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Results for “Type VII secretion”

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PubMed2000

Severe factor VII deficiency caused by a novel mutation His348 to Gln in the catalytic domain.

Factor VII is a vitamin K-dependent zymogen that plays a key role in the initiation of the extrinsic pathway. A severe factor VII deficiency was identified in a 45-year old male whose plasma factor VII antigen was less than 60 ng/ml and expressed 5.2% of normal factor VII activity. DNA sequence analysis of the patient's factor VII gene showed a thymidine to guanine transversion at nucleotide 10968 in exon VIII that results in a novel amino acid substitution of His348 to Gln. The patient was homozygous for this mutation, whereas some of his family members were heterozygous. Both wild type and mutant factor VII were transiently expressed in COS-1 cells. The level of secreted mutant factor VII antigen was only 11.0% of the level of wild type factor VII. In CHO cells stably transfected with the mutant factor VII, only 37.3% of the total labeled FVII was secreted into the conditioned media and the remainder was retained inside the cells. These data suggest this mutation leads to factor VII deficiency due to the impaired secretion of the molecule.

Amino Acid Sequence↗

PubMed2001

Synexin and GTP increase surfactant secretion in permeabilized alveolar type II cells.

We have previously suggested that synexin (annexin VII), a Ca(2+)-dependent phospholipid binding protein, may have a role in surfactant secretion, since it promotes membrane fusion between isolated lamellar bodies (the surfactant-containing organelles) and plasma membranes. In this study, we investigated whether exogenous synexin can augment surfactant phosphatidylcholine (PC) secretion in synexin-deficient lung epithelial type II cells. Isolated rat type II cells were cultured for 20-22 h with [(3)H]choline to label cellular PC. The cells were then treated with beta-escin, which forms pores in the cell membrane and releases cytoplasmic proteins including synexin. These cells, however, retained lamellar bodies. The permeabilized type II cells were evaluated for PC secretion during a 30-min incubation. Compared with PC secretion under basal conditions, the presence of Ca(2+) (up to 10 microM) did not increase PC secretion. In the presence of 1 microM Ca(2+), synexin increased PC secretion in a concentration-dependent manner, which reached a maximum at approximately 5 microg/ml synexin. The secretagogue effect of synexin was abolished when synexin was inactivated by heat treatment (30 min at 65 degrees C) or by treatment with synexin antibodies. GTP or its nonhydrolyzable analog beta:gamma-imidoguanosine-5'-triphosphate also increased PC secretion in permeabilized type II cells. The PC secretion was further increased in an additive manner when a maximally effective concentration of synexin was added in the presence of 1 mM GTP, suggesting that GTP acts by a synexin-independent mechanism to increase membrane fusion. Thus our results support a direct role for synexin in surfactant secretion. Our study also suggests that membrane fusion during surfactant secretion may be mediated by two independent mechanisms.

Animals↗

PubMed1989

Anchoring fibrils and type VII collagen are absent from skin in severe recessive dystrophic epidermolysis bullosa.

Skin of patients with severe generalized recessive dystrophic epidermolysis bullosa (SGRDEB) was studied by immunoelectron microscopy and immunoblotting with antibodies to type VII collagen, a major structural component of anchoring fibrils. In normal skin, the protein was localized to the dermoepidermal junction zone below the basement membrane and was extractable from the papillary dermis after artificial epidermolysis. In SGRDEB skin, neither immunoreactive material below the basement membrane nor identifiable anchoring fibrils could be recognized and neither the tissue form nor the specific proteolytic fragments of type VII collagen were found in extracts of SGRDEB skin. Very low amounts of type VII collagen alpha-chains could be detected in cultures of SGRDEB-fibroblasts, whereas normal fibroblasts synthesized more of this collagen. These results suggest that a genetic defect in the correct synthesis, secretion, or in the molecular assembly of type VII collagen may underlie SGRDEB.

