Impact of adopting lower-fat food choices on energy and nutrient intakes of American adults

Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical domain flanked by two noncollagenous, globular domains, NC1 and NC2. Approximately 50% of the molecular mass of the molecule is consumed by the NC1 domain. We previously demonstrated that NC1 binds to various extracellular matrix components including a complex of laminin 5 and laminin 6 (Chen et al, 1997a). In this study, we examined the interaction of NC1 with laminin 5 (a component of anchoring filaments). Both authentic and purified recombinant NC1 bound to human and rat laminin 5 as measured by enzyme-linked immunosorbant assay and by binding of 125I-radiolabeled NC1 to laminin 5-coated wells, but not to laminin 1 or albumin. NC1 bound predominantly to the beta3 chain of laminin 5, but also to the gamma2 chain when examined by a protein overlay assay. The binding of 125I-NC1 to laminin 5 was inhibited by a 50-fold excess of unlabeled NC1 or de-glycosylated NC1, as well as a polyclonal antibody to laminin 5 or a monoclonal antibody to the beta3 chain. In contrast, the NC1-laminin 5 interaction was not affected by a monoclonal antibody to the alpha3 chain. Using NC1 deletion mutant recombinant proteins, a 285 AA (residues 760-1045) subdomain of NC1 was identified as the binding site for laminin 5. IgG from an epidermolysis bullosa acquisita serum containing autoantibodies to epitopes within NC1 that colocalized with the laminin 5 binding site inhibited the binding of NC1 to laminin 5. Thus, perturbation of the NC1-laminin 5 interaction may contribute to the pathogenesis of epidermolysis bullosa acquisita.     

Class II, division 1, case with multiple treatment challenges

IgG antibodies from the sera of some patients with bullous pemphigoid (BP) react with a 180 kDa protein termed BPAg2. Antibodies in BP are directed to an extracellular noncollagenous domain of this protein termed NC16A. Our group has recently shown that a portion of the extracellular domain of BPAg2 is identical to LABD97 on the basis of amino acid sequencing. We evaluated sera from 33 patients with BP with circulating IgG antibodies on indirect immunofluorescence, which stained the epidermal side of split skin with titers ranging from 1:40 to 1:640. Immunoblotting was performed against (i) two preparations of proteins from epidermal extract, one containing BPAg2 and one containing LABD97, and (ii) the recombinant NC16A domain of the BPAg2 protein. Twelve sera reacted with the BPAg2 protein. Ten of these also reacted strongly with the NC16A domain. Nine of the 12 sera also reacted with the LABD97 antigen. Bound antibodies were eluted from the 97 kDa band and reapplied to split skin where they bound to the epidermal side. The eluted antibodies also reacted to the BPAg2 protein from the epidermal extract, but did not react with the NC16A domain on immunoblot. We conclude that these nine sera react with an epitope present within BPAg2 and LABD97 but not within the NC16A domain. This epitope is therefore distal to the previously described epitopes in BP. In BP, epitope spreading may occur and antibodies may be produced that recognize the distal portion of the BPAg2 molecule identical to LABD97 but that do not involve the NC16A domain.     

