The small bovine amelogenin LRAP fails to rescue the amelogenin null phenotype.

Amelogenins are the most abundant secreted proteins in developing dental enamel. These evolutionarily-conserved proteins have important roles in enamel mineral formation, as mutations within the amelogenin gene coding region lead to defects in enamel thickness or mineral structure. Because of extensive alternative splicing of the primary RNA transcript and proteolytic processing of the secreted proteins, it has been difficult to assign functions to individual amelogenins. To address the function of one of the amelogenins, we have created a transgenic mouse that expresses bovine leucine-rich amelogenin peptide (LRAP) in the enamel-secreting ameloblast cells of the dental organ. Our strategy was to breed this transgenic mouse with the recently generated amelogenin knockout mouse, which makes none of the amelogenin proteins and has a severe hypoplastic and disorganized enamel phenotype. It was found that LRAP does not rescue the enamel defect in amelogenin null mice, and enamel remains hypoplastic and disorganized in the presence of this small amelogenin. In addition, LRAP overexpression in the transgenic mouse (wildtype background) leads to pitting in the enamel surface, which may result from excess protein production or altered protein processing due to minor differences between the amino acid compositions of murine and bovine LRAP. Since introduction of bovine LRAP into the amelogenin null mouse does not restore normal enamel structure, it is concluded that other amelogenin proteins are essential for normal appearance and function.

Randomized, double-blind, placebo-controlled trial of autologous blood therapy for atopic dermatitis.

BACKGROUND: Autologous blood therapy (ABT) is used for treating atopic dermatitis (AD) in some European countries and is promoted on internet sites for this condition. However, there is little evidence from rigorous clinical trials to suggest that it is effective. OBJECTIVES: To test the effectiveness of ABT for the symptomatic treatment of patients with AD. METHODS: Fifty subjects responded to press advertisements, and 31 were randomized within strata of severity at recruitment. Patients were included into a double-blind, placebo-controlled trial and received ABT or placebo once weekly for 5 weeks. Assessments were performed at baseline, at weekly intervals and after a 5-week follow up. The Six Area, Six Sign AD (SASSAD) severity index was predefined as the primary outcome measure. The Dermatology Life Quality Index and patient ratings of pruritus, quality of sleep and skin appearance on 100-mm visual analogue scales were defined as secondary outcome measures. Success of patient blinding and adverse events were assessed. RESULTS: Data were analysed on an intention-to-treat basis. Analysis of covariance suggested a significant differential change of the SASSAD score between baseline and the end of the follow-up period in favour of ABT. The mean reduction in SASSAD score was 13.5 points (95% confidence interval, CI 6.6-20.4, P < 0.001) over and above placebo; the corresponding value at the end of treatment was 9.6 (95% CI 4.2-14.9, P = 0.001). No clear significant intergroup differences in any of the secondary outcome measures were found. Six patients in the ABT group and seven in the placebo group reported minor and transient adverse events. CONCLUSIONS: These data suggest that, according to the SASSAD score, ABT has beneficial effects in the treatment of AD, although this was not confirmed by the patient-rated assessments. The improvement in observer-rated skin condition suggested by this study needs confirmation in larger trials.

Postoperative minocycline pigmentation.

This paper describes an unusual case of florid postoperative pigmentation caused by minocycline which was not diagnosed for some 8 months, causing anxiety and distress to both patient and surgeon.

Adult linear IgA disease and chronic bullous disease of childhood: the association with human lymphocyte antigens Cw7, B8, DR3 and tumour necrosis factor influences disease expression.

