Tn-MUC1 DC Vaccination of Rhesus Macaques and a Phase I/II Trial in Patients with Nonmetastatic Castrate-Resistant Prostate Cancer.

MUC1 is a glycoprotein expressed on the apical surface of ductal epithelial cells. Malignant transformation results in loss of polarization and overexpression of hypoglycosylated MUC1 carrying truncated carbohydrates known as T or Tn tumor antigens. Tumor MUC1 bearing Tn carbohydrates (Tn-MUC1) represent a potential target for immunotherapy. We evaluated the Tn-MUC1 glycopeptide in a human phase I/II clinical trial for safety that followed a preclinical study of different glycosylation forms of MUC1 in rhesus macaques, whose MUC1 is highly homologous to human MUC1. Either unglycosylated rhesus macaque MUC1 peptide (rmMUC1) or Tn-rmMUC1 glycopeptide was mixed with an adjuvant or loaded on autologous dendritic cells (DC), and responses were compared. Unglycosylated rmMUC1 peptide induced negligible humoral or cellular responses compared with the Tn-rmMUC1 glycopeptide. Tn-rmMUC1 loaded on DCs induced the highest anti-rmMUC1 T-cell responses and no clinical toxicity. In the phase I/II clinical study, 17 patients with nonmetastatic castrate-resistant prostate cancer (nmCRPC) were tested with a Tn-MUC1 glycopeptide-DC vaccine. Patients were treated with multiple intradermal and intranodal doses of autologous DCs, which were loaded with the Tn-MUC1 glycopeptide (and KLH as a positive control for immune reactivity). PSA doubling time (PSADT) improved significantly in 11 of 16 evaluable patients (P = 0.037). Immune response analyses detected significant Tn-MUC1-specific CD4+ and/or CD8+ T-cell intracellular cytokine responses in 5 out of 7 patients evaluated. In conclusion, vaccination with Tn-MUC1-loaded DCs in nmCRPC patients appears to be safe, able to induce significant T-cell responses, and have biological activity as measured by the increase in PSADT following vaccination. Cancer Immunol Res; 4(10); 881-92. ©2016 AACR.

Inherited epidermolysis bullosa: updated recommendations on diagnosis and classification.

BACKGROUND: Several new targeted genes and clinical subtypes have been identified since publication in 2008 of the report of the last international consensus meeting on diagnosis and classification of epidermolysis bullosa (EB). As a correlate, new clinical manifestations have been seen in several subtypes previously described. OBJECTIVE: We sought to arrive at an updated consensus on the classification of EB subtypes, based on newer data, both clinical and molecular. RESULTS: In this latest consensus report, we introduce a new approach to classification ("onion skinning") that takes into account sequentially the major EB type present (based on identification of the level of skin cleavage), phenotypic characteristics (distribution and severity of disease activity; specific extracutaneous features; other), mode of inheritance, targeted protein and its relative expression in skin, gene involved and type(s) of mutation present, and--when possible--specific mutation(s) and their location(s). LIMITATIONS: This classification scheme critically takes into account all published data through June 2013. Further modifications are likely in the future, as more is learned about this group of diseases. CONCLUSION: The proposed classification scheme should be of value both to clinicians and researchers, emphasizing both clinical and molecular features of each EB subtype, and has sufficient flexibility incorporated in its structure to permit further modifications in the future.

Transmission electron microscopy for the diagnosis of epidermolysis bullosa.

Transmission electron microscopy (TEM) has long been the best available method for the diagnosis of epidermolysis bullosa. Today, TEM is largely superseded by immunofluorescence microscopy mapping, which is generally more available. This article discusses its continuing role in confirming or refining results obtained by other methods, or in establishing the diagnosis where other techniques have been unsuitable or have failed. It covers key steps for optimizing tissue preparation, features of analysis, recently classified epidermolysis bullosa disorders, and strengths and weaknesses of TEM.