Reactions to less common species of fire ants

The BP180 antigen, a component of the epidermal anchoring complex, has been identified as one of the major antigenic targets of autoantibodies associated with the blistering skin disease, bullous pemphigoid. Our research group has recently demonstrated that reactivity of bullous pemphigoid autoantibodies to the BP180 ectodomain is almost entirely restricted to a set of four antigenic sites clustered within the membrane-proximal noncollagenous stretch (NC16A). Using a passive transfer mouse model, antibodies to the corresponding noncollagenous region of murine BP180 were shown to trigger an inflammatory subepidermal blistering disease that closely mimics bullous pemphigoid. We now report the development of an enzyme-linked immunoabsorbent assay system that is extremely sensitive in detecting disease-specific autoantibodies in the sera of bullous pemphigoid patients. The target antigen in this assay is a recombinant form of the BP180 NC16A domain that contains all four of the well-defined bullous pemphigoid-associated antigenic sites. Of 50 randomly selected bullous pemphigoid sera tested, 47 (94%) were positive in this assay, whereas no specific reactivity was detected in any of the 107 controls. Interestingly, all three of the bullous pemphigoid sera that were negative in this assay had been obtained from patients who were already undergoing treatment. The NC16A enzyme-linked immunosorbent assay is more sensitive than any of the standard techniques for detecting circulating bullous pemphigoid autoantibodies, including other enzyme-linked immunosorbent assays, immunoblotting, and indirect immunofluorescence. Finally, the NC16A enzyme-linked immunosorbent assay provides immunologic information that cannot be obtained from direct immunofluorescence studies of skin biopsies, and that may well be relevant in the diagnosis and treatment of bullous pemphigoid.     

A case of nonscarring inflammatory epidermolysis bullosa acquisita: characterization of IgG autoantibodies by immunofluorescence, immunoblotting and immunogold electron microscopy

We report a case of nonscarring inflammatory epidermolysis bullosa acquisita in a 59-year-old Japanese woman. She developed blisters and erosions on her lip, trunk and extremities. Sodium aurothiomalate was effective for the skin lesions. The patient had been free from bullous skin lesions for the last 13 years and had shown no scarring. Indirect immunofluorescence (IF) study on 1 M NaCl-split skin revealed IgG autoantibodies against the dermal side of the split skin. Immunoblotting using normal human dermal extracts disclosed IgG autoantibodies reactive with the 290 and 145 kD antigens. Circulating IgG autoantibodies were deposited on the lamina densa by immunoelectron microscopy. IF mapping using several antibodies for the components of the basement membrane zone revealed blister formation at the lamina densa. These results suggest that the cleavage at the lamina lucida does not necessarily exclude the diagnosis of EBA and that the definite diagnosis of EBA should be confirmed by immunoblotting or immunoelectron microscopic study.     

Epidermolysis bullosa acquisita diagnosed by direct immunoelectron microscopy of the conjunctiva

OBJECTIVE: To describe for the first time the direct immunoelectron microscopic pattern of immune deposits on the conjunctival basement membrane in epidermolysis bullosa acquisita (EBA). DESIGN: Case reports. PARTICIPANTS: Two patients. INTERVENTION: Epidermolysis bullosa acquisita associated with cicatrizing conjunctivitis. MAIN OUTCOME MEASURES: Direct immunofluorescence and direct immunoelectron microscopy without freezing on conjunctival and skin biopsy specimens, indirect immunofluorescence, Western immunoblot analysis. RESULTS: Results of direct immunoelectron microscopic examination of the conjunctiva showed the presence of immune deposits in the anchoring fibril zone, just beneath the lamina densa, in both patients. This finding was the same as the direct immunoelectron microscopic pattern shown in the skin of these patients, which is known to be very specific for EBA. Direct immunofluorescence was positive in the conjunctiva of only one patient. Indirect immunofluorescence and Western immunoblot analysis failed to detect circulating autoantibodies. CONCLUSIONS: Direct immunoelectron microscopy on the conjunctiva is a useful diagnostic tool to differentiate EBA from other related autoimmune mucocutaneous blistering diseases.     

Helix-coil transitions in DNA using a pH variation method: case of a melting paradox as a function of ionic strength

We describe a 31-year-old Japanese woman with generalized pustular psoriasis treated with PUVA who subsequently developed a bullous disease. Throughout the disease course, there was no phase of psoriasis vulgaris. Although several reports describe coexistence of psoriasis vulgaris and bullous disease such as bullous periphigoid, coexistence of generalized pustular psoriasis without any phase of psoriasis vulgaris and bullous disease is rare. As for the bullous disease, direct immunofluorescence study showed IgG and C3 deposition along the basement membrane zone. Indirect immunofluorescence disclosed IgG antibasement membrane zone antibodies. Indirect immunofluorescence on 1 mol/l sodium chloride-split skin demonstrated linear IgG staining almost exclusively on the dermal side of the split. Western immunoblot analysis revealed that the antibody was directed to neither epidermolysis bullosa acquisita antigen nor bullous pemphigoid antigens. Considering the unusual clinical course, we suspect the possibility of a novel autoimmune blistering disease.     

