Marcia G. Tonnesen

Detection of a Herpes Simplex Viral Antigen in Skin Lesions of Erythema Multiforme

The commonest variety of erythema multiforme follows a lesion caused by a recurrent herpes simplex virus infection. In studying the immunopathogenesis of herpes-associated erythema multiforme, we examined skin lesions for the presence of a herpes simplex viral antigen by an indirect immunofluorescence test using a monoclonal antibody to a major type-common glycoprotein antigen, gB. Focal staining showed this antigen to be present in epidermal cells in 12 of 16 skin biopsy specimens. The staining was similar to, but less intense than, that seen in biopsy samples of lesions of recurrent herpes simplex virus infections. Similar findings were not seen in control skin biopsy specimens from lesions of other skin diseases; a control monoclonal stain also was negative in biopsy specimens. These findings suggest that the immune reaction and subsequent tissue damage of herpes-associated erythema multiforme are due to the presence of herpes antigens in the skin.

Fibronectin Growth Factor-Binding Domains Are Required for Fibroblast Survival

Fibronectin (FN) is required for embryogenesis, morphogenesis, and wound repair, and its Arg-Gly-Asp-containing central cell-binding domain (CCBD) is essential for mesenchymal cell survival and growth. Here, we demonstrate that FN contains three growth factor-binding domains (FN-GFBDs) that bind platelet-derived growth factor-BB (PDGF-BB), a potent fibroblast survival and mitogenic factor. These sites bind PDGF-BB with dissociation constants of 10-100 nM. FN-null cells cultured on recombinant CCBD (FNIII(8-11)) without a FN-GFBD demonstrated minimal metabolism and underwent autophagy at 24 hours, followed by apoptosis at 72 hours, even in the presence of PDGF-BB. In contrast, FN-null cells plated on FNIII(8-11) contiguous with FN-GFBD survived without, and proliferated with, PDGF-BB. FN-null cell survival on FNIII(8-11) and noncontiguous arrays of FN-GFBDs required these domains to be adsorbed on the same surface, suggesting the existence of a mesenchymal cell-extracellular matrix synapse. Thus, fibroblast survival required GF stimulation in the presence of a FN-GFBD, as well as adhesion to FN through the CCBD. The findings that fibroblast survival is dependent on FN-GFBD underscore the critical importance of pericellular matrix for cell survival and have significant implications for cutaneous wound healing and regeneration.

Neutrophil Emigration, Activation, and Tissue Damage

The early inflammatory phase of wound healing is characterized by a rapid accumulation of neutrophils. Despite this observation, the neutrophil does not appear to play a central or essential role in the wound healing process per se. The classic study by Simpson and Ross (1972), in which wound repair was monitored in guinea pigs depleted of neutrophils by the administration of antineutrophil serum, failed to demonstrate that either wound debridement or formation of granulation tissue is dependent on the presence of neutrophils.

Fibronectin Peptides that Bind PDGF-BB Enhance Survival of Cells and Tissue under Stress

Stressors after injury from a multitude of factors can lead to cell death. We have identified four fibronectin (FN) peptides, two from the first FN type III repeat (FNIII1), one from the 13th FN type III repeat (FNIII13), and one from FN variable region (IIICS), that when tethered to a surface acted as platelet-derived growth factor-BB (PDGF-BB) enhancers to promote cell survival. One of the FNIII1 peptides and its smallest (14mer) bioactive form (P12) were also active in solution. Specifically, P12 bound PDGF-BB (KD = 200nM), enhanced adult human dermal fibroblast (AHDF) survival under serum starvation, oxidative or endoplasmic reticulum (ER) stressors, and limited burn injury progression in a rat hot comb model. Furthermore, P12 inhibited ER stress-induced c-Jun N-terminal kinase (JNK) activation. Although many growth factors have been found to bind FN directly or indirectly, this is the first report to identify peptide sequences of growth factor-binding sites in FN. The finding of these novel peptides further delineated how the extracellular matrix protein FN can support cell survival. Since the peptide P12 is active in either soluble form or tethered to a substrate, it will have multifactorial uses as a bioactive in tissue engineering.

Loss of primary cilia in melanoma cells is likely independent of proliferation and cell cycle progression.

Elizabeth R. Snedecor1,3,9, Clifford Sung1,4,9, Alejandra Moncayo1,5, Brooke E. Rothstein1,6, Daniel C. Mockler1, Marcia G. Tonnesen2,8, Evan C. Jones2, Mayumi Fujita7, Richard A. Clark2,5, Kenneth R. Shroyer1, and Jiang Chen1,2 1Department of Pathology, Stony Brook University, Stony Brook, NY 11794 2Department of Dermatology, Stony Brook University, Stony Brook, NY 11794 3Program in Genetics, Stony Brook University, Stony Brook, NY 11794 4School of Medicine, Stony Brook University, Stony Brook, NY 11794 5Department of Biomedical Engineering, Stony Brook University, Stony Brook, NY 11794 6School of Medicine, Tufts University, Boston, MA 02111 7Department of Dermatology, University of Colorado Denver, Aurora, CO 80045 8Dermatology Section, Northport VA Medical Center, Northport, NY 11768

Curcumin myths or wonders? A systematic analysis of in vitro studies.