Survival is not enough: reflections of a long-term renal patient.

A kidney patient recalls his experience of almost 45 years of renal replacement therapy covering nearly 25 years of dialysis and 20 years with a transplant. At the beginning, patient or graft survival was a major goal and symbol of successful treatment. But for the patient, what really matters is the quality of his life, assuming he can survive.

The classification of inherited epidermolysis bullosa (EB): Report of the Third International Consensus Meeting on Diagnosis and Classification of EB.

BACKGROUND: Since publication in 2000 of the Second International Consensus Report on Diagnosis and Classification of Epidermolysis Bullosa, many advances have been made to our understanding of this group of diseases, both clinically and molecularly. At the same time, new epidermolysis bullosa (EB) subtypes have been described and similarities with some other diseases have been identified. OBJECTIVE: We sought to arrive at a new consensus of the classification of EB subtypes. RESULTS: We now present a revised classification system that takes into account the new advances, as well as encompassing other inherited diseases that should also be included within the EB spectrum, based on the presence of blistering and mechanical fragility. Current recommendations are made on the use of specific diagnostic tests, with updates on the findings known to occur within each of the major EB subtypes. Electronic links are also provided to informational and laboratory resources of particular benefit to clinicians and their patients. LIMITATIONS: As more becomes known about this disease, future modifications may be needed. The classification system has been designed with sufficient flexibility for these modifications. CONCLUSION: This revised classification system should assist clinicians in accurately diagnosing and subclassifying patients with EB.

A germline mutation in BLOC1S3/reduced pigmentation causes a novel variant of Hermansky-Pudlak syndrome (HPS8).

Hermansky-Pudlak syndrome (HPS) is genetically heterogeneous, and mutations in seven genes have been reported to cause HPS. Autozygosity mapping studies were undertaken in a large consanguineous family with HPS. Affected individuals displayed features of incomplete oculocutaneous albinism and platelet dysfunction. Skin biopsy demonstrated abnormal aggregates of melanosomes within basal epidermal keratinocytes. A homozygous germline frameshift mutation in BLOC1S3 (p.Gln150ArgfsX75) was identified in all affected individuals. BLOC1S3 mutations have not been previously described in patients with HPS, but BLOC1S3 encodes a subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1). Mutations in other BLOC-1 subunits have been associated with an HPS phenotype in humans and/or mouse, and a nonsense mutation in the murine orthologue of BLOC1S3 causes the reduced pigmentation (rp) model of HPS. Interestingly, eye pigment formation is reported to be normal in rp, but we found visual defects (nystagmus, iris transilluminancy, foveal hypoplasia, reduced visual acuity, and evidence of optic pathway misrouting) in affected individuals. These findings define a novel form of human HPS (HPS8) and extend genotype-phenotype correlations in HPS.

Mutations in ABCA12 underlie the severe congenital skin disease harlequin ichthyosis.

Harlequin ichthyosis (HI) is the most severe and frequently lethal form of recessive congenital ichthyosis. Although defects in lipid transport, protein phosphatase activity, and differentiation have been described, the genetic basis underlying the clinical and cellular phenotypes of HI has yet to be determined. By use of single-nucleotide-polymorphism chip technology and homozygosity mapping, a common region of homozygosity was observed in five patients with HI in the chromosomal region 2q35. Sequencing of the ABCA12 gene, which maps within the minimal region defined by homozygosity mapping, revealed disease-associated mutations, including large intragenic deletions and frameshift deletions in 11 of the 12 screened individuals with HI. Since HI epidermis displays abnormal lamellar granule formation, ABCA12 may play a critical role in the formation of lamellar granules and the discharge of lipids into the intercellular spaces, which would explain the epidermal barrier defect seen in this disorder. This finding paves the way for early prenatal diagnosis. In addition, functional studies of ABCA12 will lead to a better understanding of epidermal differentiation and barrier formation.