The immunofluorescence evaluation of some subepidermal bullous diseases by use of NaCl-split skin

Authors described the results of immunohistological investigation in 13 patients--12 with clinical diagnosis of bullous pemphigoid and 1 patient with clinical diagnosis of epidermolysis bullosa acquisita. 1 M NaCl solution was used to split normal human skin, used as substrates for immunohistological testing of the antilamina lucida and anti-sublamina densa antibodies.     

Modulation of disease severity of dystrophic epidermolysis bullosa by a splice site mutation in combination with a missense mutation in the COL7A1 gene

Dystrophic epidermolysis bullosa (EBD) is a clinically heterogeneous skin disorder, characterized by abnormal anchoring fibrils (AF) and loss of dermal-epidermal adherence. EBD has been linked to the COL7A1 gene at chromosome 3p21 which encodes collagen VII, the major component of the AF. Here we investigated two unrelated EBD families with different clinical phenotypes and novel combinations of recessive and dominant COL7A1 mutations. Both families shared the same recessive heterozygous 14 bp deletion at the exon-intron 115 boundary of the COL7A1 gene. The deletion caused in-frame skipping of exon 115 and the elimination of 29 amino acid residues from the pro-alpha1(VII) polypeptide chain. As a result, procollagen VII was not converted to collagen VII and the C-terminal NC-2 propeptide which is normally removed from the procollagen VII prior to formation of the anchoring fibrils was retained in the skin. All affected individuals also carried missense mutations in exon 73 of COL7A1 which lead to different glycine-to-arginine substitutions in the triple-helical domain of collagen VII. Combination of the deletion mutation with a G2009R substitution resulted in a mild phenotype. In contrast, combination of the deletion with a G2043R substitution led to a severe phenotype. The G2043R substitution was a de novo mutation which alone caused a mild phenotype. Thus, different combinations of dominant and recessive COL7A1 mutations can modulate disease activity of EBD and alter the clinical presentation of the patients.     

Lichen planus pemphigoides is a heterogeneous disease: a report of five cases studied by immunoelectron microscopy

Lichen planus pemphigoides (LPP) is a rare and controversial disease. It is characterized by bullae arising on lichen planus papules and on uninvolved skin, subepidermal bullae in histology, and linear deposits of IgG and C3 along the basal membrane zone on immunofluorescence of peribullous skin. Our goal was to identify the localization of the target antigen in cases of LPP. Five patients diagnosed with LPP on clinical, histological and immunofluorescence criteria were explored by immunoelectron microscopy and immunoblot. Our results show that the target antigen in LPP is not unique. The localization of the immune deposits was consistent with a diagnosis of bullous pemphigoid in two cases, of cicatricial pemphigoid in two cases and of epidermolysis bullosa acquisita in one case. Our study supports the view that LPP is a heterogeneous condition in which lichen planus may induce different subepidermal acquired bullous dermatoses.     

