Henoch-Schönlein purpura in pregnancy.

Henoch-Schönlein purpura (HSP) is an IgA-mediated small vessel vasculitis which commonly involves the skin, gastrointestinal system and kidneys. Numerous HSP triggers have been identified, and pregnancy has been reported as an exacerbating factor. After a pregnant woman had been diagnosed as having new-onset HSP, we reviewed all cases of immunofluorescence-proven HSP evaluated by the Department of Dermatology at the Johns Hopkins Hospital between 1990 and 2002, and report three cases of HSP occurring during pregnancy. Two patients developed new-onset HSP, one at 16 weeks gestation and one at 22 weeks, while the third developed a recurrence of HSP at 12 weeks gestation after 19 years of remission. We conclude that pregnancy may be a trigger for HSP onset or recurrence in susceptible individuals.

Wegener's granulomatosis with cardiac involvement masquerading as Lyme disease.

Cardiac conduction disease is an infrequent complication of Wegener's granulomatosis (WG). We describe a case demonstrating an association between WG and complete atrioventricular dissociation. This manifestation was initially interpreted as Lyme disease based on these cardiac findings, arthritis, myalgias and positive Lyme serology. The clinical overlap between these disorders and the appropriate use of respective serologies is discussed.

Infliximab for peristomal pyoderma gangrenosum.

Peristomal pyoderma gangrenosum (PPG) is a variant of pyoderma gangrenosum (PG) that is more refractory to treatment. It is a cause of severe morbidity and poses a therapeutic challenge for the clinician. Infliximab (Remicade(R); Centocor, Malvern, PA, USA) is a chimeric monoclonal antibody directed against tumour necrosis factor-alpha that has been proven to be effective in the treatment of inflammatory bowel disease (IBD) and rheumatoid arthritis. Currently, very few reports exist documenting its use in the treatment of PG and none in the treatment of PPG. We describe our experience of treating three patients with IBD-associated PPG with infliximab. All patients tolerated the drug without significant side-effects. Two patients with PPG recovered completely following the administration of infliximab, and one patient had a partial response to the drug. We conclude that infliximab appears to be a safe and effective therapeutic alternative in patients with PPG.

Mycophenolate mofetil may serve as a steroid-sparing agent for sarcoidosis.

Sarcoidosis is an inflammatory disease with potentially severe mucocutaneous manifestations. Mycophenolate mofetil (MMF) is an immunosuppressive drug extensively used in organ transplantation. Its use has been rapidly expanded into autoimmune and inflammatory diseases. We report the first successful and safe use of MMF in five patients with sarcoidosis.

Paraneoplastic pemphigus in children and adolescents.

BACKGROUND: Paraneoplastic pemphigus (PNP) is an autoimmune mucocutaneous disease associated with specific B-cell lymphoproliferative neoplasms. There has been an increasing number of individual reports in the childhood and adolescent population. OBJECTIVES: To examine the clinical and immunopathological features of PNP occurring in children and adolescents. PATIENTS AND METHODS: We analysed the clinical and immunopathological findings of 14 patients under the age of 18 years with a confirmed diagnosis of PNP. Sera from all patients were analysed by indirect immunofluorescence (IF) and immunoprecipitation for plakin autoantibodies, immunoblotting for detection of plectin autoantibodies, and enzyme-linked immunosorbent assay (ELISA) for the detection of desmoglein (Dsg) 1 and Dsg3 autoantibodies. RESULTS: Severe oral mucositis was observed in all patients, and lichenoid cutaneous lesions in eight of 14 patients. The average age at presentation was 13 years. Striking findings included: pulmonary destruction leading to bronchiolitis obliterans in 10 patients, association with Castleman's disease in 12 patients, and a fatal outcome in 10 patients. The underlying neoplasm was occult in 10 patients. Histological findings include lichenoid and interface dermatitis with variable intraepithelial acantholysis. Deposition of IgG and C3 in the mouth and skin by direct IF was not found in some cases, but indirect IF detected IgG autoantibodies in all cases. Immunoprecipitation revealed IgG autoantibodies against desmoplakin I, envoplakin and periplakin in all cases, and against desmoplakin II and the 170-kDa antigen in 13 and 10 patients, respectively. Dsg3 and Dsg1 autoantibodies were present in 10 and three patients, respectively, and plectin autoantibodies in 13 patients. CONCLUSIONS: PNP in children and adolescents is most often a presenting sign of occult Castleman's disease. It presents with severe oral mucositis and cutaneous lichenoid lesions. Serum autoantibodies against plakin proteins were the most constant diagnostic markers. Pulmonary injury appears to account for the very high mortality rates observed.