Adolescent↗

PubMed2000

Development and characterization of a recombinant truncated type VII collagen "minigene". Implication for gene therapy of dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous disorder of skin caused by mutations in the type VII collagen gene. The lack of therapy for DEB provides an impetus to develop gene therapy strategies. However, the full-length 9-kilobase type VII collagen cDNA exceeds the cloning capacity of current viral delivery vectors. In this study, we produced a recombinant type VII minicollagen containing the intact noncollagenous domains, NC1 and NC2, and part of the central collagenous domain using stably transfected human 293 cell clones and purified large quantities of the recombinant minicollagen VII from culture media. Minicollagen VII was secreted as correctly-folded, disulfide-bonded, helical trimers resistant to protease degradation. Purified minicollagen VII bound to fibronectin, laminin-5, type I collagen, and type IV collagen. Furthermore, retroviral-mediated transduction of the minigene construct into DEB keratinocytes (in which type VII collagen was absent) resulted in persistent synthesis and secretion of a 230-kDa recombinant minicollagen VII. In comparison with parent DEB keratinocytes, the gene-corrected DEB keratinocytes demonstrated enhanced cell-substratum adhesion, increased proliferative potential, and reduced cell motility, features that reversed the DEB phenotype toward normal. We conclude that the use of the minicollagen VII may provide a strategy to correct the cellular manifestations of gene defects in DEB.

Cell Line↗

PubMed2001

Abnormal secretion and function of recombinant human factor VII as the result of modification to a calcium binding site caused by a 15-base pair insertion in the F7 gene.

A case of a novel mutation in the F7 gene that results in factor VII coagulant activity (VII:c) of less than 1% and VII antigen (VII:Ag) levels of 10% is presented. DNA analysis revealed a homozygous 15-base pair (bp) in-frame insertion-type mutation at nucleotide 10554. This insertion consisted of a duplication of residues leucine (L)213 to aspartic acid (D)217 (leucine, serine, glutamic acid, histidine, and aspartic acid), probably arising by slipped mispairing between 2 copies of a direct repeat (GCGAGCACGAC) separated by 4 bp. Molecular graphic analyses showed that the insertion is located at the surface of the catalytic domain in an exposed loop stabilized by extensive salt-bridge and hydrogen bond formation at which the calcium binding site is located. The mutation probably interferes with protein folding during VII biosynthesis and/or diminishes functional activity through the loss of calcium binding. In vitro expression studies demonstrated that the levels of VII:Ag in lysates of cells transfected with wild type VII (VIIWT) were equivalent to those with mutant type VII (VIIMT), but the level of secreted VIIMT was 5% to 10% that of VIIWT. Pulse chase studies demonstrated that VIIMT did not accumulate intracellularly, and studies with inhibitors of protein degradation showed that recombinant VIIMT was partially degraded in the pre-Golgi compartment. Accordingly, only small amounts of VIIMT with undetectable procoagulant activity were secreted into conditioned media. These results demonstrate that a combination of secretion and functional defects is the mechanism whereby this insertion causes VII deficiency.

Base Pair Mismatch↗

PubMed1997

The Arg353Gln polymorphism reduces the level of coagulation factor VII. In vivo and in vitro studies.

