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Biomedical subjects

L A Diaz

Publications and source records attributed to L A Diaz.

At least 19 recordsLinked to original sources
PubMed1995

Molecular mapping of a pathogenically relevant BP180 epitope associated with experimentally induced murine bullous pemphigoid.

Bullous pemphigoid (BP) and herpes gestationis (HG) are subepidermal blistering diseases associated with an autoimmune response directed against BP180, an epidermal hemidesmosomal glycoprotein. The pathogenic relevance of this Ag/Ab system was established by the recent demonstration that IgG Abs reactive with the murine form of BP180 (mBP180) are capable of triggering a subepidermal blistering disease after passive transfer into neonatal BALB/c mice. The aim of the present study was to determine the fine specificity of the pathogenically relevant Abs in this experimental model of BP. Four high titer rabbit-anti-mBP180 antisera were included in this analysis--only two of which exhibited pathogenic activity in the passive transfer model. Immunoblot analysis using a panel of mBP180 deletion mutants revealed that each of the four rabbit sera reacted with at least three distinct sites on the mBP180 ectodomain; however, this technique failed to distinguish between the reactivity patterns of the pathogenic and nonpathogenic sera. An alternative technique, liquid phase immunoadsorption analysis, was used to identify one mBP180 antigenic site, comprising 9 to 12 amino acids and designated mBP1, that was specifically recognized by the two pathogenic sera. Pre-adsorption of pathogenically active IgG preparations with fusion proteins containing the mBP1 antigenic site resulted in complete blocking of immunofluorescence reactivity with the murine basement membrane zone (BMZ) and in complete neutralization of pathogenic activity. Anti-BMZ reactivity displayed by nonpathogenic Abs was not altered or diminished by pre-adsorption with this same mBP180 recombinant protein. These findings should help to elucidate the immunopathologic mechanisms responsible for human BP and HG and may have significant implications in the diagnosis and treatment of these autoimmune diseases.

Animals↗
PubMed1995

Anaerobic biodegradability testing of surfactants.

Anionic and nonionic surfactants (5-50 mg C/g solids/L medium) were screened for anaerobic microbial decomposition to methane in an automated pressure transducer serum bottle assay system at 35C using municipal digester solids as a source of anaerobic bacteria. Analysis of the headspace gas recovered from tests with linear primary alcohol sulfates (A45S and A24S) and a linear alcohol ethoxylate (LAE-8) showed that these compounds were readily degraded (60-85% of the theoretical methane, TM) after a 15-30 day lag period at 50 ppm C. The extent of degradation of a branched alkyl phenol ethoxylate (NPE-9) was lower (30-40% TM). A survey of intact nonionic and anionic surfactants present in municipal digester sludges in the U.S. showed that these materials were present at levels of 0.5-8 mg CTAS or MBAS/g dry solids. A surfactant which was slower to biodegrade (NPE-9) at 50 ppm C was readily metabolized to methane when tested at 5 and 10 mg C/g solids/L. The pressure transducer serum bottle method described may be used to test biodegradability and inhibitory effects on methanogenesis at surfactant concentrations (e.g. 5 ppm C/g solids) typically present in digesters.

Bacteria, Anaerobic↗
PubMed1995

The role of complement in experimental bullous pemphigoid.

Bullous pemphigoid (BP) is a blistering skin disease associated with an IgG autoimmune response directed against the ectodomain of the hemidesmosomal protein, BP180. An animal model of BP has recently been developed by our laboratory based on the passive transfer of rabbit antimurine BP180 antibodies into neonatal BALB/c mice. The experimental animals develop a blistering disease that reproduces all of the key immunopathological features of BP. In the present study we have investigated the role of complement in the pathogenesis of subepidermal blistering in the mouse model of BP. We demonstrate the following. (a) Rabbit anti-murine-BP180 IgG was effective in inducing cutaneous blisters in a C5-sufficient mouse strain, but failed to induce disease in the syngeneic C5-deficient strain; (b) neonatal BALB/c mice, pretreated with cobra venom factor to deplete complement, became resistant to the pathogenic effects of the anti-BP180 IgG; (c) F(ab')2 fragments generated from the anti-BP180 IgG exhibited no pathogenic activity in the mouse model; and (d) histologic evaluation of the skin of mice described in points b and c above showed minimal or no neutrophilic cell infiltration in the upper dermis. Thus, anti-BP180 antibodies trigger subepidermal blistering in this BP model via complement activation. This experimental model of BP should greatly facilitate future studies on the pathophysiology of autoantibody-mediated diseases of the dermal-epidermal junction.