Linear IgA disease and chronic bullous disease of childhood are both subepidermal autoimmune blistering diseases. Class I and II major histocompatibility locus (MHC) antigen typing was performed on 60 patients (26 chronic bullous disease of childhood, 34 adult linear IgA disease), and the findings were correlated with the clinical course. The typing was performed using a lymphocyte microcytotoxicity assay, and the results were compared with a reference population of U.K. organ donors. Analysis of the tumour necrosis factor (TNF) locus was performed using sequence-specific oligonucleotides on a dot blot in 51 patients and compared with a random control population and human lymphocyte antigen (HLA) DR3 matched controls. The disease was found to be significantly associated with HLA Cw7 (chi2 = 19.24, P = 0.001), B8 (chi2 = 9.89, P = 0.04) and DR3 (chi2 = 10.47, P = 0.014), all components of the common Caucasian haplotype. There was also a close association between the disease and possession of HLA DR2 or 3 (chi2 = 16.34, P = 0.001). A reduction in the incidence of DR1 and DR4 (alleles carrying the rheumatoid motif) was observed, which is more marked in the children (chi2 = 8.34, P = 0.039). In the childhood group there was an increased frequency of B8, DR3 and DQ2 compared with the adults which included five of 26 who were homozygous for these antigens, a feature not seen in the adults, which may account for the differences seen between the two groups. Possession of HLA B8, DR3 and DQ2 probably facilitates earlier presentation of the disease as there is no evidence from our results that the adults and children differ fundamentally in their MHC associations. The rare TNF2 allele was found in 29 of 51 patients (expected 8.2, chi2 = 18. 3, P = 0.0001). This was more marked in the children (19 of 26). Patients with the TNF2 allele had a longer disease duration (5.3 years TNF2, 3.0 years TNF1).

A comparison of the expression of known basement membrane components with the linear IgA disease antigens using the novel substrate cylindroma.

Linear IgA disease (LAD) is characterized by IgA basement membrane zone autoantibodies. Immunofluorescence, immunoblotting and immunoelectron microscopy studies have established the complexity and heterogeneity of the target antigens. We have studied the expression of the LAD antigens by cylindroma, a benign epithelial tumour that secretes abundant basement membrane, using 57 LAD sera categorized by indirect immunofluorescence on intact and salt-split skin. The expression of known components of hemidesmosomes, the anchoring filaments, extracellular matrix and the anchoring fibrils could be differentiated and were compared with the expression of the LAD antigens. The results showed that the LAD sera bound to the cylindroma tumour in two distinctive patterns. Thirty-three sera were positive on cylindroma. Twenty-seven sera bound to the basement membrane around the tumour clusters and the islets within the clusters in a thin linear band that was occasionally discontinuous. This was similar to the pattern observed with antibodies to the hemidesmosome components, the alpha6beta4 integrin and the bullous pemphigoid antigens BP230 and BP180. This pattern was observed with sera that bound to the epidermal (11) and the dermal (3) aspects of split skin or were negative (11), and with one serum which bound only to intact skin. Seven sera, all binding to the dermal aspect of split skin, bound the tumour cluster basement membrane in a thick band that was identical in appearance to that seen with antibodies to collagen VII; however, binding to the islets was either identical to collagen VII or similar but differentiated with double staining. Some sera were negative on cylindroma. Using fluorescence overlay antigen mapping we demonstrated colocalization of some epidermal-associated LAD target antigens with known hemidesmosome proteins, and colocalization of some dermal-associated LAD target antigens with anchoring fibril components. The results using cylindroma as substrate suggest that LAD autoantibodies may react with three or more target antigens. We propose from the results of this study that the autoantibodies in LAD target multiple antigens associated with hemidesmosomes and anchoring fibrils.

Comparison of upstream regions of X- and Y-chromosomal amelogenin genes.

The amelogenin genes encode abundant enamel proteins that are required for the development of normal tooth enamel. These genes are active only in enamel-forming ameloblasts within the dental organ of the developing tooth, and are part of a small group of genes that are active on both sex chromosomes. The upstream regions of the bovine X- and Y-chromosomal and the sole murine X-chromosomal amelogenin genes have been cloned and sequenced, and conservation at nearly 60% is found in the 300 bp upstream of exon 1 for the 3 genes. A region of the bovine X-chromosomal gene that has inhibitory activity when assayed by gene transfer into heterologous cells includes motifs that have a silencing activity in other genes, and may be important to the mechanism that represses amelogenin expression in non-ameloblast cells in vivo. A comparison of sequences from three genes has led to the identification of several regions with conserved motifs that are strong candidates for having positive or negative regulatory functions, and these regions can now be tested further for interaction with nuclear proteins, and for their ability to regulate expression in vivo.

DNA sequences of amelogenin genes provide clues to regulation of expression.

