Charles G. Hurst

Sulfur mustard: Its continuing threat as a chemical warfare agent, the cutaneous lesions induced, progress in understanding its mechanism of action, its long-term health effects, and new developments for protection and therapy

Although sulfur mustard (SM) has been used as a chemical warfare agent since the early twentieth century, it has reemerged in the past decade as a major threat around the world. SM is an agent that is easily produced even in underdeveloped countries and for which there is no effective therapy. This agent is a potential threat not only on the battlefield but also to civilian populations. The skin and other epithelial surfaces are the first targets as this agent is absorbed, and reactions within the skin are the subject of active research into the mechanism of action of this alkylating agent. The depletion of glutathione, generation of reactive oxygen species, and the formation of stable DNA adducts remain theoretic and demonstrated by-products of SM exposure implicated in the disease produced. However, new findings related to the effects of SM on the basement membrane zone; interest in delayed healing of the lesions induced; the inflammatory mediators, enzymes, and cytokines that result; and cellular typing of the inflammatory infiltrate will increase our understanding of the pathophysiology of the lesions caused by SM. In addition, the recent development of a topical skin protectant for SM and for other chemical warfare agents may have broad applications within dermatology.

Immunohistochemical studies of basement membrane proteins and proliferation and apoptosis markers in sulfur mustard induced cutaneous lesions in weanling pigs

Sulfur mustard (2,2-dichlorodiethyl sulfide, HD) is a chemical warfare agent that is a threat to both troops and civilians. The focus of HD research has been on intracellular adduct formation leading to apoptosis and/or necrosis in cutaneous lesions. However, there is work which suggests that HD may have a more direct effect on the basement membrane zone. Immunohistochemical staining to desmosomal proteins, cellular fibronectin, laminin 1, laminin 5, collagen IV, collagen VII, p53, Bcl-2, and PCNA was performed on weanling pig skin exposed to vesicating doses of HD, GB3, an antibody to laminin 5, showed a progressive decrease with loss of expression during the time period of clinical vesiculation. The other basement membrane proteins showed no change or inconsistent changes. PCNA, and p53 staining increased in the overlying epidermis in areas of vesiculation without significant necrosis. Bcl-2 positive cells were decreased or absent after exposure. This study implicates laminin 5 as the main basement membrane protein affected acutely by HD exposure. The patterns of staining of PCNA, Bcl-2, and p53 within the epidermis suggest that apoptosis and cellular necrosis both may play a role in cell death secondary to HD.

Depth of morphologic skin damage and viability after one, two, and three passes of a high-energy, short-pulse CO 2 laser (Tru-Pulse) in pig skin

BACKGROUND CO2 laser energy is absorbed by water, which is present in all tissue. The depth of penetration of CO2 lasers is narrow with minimal reflection, scatter, or transmission. However, thermal damage has limited the usefulness of conventional, continuous-wave CO2 lasers for debridement as demonstrated by wound healing studies. The development of high-energy CO2 lasers, with pulse durations that are less than the thermal relaxation time of tissue, have made vaporization of skin for resurfacing and wound debridement possible because of the decreased risk of thermal damage. OBJECTIVE This study was performed to evaluate thermal damage produced by a CO2 laser. METHODS Routine histopathologic examination and nitroblue-tetrazolium chloride (NBTC) staining were used to evaluate the depth of tissue damage and viability in weanling pig skin after one, two, and three passes of the laser. RESULTS At a pulse energy of 300 mJ, with a pulse duration of 60 microseconds, one pass of the laser produced vaporization of the epidermis with minimal thermal damage. Two passes produced areas of denatured collagen with loss of viable cells in the superficial papillary dermis. Three passes extended the damage into the papillary dermis. CONCLUSION Hyalinization of collagen appears to correspond well with the level of thermal damage as measured by NBTC staining. Our findings suggest that the energy necessary to vaporize the dermis may be greater than that needed to vaporize epidermis.

Increased Smooth Muscle Actin, Factor XHIa, and Vimentin-positive Cells in the Papillary Dennis of Carbon Dioxide Laser-debrided Porcine Skin

BACKGROUND. Pulsed carbon dioxide (CO2) laser debridement is now being used as therapy for photodamaged skin. It has been proposed that the long duration of erythema and a tissue scaffold, which results from tightening of the collagen helix induced by the laser heat, may lead to tightening of sagging skin and skin creases of lesser magnitude. METHODS. Weanling pigs exposed to mild and moderate erythema producing doses of sulfur mustard (bis‐2‐chloroethyl sulfide; HD) were treated with the CO2 laser (Tru‐Pulse) at 6, 24, and 48 hours after exposure. In addition to histologic examination of laser‐debrided and nondebrided biopsy specimens obtained at 14 days after exposure, immunohistochemical staining with antibodies to smooth muscle actin, Factor XIIIa, vimentin, and CD3 was performed. RESULTS. CO2 laser debridement of the HD‐exposed skin resulted in clearing of the cytologic atypia induced by this chemical carcinogen and reduced the inflammatory infiltrate. In addition laser debridement resulted in increased numbers of stromal cells within the papillary dermis, which showed immunohistochemical staining for smooth muscle actin, Factor Xllla, and vimen‐tin. CONCLUSIONS. CO2 laser debridement is effective in clearing the epidermis of cytologically damage cells in HD as well as solar‐damaged skin. In addition CO, laser debridemant may result in tightening of sagging skin and produce a decrease in skin creases initially, by inducing increased stromal cells within the papillary dermis, with prominent contractile actin filaments. The collagen produced by these stromal cells may subsequently maintain these improvements in the photoaged skin.

