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James R McMillan

Publications and source records attributed to James R McMillan.

17 recordsLinked to original sources
PubMed2007

Small-diameter porous poly (epsilon-caprolactone) films enhance adhesion and growth of human cultured epidermal keratinocyte and dermal fibroblast cells.

Autologous keratinocyte grafts provide clinical benefit by rapidly covering wounded areas, but they are fragile. We therefore developed biocompatible hexagonal-packed porous films with uniform, circular pore sizes to support human keratinocytes and fibroblasts. Cells were cultured on these porous poly (epsilon-calprolactone) films with pore sizes ranging from novel ultra-small 3 microm to 20 microm. These were compared with flat (pore-less) films. Cell growth rates, adhesion, migration, and ultrastructural morphology were examined. Human keratinocytes and fibroblasts attached to all films. Furthermore, small-pore (3-5 microm) films showed the highest levels of cell adhesion and survival and prevented migration into the pores and opposing film surface. Keratinocyte migration over small-pore film surface was inhibited. Keratinocytes optimally attached to 3-microm-pore films due to a combination of greater pore numbers (porosity), a greater circumference of the pore edge per unit surface area, and greater frequency of flat surface areas for attachment, allowing better cell-substrate and cell-cell attachment and growth. The 3-microm pore size allowed cell-cell communication, together with diffusion of soluble nutrients and factors from the culture medium or wound substrate. These characteristics are considered important in developing grafts for use in the treatment of human skin wounds.

Biocompatible Materials↗
PubMed2006

DNA-based prenatal diagnosis of harlequin ichthyosis and characterization of ABCA12 mutation consequences.

Until the identification of ABCA12 as the causative gene, prenatal diagnosis (PD) for harlequin ichthyosis (HI) had been performed by electron microscopic observation of fetal skin biopsy samples. We report the first case of HI DNA-based PD. Direct sequence analysis of ABCA12 revealed that the deceased proband was a compound heterozygote for two novel mutations. The maternal nonsense mutation p.Ser1249Term likely leads to nonsense-mediated messenger RNA decay. The paternal mutation c.7436G>A affects the last codon of exon 50 and was expected to be a splice site mutation. For their third pregnancy, the parents requested PD. Direct sequence analysis of fetal genomic DNA from amniotic fluid cells at 17 weeks gestation revealed the fetus was a compound heterozygote for both mutations. The parents requested the pregnancy to be terminated. Analysis of ABCA12 transcripts of cultured keratinocytes from the abortus showed the presence of six abnormally spliced products from the allele carrying the splice site mutation. Four of them lead to premature termination codons whereas the two others produced shortened proteins missing 21 and 31 amino acids from the second ATP-binding cassette. This report provides evidence for residual ABCA12 expression in HI, and demonstrates the efficiency of early DNA-based PD of HI.

ATP-Binding Cassette Transporters↗
PubMed2006

N-linked neutral oligosaccharides in the stratum corneum of normal and ichthyotic skin.

N-Glycan oligosaccharides are thought to play multiple, important roles in a variety of biological events. However, N-glycan profiles in the stratum corneum of human skin have not yet been studied in detail. To clarify the N-glycan profiles in the stratum corneum of normal and ichthyotic epidermis, N-glycan profiles were studied by high-performance liquid chromatography using normal human epidermal samples and scales from hyperkeratotic skin of ichthyosis patients. Chromatograms of patient scale samples showed unique alterations in three peaks eluted at 15.8, 18.8 and 26.9 min. The N-glycan profiles were significantly altered in ichthyotic hyperkeratotic skin compared with normal non-hyperkeratotic controls. These findings indicate the reduction of N-acetylglucosaminyltransferase II and fucosyltransferase 8 activities. Alteration of N-glycan structures in hyperkeratotic skin suggests the biological role of N-glycans in keratinization.

Chromatography, High Pressure Liquid↗
PubMed2006

CTACK/CCL27 accelerates skin regeneration via accumulation of bone marrow-derived keratinocytes.

