Christopher M. Hull

Activated Notch4 Inhibits Angiogenesis: Role of β1-Integrin Activation

ABSTRACT Notch4 is a member of the Notch family of transmembrane receptors that is expressed primarily on endothelial cells. Activation of Notch in various cell systems has been shown to regulate cell fate decisions. The sprouting of endothelial cells from microvessels, or angiogenesis, involves the modulation of the endothelial cell phenotype. Based on the function of other Notch family members and the expression pattern of Notch4, we postulated that Notch4 activation would modulate angiogenesis. Using an in vitro endothelial-sprouting assay, we show that expression of constitutively active Notch4 in human dermal microvascular endothelial cells (HMEC-1) inhibits endothelial sprouting. We also show that activated Notch4 inhibits vascular endothelial growth factor (VEGF)-induced angiogenesis in the chick chorioallantoic membrane in vivo. Activated Notch4 does not inhibit HMEC-1 proliferation or migration through fibrinogen. However, migration through collagen is inhibited. Our data show that Notch4 cells exhibit increased β1-integrin-mediated adhesion to collagen. HMEC-1 expressing activated Notch4 do not have increased surface expression of β1-integrins. Rather, we demonstrate that Notch4-expressing cells display β1-integrin in an active, high-affinity conformation. Furthermore, using function-activating β1-integrin antibodies, we demonstrate that activation of β1-integrins is sufficient to inhibit VEGF-induced endothelial sprouting in vitro and angiogenesis in vivo. Our findings suggest that constitutive Notch4 activation in endothelial cells inhibits angiogenesis in part by promoting β1-integrin-mediated adhesion to the underlying matrix.

Lipopolysaccharide Signals an Endothelial Apoptosis Pathway Through TNF Receptor-Associated Factor 6-Mediated Activation of c-Jun NH2-Terminal Kinase

Inflammatory mediators such as TNF and bacterial LPS do not cause significant apoptosis of endothelial cells unless the expression of cytoprotective genes is blocked. In the case of TNF, the transcription factor NF-κB conveys an important survival signal. In contrast, even though LPS can also activate NF-κB, this signal is dispensable for LPS-inducible cytoprotective activity. LPS intracellular signals are transmitted through a member of the Toll-like receptor family, TLR4. This family of receptors transduces signals through a downstream molecule, TNFR-associated factor 6 (TRAF6). In this study, we demonstrate that the C-terminal fragment of TRAF6 (TRAF6-C) inhibits LPS-induced NF-κB nuclear translocation and c-Jun NH2-terminal kinase (JNK) activation in endothelial cells. In contrast, LPS activation of p38 kinase is not inhibited by TRAF6-C. TRAF6-C also inhibits LPS-initiated endothelial apoptosis, but potentiates TNF-induced apoptosis. LPS-induced loss of mitochondrial transmembrane potential, cytochrome c release, and caspase activation are all blocked by TRAF6-C. We demonstrate that TRAF6 signals apoptosis via JNK activation, since inhibition of JNK activation using a dominant-negative mutant also inhibits apoptosis. JNK inhibition blocks caspase activation, but the reverse is not true. Hence, JNK activation lies upstream of caspase activation in response to LPS stimulation.

Elevation of IgA anti-epidermal transglutaminase antibodies in dermatitis herpetiformis

Background  Dermatitis herpetiformis (DH) is a papulovesicular eruption caused by ingestion of gluten. It is characterized by the deposition of IgA in the dermal papillae. IgA antibodies directed at tissue transglutaminase (TG2) are elevated in gluten‐sensitive diseases including DH and coeliac disease (CD). More recently, antibodies directed at epidermal transglutaminase (TG3) were identified in patients with DH, and this may be the dominant autoantigen in this disease.

IgA Anti-Epidermal Transglutaminase Antibodies in Dermatitis Herpetiformis and Pediatric Celiac Disease

leprosy through their functional roles in antigen presentation and inhibition of T-cell responses. Although predisposing major histocompatibility complex alleles may exhibit inefficient antigen presentation, the LYP-Trp620 allele may have a pathogenic role in the hyporesponsiveness of T cells owing to anomalies in early T-cell signaling, resulting in clinical manifestations of leprosy. Contrary to our expectations, a significantly higher number of tuberculoid patients had PTPN22 1858CT, suggesting that there may be early T-cell defects in these patients. This results in a compromised immune response to the infectious agent, which manifests in the milder form of the disease. Most healthy people exposed to M. leprae are resistant to the infection, and with an effective immune response they do not develop the disease (Bongiorno et al., 2008). Because the CT genotype accounts for only 15–16% of lepromatous and tuberculoid patients, other genes involved in the downregulation of T-cell responses, such as CTLA-4 and Foxp3, should be investigated for additional host factors that may be detrimental in conferring anergy to M. leprae antigens in lepromatous leprosy patients.

Contact urticaria from curcumin.