Animals↗
PubMed1994

The human 4F2 antigen: evidence for cryptic and noncryptic epitopes and for a role of 4F2 in human T lymphocyte activation.

T lymphocyte activation can be triggered through multiple distinct, but functionally related, pathways. Murine monoclonal antibodies (mAbs) have been used to characterize the surface components of several of these pathways, as well as structures whose function is not yet known. One such cell surface structure is the heterodimeric 4F2 antigen, which is expressed on activated and proliferating cells. Two new mAbs that recognize the heavy chain of the 4F2 antigen have been produced in our laboratory. One antibody, UM7F8, is comitogenic with soluble anti-CD2 and immobilized (but not soluble) anti-CD3 mAbs. The second antibody, termed UM2G12, appears to recognize a cryptic epitope on the 4F2 heavy chain and is not comitogenic for T cells. In view of the functional effects seen with UM7F8, and the highly regulated expression of the 4F2 antigen, it seems likely that 4F2 has a specific role in T cell development and activation.

Antibodies, Monoclonal↗
PubMed1994

Development of an ELISA to detect anti-BP180 autoantibodies in bullous pemphigoid and herpes gestationis.

Autoantibodies associated with the subepidermal blistering disorders bullous pemphigoid and herpes gestationis react with a 180-kD transmembrane hemidesmosomal protein, designated BP180. The BP180 ectodomain is composed of a series of interrupted collagen triple helical domains. Located on one of the noncollagenous extracellular segments of this protein is an immunodominant epitope, designated MCW-1, recognized by patient autoantibodies. In this investigation we have developed an enzyme-linked immunosorbent assay system to detect antibody reactivity against the MCW-1 epitope with the use of a bacterial fusion protein containing the BP180 autoantibody-reactive site. The following sera were assayed for reactivity with this recombinant protein: bullous pemphigoid (n = 62), herpes gestationis (n = 28), endemic pemphigus foliaceus (n = 17), lupus erythematosus (n = 15), and normal human sera (n = 22). This enzyme-linked immunosorbent assay-based protocol was shown to be highly specific (98.3%) in detecting autoantibody activity in bullous pemphigoid and herpes gestationis patients. Fifty-three percent of bullous pemphigoid sera and 71% of herpes gestations sera, but none of the control sera, yielded positive results in this assay. Of the patient sera that were known to react with full-length BP180, almost all showed reactivity with the MCW-1 antigenic site of this protein. Autoantibodies detected in this assay were predominantly of the immunoglobulin G class. The results presented here lend support to the hypothesis that this well-defined antigen/antibody system may be relevant in pathogenesis.

Autoantibodies↗
PubMed1994

Autoimmune response against the bullous pemphigoid 180 autoantigen.

An immunodominant and potentially pathogenic epitope associated with bullous pemphigoid (BP) and herpes gestationis (HG) has recently been mapped by our laboratory to a noncollagenous stretch of the extracellular domain of the human BP180 antigen. This antigenic site, designated the MCW-1 epitope, has been shown to be recognized by the majority of BP and HG sera. Interestingly, the MCW-1 epitope is absent from the murine BP180 molecule, and therefore, human autoantibodies directed against this site could not be tested for pathogenicity using the conventional passive transfer mouse model. Alternatively, rabbit antibodies were prepared against recombinant forms of the human MCW-1 epitope and the murine NC16A domain and were tested for pathogenicity by passive transfer experiments. Neonatal mice injected with rabbit antimurine BP180 IgG developed a subepidermal blistering disease that closely mimicked BP and HG at the clinical, histological and immunological levels. Rabbit IgG specific for the human MCW-1 epitope was not pathogenic. These results suggest that the autoantibodies against the MCW-1 epitope of the human BP180 antigen found in BP and HG sera may be relevant in the pathogenesis of blister formation in these patients.

Animals↗
PubMed1993

A passive transfer model of the organ-specific autoimmune disease, bullous pemphigoid, using antibodies generated against the hemidesmosomal antigen, BP180.