Tumor necrosis factor-alpha induces distinctive NF-kappa B signaling within human dermal fibroblasts.

The TNF-alpha receptor-associated factor 2 (TRAF2) and its downstream mediator, the NF-kappa B-inducing kinase (NIK), have been shown to induce NF-kappa B activation in 293 cells. Investigating the role these mediators play in human skin, we found that both NIK and TRAF2 failed to evoke transcription from NF-kappa B-dependent promoters linked to the CAT reporter in human dermal fibroblast cultures, while epidermal keratinocyte cultures demonstrated NIK-dependent signaling. Further, NF-kappa B activation by TNF-alpha was unaffected by overexpression of a dominant negative mutant NIK in fibroblasts, despite detection of endogenous TRAF2 and NIK by Western analysis. To explore alternative signaling mechanisms in dermal fibroblasts, we found that the intracellular calcium chelator, 3,4,5-trimethoxybenzoic acid, and the calpain inhibitor, N-acetyl-Leu-Leu-norleucinal, both blocked NF-kappa B activation; however, the specific proteosome inhibitor, lactacystin, failed to do so. Furthermore, TNF-alpha receptor mutants lacking a functional death domain failed to stimulate NF-kappa B, while phosphatidylcholine-phospholipase C inhibition and alkalization of endolysosomal compartments blocked its activation by TNF-alpha. These data indicate that, while epidermal keratinocytes utilize previously defined, NIK-dependent NF-kappa B pathways, dermal fibroblasts demonstrate unique NIK/TRAF2-independent signal transduction, where both acidic sphingomyelinase and calpain activity act as surrogate mediators for NF-kappa B activation.

Cytokine modulation of extracellular matrix gene expression: relevance to fibrotic skin diseases.

Fibrotic skin diseases demonstrate a spectrum of phenotypic manifestations with a unifying histopathologic hallmark, i.e. the accumulation of collagen in the lesional skin. Mechanistically, collagen deposition could result either from enhanced biosynthetic activity or reduced rate of degradation, thus leading to an imbalance in physiological turnover of collagen in the dermis. This overview summarizes the mechanisms of regulation of collagen gene expression in fibroblasts as exemplified by cytokine modulation. This summary also provides evidence in support of the notion that transcriptional activation of collagen gene expression is the underlying mechanism leading to development of fibrotic cutaneous lesions, as documented in keloids, a prototypic example of cutaneous fibrosis.

Nuclear factor-kappa B mediates TNF-alpha inhibitory effect on alpha 2(I) collagen (COL1A2) gene transcription in human dermal fibroblasts.

Among its plethora of activities as an inflammatory mediator, TNF-alpha has potent regulatory control on extracellular matrix production and degradation. Earlier studies have documented that TNF-alpha inhibits type I collagen gene (COL1A2) expression at the transcriptional level, but the characterization of the transcription factors involved has been elusive. In the present study, using transient cell transfection of human dermal fibroblasts with a battery of 5' end deletion/chloramphenicol acetyltransferase (CAT) reporter gene constructs, we have characterized the TNF-alpha response element of the COL1A2 promoter. The TNF-alpha response element was attributed to a specific region that comprises noncanonical activator protein-1 (AP-1) (CGAGTCA) and NF-kappa B (AGAGTTTCCC) binding sites. TNF-alpha effect was eliminated by a 2-bp substitution mutation in the NF-kappa B1 binding half site of the NF-kappa B cis element. Electrophoretic mobility shift assays (EMSA) showed that recombinant human NF-kappa B heterodimers as well as NF-kappa B1 and RelA homodimers, but not AP-1, were capable of binding this element. Further, EMSA with human fibroblast nuclear extracts demonstrated enhanced binding of a single, specific complex within 5 min of TNF-alpha stimulation, which reached a plateau by 1 h and was not affected by preincubation of cells with cycloheximide. Gel supershift assays identified the complex as the NF-kappa B (p50/p65) heterodimer, whereas Abs to nuclear factor of activated T cells (NF-AT) and Jun family members failed to recognize the complex. These data suggest that in fibroblasts TNF-alpha activates and initiates the nuclear translocation of NF-kappa B that binds a divergent NF-kappa B element and plays a critical role in the observed inhibition of alpha 2(I) collagen gene transcription.