The amelogenins are a heterogeneous group of enamel proteins, which have an important function in enamel formation, as mutations in the amelogenin gene result in the enamel defect amelogenesis imperfecta. The cDNAs that encode murine, bovine, human, porcine, rat and opossum amelogenins have been cloned, and as many as nine alternatively spliced messages can be produced from a single primary transcript, explaining some of the protein heterogeneity. Bovine and human amelogenin genes are found on both X and Y chromosomes, and the sexually dimorphic proteins would have 87-93% identity. A comparison of genes from human, bovine and mouse indicates that they are organized into seven exons, and sequences are highly homologous among species. Bovine, murine and human upstream regions also have similarities, with consensus sequences for potential binding of transcription factors, such as AP1 and CTF/NF1. Transgenic mouse studies have shown that 2300-3500 bp of upstream region are sufficient for expression, while 900 bp are insufficient. Analysis of DNA sequence has identified (a) major homology between species for coding exons with the exception of exon 4, (b) similarities in upstream regions likely involved in tissue specific regulation of expression, and (c) sequences at the RNA splice sites which may determine exon inclusion or skipping.

Linear IgA disease: a report of two dermal binding sera which recognize a pepsin-sensitive epitope (?NC-1 domain) of collagen type VII.

Linear IgA disease is a subepidermal blistering disease characterized by IgA autoantibody deposition at the basement membrane zone of skin and mucosa. The antigens targeted in linear IgA disease have been defined by their molecular weight and localization. It has been proposed that a minority of linear IgA disease sera that bind to the dermal aspect of salt-split skin target collagen type VII. We have identified two patients with linear IgA disease using dermal binding IgA autoantibodies on salt-split skin which recognize collagen type VII by immunoblot analysis. The reactive epitope was destroyed by the proteolytic enzymes pepsin, which destroys the NC-1 domain of collagen type VII, and protease type VIII. Localization studies compared the IgA autoantibodies from these patients with the monoclonal antibody LH7.2 to the NC-1 domain of collagen type VII and showed colocalization on a dermal cylindroma tumour tissue, and a similar distribution with immunogold electron microscopy, using purified blister fluid from one patient. We propose from these results that the IgA autoantibodies from these two patients recognize the NC-1 domain of collagen type VII, the classical immunodominant epitope for epidermolysis bullosa acquisita.

An amelogenin gene defect associated with human X-linked amelogenesis imperfecta.

Dental enamel is a product of ameloblast cells, which secrete a mineralizing organic matrix, composed primarily of amelogenin proteins. The amelogenins are thought to be crucial for development of normal, highly mineralized enamel. The X-chromosomal amelogenin gene is a candidate gene for those cases of amelogenesis imperfecta, resulting in defective enamel, in which inheritance is X-linked. In this report, a kindred is described that has a C to A mutation resulting in a pro to thr change in exon 6 of the X-chromosomal amelogenin gene in three affected individuals, a change not found in unaffected members of the kindred. The proline that is changed by the mutation is conserved in amelogenin genes from all species examined to date.

Regulation of amelogenin gene expression.

The amelogenins are found uniquely in enamel, where they constitute the predominant class of secreted matrix proteins and where they play a fundamental role in normal enamel formation. To better understand the high level of tissue-specific expression, we cloned the bovine X and Y chromosomal amelogenin genes and the murine amelogenin gene and determined the DNA sequences for the regions upstream of the transcription start sites. We observed segments of strong homology among species, and identified consensus sequences for the binding of various transcription factors, including the glucocorticoid receptor, AP1, RXR and p53. Although specific sis-elements conferring enhanced transcription have not yet been identified, elements have been localized that have silencing effect in non-ameloblast cells. Conserved sequences are likely to be involved in tissue-specific expression. Transgenic mouse studies have shown that 3.5 kb of upstream region is sufficient but 900 bp is insufficient for specific expression in vivo. Alternative splicing of the primary transcript is an effective mechanism for generating molecular heterogeneity. Amelogenin genes contain seven exons, and exons 3, 4, 5 and most of 6 can be deleted by alternative splicing. However, the pattern of exon splicing varies according to the species, and skipping of bovine exon 3 appears to be developmentally regulated. It will be important to determine whether the relative amounts of translation products differ among species as do the mRNAs, and to correlate the various protein structures with function. These findings also suggest that the regulation of amelogenin gene expression is complex and takes place at several levels.

Albumin gene expression during mouse odontogenesis.