&NA; Turmeric, a spice derived from the rhizome of the plant Curcuma longa, contains the chemical curcumin, which is responsible for turmerics taste, color, and biologic properties. Curcumin is used as a spice in foods, as a treatment in traditional medicine, as a dye for fur, and as a component in nutritional supplements. A few cases of allergic contact dermatitis from curcumin have been reported. We report two cases of contact urticaria from curcumin. These cases are mediated by two different mechanisms of contact urticaria: nonimmunologic and immunologic (immunoglobulin‐E mediated).

Dermatitis Herpetiformis Sera or Goat Anti–Transglutaminase-3 Transferred to Human Skin-Grafted Mice Mimics Dermatitis Herpetiformis Immunopathology

Dermatitis herpetiformis (DH) is characterized by deposition of IgA in the papillary dermis. However, indirect immunofluorescence is routinely negative, raising the question of the mechanism of formation of these immune deposits. Sárdy et al. (2002. J. Exp. Med. 195: 747–757) reported that transglutaminase-3 (TG3) colocalizes with the IgA. We sought to create such deposits using passive transfer of Ab to SCID mice bearing human skin grafts. IgG fraction of goat anti-TG3 or control IgG were administered i.p. to 20 mice. Separately, sera from seven DH patients and seven controls were injected intradermally. Biopsies were removed and processed for routine histology as well as direct immunofluorescence. All mice that received goat anti-TG3 produced papillary dermal immune deposits, and these deposits reacted with both rabbit anti-TG3 and DH patient sera. Three DH sera high in IgA anti-TG3 also produced deposits of granular IgA and TG3. We hypothesize that the IgA class anti-TG3 Abs are directly responsible for the immune deposits and that the TG3 is from human epidermis, as this is its only source in our model. These deposits seem to form over weeks in a process similar to an Ouchterlony immunodiffusion precipitate. This process of deposition explains the negative indirect immunofluorescence results with DH serum.

Topical Chemotherapy in Cutaneous T-cell Lymphoma: Positive Results of a Randomized, Controlled, Multicenter Trial Testing the Efficacy and Safety of a Novel Mechlorethamine, 0.02%, Gel in Mycosis Fungoides

OBJECTIVE To evaluate the efficacy and safety of a novel mechlorethamine hydrochloride, 0.02%, gel in mycosis fungoides. DESIGN Randomized, controlled, observer-blinded, multicenter trial comparing mechlorethamine, 0.02%, gel with mechlorethamine, 0.02%, compounded ointment. Mechlorethamine was applied once daily for up to 12 months. Tumor response and adverse events were assessed every month between months 1 and 6 and every 2 months between months 7 and 12. Serum drug levels were evaluated in a subset of patients. SETTING Academic medical or cancer centers. PATIENTS In total, 260 patients with stage IA to IIA mycosis fungoides who had not used topical mechlorethamine within 2 years and were naive to prior use of topical carmustine therapy. MAIN OUTCOME MEASURES Response rates of all the patients based on a primary clinical end point (Composite Assessment of Index Lesion Severity) and secondary clinical end points (Modified Severity-Weighted Assessment Tool and time-to-response analyses). RESULTS Response rates for mechlorethamine gel vs ointment were 58.5% vs 47.7% by the Composite Assessment of Index Lesion Severity and 46.9% vs 46.2% by the Modified Severity-Weighted Assessment Tool. By the Composite Assessment of Index Lesion Severity, the ratio of gel response rate to ointment response rate was 1.23 (95% CI, 0.97-1.55), which met the prespecified criterion for noninferiority. Time-to-response analyses demonstrated superiority of mechlorethamine gel to ointment (P< .01). No drug-related serious adverse events were seen. Approximately 20.3% of enrolled patients in the gel treatment arm and 17.3% of enrolled patients in the ointment treatment arm withdrew because of drug-related skin irritation. No systemic absorption of the study medication was detected. CONCLUSION The use of a novel mechlorethamine, 0.02%, gel in the treatment of patients with mycosis fungoides is effective and safe. TRIAL REGISTRATION clinicaltrials.gov Identifier:NCT00168064.

Treatment of refractory chronic urticaria with tumor necrosis factor–alfa inhibitors