Effect of nonenzymatic glycosylation on the titers of circulating autoantibodies in pemphigus and pemphigoid

Hyperglycemia is observed in some patients with autoimmune bullous diseases complicated by diabetes mellitus or treated with systemic corticosteroids. High concentrations of glucose can react with various proteins and change their structural and functional properties. We previously reported that nonenzymatic glycosylation of antibody can impair antigen-antibody binding. We ascertained whether glycosylation of autoantibody decreases the autoantibody titer by examining 30 sera from patients with pemphigus and pemphigoid. Nonenzymatic glycosylation in the physiological range was induced by incubation of sera with 1650 mM D-glucose at 4 degrees C for 7 days. The titers of sera were determined by indirect immunofluorescence (IIF). In all cases, the immunofluorescence intensity of glycosylated sera was weaker than that of nonglycosylated sera. Glycosylated sera showed a lower antibody titer by 1 doubling dilution in 18 out of 30 cases, compared with nonglycosylated sera. The ten BP patients' sera were also analyzed by immunoblotting for reactivity with the BP180-GST fusion proteins, S delta 1 and 4575. All BP sera reacted with S delta 1, and 5 out of 10 BP sera reacted with both S delta 1 and 4575. In all the sera that reacted only with S delta 1, the glycosylated sera showed a 1 doubling dilution decrease in autoantibody titer. Interestingly, in 4 out of 5 sera that reacted with both S delta 1 and 4575, there were no differences in the antibody titer between glycosylated and nonglycosylated sera. These results indicate the possibility of a false decrease in autoantibody titers of sera from patients with autoimmune bullous diseases complicated with hyperglycemia. Although the false decrease in titers of autoantibodies induced by nonenzymatic glycosylation is not dramatic, it must be considered in order not to underestimate the disease activity of pemphigus in such cases.     

Molecular biological aspects of acquired bullous diseases

Bullous diseases of the oral mucosa and skin were originally classified on the basis of clinical and histological criteria. The discovery of autoantibodies in some of these patients and the introduction of molecular biology have resulted in a new understanding of the pathological mechanisms of many of the bullous lesions. In this article, updated topics of the immune-mediated bullous lesions which involve oral mucosa and skin are reviewed. Pemphigus antigens, which are desmosomal-associated proteins and belong to the cadherin superfamily of cell adhesion proteins, have been isolated, and their genes have been cloned. The antigens which react with autoantibodies from patients with bullous pemphigoid, cicatricial pemphigoid, acquired epidermolysis bullosa, and linear IgA disease are all proteins of the hemidesmosome basement membrane complex. Interestingly, most of the antigens also appear to be the target for mutations seen in patients with the inherited type of epidermolysis bullosa in which bullous lesions are a prominent clinical feature.     

Clinical diagnosis in patients with smooth muscle antibodies. A study of a one-year material

Out of 17 109 sera tested for autoantibodies by indirect immunofluorescence, 236 contained smooth muscle antibodies (SMA) with a titre of greater than or equal to 1/25. The majority of these sera, from 190 patients, reacted both with smooth muscle and renal glomeruli and the specificity of these SMA is against actin. 91% of high-titred sera (greater than or equal to 1/100) with IgG antibodies giving this staining pattern were derived from patients with chronic inflammatory liver disease, mainly chronic active hepatitis. In the group with a titre of 1/25, non-liver diseases such as joint diseases were more common and liver conditions occurred only in 55%. Sera with SMA of IgM class weremostly derived from patients with acute viral hepatitis.     

Identification of different circulating autoantibodies in patients with bullous pemphigoid and pemphigus vulgaris by means of immunoblotting

Immunoblotting studies with salt-split human epidermis were performed on sera from 15 patients with pemphigus vulgaris and 20 patients with bullous pemphigoid by using peroxidase-labelled antihuman IgG and IgA. Eleven sera of pemphigus vulgaris antigen. Most of the sera gave additional specific bands at 210 and 80 kD, with lower intensity. The sera of 4 patients, 3 of them were in clinical remission, did not yield specific bands. Seventeen sera of the 20 bullous pemphigoid patients yielded a 220-230 kD protein band against the major bullous pemphigoid antigen and 4 of them gave another specific band at 160-180 kD. Five sera produced multiple bands (220, 130, 100 and 75 kD). IgA antibodies against the major bullous pemphigoid antigen were demonstrated in 2 cases with IgA deposits along their basement membrane, as revealed by direct immunofluorescence. The immunoblot patterns correlated only weakly with the clinical findings in bullous pemphigoid. There was a considerable diversity in both clinical findings and immunoblot patterns.     