Subepidermal blistering associated with the human skin diseases bullous pemphigoid and herpes gestationis has been thought to be an IgG autoantibody-mediated process; however, previous attempts to demonstrate the pathogenicity of patient autoantibodies have been unsuccessful. An immunodominant and potentially pathogenic epitope associated with these blistering diseases has recently been mapped to the extracellular domain of a human epidermal antigen, BP180. Patient autoantibodies that react with this well-defined antigenic site failed to crossreact with the murine form of this autoantigen and thus could not be assayed for pathogenicity in a conventional passive transfer mouse model. As an alternative, rabbit polyclonal antibodies were generated against a segment of the murine BP180 protein homologous with the human BP180 autoantibody-reactive site and were passively transferred into neonatal BALB/c mice. The injected animals developed a subepidermal blistering disease that closely mimicked bullous pemphigoid and herpes gestationis at the clinical, histological, and immunological levels. Autoantibodies that recognize the human BP180 ectodomain are therefore likely to play an initiatory role in the pathogenesis of bullous pemphigoid and herpes gestationis.

Amino Acid Sequence↗
PubMed1992

cDNA cloning of a novel human ubiquitin carrier protein. An antigenic domain specifically recognized by endemic pemphigus foliaceus autoantibodies is encoded in a secondary reading frame of this human epidermal transcript.

Autoantibodies from a patient suffering from endemic pemphigus foliaceus (EPF), a blistering skin disease, were used to screen a lambda gt11 human keratinocyte cDNA library. One immunoreactive cDNA clone (lambda EPF5) containing a 900-base pair insert was isolated and subjected to further analysis. Eight of 25 EPF sera were shown to react with the EPF5 fusion protein on immunoblots. The EPF5 cDNA insert hybridized with a 1.2-kilobase epidermal RNA transcript on a Northern blot. Sequence analysis revealed that lambda EPF5 contained the complete coding sequence for a 24-kDa polypeptide exhibiting significant sequence homology with a family of enzymes known as ubiquitin carrier proteins, or E2s, which are an essential component of the ubiquitin-protein conjugation system. The homology was particularly high in the core region containing the active site cysteine. The keratinocyte ubiquitin carrier protein expressed in bacteria, and isolated either intact or as a glutathione S-transferase fusion protein, exhibited the ability to form a thiol ester linkage with ubiquitin in a ubiquitin activating enzyme (E1)-dependent manner, a characteristic property of ubiquitin carrier proteins. The E2 enzyme encoded by clone EPF5 is the first member of this protein family to be cloned from an epidermal source. Interestingly, the EPF autoantibody-reactive epitope and the ubiquitin carrier protein were shown to be encoded in two different translational reading frames. The relevance of the cloned EPF epitope in the pathogenesis of this autoimmune disorder remains to be determined.

Amino Acid Sequence↗
PubMed1992

Pregnant women with endemic pemphigus foliaceus (Fogo Selvagem) give birth to disease-free babies.

Endemic pemphigus foliaceus (PF), also known as Fogo Selvagem (FS), is an organ-specific autoimmune disease mediated by autoantibodies. These autoantibodies are disease specific, predominantly restricted to the IgG4 subclass, and pathogenic, as demonstrated by passive transfer studies. In contrast to pemphigus vulgaris, neonatal skin disease does not appear to occur in babies born to mothers with non-endemic PF or FS. In the present study we have examined 19 mother/neonate pairs where the mother had documented FS. Mothers and neonates were examined soon after delivery and tested by immunofluorescent (IF) techniques for FS autoantibodies either in circulation (mothers' sera or babies' cord blood) or bound to the neonatal epidermis. All neonates included in this study were born with normal skin. Twelve biopsies from 17 neonates showed negative direct IF using both FITC-antihuman IgG or monoclonal anti-IgG subclass antibodies. In five biopsies the epidermal ICS of the babies showed weak staining. In 10 of the 19 cord sera tested, FS IgG autoantibodies were undetectable; in nine, these autoantibodies were present in low titers (less than 1:40). The sera of the mothers showed higher titers of FS autoantibodies, and IgG4 was the predominant IgG subclass autoantibodies. It appears that human placenta may modulate the expression of disease in the newborn by operating as a "biologic immunoadsorbent" of pathogenic autoantibodies.

Adult↗
PubMed1992

Cloning and primary structural analysis of the bullous pemphigoid autoantigen BP180.

