera with fusion proteins corresponding to the G domain substantially reduced basement membrane autoantibody titers. IgG from patients with this form of cicatricial pemphigoid recognize the portion of laminin 5 thought to play a key role in promoting keratinocyte adhesion to epidermal basement membrane.

Patients with one form of cicatricial pemphigoid have IgG antibasement membrane autoantibodies against laminin 5 (alpha3beta3gamma2). Although passive transfer of rabbit anti-laminin 5 IgG to neonatal mice has been shown to induce subepidermal blisters that mimic those in patients, it has not been possible to directly assess the pathogenic activity of human autoantibodies in this animal model because the latter do not bind murine skin. To address this question, a disease model in adult mice as well as SCID mice bearing human skin grafts was developed. Adult BALB/C mice challenged with rabbit anti-laminin 5 IgG developed, in a concentration-related fashion, erythema, erosions, and crusts surrounding injection sites, histologic evidence of noninflammatory, subepidermal blisters, and deposits of rabbit IgG and murine C3 in epidermal basement membranes. Anti-laminin 5 IgG also induced subepidermal blisters in: adult complement-, mast cell-, and immuno-deficient mice; adult BALB/C mice pretreated with dexamethasone; and human skin grafts on SCID mice. Alterations did not develop in matching controls challenged with identical amounts of purified normal rabbit IgG or bovine serum albumin. Using this adult mouse model, human skin grafts on SCID mice were challenged with purified IgG from patients with alpha subunit-specific, anti-laminin 5 autoantibodies, or normal controls. Patient (but not control) IgG induced epidermal fragility as well as noninflammatory, subepidermal blisters in grafted human (but not adjacent murine) skin. Moreover, whereas all mice that received patient autoantibodies had anti-laminin 5 IgG in their circulation, deposits of human IgG were present only in the epidermal basement membranes of grafts. Interestingly, these in situ and circulating autoantibodies were predominately of the IgG4 subclass. These studies demonstrate that human anti-laminin 5 autoantibodies are pathogenic in vivo and describe an animal model that can be used to define disease pathomechanisms and biologically important domains within this autoantigen.

BACKGROUND:Type XVII collagen promotes adhesion of basal keratinocytes to epidermal basement membrane, and is the target of disease in patients with certain inherited or acquired blistering diseases. Two forms of type XVII collagen are produced by cultured human keratinocytes: a 180-kDa full-length, transmembrane protein, and a recently identified 120-kDa soluble fragment that corresponds to its collagenous ectodomain.OBJECTIVES:We aimed to determine the incidence and pattern of reactivity of autoantibodies against the 180- and 120-kDa forms of type XVII collagen in sera from 40 patients with bullous pemphigoid (BP), pemphigoid gestationis or cicatricial pemphigoid (CP), as well as six patients with linear IgA dermatosis (LAD).METHODS:Various immunochemical techniques were used.RESULTS:These studies found that the 120-kDa fragment of type XVII collagen was bound by circulating autoantibodies in 13 of 38 patients with BP or CP and all six patients with LAD. While many pemphigoid sera had specific reactivity against one but not both forms of this protein, autoantibodies from patients with LAD bound only the soluble ectodomain.CONCLUSIONS:These findings are consistent with the presence of both neoepitopes and cross-reactive epitopes on the ectodomain of type XVII collagen. The finding that sera from patients with LAD showed specific reactivity to epidermal basement membrane suggests that such neoepitopes are present in human skin and that their targeting by autoantibodies may contribute to disease pathogenesis.

Generalized atrophic benign epidermolysis bullosa is an autosomal recessive subepidermal blistering disease typified by null mutations in COL17A1. In 1 large kindred, affected individuals were homozygous for a 2-bp deletion in COL17A1, 4003delTC, which resulted in a downstream premature termination codon, nonsense-mediated mRNA decay, and abrogation of type XVII collagen synthesis. Interestingly, 1 of these patients, although phenotypically identical to her affected siblings, showed focal expression of type XVII collagen in epidermal basement membrane in a pattern suggestive of revertant mosaicism. When studies of randomly obtained epidermal, oromucosal, and peripheral blood cells failed to identify the genetic basis of this apparent mosaicism, microscopic subpopulations of potentially revertant epidermal cells (i.e., those overlying basement membrane containing type XVII collagen) were selectively isolated using laser capture microdissection. Analysis of DNA and RNA from these cells revealed a second mutation, 4080insGG, on 1 allele of COL17A1. This 2-bp insertion corrected the reading frame just proximal to the premature termination codon, countered nonsense-mediated mRNA decay, and allowed protein production by patient keratinocytes in vivo and in vitro. These studies elucidate the molecular basis of a novel form of revertant mosaicism in humans.