Robert A. Swerlick

Definitions and outcome measures for bullous pemphigoid: recommendations by an international panel of experts

Our scientific knowledge of bullous pemphigoid (BP) has dramatically progressed in recent years. However, despite the availability of various therapeutic options for the treatment of inflammatory diseases, only a few multicenter controlled trials have helped to define effective therapies in BP. A major obstacle in sharing multicenter-based evidences for therapeutic efforts is the lack of generally accepted definitions for the clinical evaluation of patients with BP. Common terms and end points of BP are needed so that experts in the field can accurately measure and assess disease extent, activity, severity, and therapeutic response, and thus facilitate and advance clinical trials. These recommendations from the International Pemphigoid Committee represent 2 years of collaborative efforts to attain mutually acceptable common definitions for BP and proposes a disease extent score, the BP Disease Area Index. These items should assist in the development of consistent reporting of outcomes in future BP reports and studies.

Fluvastatin inhibits up‐regulation of tissue factor expression by antiphospholipid antibodies on endothelial cells

Summary.   Background: Mechanisms of thrombosis induced by antiphospholipid (aPL) antibodies include up‐regulation of tissue factor (TF) expression on endothelial cells (ECs). Statins have been shown to reduce levels of TF induced by tumor necrosis factor (TNF‐α) and lipopolysaccharide (LPS) on ECs. In a recent study, fluvastatin inhibited thrombogenic and proinflammatory properties of aPL antibodies in in vivo models. The aim of this study was to determine whether fluvastatin has an effect on aPL‐induced expression of TF on ECs. Methods: IgGs were purified from four patients with APS (IgG‐APS) and from control sera (IgG‐NHS). Cultured human umbilical vein endothelial cells (HUVEC) were treated with IgG‐APS or IgG‐NHS or with medium alone or with phorbol myristate acetate (PMA), as a positive control. In some experiments, cells were pretreated with fluvastatin (2.5, 5 or 10 µm) with and without mevalonate (100 µm). TF expression on HUVECs was measured by ELISA. Results: PMA and the four IgG‐APS preparations increased the expression of TF on EC significantly (4.9‐, 2.4‐, 4.2‐, 3.5‐ and 3.1‐fold, respectively), in a dose‐dependent fashion. Fluvastatin (10 µm) inhibited the effects of PMA and the four IgG‐APS on TF expression by 70, 47, 65, 22 and 68%, respectively, and this effect was dose‐dependent. Mevalonate (100 µm) completely abrogated the inhibitory effects of fluvastatin on TF expression induced by aPL. Conclusion: Because of the suggested pathogenic role of aPL on induction of TF on ECs, our data provide a rationale for using statins as a therapeutic tool in treatment of thrombosis in APS.

Determining force dependence of two-dimensional receptor-ligand binding affinity by centrifugation.

Analyses of receptor-ligand interactions are important to the understanding of cellular adhesion. Traditional methods of measuring the three-dimensional (3D) dissociation constant (Kd) require at least one of the molecular species in solution and hence cannot be directly applied to the case of cell adhesion. We describe a novel method of measuring 2D binding characteristics of receptors and ligands that are attached to surfaces and whose bonds are subjected to forces. The method utilizes a common centrifugation assay to quantify adhesion. A model for the experiment has been formulated, solved exactly, and tested carefully. The model is stochastically based and couples the bond force to the binding affinity. The method was applied to examine tumor cell adherence to recombinant E-selectin. Satisfactory agreement was found between predictions and data. The estimated zero-force 2D Kd for E-selectin/carbohydrate ligand binding was approximately 5 x 10(3) microm(-2), and the bond interaction range was subangstrom. Our results also suggest that the number of bonds mediating adhesion was small (<5).

Mortality of bullous pemphigoid: An evaluation of 223 patients and comparison with the mortality in the general population in the United States

BACKGROUND There are large discrepancies in reported mortality for bullous pemphigoid (BP). OBJECTIVE We sought to determine the mortality of a large cohort of patients with BP and compare this with age-matched control subjects. METHODS Data were collected on 223 patients with a new diagnosis of BP between 1998 and 2003 through our cutaneous immunofluorescence laboratory databases. The mortality of patients with BP was compared with that of age-matched control subjects in the general US population. RESULTS The 1-, 2-, and 5-year mortality was 0.23 (95% confidence interval=0.18, 0.29), 0.37 (95% confidence interval=0.31, 0.44), and 0.50 (95% confidence interval=0.42, 0.57), respectively. However, relative to age-matched control subjects, no difference in expected mortality was detected. LIMITATIONS This was a retrospective cohort analysis. CONCLUSIONS Mortality of patients with BP is more likely related to advanced age and associated medical conditions than to disease-specific factors.

Expression and Functional Significance of Adhesion Molecules on Cultured Endothelial Cells in Response to Ionizing Radiation

Objective: Upregulation of adhesion molecules on endothelial cells following irradiation has been shown, but the functional significance of this upregulation in various endothelial cell lines is not clear. We have developed an in vitro flow model to study the functional consequences of the radiation‐induced upregulation of E‐selectin and intercellular adhesion molecule (ICAM‐1).