Factor VII levels are regulated by environmental and genetic factors. Two polymorphisms, a G-to-A transversion at nucleotide 10,976 resulting in Arg353Gln and a decanucleotide insert at position -323 in the 5'-flanking region of the factor VII gene, have been associated with a 20% to 25% reduction in plasma factor VII levels. However Arg353Gln almost always segregates on alleles containing the insert in UK and Italian populations, thereby making it impossible to independently evaluate the impact of Arg353Gln on factor VII levels in these ethnic groups. We have evaluated the influence of genotype on factor VII levels in 99 healthy Polish blood donors and observed that Arg353Gln frequently occurs in the absence of the insert. In univariate analysis, the mean levels of factor VII coagulant activity (VII:C) and factor VII antigen (VII:Ag) were significantly lower in 16 people who were heterozygous for Arg353Gln and the insert compared with 72 normal subjects who had neither Arg353Gln nor the insert (88.8% of normal and 83.1% versus 102% and 100%, P = .019 and P = .0003, respectively). In nine subjects heterozygous for Arg353Gln alone, VII:C and VII:Ag were significantly decreased compared with the normal subjects (81.9% and 83%, respectively, P = .007 and P = .004). In multivariate analysis, Arg353Gln but not the insert significantly reduced VII:C and VII:Ag after adjustment for age and plasma triglycerides (P < .05 and P = .02, respectively). To evaluate the mechanism responsible for reduced factor VII levels in individuals with Arg353Gln, we performed transient transfection assays with factor VII cDNA containing the base substitution resulting in Gln353 and wild-type factor VII cDNA in COS-1 cells. The levels of VII:Ag in the cell lysates were similar, but the amino acid substitution significantly reduced factor VII secretion into the media to 74.9% of wild-type (P = .0001). Based on these in vivo and in vitro studies, we conclude that the Arg353Gln polymorphism alone can decrease plasma factor VII levels.

Animals↗

PubMedDate not supplied

Hormonal regulation of plasminogen activator in rat and mouse seminiferous epithelium.

To elucidate the possible role of plasminogen activator (PA) in spermatogenesis and spermiation in mammals, we studied the hormonal regulation of PA secretion in cultured rat and mouse seminiferous tubules during defined stages of spermatogenesis. Results indicated that: (1) under basal conditions, segments of rat seminiferous tubules released primarily urokinase-type PA (uPA) at all stages of the cycle. The highest level of PA secretion occurred at stages VIIab, VIIcd and VIII. FSH, 8-bromo cyclic AMP and forskolin (FK) stimulated PA secretion, predominantly tissue-type PA (tPA). (2) In contrast, mouse seminiferous tubules secreted only tPA under basal conditions. In the presence of 50 microM MIX, seminiferous tubules at stages VII and VIII secreted higher levels of both types of PA than at the other stages. Both tPA and uPA secretion was enhanced by addition of FSH and FK to the organ culture media. (3) Segments of both rat and mouse seminiferous tubules at stages IX-XII in which the sperm residual bodies are absorbed into the Sertoli cells were also very sensitive to the addition of FSH to the organ culture. These results suggest that tPA in rat and mouse testes may play an essential role in the process of spermatogenesis and spermiation as well as in sperm residual body absorption.

1-Methyl-3-isobutylxanthine↗

PubMed2002

Enhanced secretion and uptake of beta-glucuronidase improves adeno-associated viral-mediated gene therapy of mucopolysaccharidosis type VII mice.

Previous treatment of mucopolysaccharidosis type VII mice (Sly syndrome) with AAV vectors has resulted in increased levels of beta-glucuronidase (GUS) enzyme in some tissues with reduction of glycosaminoglycan storage granules and improved health. By adding coding sequences for secretion (Igkappa) and uptake (HIV-1 TAT) signals to the GUS gene delivered by AAV, and treating mice both intrathecally and intravenously as newborns, we have increased the GUS enzyme levels in more tissues and have improved the health of the mice so much that they are able to breed. The levels of GUS in the serum were above normal in some mice, which caused reduction of storage in the spleen, a nontransduced tissue. The heart and aorta showed therapeutic levels of GUS enzyme. AAV GUS DNA was found in brain and liver, which showed no storage. Phenotypically the treated mice were more active and showed less stunted skeletal growth. The pups born to these mice were not affected by the gene therapy, as shown by mutant levels of GUS enzyme in their tissues and the absence of AAV GUS DNA. However, they were resistant to intravenous treatment with AAV GUS due to the mother's antibodies, but not to intrathecal treatment.