Improved Healing of Sulfur Mustard-Induced Cutaneous Lesions in the Weanling Pig by Pulsed Co2 Laser Debridement

AbstractThe healing of cutaneous wounds induced by the chemical war-fare agent sulfur mustard [bis(2-chloroethyl)sulfide; HD] following pulsed CO2 laser debridement was examined in weanling pigs. Six animals (7-9 kg) were used to examine the efficacy of a Tru-Pulse CO2 laser in promoting improved healing. Each animal had 24 exposure sites on the dorsum. Eight sites were sham exposed, eight sites were exposed to saturated HD vapor for 5 min, and eight sites were exposed to saturated HD vapor for 15 min. Twelve sites on each pig were debrided using a pulsed CO2 laser. Two pigs were debrided each at 6, 24, and 48 h postexposure. The pigs were monitored during a 14-day healing period for erythema (by reflectance colorimetry), edema (Draize scoring), systemic toxicity (by hematological and clinical chemistry examinations), and lesion size (by video dermatoscopic examination and image analysis). At the end of the 14-day period, animals were euthanized and biopsies taken of all exposure sites for histopathologic...

Evaluation of cross-reacting anti-human antibodies in the euthymic hairless guinea pig model (HGP) suggests that the HGP may be a model for the study of proliferative skin disease☆

Animal models have an important role in cutaneous research. The guinea pig has proven to be a useful model in a wide spectrum of these cutaneous studies; however, its usefulness is often compromised by the need for depilation. A euthymic hairless guinea pig (HGP) model avoids the problems associated with depilation. Morphologically, as in human skin, these animals have a multi-cell-layer epidermis. Proliferation kinetic studies, as well as documentation of the degree of immunologic cross-reactivity between available antibodies to human cutaneous antigens, could extend the usefulness of this animal model. We performed a battery of anti-human antibodies on formalin fixed tissue, to a variety of antigens present within the skin and on inflammatory cells. These included CD3, UCHL-1, OPD4, L-26, KP-1, Factor XIIIa, S-100 protein, cytokeratin (AE1, AE3 and CK1), CAM 5.2, vimentin, CD 34, Factor VIII, fibronectin, SM actin, collagen IV, laminin, Bcl-2, p53, Ki-67, and PCNA. Cross-reacting antibodies included: CD3, S-100 protein, cytokeratin (AE1, AE3 and CK1), vimentin, Factor VIII, SM actin, collagen IV, p53, Ki-67, and PCNA. Although this battery of antibodies is limited, the markedly increased staining of Ki-67 and PCNA within keratinocytes in the epidermis as compared to normal human skin reflects a high proliferative rate. In addition, positive staining for p53, Ki-67, and PCNA may be useful in studying effects on cell cycle kinetics and apoptosis.

Sensitivity of cross-reacting antihuman antibodies in formalin-fixed porcine skin: including antibodies to proliferation antigens and cytokeratins with specificity in the skin

Although no animal is a perfect skin model for the study of toxicological and therapeutic agents, structurally the pig may be superior to even non-human primates. Because our work involves effects of toxicological and therapeutic agents on the skin, we wanted to identify stains which may prove useful as well as determine cross-reactivity of some newer antihuman antibodies. We performed a battery of formalin-fixed skin from weanling pigs and minipigs. The battery of antibodies included LCA, CD3, OPD-4, CD34, UCHL-1, L-26, KP-1, MAC-387, Factor XIIIa, Leu-7, S-100 protein, HMB-45, GFAP, synaptophysin, neurofilament protein, ubiquitin, vimentin, type IV collagen, laminin, fibronectin, Factor VIII related antigen, Desmin-M, smooth muscle actin, cytokeratin 7, cytokeratin 20, AEI/AE3, CAM 5.2, EMA, GCDFP, Ki-67, and PCNA. Immunohistochemical stains for CD3, Leu-7, S-100 protein, type IV collagen, laminin, Factor VIII related antigen, GFAP, synaptophysin, neurofilament protein, ubiquitin, smooth muscle actin, vimentin, Desmin-M, cytokeratin 7, cytokeratin 20, AE1/AE3, CAM 5.2, Ki-67 and PCNA showed consistent cross-reactivity. In formalin-fixed tissue, only antibodies to lymphoreticular cells showed poor cross-reactivity. A high percentage of the remaining antibodies did show good cross-reactivity but with some interesting similarities and differences in specificity.