Albumin protein is present in developing teeth of several species. Oligomer primers and cRNA probes specific for albumin were designed to perform RT-PCR, and for in situ hybridization, respectively. In situ hybridization failed to reveal albumin expression in any tooth cells, however, albumin PCR products were amplified from tissues adhering to the roots of developing teeth from four-week-old mice. It is concluded that this source is not the primary source of albumin protein found in developing enamel, because of the location and level of expression of albumin mRNA in periodontal tissue.

Analysis of amelogenin mRNA during bovine tooth development.

The amelogenins are highly conserved enamel-matrix proteins that are essential for proper mineral formation. Transcriptionally active genes encoding the bovine amelogenin proteins reside on both the X and Y chromosomes. Comparison of relative levels of amelogenin mRNAs at various stages of development indicated that the X-chromosomal amelogenin message is at least six fold more abundant than the Y. Alternative splicing generates at least seven messages, five from the X primary transcript, and two from the Y. The two most abundant X-chromosomal amelogenin messages are approx. 850 and 450 nucleotides long, and nearly 10-fold more 850-nucleotide mRNA can be measured than 450 nucleotide, which has lost most of exon 6 by splicing. The predominant small message encodes leucine-rich amelogenin protein (LRAP), and amounts of LRAP message are relatively constant during development. However, the amelogenin message from which exon 3 has been spliced declines approximately 2.3-fold, when compared to total X chromosomal amelogenin transcripts, suggesting differential regulation of alternative splicing. In addition, a new exon was identified within genomic DNA, which was shown to be expressed by the use of reverse transcriptase-polymerase chain reaction, and the exons were renamed accordingly. This new exon-4 sequence is unusual in that it is not highly conserved between species.

Bovine enamel organ cells express tissue non-specific alkaline phosphatase mRNA.

Alkaline phosphatase (AP) is expressed at high levels in all mineralizing tissues, and the isoform identified in developing enamel has biochemical properties similar to that found in bone. While the bone AP is referred to as the liver/bone/kidney or tissue non-specific (TNS) form, other APs are highly specific for tissue of expression. To determine unequivocally the AP isoform made by enamel organ cells, we constructed a fetal bovine enamel organ cDNA library, which yielded eight AP cDNA clones. In each case, the DNA sequence was homologous to the partial cDNA reported for bovine kidney AP (Garattini et al., 1987). It is concluded that enamel organ cells express the TNS-AP isoform. The extended 3' untranslated region of the cDNA has considerable homology to human TNS-AP, and the conservation of sequence suggests that the 3' end may have a role in post-transcriptional regulation of expression.

The localization of the target antigens and antibodies in linear IgA disease is heterogeneous, and dependent on the methods used.

Fifty-nine patients with linear IgA disease, 24 with onset in childhood and 35 with adult onset, were studied. Sera from all patients were tested by indirect immunofluorescence, using as substrates intact normal skin and normal skin which had been split through the lamina lucida region of the basement membrane zone by suction and by prolonged incubation with molar NaCl. This enabled the site of the target antigen for the circulating IgA antibodies to be determined. The sites of deposition of the IgA antibodies in vivo were detected by raising a suction blister in eight patients, and splitting seven patients' biopsies by prolonged incubation with molar NaCl. Eighteen sera were positive with intact skin, and 34 with split skin. Twenty-nine sera were positive with suction blisters as substrate; 14 bound to the epidermal aspect of the split skin, seven in a combined pattern (binding to the epidermis and dermis) and six to the dermal aspect. Thirty-one sera bound to salt-split skin, 24 to the epidermal side and seven on the dermal side. There was discordance between the two methods of skin splitting in 15 sera. Seven sera gave a combined pattern with suction but with salt-split skin, five of these bound epidermally, one was dermal, and one negative. Five sera showed epidermal binding on salt-split skin and were negative on suction blisters, and the reverse was seen with one serum. Two sera gave variable results on suction blisters. Direct immunofluorescence studies showed dermal binding on all eight patients with suction blisters, and epidermal binding in four and dermal binding in three patients with salt splitting.(ABSTRACT TRUNCATED AT 250 WORDS)

Severe case of pemphigoid gestationis with unusual target antigen.

