Skin and pulmonary models using coated bentonite particles for the study of the inflammation evoked by Paracoccidioides brasiliensis antigens in previously immunized mice

Bentonite particles coated with polysaccharide antigen or crude soluble antigen of Paracoccidioides brasiliensis were injected intradermally or intravenously in mice. In control animals that were not pre-immunized with P. brasiliensis antigens, coated and uncoated bentonite caused minimal and nonspecific inflammation around the cutaneous injection site or around the bentonite thrombi in small lung vessels after intravenous injection. However, in mice previously immunized with P. brasiliensis antigens, the coated bentonite particles boosted the humoral and cellular immune responses to P. brasiliensis and evoked intense inflammatory reactions. Twelve days after intradermal injection, the inflammatory reaction around the bentonite was rich in neutrophils, macrophages, lymphocytes and plasma cells associated with young granulation tissue. In intravenously injected mice, the pulmonary inflammation was maximal at day 2, and was characterized by a florid neutrophilic and macrophagic cellular infiltration around bentonite thrombi; in some foci, there was incipient organization to mature granuloma. However, in both models, there was no formation of epithelioid granulomata, demonstrating that in paracoccidioidomycosis cellular immunity alone, without the presence of intact micro-organisms, may not be enough for the development of this type of granuloma.

Avaliação da ação sistêmica de extrato liofilizado de Pfaffia glomerata na carcinogênese quimicamente induzida pelo DMBA em pele de camundongos hairless

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Liquid Crystal Nanodispersions Enable the Cutaneous Delivery of Photosensitizer for Topical PDT: Fluorescence Microscopy Study of Skin Penetration

Topical photodynamic therapy (PDT) has been applied to almost all types of nonmelanoma skin cancer and numerous superficial benign skin disorders. Strategies to improve the accumulation of photosensitizer in the skin have been studied in recent years. Although the hydrophilic phthalocyanine zinc compound, zinc phthalocyanine tetrasulfonate (ZnPcSO4) has shown high photodynamic efficiency and reduced phototoxic side effects in the treatment of brain tumors and eye conditions, its use in topical skin treatment is currently limited by its poor skin penetration. In this study, nanodispersions of monoolein (MO)-based liquid crystalline phases were studied for their ability to increase ZnPcSO4 uptake by the skin. Lamellar, hexagonal and cubic crystalline phases were prepared and identified by polarizing light microscopy, and the nanodispersions were analyzed by dynamic light scattering. In vitro skin penetration studies were performed using a Franz's cell apparatus, and the skin uptake was evaluated in vivo in hairless mice. Aqueous dispersions of cubic and hexagonal phases showed particles of nanometer size, approximately 224 +/- 10 nm and 188 +/- 10 nm, respectively. In vitro skin retention experiments revealed higher fluorescence from the ZnPcSO4 in deeper skin layers when this photosensitizer was loaded in the hexagonal nanodispersion system when compared to both the cubic phase nanoparticles and the bulk crystalline phases (lamellar, cubic and hexagonal). The hexagonal nanodispersion showed a similar penetration behavior in animal tests. These results are important findings, suggesting the development of MO liquid crystal nanodispersions as potential delivery systems to enhance the efficacy of topical PDT.

The choice of anesthesia influences oxidative energy metabolism and tissue survival in critically ischemic murine skin

In surgical animal studies anesthesia is used regularly. Several reports in the literature demonstrate respiratory and cardiovascular side effects of anesthesiologic agents. The aim of this study was to compare two frequently used anesthesia cocktails (ketamine/xylazine [KX] versus medetomidine/climazolam/fentanyl [MCF]) in skin flap mouse models. Systemic blood values, local metabolic parameters, and surgical outcome should be analyzed in critical ischemic skin flap models. Systemic hypoxia was found in the animals undergoing KX anesthesia compared with normoxia in the MCF group (sO(2): 89.2% +/- 2.4% versus 98.5% +/- 1.2%, P < 0.01). Analysis of tissue metabolism revealed impaired anaerobic oxygen metabolism and increased cellular damage in critical ischemic flap tissue under KX anesthesia (lactate/pyruvate ratio: KX 349.86 +/- 282.38 versus MCF 64.53 +/- 18.63; P < 0.01 and glycerol: KX 333.50 +/- 83.91 micromol/L versus MCF 195.83 +/- 29.49 micromol/L; P < 0.01). After 6 d, different rates of flap tissue necrosis could be detected (MCF 57% +/- 6% versus KX 68% +/- 6%, P < 0.01). In summary we want to point out that the type of anesthesia, the animal model and the goal of the study have to be well correlated. Comparing the effects of KX and MCF anesthesia in mice on surgical outcome was a novel aspect of our study.