Reduced expression of insulin-like growth factor-binding protein-3 (IGFBP-3) in Squamous cell carcinoma complicating recessive dystrophic epidermolysis bullosa.

Squamous cell carcinoma (SCC) is a common complication in individuals with recessive dystrophic epidermolysis bullosa (RDEB). For the severe Hallopeau-Siemens subtype, the mortality rate from SCC is over 55% by the age of 40 y. Currently, little is known about the molecular pathology or cell biology of SCC in RDEB. In this study, we compared gene expression in RDEB SCC (n=3) and non-EB SCC (n=3) with corresponding RDEB and non-EB peri-tumoral skin, with microarray analysis using DermArray membranes as well as semi-quantitative and real-time RT-PCR. Both tumor sets showed downregulation of epidermal differentiation markers (e.g., profilaggrin, keratins 1 and 10) as well as certain pro-apoptotic genes (e.g., death-associated kinase-3 or ZIP kinase). Likewise, in both groups there was upregulation of matrix metalloproteinase 1 and laminin 5 in the tumors. But we found that the expression of insulin-like growth factor-binding protein-3 (IGFBP-3) was lower (mean of 5.8-fold) in RDEB SCC compared with non-EB SCC. These data were verified by immunohistochemistry. IGFBP-3 has an important role in cancer cell apoptosis mediated via the nuclear retinoid X receptor alpha (RXRalpha). Reduced expression of IGFBP-3 in RDEB SCC may provide a partial explanation for the aggressive behavior and poor prognosis of these tumors in this genodermatosis.

Recurrent mutations in kindlin-1, a novel keratinocyte focal contact protein, in the autosomal recessive skin fragility and photosensitivity disorder, Kindler syndrome.

Kindler syndrome (OMIM 173650) is a rare autosomal recessive disorder characterized by trauma-induced blister formation (especially in childhood) and photosensitivity. Other features include mucocutaneous scarring and progressive poikiloderma. There is also an increased risk of skin and mucous membrane malignancy. The disorder was recently mapped to 20p12.3 and pathogenic mutations were identified in a new gene, KIND1. This gene encodes a 677 amino acid protein, kindlin-1, a component of focal contacts in keratinocytes. In this study, we identified four new recurrent mutations in KIND1 in 16 individuals with Kindler syndrome from 13 families of Pakistani (676insC), UK Caucasian (E304X), Omani (W616X), or Italian (958-1G > A) origins. Haplotype analysis demonstrated common ancestral mutant alleles for each mutation, apart from one of the six Pakistani families in which the mutation 676insC (which occurs in a repeat of seven cytosines) was present on a different genetic background. All mutations were homozygous, apart from the three UK Caucasian cases that were all compound heterozygotes (second allele mutations: L302X, 1161delA, 1909delA). All mutations were associated with markedly reduced or absent skin immunostaining with an antikindlin-1 antibody. These loss-of-function KIND1 mutations demonstrate the importance of kindlin-1 in maintaining epithelial integrity, although the mechanism linking this mutant protein to photosensitivity and poikiloderma remains to be determined. Delineation of these recurrent mutations is also relevant to optimizing mutation detection strategies in Kindler syndrome patients from particular ethnic backgrounds.

Efficacy and safety of endoscopic dilation of esophageal strictures in epidermolysis bullosa.

BACKGROUND: Epidermolysis bullosa is a rare genetically determined disorder of the stratified squamous epithelium. Patients with the most severe forms develop scarring of the esophagus after ingestion of food. This results in dysphagia, which severely compromises the ability to eat. Maintenance of adequate nutritional intake is a central aim, but the most appropriate method is unknown. METHODS: The results of endoscopic through-the-scope balloon dilation under propofol anesthesia in 53 patients with epidermolysis bullosa and esophageal strictures are reported. RESULTS: Seventy-five percent of patients had a single stricture (range 1 to 6 strictures), most often in the proximal esophagus (median 20 cm from incisors). A total of 182 dilations were performed (median two per patient) over a median follow-up period of 3.5 years. For all but 3 patients, there was an improvement in the dysphagia score. There was a mean increase in weight after the procedure of 2.9 kg: 95% CI[2.0, 3.8]; p<0.001, over a median 29 days. There was no significant post-procedure morbidity. CONCLUSIONS: Endoscopic balloon dilation is a safe and effective treatment for the esophageal strictures of epidermolysis bullosa. In the majority of patients, dilation relieves dysphagia and improves nutritional status.

