Novel application of high-dose rate brachytherapy for severe, recalcitrant palmoplantar pustulosis.

Palmoplantar pustulosis (PPP) is a chronic pustular dermatitis of the palms and soles, which is frequently associated with significant pruritus and pain, often limiting daily activities. We present the case of a 36-year-old man with severe PPP who had treatment failure with multiple medical therapies but showed marked improvement with high-dose rate brachytherapy. Brachytherapy has the advantage of providing a conformal dose distribution over complex curved surfaces, such as the foot and ankle. Our observations suggest that brachytherapy may be a well-tolerated treatment option for patients with severe, refractory PPP.

Outcome assessment in cellulitis clinical trials: is telephone follow up sufficient?

The US Food and Drug Administration has scrutinized clinical trial methodology in cellulitis, partly because the definition and timing of cure are debatable. We analysed the validity of telephone self-report as a proxy for in-person follow up in a cellulitis treatment trial comparing cephalexin alone with cephalexin-plus-trimethoprim/sulfamethoxazole. Our results demonstrate poor agreement between these two methods of outcome determination and have implications for future cellulitis clinical trial design and clinical management.

1,24-Dihydroxyvitamin D3 (tacalcitol) prevents skin T-cell infiltration.

BACKGROUND: 1,24-Dihydroxyvitamin D3 (tacalcitol), a vitamin D(3) compound, has been used to treat T cell-mediated inflammatory skin diseases such as psoriasis, prurigo and vitiligo. The best-known mechanism of action of this compound is inhibition of the abnormal proliferation of keratinocytes and subsequent maturation; however, its effects on skin T-cell recruitment have not yet been evaluated. Cutaneous lymphocyte-associated antigen (CLA), a surface glycoprotein expressed on T cells, plays a critical role in skin T-cell infiltration. We recently reported that 1,25-dihydroxyvitamin D3 inhibits skin infiltration of CD4+ T cells by suppressing CLA expression on T cells. OBJECTIVES: In this study, we investigated the effect of tacalcitol on CLA epitope decoration and on the levels of gut or lymph node homing receptor expression in human T cells. METHODS: We cultured human T cells with tacalcitol and analysed the effect on CLA expression and skin-homing ability, and evaluated glycosyltransferase mRNAs. We also performed an in vivo study using an antigen-dependent delayed-type hypersensitivity (DTH) mouse model and investigated the effect of tacalcitol on skin-infiltrating CD4+ T cells. RESULTS: Tacalcitol downregulated the expression of CLA and, in parallel, the E- and P-selectin ligand function; however, it exerted no effect on other homing receptors. Subcutaneously and intraperitoneally administered tacalcitol downregulated skin infiltration of effector CD4+ T cells in an in vivo DTH mouse model. CONCLUSIONS: These findings suggest that tacalcitol reduces skin inflammation by partially downregulating CLA expression levels.

Expression of cell-cycle regulator CDK2-associating protein 1 (p12CDK2AP1) in transgenic mice induces testicular and ovarian atrophy in vivo.

The novel cell-cycle regulator p12(CDK2AP1) (p12) gene encodes a cyclin-dependent kinase 2 (CDK2) partner that participates in cell-cycle regulation, apoptosis, and proliferation. CDK2 has been implicated in maintenance of gonadal homeostasis, as knockout mice display reproductive abnormalities. To investigate the role of p12 in homeostasis of gonadal tissues in vivo, we generated a transgenic mouse model driven by the human keratin 14 promoter, reported to target transgene expression to gonadal tissues and also stratified epithelia. Overexpression of the transgene was associated with a gonadal atrophy phenotype in mice of both sexes, yet fertility was not impaired. Histological evaluation of testes showed seminiferous tubule degeneration and decreased tubule diameter. Female transgenic mice had small ovaries, with a higher number of atretic follicles/mm(2) as compared to control nontransgenic mice. Also observed was increased germ cell apoptosis in both sexes (TUNEL). These results suggest that overexpression of p12 leads to testicular and ovarian abnormalities, a phenotype closely related to that of cdk2-/- mice. In combination, these observations suggest that the p12/CDK2 signaling pathways are carefully orchestrated to maintain proper gonadal tissue homeostasis. We suggest that the mechanisms of this regulation may be through p12-mediated altered expression of gonadal-specific genes and apoptotic pathways.

Oral epithelial overexpression of IL-1alpha causes periodontal disease.