Microvascular response to shock wave application in striated skin muscle

This study aims to quantify by intravital microscopy the microhemodynamic response after extracorporeal shock wave application (ESWA) to the physiologic microcirculation of the mouse dorsal skinfold chamber.

CD69 modulates sphingosine-1-phosphate-induced migration of skin dendritic cells

In this study, we have investigated the role of CD69, an early inducible leukocyte activation receptor, in murine dendritic cell (DC) differentiation, maturation, and migration. Skin DCs and DC subsets present in mouse lymphoid organs express CD69 in response to maturation stimuli. Using a contact sensitization model, we show that skin DCs migrated more efficiently to draining lymph nodes (LNs) in the absence of CD69. This was confirmed by subcutaneous transfer of CD69-/- DCs, which presented an increased migration to peripheral LNs. Two-photon microscopy analysis showed that once DCs reached the LNs, CD69 deficiency did not alter DC interstitial motility in the LNs. Chemotaxis to sphingosine-1-phosphate (S1P) was enhanced in CD69-/- DCs compared with wild-type DCs. Accordingly, we detected a higher expression of S1P receptor type-1 (S1P(1)) by CD69-/- DCs, whereas S1P(3) expression levels were similar in wild-type and CD69-/- DCs. Moreover, in vivo treatment with S1P analogs SEW2871 and FTY720 during skin sensitization reduced skin DC migration to peripheral LNs. These results suggest that CD69 regulates S1P-induced skin DC migration by modulating S1P(1) function. Together, our findings increase our knowledge on DC trafficking patterns in the skin, enabling the development of new directed therapies using DCs for antigen (Ag) delivery.

Botulinum toxin A and B raise blood flow and increase survival of critically ischemic skin flaps

BACKGROUND Botulinum toxin (BTX) A and B are commonly used for aesthetic indications and in neuromuscular disorders. New concepts seek to prove efficacy of BTX for critical tissue perfusion. Our aim was to evaluate BTX A and B in a mouse model of critical flap ischemia for preoperative and intraoperative application. METHODS BTX A and B were applied on the vascular pedicle of an axial pattern flap in mice preoperatively or intraoperatively. Blood flow, tissue oxygenation, tissue metabolism, flap necrosis rate, apoptosis assay, and RhoA and eNOS expression were endpoints. RESULTS Blood-flow measurements 1 d after the flap operation revealed a significant reduction to 53% in the control group, while flow was maintained or increased in all BTX groups (103%-129%). Over 5 d all BTX groups showed significant increase in blood flow to 166-187% (P < 0.01). Microdialysis revealed an increase of glucose and reduced lactate/pyruvate ratio and glycerol levels in the flap tissue of all BTX groups. This resulted in significantly improved tissue survival in all BTX groups compared with the control group (62% ± 10%; all P < 0.01): BTX A preconditioning (84% ± 5%), BTX A application intraoperatively (88% ± 4%), BTX B preconditioning (91% ± 4%), and intraoperative BTX B treatment (92% ± 5%). This was confirmed by TUNEL assay. Immunofluorescence demonstrated RhoA and eNOS expression in BTX groups. All BTX applications were similarly effective, despite pharmacologic dissimilarities and different timing. CONCLUSIONS In conclusion, we were able to show on a vascular, tissue, cell, and molecular level that BTX injection to the feeding arteries supports flap survival through ameliorated blood flow and oxygen delivery.

Detailed analysis of sequence changes occurring during vlsE antigenic variation in the mouse model of Borrelia burgdorferi infection.