Animals↗

PubMed2005

HIP/PAP accelerates liver regeneration and protects against acetaminophen injury in mice.

Human hepatocarcinoma-intestine-pancreas/pancreatic-associated protein HIP/PAP is a secreted C-type lectin belonging to group VII, according to Drickamer's classification. HIP/PAP is overexpressed in liver carcinoma; however, its functional role remains unclear. In this study, we demonstrate that HIP/PAP is a paracrine hepatic growth factor promoting both proliferation and viability of liver cells in vivo. First, a low number of implanted hepatocytes deriving from HIP/PAP-transgenic mice (<1:1,000) was sufficient to stimulate overall recipient severe combined immunodeficiency liver regeneration after partial hepatectomy. After a single injection of HIP/PAP protein, the percentages of bromodeoxyuridine-positive nuclei and mitosis were statistically higher than after saline injection, indicating that HIP/PAP acts as a paracrine mitogenic growth factor for the liver. Comparison of the early events posthepatectomy in control and transgenic mice indicated that HIP/PAP accelerates the accumulation/degradation of nuclear phospho-signal transducer activator transcription factor 3 and tumor necrosis factor alpha level, thus reflecting that HIP/PAP accelerates liver regeneration. Second, we showed that 80% of the HIP/PAP-transgenic mice versus 25% of the control mice were protected against lethal acetaminophen-induced fulminate hepatitis. A single injection of recombinant HIP/PAP induced a similar cytoprotective effect, demonstrating the antiapoptotic effect of HIP/PAP. Comparison of Cu/Zn superoxide dismutase activity and glutathione reductase-like effects in control and transgenic liver mice indicated that HIP/PAP exerts an antioxidant activity and prevents reactive oxygen species-induced mitochondrial damage by acetaminophen overdose. In conclusion, the present data offer new insights into the biological functions of C-type lectins. In addition, HIP/PAP is a promising candidate for the prevention and treatment of liver failure.

Acetaminophen↗

PubMed1999

Mechanism underlying factor VII deficiency in Jewish populations with the Ala244Val mutation.

We investigated a Sephardic Jewish patient with a mild bleeding diathesis whose plasma levels of factor VII coagulant activity and factor VII antigen were 7% and 9% of normal, respectively. Sequencing demonstrated homozygosity for the Ala244Val mutation and the Arg353Gln polymorphism, which is associated with a modest decrease in factor VII levels. To elucidate the mechanism by which Ala244Val reduced factor VII levels in this patient, transient transfections were performed in COS-1 cells with wild type and mutant factor VII cDNAs and factor VII antigen levels in cell lysates and conditioned media were measured. The secretion of the mutant protein (FVII244V) into the media was 20% of wild type (FVIIwt), and intracellular levels of FVII244V were 60% of FVIIwt. A construct encoding Ala244Val along with the Arg353Gln polymorphism decreased the factor VII level in the media to that observed in the patient's plasma. Pulse-chase experiments demonstrated that FVII244V did not accumulate intracellularly and that low levels of the abnormal protein were maintained throughout the chase. To test the hypothesis that FVII244V results in an unstable molecule, amino acids with smaller (Gly) or larger (Phe) side chains were substituted for Val244 by site-directed mutagenesis. Transient transfection assays with these constructs demonstrated that the side chain of amino acid 244 is crucial in maintaining a proper conformation of the molecule. We conclude that Ala244Val results in a factor VII molecule that is unstable and is probably degraded intracellularly.

Alanine↗

PubMed1997

Decreased lysosomal storage in the adult MPS VII mouse brain in the vicinity of grafts of retroviral vector-corrected fibroblasts secreting high levels of beta-glucuronidase.