Periodontal disease is a bacterial infection that results in inflammatory destruction of tissues that support the teeth, including connective tissue and bone. In this study, we report that transgenic mice that overexpress the 17-kDa form of IL-1alpha in the basal layer of oral mucosal epithelium develop a syndrome that possesses all of the cardinal features of periodontal disease, including epithelial proliferation and apical migration, loss of attachment, and destruction of cementum and alveolar bone. In this model, bacterial colonization and infection were not required, since levels of periodontal bacteria were equivalent in transgenic and wild-type mice, and continuous treatment with antibiotics from birth did not ameliorate the disease. Our findings therefore indicate that elevated levels of IL-1alpha in the oral micro-environment can mediate all of the clinical features of periodontal disease.

Gene therapy to target dendritic cells from blood to lymph nodes.

Peripheral lymph nodes (PLN) are strategic microenvironments where antigen-presenting dendritic cells (DC), loaded with environmental antigens, and naive lymphocytes meet to initiate immune responses. The unique capacity of DC to induce primary immune responses has led to their use in clinical medicine; however, delivering DC to lymph nodes is problematic. Intravenously injected DC cannot access to PLN, while DC injected into tissue migrate inefficiently through lymphatics to PLN. We achieved DC targeting to T-cell areas of PLN by endowing DC with a novel receptor for peripheral node addressin (PNAd), an adhesion molecule present on the lymph node venular endothelium. This novel receptor is a chimeric E/L-selectin (ELS) that, we have previously shown, binds to PNAd. DC were genetically modified by retroviral transduction to express ELS. ELS expression was targeted to tips of microvilli, and mediated rolling of DC on PNAd both in vivo and in vitro. Such genetically engineered DC could extravasate directly from blood through the lymph node endothelium as opposed to nontransduced DC. This study provides evidence that the trafficking of DC can be modified using gene transfer technologies. More efficient delivery of DC to PLN should assist the development of improved vaccination strategies.

Tonsillar B cells do not express PSGL-1, but a significant fraction displays the cutaneous lymphocyte antigen and exhibits effective E- and P-selectin ligand activity.

Skin-homing T cells are defined by the expression of the cutaneous lymphocyte-associated antigen (CLA) which enables the cells to selectively bind to vascular endothelial E-selectin close to sites of cutaneous inflammation, an initial step in the effective extravasation from blood into the inflamed tissue. Essentially all CLA on T cells decorates the backbone of the P-selectin glycoprotein ligand-1 (PSGL-1). In this study we show that human peripheral blood B cells (PBBC) and tonsillar B cells (TBC) do not display PSGL-1 in fluorescence-activated cell sorter analysis using different murine monoclonal antibodies and polyclonal rabbit anti-PSGL-1 antiserum. A significant population of TBC, however, expresses a HECA-452-reactive epitope. These cells represent nonactivated IgM(+)/IgG(-) mature B lymphocytes. Up to 50% of the TBC in a given preparation strongly bind to E- and up to 79% to P-selectin. The shear stress resistance in a parallel-plate flow chamber system was high. Neuraminidase treatment of TBC totally and O-sialoglycoprotein endopeptidase partially diminished HECA-452 reactivity and reduced E- but not P-selectin ligand activities. Mocarhagin had no effect in the assays. The data suggest a different ligand for P-selectin and a distinct glycoprotein carrier for the E-selectin ligand as compared to T cells or other leukocytes. Adhesion to P-selectin, however, still required sulfation of the ligand for function. Western blots of TBC cell lysates detected a >240-kD HECA-452-reactive material that was resistant to reducing conditions. Anti-PSGL-1 did not reveal immunoreactive material in these cell lysates. B cell activation did neither significantly change HECA positivity nor induce PSGL-1 expression. Cultured, activated TBC, however, maintained expression of the integrin alpha4beta7. Human peripheral blood B cells had similar cell surface characteristics to TBC. Our observations suggest that several adhesion molecules may be involved in B cell homing which include CLA, the P-selectin ligand, and structures such as alpha4beta7.

Inflammatory skin disease in K14/p40 transgenic mice: evidence for interleukin-12-like activities of p40.

