Demonstration of intra- and extracellular localization of bullous pemphigoid antigen using cryofixation and freeze substitution for postembedding immunoelectron microscopy

Shimizu, Hiroshi; McDonald, Jared; Kennedy, Andrew R.; Eady, R.A.J.

doi:10.1007/bf00510078

Cited by 119 publications

(69 citation statements)

References 22 publications

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“…Normal human skin samples were obtained from skin surgical operations under fully informed consent and were processed for postembedding immunoelectron microscopy as previously described (36). Cyrofixed, cryosubstituted samples were embedded in Lowicryl K11M resin (Electron Microscopy Sciences).…”

Section: Methodsmentioning

confidence: 99%

Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer

Akiyama¹

2005

Journal of Clinical Investigation

354

437

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Harlequin ichthyosis (HI) is a devastating skin disorder with an unknown underlying cause. Abnormal keratinocyte lamellar granules (LGs) are a hallmark of HI skin. ABCA12 is a member of the ATP-binding cassette transporter family, and members of the ABCA subfamily are known to have closely related functions as lipid transporters. ABCA3 is involved in lipid secretion via LGs from alveolar type II cells, and missense mutations in ABCA12 have been reported to cause lamellar ichthyosis type 2, a milder form of ichthyosis. Therefore, we hypothesized that HI might be caused by mutations that lead to serious ABCA12 defects. We identify 5 distinct ABCA12 mutations, either in a compound heterozygous or homozygous state, in patients from 4 HI families. All the mutations resulted in truncation or deletion of highly conserved regions of ABCA12. Immunoelectron microscopy revealed that ABCA12 localized to LGs in normal epidermal keratinocytes. We confirmed that ABCA12 defects cause congested lipid secretion in cultured HI keratinocytes and succeeded in obtaining the recovery of LG lipid secretion after corrective gene transfer of ABCA12. We concluded that ABCA12 works as an epidermal keratinocyte lipid transporter and that defective ABCA12 results in a loss of the skin lipid barrier, leading to HI. Our findings not only allow DNA-based early prenatal diagnosis but also suggest the possibility of gene therapy for HI.

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Section: Methodsmentioning

confidence: 99%

Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer

Akiyama¹

2005

Journal of Clinical Investigation

354

437

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“…Four samples of human skin were cryofixed and processed for postembedding IEM according to the previously described methods (Shimizu et al 1989(Shimizu et al ,1990. Samples were washed in PBS and cryoprotected in 20% glycerol (in PBS) for up to 1 hr at 4C.…”

Section: Immunogold Electron Microscopymentioning

confidence: 99%

Colocalization of Multiple Laminin Isoforms Predominantly beneath Hemidesmosomes in the Upper Lamina Densa of the Epidermal Basement Membrane

McMillan

Akiyama

Nakamura

et al. 2006

J Histochem Cytochem.

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S U M M A R Y Multiple laminin isoforms including laminins 5 (a3 b3 g2), 6 (a3 b1 g1), 10 (a5 b1 g1), and possibly laminins 7 (a3 b2 g1) and 11 (a5 b2 g1) are present in the epidermal basement membrane. However, only the precise epidermal ultrastructural localization of laminin 5 (a3 b3 g2) has been elucidated. We therefore determined the precise expression and ultrastructural localization of the a5, b1, b2, and g1 chains in the epidermis. The expression of laminin chains in skin samples was analyzed from patients with epidermolysis bullosa (EB, n515) that harbor defects in specific hemidesmosome (HD)-associated components. The expression of the a5, b1, and g1 chains (present in laminins 10/11) and b2 chain (laminins 7/11) was unaffected in all intact (unseparated) skin of EB patients including Herlitz junctional EB with laminin-5 defects (n56). In the basement membrane of human epidermis, the a5, b1, b2, and g1 chains were expressed but also localized to the dermal vessels. Immunogold electron microscopy of normal human epidermis localized the a5, b1, b2, and g1 chains to the upper lamina densa, with between 84%

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“…Ultrathin sections were cut and immunogold stained using a 5 nm gold conjugated secondary (British Biocell, Cardiff, UK). 17 The antihuman HBD3 antibody and the anti-human cathepsin D rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as primary antibodies and then a goat anti-rabbit IgG gold-conjugated secondary antibody (British Biocell, Cardiff, UK) was used. Gold particles were observed in the intercellular spaces of the stratum corneum (-) and in lamellar bodies of intracellular spaces (.)…”

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confidence: 99%

Beta defensin-3 engineered epidermis shows highly protective effect for bacterial infection

et al. 2005

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Defensins are small cationic proteins that harbor broadspectrum microbicidal activity against bacteria, fungi and viruses. This study examines the effects on pathogens of the epidermis engineered to express human beta-defensin 3 (HBD3) to combat bacterial infections. First, we examined the localization of HBD3 in the epidermis and observed HBD3 in the intercellular spaces and lamellar bodies of the upper epidermal layers. This result showed HBD3 expressed and assembled in the outer layers of the epidermis was suspected to counter the invading microorganisms. Next, we established a keratinocyte cell line that stably expressed HBD3 and found that the culture medium showed antibacterial activity. Furthermore, we prepared an epidermal sheet of these cells with the HBD3 gene and grafted this onto a dermal wound on a nude rat. The HBD3 engineered epidermis demonstrated significant antimicrobial activity. Skin ulcers without epidermis are constantly exposed to invading microorganisms. Biopsy samples of re-epithelizing epidermis from patients with skin ulcers were collected, and HBD3 mRNA level measured in the epidermis. The epidermal samples from the ulcer skin expressed 2.5 times higher levels of HBD3 transcript than those in the control skin. These results, taken together, indicate that the therapeutic introduction of the HBD3 gene into somatic cells may provide a new gene therapy strategy for intractable infectious diseases. Gene Therapy (2005) 12, 857-861.

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Demonstration of intra- and extracellular localization of bullous pemphigoid antigen using cryofixation and freeze substitution for postembedding immunoelectron microscopy

Cited by 119 publications

References 22 publications

Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer

Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer

Colocalization of Multiple Laminin Isoforms Predominantly beneath Hemidesmosomes in the Upper Lamina Densa of the Epidermal Basement Membrane

Beta defensin-3 engineered epidermis shows highly protective effect for bacterial infection

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