Desmosomal proteins, including desmoglein 3, serve as novel negative markers for epidermal stem cell-containing population of keratinocytes.

No single method has been universally adopted for identifying and isolating epidermal stem/progenitor cells, and the emergence of new markers of stem cell populations is worth exploring. Here we report, for the first time, that clusters of basal keratinocytes at the tips of the rete ridges in human palm, previously recognised as a major repository of stem cells, had very low levels of desmoplakin protein and mRNA expression, compared with cells at the sides of the ridges or above the dermal papillae. We found that in populations of palm keratinocytes, selected by their ability to adhere rapidly to type IV collagen, there were significantly reduced levels of desmoplakin and other major desmosome proteins. We then showed that a low desmoglein 3 (Dsg3) expression on the cell surface could be used to enrich for a cell population with high clonogenecity, colony forming efficiency and enhanced proliferative potential, but with a low ability to form the abortive clones, compared with populations with a higher Dsg3 expression. Moreover, stringent sorting of populations showing both beta1 integrin-bright and Dsg3-dull expression enabled even further enrichment of a population containing the putative epidermal stem cells. These findings provide the basis for a new strategy for epidermal stem/progenitor cell enrichment, and encourage further study of the role of desmosomes in stem cell biology.

Desmosomes exhibit site-specific features in human palm skin.

Hereditary skin disorders resulting from desmosome gene pathology may preferentially involve the palms and soles. Why this is so is not clear. Moreover, even in normal control skin it is unknown whether there are differences in desmosome number, size or structural organization in palmoplantar sites compared with skin from other body regions. Therefore, we sought evidence for such differences by examining desmosome expression in relation to epidermal differentiation in both epidermis and cultured keratinocytes from normal human palm and breast skin samples. Confocal microscopy of skin biopsy material showed relative differences in the expression profiles of several desmosomal proteins (desmogleins, desmocollins, desmoplakin, plakoglobin and plakophilin 1) between the two sites. Western blotting revealed a higher expression level of all five proteins in palm compared with breastcultured keratinocytes. Staining for the differentiation-associated component, involucrin, suggested an earlier onset of synthesis of this protein in palm epidermis, and a suspension-induced differentiation assay showed that involucrin synthesis began earlier in palm keratinocytes than in breast cells. At 4-8 h, the number of involucrin-positive cells in palm keratinocytes was almost twice that in breast. Morphometric analysis showed that, overall, desmosomes were larger but of similar population density in the palm compared with breast skin. These findings demonstrate differences in desmosome structure and protein expression between the two sites, possibly reflecting the needs of palms and soles to withstand constant mechanical stress. They may also help to explain the preferential involvement of this region in certain hereditary disorders (palmoplantar keratodermas), associated with mutations in desmoplakin or desmoglein 1.

An unusual N-terminal deletion of the laminin alpha3a isoform leads to the chronic granulation tissue disorder laryngo-onycho-cutaneous syndrome.