Lyme disease Borrelia can infect humans and animals for months to years, despite the presence of an active host immune response. The vls antigenic variation system, which expresses the surface-exposed lipoprotein VlsE, plays a major role in B. burgdorferi immune evasion. Gene conversion between vls silent cassettes and the vlsE expression site occurs at high frequency during mammalian infection, resulting in sequence variation in the VlsE product. In this study, we examined vlsE sequence variation in B. burgdorferi B31 during mouse infection by analyzing 1,399 clones isolated from bladder, heart, joint, ear, and skin tissues of mice infected for 4 to 365 days. The median number of codon changes increased progressively in C3H/HeN mice from 4 to 28 days post infection, and no clones retained the parental vlsE sequence at 28 days. In contrast, the decrease in the number of clones with the parental vlsE sequence and the increase in the number of sequence changes occurred more gradually in severe combined immunodeficiency (SCID) mice. Clones containing a stop codon were isolated, indicating that continuous expression of full-length VlsE is not required for survival in vivo; also, these clones continued to undergo vlsE recombination. Analysis of clones with apparent single recombination events indicated that recombinations into vlsE are nonselective with regard to the silent cassette utilized, as well as the length and location of the recombination event. Sequence changes as small as one base pair were common. Fifteen percent of recovered vlsE variants contained "template-independent" sequence changes, which clustered in the variable regions of vlsE. We hypothesize that the increased frequency and complexity of vlsE sequence changes observed in clones recovered from immunocompetent mice (as compared with SCID mice) is due to rapid clearance of relatively invariant clones by variable region-specific anti-VlsE antibody responses.

Detailed analysis of sequence changes occurring during vlsE antigenic variation in the mouse model of Borrelia burgdorferi infection.

Lyme disease Borrelia can infect humans and animals for months to years, despite the presence of an active host immune response. The vls antigenic variation system, which expresses the surface-exposed lipoprotein VlsE, plays a major role in B. burgdorferi immune evasion. Gene conversion between vls silent cassettes and the vlsE expression site occurs at high frequency during mammalian infection, resulting in sequence variation in the VlsE product. In this study, we examined vlsE sequence variation in B. burgdorferi B31 during mouse infection by analyzing 1,399 clones isolated from bladder, heart, joint, ear, and skin tissues of mice infected for 4 to 365 days. The median number of codon changes increased progressively in C3H/HeN mice from 4 to 28 days post infection, and no clones retained the parental vlsE sequence at 28 days. In contrast, the decrease in the number of clones with the parental vlsE sequence and the increase in the number of sequence changes occurred more gradually in severe combined immunodeficiency (SCID) mice. Clones containing a stop codon were isolated, indicating that continuous expression of full-length VlsE is not required for survival in vivo; also, these clones continued to undergo vlsE recombination. Analysis of clones with apparent single recombination events indicated that recombinations into vlsE are nonselective with regard to the silent cassette utilized, as well as the length and location of the recombination event. Sequence changes as small as one base pair were common. Fifteen percent of recovered vlsE variants contained "template-independent" sequence changes, which clustered in the variable regions of vlsE. We hypothesize that the increased frequency and complexity of vlsE sequence changes observed in clones recovered from immunocompetent mice (as compared with SCID mice) is due to rapid clearance of relatively invariant clones by variable region-specific anti-VlsE antibody responses.

Transcriptional regulation of the mouse $\alpha$2(I) collagen gene

The mouse $\alpha$2(I) collagen gene is specifically expressed in a limited number of cell types in the body including fibroblasts and osteoblasts. We had previously shown that a promoter containing the sequences between $-$350 and +54 bp was expressed at low levels in a cell- and tissue-specific fashion in transgenic mice. Further studies suggested that the sequence between $-$315 and $-$284 bp could mediate cell- and tissue-specific expression of reporter genes in cell culture and in transgenic mice. We report here characterization of the proteins binding to this segment and propose a model for the cell-specific expression conferred by this sequence. In this study we also identified a strong enhancer for the mouse $\alpha$2(I) collagen gene located approximately 13.5 to 19.5 kb upstream of the transcriptional start site. This enhancer segment is characterized by the presence of three cell-specific hypersensitive sites and can drive high levels of cell-specific expression of a heterologous 220-bp mouse $\alpha$1(I) collagen promoter. In the course of this study, we identified a novel zinc finger transcription factor (designated murine epithelial zinc finger, mEZF) which was transiently expressed in the mesenchymal cells which give rise to the skeletal primordia and the metanephric kidney during the early stages of embryogenesis. In newborn mice, the mEZF gene is expressed at high levels in differentiated epithelial cells of the skin, oral mucosa, tongue, esophagus, stomach and colon. Chromosomal mapping suggested that the mEZF gene mapped to mouse Chromosome 4 and that the human homolog of mEZF would likely map to human Chromosome 9q31. This region of the human genome contains tumor suppressor genes for basal cell carcinomas of the skin as well as for squamous cell carcinomas of various organs. We cloned and characterized the human homolog of mEZF and mapped its chromosomal position as a first step in determining whether or not this gene plays a role in the development of these tumors. ^

