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Pemphigus vulgaris (PV) is an autoimmune intraepithelial bullous disease. Associations with the class II human leukocyte antigen (HLA) alleles and pemphigus vulgaris have been described. Furthermore, an association between the single nucleotide polymorphism of the ST18 gene and pemphigus vulgaris has been reported. We report two pairs of siblings from two unrelated Italian families affected by pemphigus vulgaris, characterizing their genetic and immunological profile. In order to assess the genetic background, HLA-DQA1, HLA-DQB1, HLA-DRB1 and a relevant ST18 polymorphism were investigated. As for the immunological profiles, anti-desmoglein antibodies were analyzed. In family A, the two pemphigus vulgaris patients had the same HLA genetic profile: HLA-DQA1 *01:04/*03:01, HLA-DQB1 *03:02/*05:03 and HLA-DRB1 *04:02/*14:01. The male patient was heterozygous for the ST18 mutation while the female patient had a wild genotype. In family B, the two pemphigus vulgaris patients were both wild type for the ST18 mutation and showed the same HLA genotype: HLA-DQA1 *03:01/*05:08, HLA-DQB1 *03:01/*03:03 and HLA-DRB1 *04:02/*11:01. Our data show a relevant relationship between the HLA profile and pemphigus vulgaris in our Italian families. In family A, all six alleles are frequently associated with pemphigus vulgaris and were expressed only in the two pemphigus patients; and in family B, two of the six alleles are frequently associated with pemphigus vulgaris. No relevant relationship was found between ST18 polymorphism and pemphigus disease.

The clinical features of bullous pemphigoid are extremely polymorphous. Several atypical forms of bullous pemphigoid have been described, and the diagnosis critically relies on immunopathological findings. We describe three bullous pemphigoid patients characterized by palmoplantar keratoderma, diffused hyperkeratotic cutaneous lesions and extremely high levels of immunoglobulin E serum. The diagnosis of bullous pemphigoid should be taken into account in patients presenting diffused hyperkeratotic cutaneous lesions and palmoplantar keratoderma, even in the absence of blisters. Alteration of the keratinization process, that could occur in patients with genetic mutations in desmosomal and hemidesmosomal genes, may also be due to circulating autoantibodies against hemidesmosomal proteins in these bullous pemphigoid patients.

Pemphigus vulgaris (PV) is an autoimmune bullous disease of the skin and mucous membranes characterized by the presence of circulating and tissue-bound autoantibodies against keratinocyte cell surface antigens, specifically desmoglein (Dsg) 1 and 3. The pathogenic role of anti-Dsg antibodies is well-established, while the mechanism of blister formation is only partly defined. We have applied a previously developed method for the efficient immortalization of IgG+ memory B cells to identify novel target antigens in PV. A human monoclonal antibody reactive with a hitherto unreported non-Dsg antigen was isolated. Immunoprecipitation and immunoblotting studies with keratinocyte extracts indicated α-catenin as the putative antigen, then confirmed by immunoblotting on the recombinant protein. Four of ten PV sera reacted with recombinant α- catenin. Although the isolated human monoclonal antibody was per se unable to dissociate keratinocyte monolayers and also to synergize with a pathogenic antibody in vitro, further studies are warranted to assess its possible in vivo contribution in the multifactorial pathogenesis and heterogeneous manifestations of PV disease.

SPINK5, encoding the putative multi-domain serine protease inhibitor LEKTI, was recently identified as the defective gene in the severe autosomal recessive ichthyosiform skin condition, Netherton syndrome (NS). Using monoclonal and polyclonal antibodies, we show that LEKTI is a marker of epithelial differentiation, strongly expressed in the granular and uppermost spinous layers of the epidermis, and in differentiated layers of stratified epithelia. LEKTI expression was also demonstrated in normal differentiated human primary keratinocytes (HK) through detection of a 145 kDa full-length protein and a shorter isoform of 125 kDa. Both proteins are N-glycosylated and rapidly processed in a post-endoplasmic reticulum compartment into at least three C-terminal fragments of 42, 65 and 68 kDa, also identified in conditioned media. Processing of the 145 and 125 kDa precursors was prevented in HK by treatment with a furin inhibitor. In addition, in vitro cleavage of the recombinant 145 kDa precursor by furin generated C-terminal fragments of 65 and 68 kDa, further supporting the involvement of furin in LEKTI processing. In contrast, LEKTI precursors and proteolytic fragments were not detected in differentiated HK from NS patients. Defective expression of LEKTI in skin sections was a constant feature in NS patients, whilst an extended reactivity pattern was observed in samples from other keratinizing disorders, demonstrating that loss of LEKTI expression in the epidermis is a diagnostic feature of NS. The identification of novel processed forms of LEKTI provides the basis for future functional and structural studies of fragments with physiological relevance.