Bullous pemphigoid (BP) is an autoimmune skin disease that is characterized by the presence of subepidermal blisters resulting from a disruption of the adhesive interactions between basal keratinocytes and the cutaneous basement membrane. Autoantibodies from patients suffering from this disorder recognize two epidermal antigens, BP180 and BP230, both of which have been localized to the hemidesmosome, a transmembrane structure of stratified, squamous epithelia that functions in cell-matrix adhesion. In the present study we report the primary structural analysis of BP180 based on the sequence of a series of overlapping cDNA clones encompassing 4,669 bases of the BP180 transcript. A polymerase chain reaction-based protocol was used to confirm the contiguity of the cDNA segments. This cloned portion of the BP180 transcript was found to contain one long open reading frame (ORF) 4.596 bases in length. This ORF encodes a polypeptide of 155,000 Daltons with an isoelectric point of 9.7. The carboxy-terminal half of BP180, a stretch of 916 amino acids, consists of 15 collagen domains of variable length (15 to 242 amino acids) that are separated from one another by short stretches of non-collagen sequences. Located 76 amino acids upstream of the collagenous region is a putative transmembrane domain, a structural feature that distinguishes BP180 from all of the well-characterized members of the collagen family. This membrane-spanning domain is predicted to function as a signal-anchor sequence, directing the C-terminal collagenous segment of this protein to the exterior of the cell. The putative intracellular domain is highly basic with an isoelectric point of 10.37. This molecular analysis predicts that the BP180 antigen is an integral membrane protein of the hemidesmosome that contains a long extracellular collagenous tail. This combination of structural features suggests that BP180 may function as a cell-matrix adhesion molecule, with the collagenous region acting as a potential site of interaction with basement membrane components. Autoantibody-mediated disruption of such an adhesive interaction may play a critical role in the development of sub-epidermal blisters in BP patients.

Amino Acid Sequence↗
PubMed1992

Cloning of partial cDNA for mouse 180-kDa bullous pemphigoid antigen (BPAG2), a highly conserved collagenous protein of the cutaneous basement membrane zone.

One-hundred-eighty kilodalton bullous pemphigoid antigen (BPAG2) is recognized by autoantibodies in the sera of patients with blistering skin diseases, bullous pemphigoid (BP), and herpes gestationis (HG). In this study, we have screened a mouse epidermal keratinocyte cDNA library with a 1.0-kb human BPAG2 cDNA, which has been shown to correspond to two collagenous domains (Giudice et al: J Clin Invest 87:734-738, 1991). Screening of the mouse library identified two cDNA clones, the larger one being 1.8 kb in size. Comparison of the mouse amino acid sequences, as deduced from cDNA, with the corresponding human sequences revealed 86% homology. Furthermore, Northern hybridizations of mouse epidermal RNA with these cDNA revealed the presence of an mRNA transcript of approximately 6 kb, the size of the human BPAG2 mRNA. Elucidation of the deduced amino-acid sequences revealed the presence of definitely one and possibly two putative membrane-associated segments, suggesting that the 180-kDa BP antigen is a transmembrane protein. The sequence analysis also identified a 7-amino-acid segment that was predicted by computer analysis to be antigenic. Elucidation of the divergence between the mouse and previously published human and chicken BPAG2 sequences indicated that this protein segment was relatively well conserved. These data suggest, therefore, that the 180-kDa bullous pemphigoid antigen associated with hemidesmosomes is a well-conserved transmembrane protein that may play a critical role in the attachment of epidermis to the underlying basement membrane.

Amino Acid Sequence↗
PubMed1991

Genomic organization of collagenous domains and chromosomal assignment of human 180-kDa bullous pemphigoid antigen-2, a novel collagen of stratified squamous epithelium.

We have recently isolated a 1.0-kilobase (kb) cDNA encoding 180-kDa bullous pemphigoid antigen (BPAG2), an autoantigen in blistering skin disease, bullous pemphigoid (Giudice, G. J., Squiquera, H. L., Elias, P. M., and Diaz, L. (1991) J. Clin. Invest. 87, 734-738). The deduced amino acid sequence identified two collagenous domains characterized by Gly-X-Y repeats. In this study we have elucidated the genomic organization of the corresponding segment in the human BPAG2 gene. Screening of a genomic lambda-phage DNA library yielded six overlapping clones, and the sequences corresponding to the 1.0-kb cDNA spanned approximately 12 kb of genomic DNA. The coding segment consisted of 19 separate exons varying in size from 27 to 222 base pairs. The organization of these exons and the splice sites at the intron-exon junctions were clearly different from other fibrillar and nonfibrillar collagen genes described to date. The BPAG2 gene was mapped by chromosomal in situ hybridization to the long arm of human chromosome 10, at locus 10q24.3; this location is distinct from any previously mapped collagens, and it also distinguishes BPAG2 from BPAG1, a 230-kDa bullous pemphigoid antigen, which has been mapped to chromosome 6p (Sawamura, D., Nomura, K., Sugita, Y., Mattei, M.-G., Chu, M.-L., Knowlton, R.G., and Uitto, J. (1990) Genomics 8, 722-726). These data suggest that BPAG2 is a novel collagen present in stratified squamous epithelia.