Cooperation between SMAD and NF-kappaB in growth factor regulated type VII collagen gene expression.

We have previously demonstrated that transforming growth factor-beta (TGF-beta) and pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta, synergistically enhance the expression of type VII collagen gene (COL7A1) in human dermal fibroblasts in culture (Mauviel et al., 1994). Recently, we identified a SMAD-containing complex, rapidly induced by TGF-beta and binding the region [-496/-444] of the COL7A1 promoter, responsible for COL7A1 gene transactivation (Vindevoghel et al., 1998a). In this report, we demonstrate that TGF-beta and TNF-alpha response elements are distinct entities within the COL7A1 promoter. In particular, we demonstrate that the TNF-alpha effect is mediated by NF-kappaB1/RelA (p50/p65) and RelA/RelA (p65/p65) NF-kappaB complexes binding the TNF-alpha response element (TaRE) located in the region [-252/-230], with RelA acting as the transcriptional activator. Finally, we provide definitive evidence for the role of both TGF-beta and TNF-alpha response elements as enhancer sequences, functioning in the context of a heterologous promoter in an additive manner in response to TGF-beta and TNF-alpha. This study provides the first identification of a functional interaction between the two immediate-early transcription factors, SMAD and NF-kappaB, to activate the expression of an extracellular matrix-related gene, COL7A1.

A proximal element within the human alpha 2(I) collagen (COL1A2) promoter, distinct from the tumor necrosis factor-alpha response element, mediates transcriptional repression by interferon-gamma.

Previous studies have shown that interferon-gamma (IFN-gamma) inhibits type I collagen gene expression through both transcriptional and post-transcriptional mechanisms (Kahäri et al., 1990). In the present study, using transient cell transfections of human dermal fibroblast cultures with a series of 5' deletion promoter/CAT reporter gene constructs, we have identified the IFN-gamma-response element of the human alpha 2(I) collagen gene (COL1A2) promoter. Specifically, we have identified a segment of the proximal promoter region, located between nucleotides -161 and -125 relative to the transcription start site, as critical for down-regulation of COL1A2 promoter activity by IFN-gamma. This IFN-gamma response element (IgRE) is clearly distinct from the previously described tumor necrosis factor-alpha response element (TaRE) located between nucleotides -265 and -241 of the COL1A2 promoter, a difference which is likely to explain the additive inhibitory effect of these two cytokines. The inhibitory effect of IFN-gamma was dose-dependent and rapidly induced, requiring less than 5 min exposure of fibroblast cultures. Gel mobility shift assays indicated that a highly specific nuclear protein complex bound to this 37-base pair region of promoter. Competition experiments with oligonucleotides spanning discrete segments of this promoter region mapped the binding element within a distinctive pyrimidine-rich sequence. Point mutations within the latter revealed that this element plays a crucial role not only in the IFN-gamma response, but also in the basal activity of the proximal promoter. Substitution mutations within the IgRE of the -161/CAT construct attenuated the promoter response to IFN-gamma, as measured in transient cell transfections, and eliminated specific DNA/protein complex formation, as measured by gel mobility shift assay. UV-crosslinking experiments indicated that two DNA/protein complexes were formed with the IgRE, with molecular weights around 55 kDa and 30 kDa, corresponding to proteins of approximately 30 kDa and approximately 6 kDa, respectively. Our results further clarify the molecular mechanisms involved in the regulation of type I collagen gene expression by IFN-gamma.

A GT-rich sequence binding the transcription factor Sp1 is crucial for high expression of the human type VII collagen gene (COL7A1) in fibroblasts and keratinocytes.