To the Editor: Chronic urticaria is a physically and emotionally debilitating condition that often fails to respond adequately to antihistamines. Systemic therapy with glucocorticoids, sulfasalazine, colchicine, intravenous immunoglobulin, dapsone, or other immunosuppressants including cyclosporine, cyclophosphamide, and methotrexate are used in many patients with the associated risks of renal and hepatic toxicity and anemia. Unfortunately, some patients have disease recalcitrant to these treatments. The pathogenic mechanisms of chronic urticaria are not well understood, but the primary effector cell is likely the mast cell. Activated mast cells release mediators, including tumor necrosis factorealfa (TNF-a), that recruit other immune cells and play a role in perpetuating an urticarial response. TNF-a is known to exist preformed in mast cells and to be newly synthesized upon mast cell activation. Several preclinical investigations have shown an upregulation of TNF-a in patients with chronic urticaria. Magerl et al described a patient with severe psoriasis and delayed pressure urticaria who became free of urticarial lesions after 4 days of treatment with etanercept. Based on these preclinical investigations and the case report, we treated six of our most refractory chronic urticaria patients with TNF-a inhibitors. The choice of TNF-awas primarily based on the patient’s insurance and our experience using the medications. When possible, we used doses tested for other systemic inflammatory conditions, such as psoriasis. A brief summary of each patient’s background and treatment course is provided in Table I. The theoretical basis for the use of TNF-ae targeting therapy to treat urticaria is supported by studies that have shown the upregulation of TNF-a in chronic urticaria. In a study of 19 chronic urticaria patients and 15 healthy controls, cytokine and chemokine production after autologous serum skin testing was analyzed and TNF-a, interleukin (IL)-10, RANTES, and macrophage inflammatory protein1a were upregulated in chronic urticaria patients but not controls. In another study that compared levels of TNF-a in lesional and nonlesional skin of chronic urticaria patients, TNF-a was found to be expressed throughout the epidermis in lesional and nonlesional chronic urticaria skin but not in control skin. Furthermore, a study by Piconi et al revealed similar immunologic profiles in patients with chronic urticaria and with rheumatoid arthritis, psoriasis, and psoriatic arthritis, with increased expression of TNF-a and IL-10, and decreased expression of IL-2 and interferon gamma. Interestingly, these diseases, along with chronic urticaria, tend to worsen with infection. Perhaps a common linkmay be an aberrant response of innate immunity mediated through TNF-a expression. In our series of six patients who were recalcitrant to all other immunosuppressive therapies, we observed dramatic improvement with TNF-a inhibitors. In most cases, the improvement was durable, lasting for several years, and in three cases the patients were subsequently tapered off all systemic treatment and their urticaria remains in remission. These results suggest that targeted TNF-a therapy may be useful for disease refractory to conventional or immunosuppressive therapy. Larger, controlled studies with longer followeup periods are needed to further confirm the efficacy and safety of TNF-a inhibitors in the management of patients with chronic urticaria.

Novel screening assay performance in pediatric celiac disease and adult dermatitis herpetiformis.

Objectives: Several serologic assays are commercially available to aid in the diagnosis of gluten-sensitive enteropathy (GSE). Our objective in this study was to assess the performance of a novel combined antigen-screening assay for GSE. Patients and Methods: Deidentified sera from 111 pediatric patients suspected of having celiac disease (CD), 130 adults diagnosed with dermatitis herpetiformis (DH), and 77 pediatric and 49 adult normal controls were included in the study. Sera from 10 patients submitted to our laboratory for GSE testing with IgA deficiency and IgG antibodies against 1 or more of the traditional serologic markers associated with GSE were also included. All sera were screened for antibodies (IgA and IgG) against tissue transglutaminase (tTG) and deamidated gliadin peptides (DGP) by enzyme immunoassay (EIA) in a single test well. In addition, all sera were assessed for each individual marker and isotype using separate EIAs. Results: The IgA/IgG anti-tTG/DGP EIA screen was 92.6% sensitive and 94.3% specific in pediatric CD and detected 1 patient (Marsh 3c) who was IgA anti-tTG negative; this patient was not IgA deficient (<7.0 mg/dL). All 10 IgA-deficient sera gave positive results by the tTG/DGP EIA screen. Sensitivity and specificity of the tTG/DGP EIA screen in retrospective and prospective DH were 65% and 100% versus 62% and 100%, respectively. Conclusions: The new IgA/IgG anti-tTG/DGP EIA screen was slightly more sensitive than IgA anti-tTG alone in pediatric CD. This novel screening assay may allow the current recommendation of measuring total serum IgA in suspected GSE patients to be eliminated.

Results of an Investigator‐Initiated Single‐Blind Split‐Face Comparison of Photodynamic Therapy and 5% Imiquimod Cream for the Treatment of Actinic Keratoses

Background Topical photodynamic therapy (PDT) with aminolevulinic acid (ALA) and 5% imiquimod cream are effective therapies for the treatment of actinic keratoses (AKs), but no split‐face studies directly comparing these treatment options are available in the literature. Objective To compare the efficacy and tolerability of ALA‐PDT and imiquimod 5% cream for the treatment of AKs. Results Sixty‐one patients were enrolled from the Salt Lake City Veterans Affairs Hospital; 51 completed the study and were included in the analysis. All patients were randomized to receive half of a sachet of imiquimod 5% cream twice weekly on half of their face and two sessions of PDT with 20% solution of ALA applied for 1 hour to the other side of the face. The 75% AK clearance rate was 34.6% for ALA‐PDT and 25% for imiquimod 5% cream (p = .30). The mean reduction in AK count was 59.2% for ALA‐PDT and 41.4% for imiquimod 5% cream (p = .002). Dermatology Life Quality Index (DLQI) scores were assessed for each treatment modality at week 4 and were 1.95 and 1.38, respectively (p = .20). Limitations The sample size was small, and patients applied a small amount of imiquimod 5% cream (half a sachet) to a large surface area. Conclusion There was no statistically significant difference in treatment response when the 100% or 75% clearance rate cutoff was used, but our secondary outcome suggests that two sessions of ALA‐PDT is superior to imiquimod 5% cream for the treatment of AKs. There was no statistically significant difference in effect on quality of life as assessed using the DLQI.