The proinflammatory cytokine interleukin-12, a p35/p40 heterodimer, is produced by resident cells in skin and has been implicated as a pathogenetic factor in T-cell-mediated skin diseases. Secretion of heterodimeric interleukin-12 is always accompanied by production of p40 monomer and p40/p40 homodimer. To investigate the possible in vivo role of p40 per se, we generated mice that constitutively express monomeric and homodimeric p40 in basal keratinocytes. These mice spontaneously developed an eczematous skin disease that was characterized by hyperkeratosis, focal epidermal spongiosis, and a mixed inflammatory infiltrate composed of T cells (CD4+), macrophages, eosinophils, mast cells, and few neutrophils. Fluorescence-activated cell sorter analysis of transgenic epidermal cell suspensions revealed induction of major histocompatibility complex class II molecules on keratinocytes and a 2-3-fold increase in the content of Langerhans cells. Cytokines produced by these activated epidermal cells include interleukin-1alpha and tumor necrosis factor alpha. The skin disease in K14/p40 mice was similar to that of littermate mice that received injections of interleukin-12, suggesting overlapping in vivo functional properties. As induction of interferon-gamma is a major function of interleukin-12, we tested the in vitro ability of transgenic p40 to induce interferon-gamma. In contrast to interleukin-12, transgenic p40 did not stimulate interferon-gamma secretion by cultured splenocytes. We conclude that transgenic p40 and interleukin-12 are equally capable of initiating cutaneous inflammation. Despite these in vivo similarities, there is a clear functional difference between interleukin-12 and transgenic p40 in vitro, suggesting that interferon-gamma is not a major factor contributing to interleukin-12-like activities of transgenic p40.

Neutrophils, monocytes, and dendritic cells express the same specialized form of PSGL-1 as do skin-homing memory T cells: cutaneous lymphocyte antigen.

Memory T cells in inflamed skin express the cutaneous lymphocyte-associated antigen (CLA), a glycosylated epitope defined by the mAb HECA-452. We previously reported that on T cells, CLA occurs almost exclusively on the protein backbone of P-selectin glycoprotein ligand-1 (PSGL-1). T cells exhibiting the CLA isoform of PSGL-1 can tether and roll on both E- and P-selectin, while T cells expressing PSGL-1 without the CLA epitope do not bind E-selectin, though they may bind P-selectin. We show here that circulating neutrophils and monocytes, and cultured blood dendritic cells, also express CLA almost entirely as an isoform of PSGL-1. These cells all tether and roll on both E- and P-selectin. A chimeric fusion protein incorporating the 19 N-terminal amino acids of mature PSGL-1 exhibited HECA-452 immunoreactivity and supported rolling of CHO cells expressing either E- or P-selectin. These findings indicate a site for the CLA modification within the distal tip of PSGL-1, previously shown to be critical for P-selectin binding and to mediate some, but not all, of the E-selectin binding of PSGL-1. We hypothesize that the types of circulating leukocytes discussed above all use CLA/PSGL-1 to tether and roll on E- and P-selectin along the vascular endothelium.

Cytokines: IL-20 - a new effector in skin inflammation.

The newly discovered cytokine interleukin-20 (IL-20) is structurally related to IL-10, yet it appears to be an autocrine factor for keratinocytes that regulates their participation in inflammation.

Targeted expression of bcl-2 to murine basal epidermal keratinocytes results in paradoxical retardation of ultraviolet- and chemical-induced tumorigenesis.

The antiapoptotic protein bcl-2 is found up-regulated in a number of malignant and premalignant skin conditions of keratinocyte origin, but in normal skin, it is expressed at low levels only in interfollicular epidermis. To investigate whether unregulated bcl-2 expression could affect the incidence of epidermal tumors, we have generated a mouse line that over-expresses human bcl-2 in the basal layer of epidermis under the control of the human keratin 14 promoter. These mice were subjected to both UVB photocarcinogenesis and classical two-stage chemical carcinogenesis. Although transgenic bcl-2 in these mice reduces the formation of sunburn cells after short-term UVB irradiation, chronically UVB irradiated K14/bcl-2 mice were protected against tumor development, because transgenic mice developed tumors much later and at a significantly lower frequency than controls. Immunohistochemical analyses of the UVB-induced tumors revealed no significant differences in the degree of inflammatory cell infiltrates. When either K14/bcl-2 mice or F(1) progeny of matings with mice expressing an activated Ha-ras oncogene (K14/bcl-2/ras) were treated with 9,10-dimethyl-1,2-benzanthracene/phorbol 12-myristate 13-acetate, the latency of first papilloma appearance was the same in transgenic mice and controls, but further papillomas developed more slowly in the mutant mice. Moreover, the K14/bcl-2/ras mice developed far fewer albeit larger tumors/mouse than did the ras/+ controls. The rate of conversion to malignant carcinomas, the carcinoma grade, and the frequency of lymph node metastases were not significantly different between mutants and controls. We conclude that, despite its antiapoptotic function, bcl-2, overexpressed in basal epidermal keratinocytes, exerts a paradoxical retardation on the development of skin tumors induced by chemical carcinogens and particularly by UVB.