Laryngo-onycho-cutaneous (LOC or Shabbir) syndrome (OMIM 245660) is an autosomal recessive epithelial disorder confined to the Punjabi Muslim population. The condition is characterized by cutaneous erosions, nail dystrophy and exuberant vascular granulation tissue in certain epithelia, especially conjunctiva and larynx. Genome-wide homozygosity mapping localized the gene to a 2 Mb region on chromosome 18q11.2 with an LOD score of 19.8 at theta=0. This region includes the laminin alpha3 gene (LAMA3), in which loss-of-expression mutations cause the lethal skin blistering disorder Herlitz junctional epidermolysis bullosa. Detailed investigation showed that this gene possesses a further 38 exons (76 exons in total) spanning 318 kb of genomic DNA, and encodes three distinct proteins, designated laminin alpha3a, alpha3b1 and alpha3b2. The causative mutation in 15 families was a frameshift mutation 151insG predicting a stop codon 7 bp downstream in an exon that is specific to laminin alpha3a. This protein is secreted only by the basal keratinocytes of stratified epithelia, implying that LOC is caused by dysfunction of keratinocyte-mesenchymal communication. Surprisingly, the 151insG mutation does not result in nonsense-mediated mRNA decay due to rescue of the transcript by an alternative translation start site 6 exons downstream. The resultant N-terminal deletion of laminin alpha3a was confirmed by immunoprecipitation of secreted proteins from LOC keratinocytes. These studies show that the laminin alpha3a N-terminal domain is a key regulator of the granulation tissue response, with important implications not only in LOC but in a range of other clinical conditions associated with abnormal wound healing.

Alterations in desmosome size and number coincide with the loss of keratinocyte cohesion in skin with homozygous and heterozygous defects in the desmosomal protein plakophilin 1.

Recessive mutations in the desmosomal plaque protein plakophilin 1 (PkP1) underlie ectodermal dysplasia/skin fragility syndrome (MIM 604536). We undertook an immunohistochemical and quantitative electron microscopic examination of suprabasal desmosomes from 4 skin samples from 3 PkP1 deficient patients, an unaffected carrier with a PKP1 heterozygous acceptor splice site mutation and 5 healthy control subjects. Desmosomal plaque size (>50 desmosomes per individual) and frequency (>20 high power fields, HPF) were assessed. Compared with controls, desmosomes were reduced dramatically both in size (49%) and frequency (61%) in the lower suprabasal layers (LSB) in PkP1 null patients (P<0.01). In the LSB compartment of the heterozygous carrier, corresponding reductions were 37% and 20%, respectively (P<0.01). Surprisingly, the PkP1 null patient's upper suprabasal layer, (USB), desmosome size was larger (59%, P<0.01) than the control value, and showed increased desmoglein 1 and PkP2 USB staining. The USB desmosome frequency in PKP1 null patients was similar to the LSB compartment (but reduced by 43% compared to USB controls). The carrier showed no difference in the USB desmosome size and frequency compared with the controls (P>0.05). The PKP1 null patients showed poorly developed inner and outer desmosomal plaques. Thus, both the patients and unaffected carrier showed reductions in the LSB desmosome size and number; despite only PkP1 null patients exhibiting any phenotype. These findings attest to the molecular recruiting and stabilizing roles of PkP1 in desmosome formation, particularly in the LSB compartment.

Lack of plakophilin 1 increases keratinocyte migration and reduces desmosome stability.

Ablation of the desmosomal plaque component plakophilin 1 underlies the autosomal recessive genodermatosis, skin fragility-ectodermal dysplasia syndrome (OMIM 604536). Skin from affected patients is thickened with increased scale, and there is loss of adhesion between adjacent keratinocytes, which exhibit few small, poorly formed desmosomes. To investigate further the influence of plakophilin 1 on keratinocyte adhesion and desmosome morphology, we compared plakophilin 1-deficient keratinocytes (vector controls) with those expressing recombinant plakophilin 1 introduced by retroviral transduction. We found that plakophilin 1 increases desmosomal protein content within the cell rather than enhancing transcriptional levels of desmosomal genes. Re-expression of plakophilin 1 in null cells retards cell migration but does not alter keratinocyte cell growth. Confluent sheets of plakophilin 1-deficient keratinocytes display fewer calcium-independent desmosomes than do plakophilin 1-deficient keratinocytes expressing recombinant plakophilin 1 or keratinocytes expressing endogenous plakophilin 1. In addition electron microscopy studies show that re-expression of plakophilin 1 affects desmosome size and number. Collectively, these results demonstrate that restoration of plakophilin 1 function in our culture system influences the transition of desmosomes from a calcium-dependent to a calcium-independent state and this correlates with altered keratinocyte migration in response to wounding. Thus, plakophilin 1 has a key role in increasing desmosomal protein content, in desmosome assembly, and in regulating cell migration.

Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellular-matrix linker protein UNC-112, causes Kindler syndrome.

Kindler syndrome is an autosomal recessive disorder characterized by neonatal blistering, sun sensitivity, atrophy, abnormal pigmentation, and fragility of the skin. Linkage and homozygosity analysis in an isolated Panamanian cohort and in additional inbred families mapped the gene to 20p12.3. Loss-of-function mutations were identified in the FLJ20116 gene (renamed "KIND1" [encoding kindlin-1]). Kindlin-1 is a human homolog of the Caenorhabditis elegans protein UNC-112, a membrane-associated structural/signaling protein that has been implicated in linking the actin cytoskeleton to the extracellular matrix (ECM). Thus, Kindler syndrome is, to our knowledge, the first skin fragility disorder caused by a defect in actin-ECM linkage, rather than keratin-ECM linkage.

Changes in gap junction distribution and connexin expression pattern during human fetal skin development.

Gap junctions are intercellular channels composed of connexin subunits that mediate cell-cell communication. The functions of gap junctions are believed to be associated with cell proliferation and differentiation and to be important in maintaining tissue homeostasis. We therefore investigated the expression of connexins (Cx)26 and 43, the two major connexins in human epidermis, and examined the formation of gap junctions during human fetal epidermal development. By immunofluorescence, Cx26 expression was observed between 49 and 96 days' estimated gestational age (EGA) but was not present from 108 days' EGA onwards. Conversely, Cx43 expression was observed from 88 days' EGA onwards. Using electron microscopy, the typical structure of gap junctions was observed from 120 days' EGA. The number of gap junctions increased over time and they were more common in the upper layers, within the periderm and intermediate keratinocyte layers rather than the basal layer. Immunoelectron microscopy revealed Cx43 labeling on the gap junction structures after 105 days' EGA. Formation of gap junctions increased as skin developed, suggesting that gap junctions may play an important role in fetal skin development. Furthermore, the changing patterns of connexin expression suggest that Cx26 is important for early fetal epidermal development.

Frameshift mutation in the V2 domain of human keratin 1 results in striate palmoplantar keratoderma.

The striate form of palmoplantar keratoderma is a rare autosomal dominant disorder affecting palm and sole skin. Genetic heterogeneity of striate palmoplantar keratoderma has been demonstrated with pathogenic mutations in the desmosomal proteins desmoplakin and desmoglein 1. We have studied a four-generation family of British descent with striate palmoplantar keratoderma. Ultrastructural studies show that intermediate filaments of suprabasal keratinocytes are finer than those of the basal layer. In addition, desmosome numbers are normal, but their inner plaques and midline structures are attenuated. Microsatellite markers were used to screen candidate loci including the epidermal differentiation complex on 1q, the desmoplakin locus on 6p, the type I and II keratin gene clusters on chromosomes 12q and 17q, and the desmosomal cadherin gene cluster on chromosome 18q. Significant genetic linkage to chromosome 12q was observed using marker D12S368, with a maximum two-point lod score of 3.496 at a recombination fraction of 0. Direct sequencing of the keratin 1 gene revealed a frameshift mutation in exon 9 that leads to the partial loss of the glycine loop motif in the V2 domain and the gain of a novel 70 amino acid peptide. Using expression studies we show that the V2 domain is essential for normal function of keratin intermediate filaments.