Recent studies have suggested that bone marrow (BM) cells transdifferentiate to regenerate a variety of cellular lineages. Due to the relatively small population of BM-derived cells in each organ, it is still controversial whether these BM-derived cells are really present in sufficient numbers for effective function. Conversely, it is speculated that chemokine/chemokine receptor interactions mediate this migration of the tissue-specific precursor cells from BM into the target tissue. Here, we show that cutaneous T-cell attracting chemokine (CTACK)/CCL27 is the major regulator involved in the migration of keratinocyte precursor cells from BM into skin. By screening various chemokine expression patterns, we demonstrated that CTACK is constitutively expressed in normal skin and upregulated in wounds and that approximately 20% of CD34(+) BM cells expressed CCR10, the ligand for CTACK. Intradermal injection of CTACK/CCL27 into the periphery of skin wounds significantly enhanced BM-derived keratinocyte (BMDK) migration, and CTACK/CCL27 neutralizing antibody inhibited this BMDK migration. Furthermore, increased BMDK migration caused by CTACK/CCL27 significantly accelerated the wound-healing process without any influence over either angiogenesis or keratinocyte proliferation. These results provide direct evidence that recruitment of BM keratinocyte precursor cells to the skin is regulated by specific chemokine/chemokine receptor interactions, making possible the development of new regenerative therapeutic strategies.

Animals↗
PubMed2006

Targeted skipping of a single exon harboring a premature termination codon mutation: implications and potential for gene correction therapy for selective dystrophic epidermolysis bullosa patients.

This study examined the feasibility of antisense oligoribonucleotide (AON) therapy for dystrophic epidermolysis bullosa (DEB). AON was designed to induce skipping of a targeted exon containing a premature termination codon mutation, resulting in restoration of the open reading frame. We targeted exon 70 of COL7A1, as a recurrent mutation 5818delC in Japanese DEB patients was localized to exon 70. We found that one AON induced effective skipping of normal exon 70 containing 16 amino acids. Attachment and migration analyses showed that recombinant collagen without contribution of exon 70 was similar in effect to normal type VII collagen. Next, we synthesized mutation-specific AON by deleting cytosine at 5818. Introduction of this AON into DEB keratinocytes harboring 5818delC showed that the AON induced skipping of exon 70 in the abnormal 5818delC allele. Furthermore, 6.2% of DEB keratinocytes started to express type VII collagen in vitro after application of the mutation-specific AON. Injection of the AON into rat model grafted with DEB keratinocytes and fibroblasts induced a low amount of type VII collagen expression. We conclude that skipping of targeted exons using mutation-specific AON may show potential for future gene therapy for DEB patients.

Animals↗
PubMed2006

Compound heterozygous mutations including a de novo missense mutation in ABCA12 led to a case of harlequin ichthyosis with moderate clinical severity.

Harlequin ichthyosis (HI) is one of the most devastating genodermatoses. Recently, ABCA12 mutations were identified as the cause of HI. A newborn Japanese male demonstrated the typical features of HI. The patient was treated with oral etretinate and his general condition has been good (now aged 1.5 years). This patient with moderate clinical severity was compound heterozygous for a novel de novo missense mutation 1160G > A (S387N) in exon 10 and a maternal deletion mutation 4158_4160delTAC (T1387del) in exon 28 of ABCA12. T1387del was a deletion of a highly conserved threonine residue within the first adenosine 5' triphosphate-binding domain and is thought to seriously affect the function of the ABCA12 protein. Conversely, the residue 387 is located outside the known active sites of ABCA12 and S387N is predicted not to lead to a serious functional deficiency in ABCA12. Electron microscopy revealed abnormal lamellar granules in the granular layer cells and a moderate number of lipid vacuoles in the cornified cells. Disturbed glucosylceramide transport was confirmed in the cultured keratinocytes from the patient. No de novo mutation in ABCA12 has yet been reported either in HI or lamellar ichthyosis. The present case suggested that a de novo ABCA12 mutation might underlie HI.

ATP-Binding Cassette Transporters↗
PubMed2005

Colocalization of multiple laminin isoforms predominantly beneath hemidesmosomes in the upper lamina densa of the epidermal basement membrane.

Multiple laminin isoforms including laminins 5 (alpha3 beta3 gamma2), 6 (alpha3 beta1 gamma1), 10 (alpha5 beta1 gamma1), and possibly laminins 7 (alpha3 beta2 gamma1) and 11 (alpha5 beta2 gamma1) are present in the epidermal basement membrane. However, only the precise epidermal ultrastructural localization of laminin 5 (alpha3 beta3 gamma2) has been elucidated. We therefore determined the precise expression and ultrastructural localization of the alpha5, beta1, beta2, and gamma1 chains in the epidermis. The expression of laminin chains in skin samples was analyzed from patients with epidermolysis bullosa (EB, n=15) that harbor defects in specific hemidesmosome (HD)-associated components. The expression of the alpha5, beta1, and gamma1 chains (present in laminins 10/11) and beta2 chain (laminins 7/11) was unaffected in all intact (unseparated) skin of EB patients including Herlitz junctional EB with laminin-5 defects (n=6). In the basement membrane of human epidermis, the alpha5, beta1, beta2, and gamma1 chains were expressed but also localized to the dermal vessels. Immunogold electron microscopy of normal human epidermis localized the alpha5, beta1, beta2, and gamma1 chains to the upper lamina densa, with between 84% and 92% of labeling restricted to beneath the HDs, similar to laminin 5 (n> or =200 gold particles per sample, sample number n=4) but distinct from collagen IV labeling (with only 63% labeling beneath HDs, p<0.001). Taken together, the majority of the alpha5beta1/beta2gamma1 laminin chains are located beneath HDs. This suggests that laminin-10-associated chains have specific functions or molecular interactions beneath HDs in the epidermal basement membrane.