CULTURE AND CHARACTERIZATION OF SINUSOIDAL ENDOTHELIAL CELLS ISOLATED FROM HUMAN LIVER

SummaryAlthough most vascular models use large vessel endothelial cells from human umbilical veins, there is marked heterogeneity among endothelial cells from different vascular beds and organs. More accurate modeling of endothelial involvement in liver diseases, including metastasis, may result from the use of human hepatic sinusoidal endothelial cells. Liver resection specimens were sectioned, then treated with a 1.2 U/ml dispase solution. The tissue slurry was mechanically disaggregated and separated by centrifugation on a Percoll density gradient. Cells were then cultured in an endothelial-specific media with growth factors. These techniques resulted in a homogeneous monolayer consistent with endothelial cells by light microscopy. An endothelial origin was further confirmed by the expression of Factor VIII, binding of Ulex lectin, and uptake of acetylated low density lipoprotein. Electron microscopy showed transcellular fenestrations consistent with a sinusoidal origin. These human hepatic sinusoidal endothelial cells were then studied for expression of the adhesion molecules CD31/PECAM, CD34, E-selectin, ICAM-1, L-selectin, LFA-3, P-selectin, and VCAM-1 plus the binding of wheat germ agglutinin lectin. The patterns of adhesion molecule expression and lectin binding by these cells are characteristic of hepatic sinusoidal endothelia. In this paper, we have described a method for isolation and culture of human cells with the morphologic and phenotypic characteristics of hepatic sinusoidal endothelia.

Integrins: Role in Cell Adhesion and Communication

Adhesive interactions are crucial for the integrity and function of all cells and tissues. As one of the major families of cell adhesion receptors, the integrins have been the focus of scientific interest for more than a decade. The resulting studies have tremendously enhanced the understanding of integrin-mediated adhesive interactions and have unveiled novel integrin functions in the cytoskeletal organization of microfilaments and in the activation of diverse signaling pathways. These functions are critically involved in the regulation of multiple processes, such as tissue development, inflammation, tumor cell growth and metastasis, and programmed cell death. The global view of integrin receptor biology has radically changed and has become much more subtle and elaborate. The enormous complexity of integrin function is determined by the heterodimeric formation of more than 20 functional integrin receptors, the cell type-specific distribution, the receptor activation state, the presence of different activation and deactivation signals, and the subsequent employment of distinct cytoskeletal and signaling complexes within a more dimensional network of time and space. This article summarizes the structural and functional properties of the integrin receptors and emphasizes some of the major achievements made in the past to enhance the understanding of integrin biology.

Cutaneous Vasculitis: Its Relationship to Systemic Disease

Necrotizing vasculitis may be localized to the skin or may involve multiple organs. Although the etiology of cutaneous necrotizing vasculitis is unknown, evidence suggests that circulating immune complexes play an important role. The most common clinical lesion seen is palpable purpura, which histologically demonstrates leukocytoclastic vasculitis. The majority of patients affected with cutaneous necrotizing vasculitis have a benign course.

Neutralization of TNF by the Antibody cA2 Reveals Differential Regulation of Adhesion Molecule Expression on TNF-Activated Endothelial Cells

Upregulation of adhesion proteins plays an important role in mediating inflammation. The induction of adhesive molecules has been well studied, but the reversibility of their expression has not been well characterized. A neutralizing anti-TNF monoclonal antibody (cA2) was used to study the down regulation of TNF-induced E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on cultured human umbilical vein endothelial cells (HUVECs). Addition of cA2 following TNF stimulation of HUVECs enhanced the rate of E-selectin and VCAM-1 down-regulation from the cell surface and also reduced steady state E-selectin and VCAM-1 mRNA levels. The cA2-mediated disappearance of E-selectin, but not VCAM-1 protein was microtubule and not microfilament dependent. Neutralization of TNF only slightly reduced ICAM-1 cell surface levels following initial TNF stimulation, suggesting a slower turnover of ICAM-1 compared to E-selectin and VCAM-1. Microtubule inhibition during TNF stimulation partially inhibited E-selectin, VCAM-1 and ICAM-1 mRNA upregulation. VCAM-1 and ICAM-1 cell surface expression were similarly partially inhibited, however, E-selectin levels were unaffected, presumably due to the dual, opposing effect of inhibiting protein expression and inhibiting internalization. Microfilament inhibition during protein induction specifically inhibited the maximal expression of VCAM-1 protein and mRNA, without affecting E-selectin or ICAM-1. These data support the notion that E-selectin, VCAM-1, and ICAM-1 expression are differentially regulated on HUVECs and suggest that TNF neutralizing therapies may be effective because of their ability to reduce the levels of pre-existing adhesion proteins.

Discordance of histopathologic parameters in cutaneous melanoma: Clinical implications

BACKGROUND Histopathologic analysis remains the gold standard for the diagnosis of melanoma, however previous studies have shown a substantial rate of interobserver variability in the evaluation of melanocytic lesions. OBJECTIVE We sought to evaluate discordance in the histopathological diagnosis and microstaging parameters of melanoma and subsequent impact on clinical management. METHODS This was a retrospective review of 588 cases of cutaneous melanoma and melanoma in situ from January 2009 to December 2014 that were referred to Emory University Hospital, Atlanta, GA, for treatment. Per institutional policy, all outside melanoma biopsy specimens were reviewed internally. Outside and institutional reports were compared. RESULTS Disagreement between outside and internal reports resulted in a change in American Joint Committee on Cancer pathologic stage in 114/588 (19%) cases, resulting in a change in management based on National Comprehensive Cancer Network guidelines in 105/588 (18%) cases. LIMITATIONS Given the retrospective nature of data collection and the bias of a tertiary care referral center, cases in this study may not be representative of all melanoma diagnoses. CONCLUSION These findings confirm consistent subjectivity in the histopathologic interpretation of melanoma. This study emphasizes that a review of the primary biopsy specimen may lead to significant changes in tumor classification, resulting in meaningful changes in clinical management.