To the Editor: The medical literature abounds with data supporting antioxidant, anti-inflammatory, and anticarcinogenic properties of curcumin, an ethanol extract of turmeric, the powdered root of Curcuma longa. As a result, there is renewed scientific interest in curcumin’s potential to prevent and treat skin disease. For example, curcumin has been reported to hasten wound healing in rodent full-thickness cutaneous wound models. A phase II clinical trial examining oral curcumin treatment for patients with plaque psoriasis showed promise in some patients but a low response rate. The chemotherapeutic possibilities of curcumin for skin cancer are also being investigated. However, the literature is difficult to interpret and impossible to integrate. This letter is meant to elucidate in part why this is so, at least at the cell culture level. In cell culture studies curcumin has been reported to scavenge reactive oxygen species (ROS). However, other studies have shown that curcumin also possesses prooxidant effects. Banerjee et al. reported that curcumin acted as an antioxidant at low concentrations (<10 mM) and prooxidant at high concentrations (>10 mM). Curcumin has also been ascribed antiinflammatory properties via interactions with numerous molecular targets. For example, inhibition of proinflammatory enzymes and cytokines has been attributed to curcumin’s suppression of nuclear factor-kappa B (NF-KB) activation. Finally, curcumin has been credited with anticarcinogenic activity through numerous molecular targets, 40 of which lead to apoptosis. For example, in melanoma cells, apoptosis appeared to be induced through a Fas receptor/ caspase-8 pathway, independent of p53. In contrast, Mukhopadhyay et al. reported that curcumin inhibited the proliferation of squamous cell carcinoma cell lines by downregulating cyclin D1 expression. Since many results from the curcumin literature appear to be inconsistent, it has been impossible to make an overarching hypothesis as to curcumin’s mechanism(s) of action. Furthermore, it is difficult to discern whether any biological actions of curcumin result from direct inhibition of ROS or enzymes, or activation of specific receptors. The situation is further complicated by the fact that many papers did not completely delineate cell culture conditions. In fact, a Task Force on Good Cell Culture Practice has stated that “clear documentation of the systems used and procedures followed is mandatory, in order to permit the traceability, interpretation, and repetition of the work.” We believe that clear delineation of cell viability, cell type, culture medium, presence or absence of serum/albumin, and time course of exposure is critical for cross-study comparisons for the following reasons. Curcumin has been shown to be cytotoxic to cells with evidence of decreased cell viability and growth inhibitory effects at levels as low as 5 mM. Thus, it is critical to ensure that any change in bioactivity is not due to the fact that cultured cells are dead or dying. Different cell types often respond differently to bioactives including curcumin. Some media contain antioxidants, e.g., pyruvate, phenol red, vitamin C or E, while others do not. Curcumin solubility in water varies depending on the presence or absence of albumin or serum, as curcumin is extremely hydrophobic but has a high affinity for albumin. Bioactivities of curcumin vary greatly by simply differing the time course of exposure. For example, Mackenzie et al. demonstrated that 2.5 mM curcumin caused significant decreased viability in Hodgkin and Reed-Sternberg KM-H2 cells after 72 hours whereas 25 mM curcumin decreased cell viability within a 24-hour incubation. Therefore, the inclusion of the aforesaid parameters were determined in curcumin cell culture studies obtained from a literature search using MEDLINE/PubMed databases for “cell” and “curcumin” from December 2006 through July 2009. Studies were limited to those published in the English language, and excluded review articles and editorials. A subgroup analysis of relevant papers was conducted on curcumin’s properties related to ROS using the five parameters from the initial analysis as well as two additional parameters: presence or absence of a potent antioxidant pyruvate in culture medium and cell density. Pyruvate is an extracellular scavenger of ROS, and Kim et al. recently demonstrated that increasing cell densities had greater resistance to oxidative stress. Two hundred fourteen papers met inclusion criteria for “cell” and “curcumin.” Analysis showed that only 58% included complete information on all five experimental parameters (Figure 1). All studies included information about cell type; however, over one third of papers lacked or did not fully present cell viability data. Conclusions cannot be made about the anti-inflammatory, antiproliferative, or other effects of curcumin if the cytotoxicity is not measured due to the

Blood vessel occlusion with erythrocyte aggregates causes burn injury progression-microvasculature dilation as a possible therapy

Burns are dynamic injuries characterized by progressive tissue death and continuous severe pain over the course of several days. The extent of burn injury progression determines the ultimate patient outcome. Initial burns result in a central zone of necrosis surrounded by a potentially viable zone of ischemia. Several mechanisms have been proposed to explain injury progression, including oxidant and cytokine stress resulting from either ischemia/reperfusion and/or inflammation, but no proven therapy has emerged. To address the unmet need to limit burn injury progression, the root cause of this process must be delineated. For this reason, we have recently focused on post‐burn blood vessel occlusion, currently ascribed to microthrombi. We have found that blood vessel occlusion is initially, mainly and persistently caused by erythrocyte aggregation. Although thermal‐induced cell necrosis is the immediate cause of cell death, apoptotic cells from persistent ischemia/anoxia, admixed with inflammatory cells, form a band between viable and nonviable tissue 24 hours later. The delayed cell death by apoptosis appears to be the main attractant for inflammatory cells. Finally, we posit that fibrinogen elevation arising from inflammation provides stimulus for additional erythrocyte aggregation, further extending blood vessel occlusion. In our view this persistent occlusion with resultant prolonged tissue ischemia/anoxia, not ischemia/reperfusion, is the root cause of burn injury progression concomitant with associated severe and persistent pain. Epiviosamines, a new class of peptides, appear to selectively dilate microvasculature, and may provide therapy for burn injury progression.