Resident cell populations of the skin contribute to the inflammatory response by producing an array of chemokines, which attract leukocytes from the circulation. TNF-α is a major inducer of proinflammatory mediators in keratinocytes. We have recently observed that epidermal growth factor receptor (EGFR) signaling affects TNF-α-driven chemokine expression in epidermal keratinocytes, and its functional impairment increases the levels of crucial chemoattractants such as CCL2/MCP-1, CCL5/RANTES, and CXCL10/IFN-γ-inducible protein-10. In this study, we report evidence that EGFR-dependent ERK1/2 activity is implicated in this mechanism. Abrogation of ERK1/2 activity with specific inhibitors increased chemokine expression in keratinocytes by enhancing mRNA stabilization. In mouse models, inflammatory response to irritants and T cell-mediated contact hypersensitivity were both aggravated when elicited in a skin area previously treated with an EGFR or a MAPK kinase 1/2 inhibitor. In contrast, impairment of p38αβ MAPK phosphorylation markedly attenuated these responses. Our data indicate that EGFR-dependent ERK1/2 activity in keratinocytes takes part to a homeostatic mechanism regulating inflammatory responses, and emphasize the distinct role of MAPKs as potential targets for manipulating inflammation in the skin.

Bullous pemphigoid is a subepidermal bullous disease of skin and mucosae associated with autoantibodies to BP180. To characterize the humoral response to BP180, we generated a random BP180 epitope library displayed on lambda bacteriophage. After validation of the library by epitope mapping of three BP180-specific monoclonal antibodies, 15 novel or known BP180 epitopes were identified using 10 bullous pemphigoid serum samples. Fifty-seven bullous pemphigoid and 81 control sera were then assayed against the selected epitopes. Thirty-one out of 57 (54%) bullous pemphigoid sera reacted with at least an additional antigenic site other than the NC16A, within the extracellular (37%) and intracellular (28%) domains of BP180. In addition, the reactivity with extracellular epitopes of BP180 contained within the residue stretches 508-541 and 1331-1404 appeared to be related to the presence of both skin and mucosal involvement. Finally, a preliminary analysis of the epitope pattern in the disease course indicated that bullous pemphigoid patients exhibit a specific reactivity pattern, and that binding to intracellular epitopes of BP180, in addition to NC16A, may be detectable at an early clinical stage. Our findings provide novel insights into the pathophysiology of bullous pemphigoid and show the potential of the utilized approach as a tool for a rapid diagnosis of bullous pemphigoid patients and their management.

The biosynthesis of syringomycin (SR) and syringopeptin 22 (SP22), bioactive lipodepsipeptides of the phytopathogenic bacterium Pseudomonas syringae pv. syringae , was studied by feeding 14 C-labeled precursors to chloramphenicol-containing bacterial suspensions. The preferential sites of incorporation were determined by comparing the specific activities of the intact radiolabeled metabolites and their single structural elements, obtained by hydrolytic degradation followed by derivatization and isolation by high performance liquid chromatography. The results show that, upon feeding L -[ 14 C(U)]-Thr, 35.0 and 31.0% of the SR radioactivity is retained in 2,3-dehydro-2-aminobutyric acid (Dhb) and 4-chlorothreonine (Thr(4-Cl)), respectively. L -[ 14 C(U)]-Asp labels the same sites, though less efficiently, and is also incorporated in 2,4-diaminobutyric acid (Dab) and 3-hydroxyaspartic acid (Asp(3-OH)). Dhb is also labeled by Thr and Asp in SP22. These are the first data on the biosynthetic origin of the modified residues in P. syringae lipopeptides.

Summary Background The NC16A immunodominant region of the bullous pemphigoid (BP) antigen BP180 has been used to develop several enzyme-linked immunosorbent assays (ELISAs) as diagnostic tools for BP autoantibody detection. Objectives Because BP180 autoantibody reactivity is not restricted to NC16A, we have investigated the possibility of developing an ELISA based on selected epitopes additional to this immunodominant region. Methods Initially 78 BP sera were tested using an NC16A ELISA and IgG reactivity was detected in 64 BP sera (82%). The 14 NC16A-negative BP sera were then analysed by immunological screening against seven BP180-specific epitopes. Recombinant phages displaying BP180 epitopes were grown as plaques, blotted onto a nitrocellulose filter and incubated with BP sera. Results Three and five NC16A-negative BP sera reacted with epitopes AA 1080–1107 and AA 1331–1404 of the BP180 ectodomain, respectively. Thus, a novel ELISA with GST-1080 and GST-1331 (GST-1080/1331) was developed: 32 of 78 BP sera (41%) proved positive by this assay. The combined use of ELISAs with GST-NC16A and GST-1080/1331 detected IgG reactivity in 72 of 78 BP sera, increasing the sensitivity from 82% to 92%. In addition, autoreactivity against the three extracellular epitopes appeared to be related to the presence of both skin and mucosal involvement as assessed by Fisher's exact probability test. Conclusions Our findings further characterize the autoimmune response in BP by identifying a subgroup of NC16A-negative patients who react with different BP180 extracellular epitopes. The developed ELISA system appears more sensitive than the ELISA based on NC16A alone and also informative about the epitope profile of BP patients.