Amino Acid Sequence↗
PubMed1991

Biological characterization of the lytic cycle of actinophage phi A7 in Streptomyces antibioticus.

Some basic parameters of the lytic development of phage phi A7 in Streptomyces antibioticus are described. One-step growth experiments demonstrated that at 28 degrees C phi A7 has a latent period of about 60 min and an exponential growth period of about 35 min. The average burst size ranged from 70-100 plaque forming units per infected cell. At the same temperature 50% of the virions were adsorbed to germ tubes of S. antibioticus in about 10 min. This corresponds to an adsorption constant of 6.5 x 10(-10) ml/min. The phage was unable to adsorb the host at other stages of the life cycle (spores or mycelium). Divalent cations are not required for phi A7 stability but Ca2+ proved to be essential for adsorption and also for a later stage of the vegetative development of the phage.

Adsorption↗
PubMed1991

HLA antigens and risk for development of pemphigus foliaceus (fogo selvagem) in endemic areas of Brazil.

Endemic pemphigus foliaceus (EPF), is an autoimmune disease associated with production of IgG antibodies against epidermal antigens. We have tested 38 patients and 50 control subjects living in endemic areas to investigate whether HLA genes are associated with host factors that determine whether or not exposed individuals will develop this disease. A variant of HLA-DR1, an antigen common in Blacks (DRB1*0102), was found to be the main susceptibility factor (relative risk = 7.3, P less than 0.0002). Two amino acids, in positions 85 and 86 of DRB1, distinguish DRB1*0102 from DRB1*0101. These residues appear to be involved in the formation of a functional epitope that causes T cell recognition and determines disease susceptibility. Moreover, subjects having DQw2 did not develop the disease, while the frequency of DQw2 in controls was 22% (RR = 0.04, P less than 0.006). Thus HLA genes appear to play a crucial role in the response to an environmental factor which in this setting frequently leads to the development of autoimmune disease. An HLA-DQ allele, DQw2, appears to be associated with factors that prevent the development of the disease in exposed individuals.

Autoimmune Diseases↗
PubMed1991

Bullous pemphigoid antigens (BPAGs): identification of RFLPs in human BPAG1 and BPAG2, and exclusion as candidate genes in a large kindred with dominant epidermolysis bullosa simplex.

Bullous pemphigoid antigens (BPAGs) are defined as autoantigens in a blistering skin disease, bullous pemphigoid. Two of the BPAGs, a 230-kD (BPAG1) and a 180-kD (BPAG2) protein, have been localized to hemidesmosomes, attachment structures at the basal keratinocyte-basement membrane interphase. We have recently cloned cDNAs corresponding to human BPAG1 and BPAG2, and mapped the corresponding genes to human chromosomes 6p and 10q, respectively. These cDNAs have now been used in a search for RFLPs in the corresponding genes. Southern hybridizations of genomic DNA from normal unrelated individuals with a BPAG1 cDNA detected an informative MspI RFLP, and similar hybridizations with a BPAG2 cDNA revealed an informative TaqI RFLP. These RFLPs were applied to a large kindred with epidermolysis bullosa simplex (EBS), generalized (Koebner) type, consisting of 14 affected and 12 unaffected individuals in three generations. Linkage analysis excluded the EBS locus in this pedigree approximately 9 cM and approximately 5 cM on either side of the BPAG1 and BPAG2 loci, respectively, when a lod score of -2.0 was taken as the limit of exclusion. This study demonstrates that mutations in the BPAG1 or BPAG2 genes are not the primary genetic defect in this family with EBS.

Autoantigens↗
PubMed1991

The relevance of immunohistochemical techniques in the differentiation of subepidermal bullous diseases.

There are several subepidermal bullous diseases. In some, the clinicopathological features are distinctive, whereas in others there is a variable degree of overlap that necessitates the use of ultrastructural and biochemical studies to distinguish the different diseases. In this paper, we review the literature and describe our experience using simplified immunological techniques in the diagnosis of subepidermal bullous diseases.

Diagnosis, Differential↗