Adolescent↗
PubMed2005

Epidermolysis bullosa simplex associated with pyloric atresia is a novel clinical subtype caused by mutations in the plectin gene (PLEC1).

Epidermolysis bullosa (EB) is an inherited mechano-bullous disorder of the skin, and is divided into three major categories: EB simplex (EBS), dystrophic EB, and junctional EB (JEB). Mutations in the plectin gene (PLEC1) cause EBS associated with muscular dystrophy, whereas JEB associated with pyloric atresia (PA) results from mutations in the alpha6 and beta4 integrin genes. In this study, we examined three EB patients associated with PA from two distinct families. Electron microscopy detected blister formation within the basal keratinocytes leading to the diagnosis of EBS. Surprisingly, immunohistochemical studies using monoclonal antibodies to a range of basement membrane proteins showed that the expression of plectin was absent or markedly attenuated. Sequence analysis demonstrated four novel PLEC1 mutations. One proband was a compound heterozygote for a nonsense mutation of Q305X and a splice-site mutation of 1344G-->A. An exon-trapping experiment suggested that the splice-site mutation induced aberrant splicing of the gene. The second proband harbored a heterozygous maternal nonsense mutation, Q2538X and homozygous nonsense mutations R1189X. Analysis of the intragenic polymorphisms of PLEC1 suggested that R1189X mutations were due to paternal segmental uniparental isodisomy. These results indicate that PLEC1 is a possible causative gene in this clinical subtype, EBS associated with PA. Furthermore, two patients out of our three cases died in infancy. In terms of clinical prognosis, this novel subtype is the lethal variant in the EBS category.

Epidermolysis Bullosa Simplex↗
PubMed2005

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

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.

ATP-Binding Cassette Transporters↗
PubMed2004

Characterization of Kdap, a protein secreted by keratinocytes.

Using a signal sequence-trap we identified a human gene encoding a polypeptide of 99 amino acids with a putative signal sequence. The gene was identical to keratinocyte differentiation-associated protein (Kdap), which was reported previously by Oomizu et al (Gene 256: 19-27, 2000) to be expressed in embryonal rat epidermis at the mRNA level. In humans, we found Kdap mRNA expression to be restricted to epithelial tissue at high levels. The 12.5 kDa protein was detected in culture supernatant of keratinocytes and those transfected adenovirally with the Kdap gene. In normal skin, Kdap protein was found exclusively within lamellar granules of granular keratinocytes and in the intercellular space of the stratum corneum. By contrast, in lesional skin of patients with psoriasis, Kdap was expressed more widely throughout suprabasal keratinocytes. When induced to differentiate in vitro, keratinocytes showed marked upregulation of Kdap mRNA expression similar to that of involucrin mRNA, but with differing kinetics. Finally, a spliced variant of Kdap mRNA was generated by alternative splicing mechanisms. Our studies indicate that human Kdap resembles rat Kdap with respect to tissue and cell expression at the mRNA level and that Kdap is a low-molecular-weight protein secreted by keratinocytes. Thus Kdap may serve as a soluble regulator of keratinocyte differentiation.

Alternative Splicing↗
PubMed2003

Epidermal basement membrane zone components: ultrastructural distribution and molecular interactions.

The epidermal basement membrane zone (BMZ) comprises a multiprotein complex that aids the attachment of epidermal keratinocytes to the underlying dermis. In the last 5 years, our understanding of epidermal BMZ morphology, structure and function has dramatically improved. A complex network of molecular interactions has recently been identified that strengthen dermal-epidermal adhesion. Special attention will be paid to the recently identified network of interactions between BMZ components, including alpha6beta4 integrin interactions, the extracellular hook domain of collagen XVII and the looping structure of collagen VII molecules. We summarize some of the recent advances in the understanding of the biology and interactions of BMZ components from an ultrastructural and molecular perspective.

Autoantigens↗
PubMed2003

Immunomapping of EBA sera to multiple epitopes on collagen VII: further evidence that anchoring fibrils originate and terminate in the lamina densa.

Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disease with circulating antibodies to type VII collagen, a major component of anchoring fibrils located at the dermal-epidermal junction. The purpose of this study was to further confirm the ultrastructural organisation of anchoring fibrils and to assess the relationship between the clinical phenotype of EBA and target site of their autoantibodies on anchoring fibrils. We studied the ultrastructural binding site of circulating autoantibodies from two patients with atypical clinical features who predominantly presented with oral lesions, and compared this with two patients with clinically typical forms of EBA. Immunoblotting of whole dermal extracts showed labelling of 290-kDa bands consistent with that of type VII collagen as well as the non-collagenous (NC-1) domain fusion protein in three out of four patients' sera. Postembedding immunoelectron microscopy (IEM) using Lowicryl K11M embedded normal human skin and patients' sera demonstrated the majority of labelling within the lamina densa, not below the lamina densa. We conclude that EBA autoantibodies in these patient's sera bind to the NC-1 domain of collagen VII situated in the lamina densa of the epidermal basement membrane, regardless of the EBA clinical phenotype. This confirms the previous notion that anchoring fibrils originate and terminate in the lamina densa.

Adult↗
PubMed2003

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.

Adolescent↗
PubMed2003

Ultrastructural orientation of laminin 5 in the epidermal basement membrane: an updated model for basement membrane organization.

Laminin 5 is a trimeric glycoprotein involved in cell adhesion in the epidermal basement membrane. To determine the precise orientation of laminin 5 in adult human skin, we used plural epitope-specific monoclonal antibodies, a polyclonal antiserum, and postembedding immunogold electron microscopy (IEM). Immunogold labeling distances from the basal keratinocyte plasma membrane (PM) were measured for each gold particle (>200 particles) and the mean distance (nm) calculated. Antibodies included BM165 (recognizing the alpha 3-chain first globular domain) that was measured at 35.40 +/- 2.20 nm from the keratinocyte PM, K140 (recognizing a region adjacent to the beta 3-chain globular domain IV) that measured 45.20 +/- 3.60 nm from the PM, and an anti-laminin 5 polyclonal antiserum that was 43.43 +/- 6.28 nm from the PM. The laminin 5 gamma 2-chain short arm hinge domain was previously localized to the lower lamina densa (LD) at approximately 56.30 +/- 1.65 nm from the keratinocyte PM. Taken together with previous gamma 2-chain data and the distribution of the polyclonal antisera, we estimate that the long axis of laminin 5 is oriented at an angle of approximately 27 degrees from the horizontal lamina lucida (LL)/LD border and propose that the gamma 2-chain lies farthest from the PM. This novel orientation, with the majority of the laminin 5 molecule lying obliquely along the LL/LD border and not perpendicularly, as was first thought, sheds new light on the organization of the basement membrane and likely molecular interactions.

Antibodies, Monoclonal↗
PubMed2002

Dominant and recessive compound heterozygous mutations in epidermolysis bullosa simplex demonstrate the role of the stutter region in keratin intermediate filament assembly.

Keratin intermediate filaments are important cytoskeletal structural proteins involved in maintaining cell shape and function. Mutations in the epidermal keratin genes, keratin 5 or keratin 14 lead to the disruption of keratin filament assembly, resulting in an autosomal dominant inherited blistering skin disease, epidermolysis bullosa simplex (EBS). We investigated a large EBS kindred who exhibited a markedly heterogeneous clinical presentation and detected two distinct keratin 5 mutations in the proband, the most severely affected. One missense mutation (E170K) in the highly conserved helix initiation peptide sequence of the 1A rod domain was found in all the affected family members. In contrast, the other missense mutation (E418K) was found only in the proband. The E418K mutation was located in the stutter region, an interruption in the heptad repeat regularity, whose function as yet remains unclear. We hypothesized that this mutated stutter allele was clinically silent when combined with the wild type allele but aggravates the clinical severity of EBS caused by the E170K mutation on the other allele. To confirm this in vitro, we transfected mutant keratin 5 cDNA into cultured cells. Although only 12.7% of the cells transfected with the E170K mutation alone showed disrupted keratin filament aggregations, significantly more cells (30.0%) cotransfected with both E170K and E418K mutations demonstrated keratin aggregation (p < 0.05). These transfection assay results corresponded to the heterogeneous clinical findings of the EBS patient in this kindred. We have identified the first case of both compound heterozygous dominant (E170K) and recessive (E418K) mutations in any keratin gene and confirmed the significant involvement of the stutter region in the assembly and organization of the keratin intermediate filament network in vitro.

Adult↗
PubMed2002

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.

Connexin 26↗