New insights into the immunoultrastructural organization of cutaneous basement membrane zone molecules

The epidermal basement membrane zone (BMZ) is composed of various molecules, each of which plays an important role in dermo-epidermal adhesion. Genetic abnormality of certain BMZ molecules leads to an inherited group of skin diseases collectively referred to as epidermolysis bullosa, whose hallmark is skin fragility of varying degrees. Furthermore, development of autoantibodies to certain BMZ molecules leads to the onset of a number of acquired autoimmune blistering diseases in which dermo epidermal separation occurs, including bullous pemphigoid and epidermolysis bullosa acquisita. The ultrastructural location of each BMZ molecule has been studied using a range of immunoelectron microscopy (immuno-EM) techniques. Recent technical advances in immuno-EM and in molecular engineering for production of epitope-specific antibodies have enabled a more correct and precise elucidation of the native ultrastructural molecular organization of the respective molecules and their relationship to each other. These recent studies have also revealed several misinterpretations in the previously established model of the immunoultrastructural organization of BMZ molecules. In response to these findings, this review focuses on three major BMZ-related molecules, type VII collagen, BPAG2 and laminin 5, for which recent immuno-EM studies have produced a revision in the accepted dogma on their ultrastructural distribution at the BMZ.     

Use of in vivo 13C nuclear magnetic resonance spectroscopy to follow sugar uptake in Zymomonas mobilis

An image analyzer was applied to pre- and post-embedding immunogold labeling with 5-nm gold probes in electron micrographs of several skin basement membrane antigens to improve the visualization of immunolabeling. With a TV camera connected to a color image analyzer, an image of an original immunoelectron micrograph was projected on a TV screen. The image was recorded in the analyzer as Record 1. After the floating threshold method procedure to reduce the contrast of the skin structure, electron-dense 5-nm gold particles could be easily detected. With the analyzer, these particles were then suitably enhanced in color and in size and their image was recorded as Record 2. Records 1 and 2 were then overlapped on the TV screen to build up a double-image picture. Compared with the small, electron-dense 5-nm gold particles in the original electron micrograph, ultrastructural localization of bullous pemphigoid antigen, epidermolysis bullosa acquisita antigen, and the collagenous part of Type VII collagen were more clearly and easily seen, even on low-magnification electron micrographs. The level of background labeling could also be accurately and objectively evaluated. By counting the number of gold particles labeling a certain area and using the analyzer to interpret the result as a diagram, quantitative analysis was also possible. We believe that this technique should be widely applicable to immunogold electron microscopy, not only of skin but also of other substrates of interest.     

Comparison of somatic development in children born at term or prematurely

Ultrastructural examination was performed in 9 biopsy specimens from 4 patients with the Cockayne-Touraine type of epidermolysis bullosa dystrophica dominans. The specimens were taken from: 1. clinically normal skin from the blister-nonpredilected sites (trunk) as well as 2. atrophic, 3. intact, and 4. experimentally frictioned skin regions from the blister-predilected sites (extremities). In the frictioned skin a dermolytic blister formations was observed. Development of anchoring fibrils showed a marked regional difference, the counts of fibrils being significantly lower (40%) in the predilection sites than in the nonpredilection sites. In addition the anchoring fibrils showed a variable degree of abnormal structure. The low frequency of often abnormally structured anchoring fibrils in the blister-preferred sites provides a good explanation for the clinical features. More studies are needed to see if regional differences in fibril frequency is a feature also of normal skin, in which case the dominant epidermolysis gene may represent a mutated structural anchoring fibril gene.     