Skin-specific caspase-1-transgenic mice show cutaneous apoptosis and pre-endotoxin shock condition with a high serum level of IL-18.

To study the pathophysiological roles of overexpressed caspase-1 (CASP1), originally designated as IL-1 beta-converting enzyme, we generated transgenic mice in which human CASP1 is overexpressed in their keratinocytes. The transgenic mice spontaneously developed recalcitrant dermatitis and skin ulcers, characterized by the presence of massive keratinocyte apoptosis. The skin of the mice contained the active form of human CASP1 and expressed mRNA for caspase-activated DNase, an effector endonuclease responsible for DNA fragmentation. Their skin and sera showed elevated levels of mature IL-18 and IL-1 beta, but not of IFN-gamma. The plasma from these animals induced IFN-gamma production by IL-18-responsive NK cells. Administration of heat-killed Propionibacterium acnes, a potent in vivo type 1 cell inducer, caused IFN-gamma-mediated lethal liver injury in the transgenic mice, which was completely inhibited by treatment with neutralizing anti-IL-18 Ab. These results indicated that in vivo overexpression of CASP1 caused spontaneous apoptotic tissue injury and rendered mice highly susceptible to exogenous type 1 cell-inducing condition in collaboration with endogenously accumulated proinflammatory cytokines.

Overexpression of Bcl-2 protects from ultraviolet B-induced apoptosis but promotes hair follicle regression and chemotherapy-induced alopecia.

Hair follicle (HF) growth and regression is an exquisitely regulated process of cell proliferation followed by massive cell death and is accompanied by cyclical expression of the apoptosis regulatory gene pair, Bcl-2 and Bax. To further investigate the role of Bcl-2 expression in the control of hair growth and keratinocyte apoptosis, we have used transgenic mice that overexpress human Bcl-2 in basal epidermis and in the outer root sheath under the control of the human keratin-14 promoter (K14/Bcl-2). When irradiated with ultraviolet B (UVB) light, K14/Bcl-2 mice developed about 5-10-fold fewer sunburn cells (ie, apoptotic keratinocytes) in the basal layer of the epidermis, compared to wild-type mice, whereas cultures of primary keratinocytes from transgenic mice were completely resistant to UVB-induced histone formation, at doses that readily induced histone release from wild-type cells. K14/Bcl-2 mice show no alteration of neonatal hair follicle morphogenesis or of the onset of the first wave of HF regression (catagen). However, compared to wild-type controls, K14/Bcl-2 mice subsequently displayed a significant acceleration of spontaneous catagen progression. During chemotherapy-induced alopecia, follicular dystrophy was promoted in K14/Bcl-2 mice. Thus, although K14-driven overexpression of Bcl-2 protected murine epidermal keratinocytes from UVB-induced apoptosis, it surprisingly promoted catagen- and chemotherapy-associated keratinocyte apoptosis.

A randomized, 12-year primary-prevention trial of beta carotene supplementation for nonmelanoma skin cancer in the physician's health study.

CONTEXT: Although basic research provides plausible mechanisms for benefits of beta carotene supplementation on nonmelanoma skin cancer (NMSC) primarily consisting of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), observational studies are inconsistent. Randomized trial data are limited to 1 trial of secondary prevention that showed no effect of beta carotene on the incidence of NMSC after 5 years. OBJECTIVE: To test whether supplementation with beta carotene reduces the risk for development of a first NMSC, including BCC and SCC. DESIGN: Randomized, double-blind, placebo-controlled trial with 12 years of beta carotene supplementation and follow-up. SETTING: Physicians' Health Study in the United States. PARTICIPANTS: Apparently healthy male physicians aged 40 to 84 years in 1982 (N = 22 071). INTERVENTION: Beta carotene, 50 mg, on alternate days. MAIN OUTCOME MEASURE: Relative risk (RR) and 95% confidence interval (CI) for a first NMSC, BCC, and SCC. RESULTS: After adjusting for age and randomized aspirin assignment, there was no effect of beta carotene on the incidence of a first NMSC (RR, 0.98; 95% CI, 0.92-1.05), BCC (RR, 0.99; 95% CI, 0.92-1.06), or SCC (RR, 0.97; 95% CI, 0.84-1.13). There was also no significant evidence of beneficial or harmful effects of beta carotene on NMSC by smoking status (current, past, or never). CONCLUSION: This large-scale, randomized, primary prevention trial among apparently healthy well-nourished men indicates that an average of 12 years of supplementation with beta carotene does not affect the development of a first NMSC, including BCC and SCC.