Type VII collagen is the major component of anchoring fibrils, structural elements that stabilize the attachment of the basement membrane to the underlying dermis. In this study, we have dissected the human type VII collagen gene (COL7A1) promoter to characterize the cis-elements responsible for the expression of the gene in cultured fibroblasts and keratinocytes. Using transient cell transfections with various 5' end deletion COL7A1 promoter/chloramphenicol acetyltransferase reporter gene plasmid constructs, we determined that the region between nucleotides -524 and -456, relative to the transcription start site, is critical for high promoter activity in both cell types studied. Gel mobility shift assays using several DNA fragments spanning this region identified a GT-rich sequence between residues -512 and -505, necessary for the binding of nuclear proteins to this region of the promoter. Point mutations abolished the binding of nuclear proteins in gel shift assays and drastically diminished the activity of the promoter in transient cell transfections. Supershift assays with antibodies against various transcription factors including Sp1, Sp3, c-Jun/AP-1, and AP-2, and competition experiments with oligonucleotides containing consensus sequences for Sp1 and AP-1 binding identified Sp1 as the transcription factor binding to this region of the COL7A1 promoter. Indeed, recombinant human Sp1 was shown to bind the COL7A1 promoter GT-rich element but not its mutated form in gel mobility shift assays. In addition, co-transfection of pPacSp1, an expression vector for Sp1, together with the COL7A1 promoter/chloramphenicol acetyltransferase construct into Sp1-deficient Drosophila Schneider SL2 cells unequivocally demonstrated that Sp1 is essential for high expression of the COL7A1 gene. These data represent the first in-depth analysis of the human COL7A1 promoter transcriptional control.

Cloning of the gene for human pemphigus vulgaris antigen (desmoglein 3), a desmosomal cadherin. Characterization of the promoter region and identification of a keratinocyte-specific cis-element.

Pemphigus vulgaris antigen is a cadherin-like desmosomal cell adhesion molecule expressed primarily in suprabasal keratinocytes within the epidermis. Previously characterized structural features have defined this molecule as a desmoglein, DSG3. In this study, we have cloned the human DSG3 gene and examined the transcriptional regulation of its expression. The total gene consisted of 15 exons and was estimated to span >23 kilobases. Comparison of exon-intron organization of DSG3 with bovine DSG1 and several classical cadherin genes revealed striking conservation of the structure. Up to 2.8 kilobases of the upstream genomic sequences were sequenced and found to contain several putative cis-regulatory elements. The promoter region was GC-rich and TATA-less, similar to previously characterized mammalian cadherin promoters. The putative promoter region was subcloned into a vector containing chloramphenicol acetyl transferase reporter gene. Transient transfections with a series of deletion clones indicated that the DSG3 promoter demonstrated keratinocyte-specific expression, as compared with dermal fibroblasts examined in parallel, and fine mapping identified a 30-base pair segment at -200 to -170 capable of conferring epidermal specific expression. The results provide evidence for the transcriptional regulation of the pemphigus vulgaris antigen gene, potentially critical for development of the epidermis and physiologic terminal differentiation of keratinocytes.

Regulation of mucin gene expression in secretory epithelial cells.

To gain insight into the regulation of mucin biosynthesis and secretion at a molecular level, we studied the expression of four mucin genes in several secretory cell types. Oligonucleotide probes for MUC1, MUC2, MUC3, and MUC4 were used in Northern blot analysis of total RNA from the cells. It was shown that MUC4 was constitutively expressed in the endometrial cell line (Ishikawa), which we use as a model system for studying airway mucin. Expression was enhanced by secretagogues. Cultured bronchial epithelial cells gave identical results. MUC1, MUC2, and MUC3 were not detected in either cell system. Secretory phase endometrial tissues also expressed only MUC4, but proliferative tissues expressed both MUC4 and MUC1. The data indicate that the mucin genes are differentially expressed in various cells and suggest a possible regulatory role for steroid hormones.

Primary care providers: managing today's prepaid risk.

Participating in managed care systems involves the issues of enrollment, marketing, referral patterns and information systems among others. The most important aspect, writes David Kouba, is reimbursement. Kouba offers several ideas for successfully managing the associated risk.