Dependence of apparent diffusion coefficients on axonal spacing, membrane permeability, and diffusion time in spinal cord white matter

Paraneoplastic pemphigus (PNP) is an autoimmune blistering disease that occurs in association with underlying neoplasms. Patients with PNP develop characteristic IgG autoantibodies directed against multiple antigens, most of which have been identified as cytoplasmic proteins of the plakin family (desmoplakin I, II, BPAG1, envoplakin, and periplakin). This study identified cell surface target antigens of PNP. We focused on desmoglein (Dsg) 3 and Dsg1, the autoantigens of pemphigus vulgaris and pemphigus foliaceus. ELISA using baculovirus-expressed recombinant Dsgs (rDsg3, rDsg1) has revealed that 25 out of 25 PNP sera tested were positive against Dsg3 and 16 of 25 were positive against Dsg1. All of 12 PNP sera tested immunoprecipitated Dsg3. Removal of anti-Dsg3 autoantibodies by immunoadsorption was sufficient to eliminate the ability of PNP sera to induce cutaneous blisters in neonatal mice in vivo. Furthermore, anti-Dsg3-specific antibodies that were affinity purified from PNP sera were proven to be pathogenic and caused blisters in neonatal mice. These findings indicate that Dsg3 and Dsg1 are the cell surface target antigens in PNP and that IgG autoantibodies against Dsg3 in PNP sera play a pathogenic role in inducing loss of cell adhesion of keratinocytes and causing blister formation.     

Phenytoin penetration into brain after administration of phenytoin or fosphenytoin

Paraneoplastic pemphigus (PNP) is an autoimmune blistering disease that occurs in association with underlying neoplasms. PNP patients develop characteristic autoantibodies directed against multiple antigens, mostly identified as members of the plakin family of cytoplasmic proteins (desmoplakin I and II, bullous pemphigoid antigen I, envoplakin, and periplakin). HD1/plectin, another member of the plakin family, has not previously been detected in the characteristic PNP antigen complex, which may relate to practical difficulties associated with its large size (molecular weight approximately 500 kDa). In this study, a combination of immunoprecipitation and immunoblot is used to demonstrate that HD1/plectin is also recognized by sera from PNP patients. Thirteen of 16 PNP sera tested were positive for HD1/plectin compared with none of 43 control sera (11 pemphigus vulgaris, 11 pemphigus foliaceus, 11 bullous pemphigoid, and 10 normal individuals). Combined with our recent finding that desmoglein 3 and desmoglein 1 are cell surface target antigens in PNP, this demonstration of plectin/HD1 as another component of the antigen complex in PNP confirms that PNP is an autoimmune disease against desmoglein and plakin family molecules.     

Some, but not all, glycine substitution mutations in COL7A1 result in intracellular accumulation of collagen VII, loss of anchoring fibrils, and skin blistering

COL7A1 gene mutations cause dystrophic epidermolysis bullosa, a skin blistering disorder. The phenotypes result from defects of collagen VII, the major component of the anchoring fibrils at the dermo-epidermal junction; however, the molecular mechanisms underlying the phenotypes remain elusive. We investigated naturally occurring COL7A1 mutations and showed that some, but not all, glycine substitutions in collagen VII interfered with biosynthesis of the protein in a dominant-negative manner. Three point mutations in exon 73 caused glycine substitutions G2006D, G2034R, and G2015E in the triple helical domain of collagen VII and interfered with its folding and secretion. Confocal laser scanning studies and semiquantitative immunoblotting determined that dystrophic epidermolysis bullosa keratinocytes retained up to 2.5-fold more procollagen VII within the rough endoplasmic reticulum than controls. Limited proteolytic digestions of mutant procollagen VII produced aberrant fragments and revealed reduced stability of the triple helix. In contrast, the glycine substitution G1519D in another segment of the triple helix affected neither procollagen VII secretion nor anchoring fibril function and remained phenotypically silent. These data demonstrate that collagen VII presents a remarkable exception among collagens in that not all glycine substitutions within the triple helix exert dominant-negative interference and that the biological consequences of the substitutions probably depend on their position within the triple helix.