The superfamily of chemokines comprises numerous small, cytokine-like chemotactic proteins, which have a fundamental role in the regulation of leukocyte trafficking. The chemokine-chemokine receptor system is highly redundant and promiscuous, and forms a complex network relevantly involved in the expression of chronic inflammatory skin diseases, including allergic contact dermatitis, atopic dermatitis and psoriasis. The pattern of chemokine expression shows overlapping features but also important differences in these diseases due to distinct sources and types of pro-inflammatory signals involved in chemokine induction, and the inherent capacity of resident skin cells to produce chemokines. Chemokine receptors (G-protein coupled receptors) rather than chemokines appear the appropriate therapeutic targets as they are more chemically tractable and play less redundant functions.

Bullous pemphigoid is a subepidermal bullous disease of skin and mucosae associated with autoantibodies to BP180. To characterize the humoral response to BP180, we generated a random BP180 epitope library displayed on lambda bacteriophage. After validation of the library by epitope mapping of three BP180-specific monoclonal antibodies, 15 novel or known BP180 epitopes were identified using 10 bullous pemphigoid serum samples. Fifty-seven bullous pemphigoid and 81 control sera were then assayed against the selected epitopes. Thirty-one out of 57 (54%) bullous pemphigoid sera reacted with at least an additional antigenic site other than the NC16A, within the extracellular (37%) and intracellular (28%) domains of BP180. In addition, the reactivity with extracellular epitopes of BP180 contained within the residue stretches 508-541 and 1331-1404 appeared to be related to the presence of both skin and mucosal involvement. Finally, a preliminary analysis of the epitope pattern in the disease course indicated that bullous pemphigoid patients exhibit a specific reactivity pattern, and that binding to intracellular epitopes of BP180, in addition to NC16A, may be detectable at an early clinical stage. Our findings provide novel insights into the pathophysiology of bullous pemphigoid and show the potential of the utilized approach as a tool for a rapid diagnosis of bullous pemphigoid patients and their management.

Two strains (B728a and Y37) of the phytopathogenic bacterium Pseudomonas syringae pv. syringae isolated from bean (Phaseolus vulgaris) plants were shown to produce in culture both syringomycin, a lipodepsinonapeptide secreted by the majority of the strains of the bacterium, and a new form of syringopeptin, SP22Phv. The structure of the latter metabolite was elucidated by the combined use of mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy and chemical procedures. Comparative phytotoxic and antimicrobial assays showed that SP22Phv did not differ substantially from the previously characterized syringopeptin 22 (SP22) as far as toxicity to plants was concerned, but was less active in inhibiting the growth of the test fungi Rhodotorula pilimanae and Geotrichum candidum and of the Gram-positive bacterium Bacillus megaterium. D 2002 Elsevier Science B.V. All rights reserved.

The biosynthesis of the lipodepsipeptides syringomycin and syringopeptin 22, secondary metabolites produced by the strain B301D-R of the phytopathogenic bacterium Pseudomonas syringae pv. syringae, was investigated by feeding radioactively labelled precursors to chloramphenicol containing bacterial suspensions. The production of labelled metabolites in the presence of a protein biosynthesis inhibitor showed that non ribosomal mechanism is operating in their synthesis. The origin of the modified amino acidic residues was studied by comparing the specific activities of the intact lipodepsipeptides, deriving from the feedings of L-[14C(U)]-Thr or L-[14C(U)3-Asp, to those of the costituent amino acids, obtained by hydrolytic degradation of the peptides, followed by appropriate derivatizations and isolation of the single structural elements by RP-HPLC. The results show that L-threonine is the precursor of dehydrobutyrin in syringomycin and in syringopeptin 22 and of 4-chlorothreonine in syringomycin. These two residues are also labelled by aspartic acid which is the biosynthetic precursor of threonine. Moreover, aspartic acid is aiso incorporated into 2,4-diaminobutyric and 3-hydroxyaspartic acid in syringomycin.