We report a severe case of pemphigoid gestationis (PG) with an unusual target antigen. The patient developed a severe itchy macular rash on the abdomen during her fifth pregnancy, at 38 weeks' gestation. The disease persisted for 8 months post-partum, and could only be controlled with high doses of systemic corticosteroids and dapsone. The diagnosis of PG was confirmed by direct and indirect immunofluorescence. A linear band of IgG and C3 was detected at the epidermal aspect of salt-split skin. Immunoblotting studies revealed circulating IgG antibodies binding to a single 200-kDa protein in epidermal extracts. Immunofluorescence studies using trypsinized human keratinocytes demonstrated uniform staining around the cell peripheries, suggesting that the target antigen was not associated with hemidesmosomes. These findings differ from the usual immunological features seen in PG, and suggest further heterogeneity of the antigens involved in PG.

Linear IgA disease and pregnancy.

BACKGROUND: Although many patients with linear IgA dermatosis (LAD) are young and have persistent disease, little is known about the interactions between LAD and pregnancy. OBJECTIVE: Our purpose was to study the effects of LAD on pregnancy, and vice versa. METHODS: Our study included 12 patients with LAD who underwent a total of 19 pregnancies. RESULTS: In all patients the disease improved during pregnancy, enabling therapy to be reduced or stopped. Dapsone was taken by patients during 11 pregnancies, and no adverse effects were seen. No patients had problems in labor. Most patients had a relapse approximately 3 months post partum, even if they had previously been in remission. In two patients, disease started within 3 months of delivery. Fetal outcome was unaffected in all but one fetus, who had a single transient blister. CONCLUSION: We found no contraindication to pregnancy in patients with LAD. We recommend that therapy be reduced or stopped whenever possible during pregnancy and that patients be counseled about the possibility of a relapse post partum.

Pseudopelade of Brocq occurring in two brothers in childhood.

The cases of two brothers who both developed pseudopelade at the age of 7 years, with progressive patchy hair loss, are reported. Biopsy in the elder brother showed typical changes with reduced numbers of hair follicles and sebaceous glands, a sparse lymphocytic infiltrate and an absence of widespread scarring. A third family member may have been affected. The absence of a family history in most cases suggests our patients may have a different aetiology. This report of two, possibly three, family members with pseudopelade suggests genetic factors may play a role in this family. Pseudopelade is very rare in children and we are not aware of any reports of familial occurrence. Atrophy rather than scarring is the primary feature of this condition and pseudopelade of Brocq should not be classified as a cicatricial alopecia.

Histologic, pharmacologic, and immunocytochemical effects of injection of bleomycin into viral warts.

BACKGROUND: The plasma concentration of bleomycin after injection of bleomycin into warts is unknown, as is the long-term stability of bleomycin solution. OBJECTIVE: Our purpose was to measure plasma bleomycin concentration after injection of bleomycin into warts, to relate histologic and immunocytochemical changes in warts to possible mechanisms of action of bleomycin, and to asses the long-term stability of stored frozen bleomycin solution. METHODS: One milligram of bleomycin was injected into warts on the hands of seven men. Blood samples were taken 15 to 120 minutes after injection, and plasma bleomycin was measured by radioimmunoassay. Warts were removed 2 hours and 48 hours after treatment and studied histologically by light microscopy and for the presence of bleomycin by immunocytochemistry. The bleomycin concentration in 8 aliquots of solution stored at -20 degrees C for varying periods was measured by radioimmunoassay. RESULTS: Peak levels of bleomycin of 7 to 113 ng/ml were reached by 45 minutes after injection. Plasma bleomycin exposure ranged from 515 to 5137 ng/ml/min between 15 and 120 minutes after injection. The most pronounced histologic changes at 48 hours were individual keratinocyte apoptosis throughout the epidermis merging into areas of complete epidermal necrosis, diffuse neutrophil accumulation, and microabscess formation at the granular layer. Immunocytochemistry demonstrated tissue-fixed bleomycin in all levels of the epidermis except the basal layer and most prominently in the granular layer. Bleomycin in solution stored for up to 27 months at -20 degrees C in glass showed no significant loss of immunoreactivity. CONCLUSION: The use of bleomycin for the treatment of warts results in significant systemic drug exposure; thus it would be prudent to exclude pregnancy before treating women of child-bearing age. Bleomycin probably has a direct toxic effect on keratinocytes. Dilute bleomycin solution stored at -20 degrees C in glass is stable.