A deficiency of beta-glucuronidase (GUSB) causes the multisystem progressive degenerative syndrome, mucopolysaccharidosis (MPS) type VII (Sly disease), which includes mental retardation. Animal homologues of MPS VII (ref. 3, 4) are models for testing somatic gene transfer approaches to treat the central nervous system in this and other lysosomal storage disorders. Previous attempts to correct murine MPS VII by gene therapy have successfully treated lesions in some organs but not in the brain. Other experimental modalities have forestalled some disease progression in the brain, but only if done at birth, before the onset of severe lesions, when the animals are phenotypically normal. We tested whether therapeutic amounts of GUSB could be delivered to the diseased adult brain by transplanting cells engineered to super-secrete the normal enzyme for export to surrounding neural tissues. Lysosomal distention was cleared from neurons and glial cells in the vicinity of the grafts, showing that the secreted enzyme could reach the diseased cells and reverse lesions in the severely diseased brain. The ability to correct established lesions will be important for the treatment of many lysosomal storage diseases affecting the brain, because most patients are not diagnosed until lesions are advanced enough to affect phenotype or developmental milestones in early childhood, and some forms of the diseases do not become apparent until later in life.

Animals↗

PubMed2004

A novel gene mutation in the 60s loop of human coagulation factor VII - inhibition of interdomain crosstalk.

A novel mutation in the factor VII gene resulting in procoagulant activity of 7.5% and antigen levels of 23% is presented. Single-stranded conformational polymorphism and DNA sequencing analysis revealed heterozygous shifts, and mutations were detected in exons 5, 7 and 8. The mutant L204P in exon 7 was novel, while the common polymorphisms, H115H and R353Q, were located in exons 5 and 8, respectively. The molecular effect of the L204P mutation was characterized using recombinant mammalian expression in Chinese hamster ovary cells. Low levels (4 ng/ml) of secreted mutant protein were found in transiently transfected cells compared to wild-type factor VII (83 ng/ml). Metabolic labeling demonstrated that the rate of mutant protein synthesis was similar to that of wild-type FVII, and the mutant protein accumulated intracellularly with no signs of increased degradation during a four-hour chase. No interaction between secreted P204 protein and immobilized soluble tissue factor was detected using surface plasmon reso-nance. The activation rate of recombinant mutant FVII protein was strongly reduced compared to wild-type FVII. A 9-fold reduction in the rate of FX activation was detected whereas Km was nearly the same for wild-type and the mutant. This slow rate was caused by a correspondingly lowered rate of P204 activation. A synthetic peptide sequence comprising amino acids 177-206 blocked binding of FVIIa to the TF-chip, and the subsequent factor X activation with an IC(50) value of 0.5 micro M in a chromogenic factor Xa assay. Additionally, evaluation of the peptide by surface plasmon resonance analysis resulted in inhibition of complex formation with an apparent K(I) of 7 micro M.

Acetylcysteine↗

PubMed1987

Anchoring fibrils contain the carboxyl-terminal globular domain of type VII procollagen, but lack the amino-terminal globular domain.

Type VII procollagen has been characterized as a product of epithelial cell lines. As secreted, it contains a large triple-helical domain terminated by a multi-globular-domained carboxyl terminus (NC-1), and a smaller amino-terminal globule (NC-2). The triple helix and the NC-1 domain have previously been identified in anchoring fibril-containing tissues by biochemical and immunochemical means, leading to the conclusion that type VII collagen is a major component of anchoring fibrils. In order to better characterize the tissue form of type VII collagen, we have produced a panel of monoclonal antibodies which recognize the NC-1 domain. Peptide mapping of these epitopes indicate that they are independent and span approximately 125,000 kDa of the total 150,000 kDa of each alpha chain contained in NC-1. All these antibodies elicit immunofluorescent staining of the basement membrane zone in tissues. Type VII collagen has been extracted from tissues. As previously reported, it is smaller than type VII procollagen, (Woodley, D. T., Burgeson, R. E., Lunstrum, G. P., Bruckner-Tuderman, L., and Briggaman, R. A., submitted for publication), and we now find that it predominantly occurs as a dimer. Following clostridial collagenase digestion, intact NC-1 has been recognized, indicating that the difference in apparent Mr between the tissue form of the molecule and type VII procollagen results from modification of the amino terminus. The size of the amino-terminal globule has been determined to be between approximately 96 and 102 kDa. Rotary shadowing analyses of extracted molecules indicate that dimeric molecules contain the NC-1 domain, but are missing intact NC-2. We propose that the tissue form monomer, Mr = 960,000, be referred to as "type VII collagen." These studies strongly suggest that anchoring fibrils contain dimeric molecules with intact NC-1 domains. The data also support the previous suggestion that the NC-2 domain is involved in the formation of disulfide bond-stabilized type VII collagen dimers, and is subsequently removed by physiological proteolytic processing.