Interleukin-1 and cutaneous inflammation: a crucial link between innate and acquired immunity.

As our primary interface with the environment, the skin is constantly subjected to injury and invasion by pathogens. The fundamental force driving the evolution of the immune system has been the need to protect the host against overwhelming infection. The ability of T and B cells to recombine antigen receptor genes during development provides an efficient, flexible, and powerful immune system with nearly unlimited specificity for antigen. The capacity to expand subsets of antigen-specific lymphocytes that become activated by environmental antigens (memory response) is termed "acquired" immunity. Immunologic memory, although a fundamental aspect of mammalian biology, is a relatively recent evolutionary event that permits organisms to live for years to decades. "Innate" immunity, mediated by genes that remain in germ line conformation and encode for proteins that recognize conserved structural patterns on microorganisms, is a much more ancient system of host defense. Defensins and other antimicrobial peptides, complement and opsonins, and endocytic receptors are all considered components of the innate immune system. None of these, however, are signal-transducing receptors. Most recently, a large family of cell surface receptors that mediate signaling through the NF-kappaB transcription factor has been identified. This family of proteins shares striking homology with plant and Drosophila genes that mediate innate immunity. In mammals, this family includes the type I interleukin-1 receptor, the interleukin-18 receptor, and a growing family of Toll-like receptors, two of which were recently identified as signal-transducing receptors for bacterial endotoxin. In this review, we discuss how interleukin-1 links the innate and acquired immune systems to provide synergistic host defense activities in skin.

T cells, immunosurveillance, and cutaneous immunity.

This presentation deals with the key role of the skin in immune function, briefly reviewing what is known about the cell trafficking patterns and molecular events involved in the generation of a specific immune response in the skin. Such information provides a basis for understanding the pathogenesis of autoimmunity-related diseases, and may provide clues to eventual therapeutic approaches.

Functional cutaneous lymphocyte antigen can be induced in essentially all peripheral blood T lymphocytes.

The cutaneous lymphocyte-associated antigen (CLA) is a skin-homing receptor expressed on a minority of memory-type peripheral blood T (PBT) lymphocytes. Induction of high-level CLA expression in PBT has previously been difficult to accomplish in vitro. Here we report that constitutive CLA expression could be readily induced in virtually all PBT by various polyclonal activators. There was no requirement for accessory cells or addition of other mediators except for IL-2 for maintaining cell survival. Absence of serum in the culture medium was important for optimal induction of CLA. The number of T cells adhering to E-selectin as well as tethering and shear stress resistance under hydrodynamic flow increased in correlation with the level of cell surface CLA expressed. Clonal analysis of CLA induction revealed that in serum-containing medium, which permits the majority of T cells to expand, only a minority of clones did not express CLA. Such T cells could be induced to highly express CLA within 8 days by switching from serum-containing to serum-free medium. This cell-surface phenotype change was closely associated with acquisition of E-selectin ligand activity. Fucosyltransferase VII, which is believed to be important for the generation of the CLA epitope on the P-selectin glycoprotein ligand-1 (PSGL-1) backbone, was shown to be significantly increased in CLA-positive versus CLA-negative T cell populations by PCR analysis. Our findings are consistent with the idea that restriction of CLA expression after activation, rather than positive selection of predetermined T cell subpopulations exposed to restrictive stimulatory conditions in unique microenvironments, may be important in vivo.

Interaction of dendritic cells with skin endothelium: A new perspective on immunosurveillance.

The goal of this study was to determine the mechanisms by which dendritic cells (DCs) in blood could interact with endothelium, a prerequisite to extravasation into tissues. Our results indicate that DCs express both HECA-452-reactive and nonreactive isoforms of P-selectin glycoprotein ligand 1 (PSGL-1) and can tether and roll efficiently on E- and P-selectin under flow conditions in vitro. Freshly isolated blood DCs were further observed to roll continuously along noninflamed murine dermal endothelium in vivo. This interaction is strictly dependent on endothelial selectins, as shown by experiments with blocking antibodies and with E- and P-selectin-deficient mice. We hypothesize that DCs in blood are constitutively poised at the interface of blood and skin, ready to extravasate upon induction of inflammation, and we showed that cutaneous inflammation results in a rapid recruitment of DCs from the blood to tissues. We propose that this is an important and previously unappreciated element of immunosurveillance.