Amnion↗

PubMed2001

Engraftment of genetically engineered amniotic epithelial cells corrects lysosomal storage in multiple areas of the brain in mucopolysaccharidosis type VII mice.

Cell-mediated gene therapy for visceral lesions of lysosomal storage diseases is promising; however, the treatment of central nervous system (CNS) lesions remains a challenge. In this study, we generated rat amniotic epithelial cells (AEC) that overexpress and secrete human beta-glucuronidase (GUSB) following transduction with an adenoviral vector encoding human GUSB. The AEC were used as donor cells for cell-mediated gene therapy of CNS lesions in mice with mucopolysaccharidosis type VII (MPSVII), a lysosomal storage disorder caused by an inherited deficiency of GUSB activity. After confirmation that the secreted GUSB was taken up mainly via mannose 6-phosphate receptors in primary cultured neurons, the AEC were transplanted into the brains of adult MPSVII mice. Histochemical analysis showed extensive GUSB activity throughout the ipsilateral hemisphere of the recipient brains, and pathological improvement of the lysosomal storage was observed even in regions far from the site of injection. These results suggest that intracerebral transplantation of genetically engineered AEC has therapeutic potential for the treatment of CNS lesions in lysosomal storage disorders.

Adenoviridae↗

PubMed1997

Microcapsules as bio-organs for somatic gene therapy.

Current human gene therapy relies on genetic modification of the patient's own cells. An alternate non-autologous approach is to use universal cell lines engineered to secrete therapeutic products. Protection with immuno-isolation devices would allow the same recombinant cell line to be used for different patients, thus potentially lowering the cost of treatment. The feasibility of this idea has now been demonstrated in vitro and in vivo. Recombinant gene products with potential therapeutic applications (human growth hormone, factor IX, lysosomal enzymes, adenosine deaminase) have been expressed from genetically modified cells after encapsulation with alginate-poly-L-lysine-alginate or hydroxyethyl methacrylate-methyl methacrylate. We have also demonstrated the feasibility of this idea in vivo. After intraperitoneal implantation, genetically modified mouse Ltk- fibroblasts or C2C12 myoblasts encapsulated in alginate-poly-L-lysine-alginate could deliver recombinant gene products (human growth hormone, human factor IX) to the systemic circulation of mice. The clinical efficacy of this novel approach to gene therapy has now been shown in murine models of human diseases. In the Snell dwarf mice deficient in growth hormone production, implantation of encapsulated mouse myoblasts engineered to secrete mouse growth hormone resulted in increases in body weight, length and organ sizes, some to > 25% above those of the controls. In the Gus/Gus mice suffering from the lysosomal storage disease mucopolysaccharidosis type VII due to deficient beta-glucuronidase, implantation of encapsulated mouse fibroblasts engineered to secrete mouse beta-glucuronidase resulted in delivery of normal levels of the enzyme in the plasma and significant correction of the organ histopathology. Hence, delivery of recombinant gene products through bioartificial devices appears to be a promising strategy for the treatment of genetic diseases.

Alginates↗

PubMed1997

Ultraviolet A irradiation upregulates type VII collagen expression in human dermal fibroblasts.

Type VII collagen, a major component of skin-anchoring fibrils, is synthesized by both fibroblasts and keratinocytes, the two principal cell types in the skin. In this study, we examined the effects of ultraviolet A (UVA) irradiation on the expression of type VII collagen in human fibroblasts. UVA irradiation (0-15 J/cm2) caused a dose-dependent increase (5- to 10-fold) in type VII collagen mRNA levels as detected by northern blot analysis. The UVA-induced enhancement of type VII collagen gene expression correlated with an increase in its protein level by immunoblot analysis of proteins secreted into the conditioned medium. The effect of UVA was observed at 12 h and reached its maximum by 18 h. Under these conditions, however, the expression of fibronectin, a major dermal matrix protein, remained unchanged, suggesting that the induction of type VII collagen expression was selective. Actinomycin D, a transcription inhibitor, blocked the UVA-mediated induction of type VII collagen gene expression, whereas cycloheximide, a protein synthesis inhibitor, superinduced the expression of type VII collagen, suggesting that de novo protein synthesis was not required for the action of UVA. Interestingly, in contrast to the increased type VII collagen expression in fibroblasts in response to UVA, a slight decrease in type VII collagen mRNA level was observed in the UVA-irradiated keratinocytes, suggesting that the effect of UVA on the type VII collagen expression is cell type specific.

Adult↗

PubMed1990

Epithelial origin of cutaneous anchoring fibrils.

Anchoring fibrils are essential structural elements of the dermoepidermal junction and are crucial to its functional integrity. They are composed largely of type VII collagen, but their cellular origin has not yet been confirmed. In this study, we demonstrate that the anchoring fibrils are primarily a product of epidermal keratinocytes. Human keratinocyte sheets were transplanted to a nondermal connective tissue graft bed in athymic mice. De novo anchoring fibril formation was studied ultrastructurally by immunogold techniques using an antiserum specific for human type VII procollagen. At 2 d after grafting, type VII procollagen/collagen was localized both intracellularly within basal keratinocytes and extracellularly beneath the discontinuous basal lamina. Within 6 d, a subconfluent basal lamina had developed, and newly formed anchoring fibrils and anchoring plaques subjacent to the xenografts were labeled. Throughout the observation period of the experiment, the maturity, population density, and architectural complexity of anchoring fibrils beneath the human epidermal graft continuously increased. Identical findings were obtained using xenografts cultivated from cloned human keratinocytes, eliminating the possibility of contributions to anchoring fibril regeneration from residual human fibroblasts. Immunolabeling was not observed at the mouse dermoepidermal junction at any time. These results demonstrate that the type VII collagen of human cutaneous anchoring fibrils and plaques is secreted by keratinocytes and can traverse the epidermal basal lamina and that the fibril formation can occur in the absence of cells of human dermal origin.

Animals↗

PubMed1990

Intra-epidermal retention of type VII collagen in a patient with recessive dystrophic epidermolysis bullosa.

An infant born with severe blisters on the limbs, face, trunk, and oral mucosa was diagnosed by light and electron microscopy to have recessive dystrophic epidermolysis bullosa. Transmission electron microscopy showed that the basal lamina remained with the epidermis and that the floor of the blister was exposed collagen of the papillary dermis. No banded anchoring fibrils were observed along either the roof or the floor of the blister; however, small filamentous structures, possibly immature anchoring fibrils, extended down from the lamina densa along the blister roof. Some basal and suprabasal keratinocytes contained large vesicles filled with filamentous matrix of variable electron density. Immunofluorescent staining of skin for type VII collagen showed sparse and irregular staining of type VII collagen along the blister roof, and intense intracellular labeling for type VII collagen in clusters of epidermal cells in basal and suprabasal layers. Type VII collagen appeared to be synthesized by keratinocytes but not secreted.

Antibodies, Monoclonal↗