Hairless Streaks in Cattle Implicate TSR2 in Early Hair Follicle Formation

Four related cows showed hairless streaks on various parts of the body with no correlation to the pigmentation pattern. The stripes occurred in a consistent pattern resembling the lines of Blaschko. The non-syndromic hairlessness phenotype observed occurred across three generations of a single family and was compatible with an X-linked mode of inheritance. Linkage analysis and subsequent whole genome sequencing of one affected female identified two perfectly associated non-synonymous sequence variants in the critical interval on bovine chromosome X. Both variants occurred in complete linkage disequilibrium and were absent in more than 3900 controls. An ERCC6L missense mutation was predicted to cause an amino acid substitution of a non-conserved residue. Analysis in mice showed no specific Ercc6l expression pattern related to hair follicle development and therefore ERCC6L was not considered as causative gene. A point mutation at the 5'-splice junction of exon 5 of the TSR2, 20S rRNA accumulation, homolog (S. cerevisiae), gene led to the production of two mutant transcripts, both of which contain a frameshift and generate a premature stop codon predicted to truncate approximately 25% of the protein. Interestingly, in addition to the presence of both physiological TSR2 transcripts, the two mutant transcripts were predominantly detected in the hairless skin of the affected cows. Immunohistochemistry, using an antibody against the N-terminal part of the bovine protein demonstrated the specific expression of the TSR2 protein in the skin and the hair of the affected and the control cows as well as in bovine fetal skin and hair. The RNA hybridization in situ showed that Tsr2 was expressed in pre- and post-natal phases of hair follicle development in mice. Mammalian TSR2 proteins are highly conserved and are known to be broadly expressed, but their precise in vivo functions are poorly understood. Thus, by dissecting a naturally occurring mutation in a domestic animal species, we identified TSR2 as a regulator of hair follicle development.

Molecular mechanism of PUVA-induced apoptosis in mouse epidermal cells

A combination of psoralens and ultraviolet-A radiation referred to as PUVA, is widely used in the treatment of psoriasis. PUVA therapy is highly effective in killing hyperproliferative cells, but its mechanism of action has not been fully elucidated. Psoralen binds to DNA, and upon photoactivation by UVA, it forms monofunctional adducts and interstrand cross-links. PUVA treatment has been shown to be mutagenic and to produce tumors in animals. In addition, epidemiological studies have reported a 10 to 15 percent increased risk of developing squamous cell carcinoma in individuals treated chronically with PUVA. However, it remains a treatment for skin disorders such as psoriasis because its benefits outweigh its risks. The widespread use of PUVA therapy and its associated cancer risk requires us to understand the molecular mechanisms by which PUVA induces cell death. Immortalized JB6 mouse epidermal cells, p53−/− mice, and Fas Ligand−/− (gld) mice were used to investigate the molecular mechanism by which PUVA kills cells. Treatment of JB6 cells with 10 μg/ml 8-methoxypsoralen followed by irradiation with 20 kJ/m2 UVA resulted in cell death. The cells exhibited morphological and biochemical characteristics of apoptosis such as chromatin condensation, DNA ladder formation, and TUNEL-positivity. PUVA treatment stabilized and phosphorylated p53 leading to its activation, as measured by nuclear localization and induction of p21Waf/Cip1, a transcriptional target of p53. Subsequent in vivo studies revealed that there was statistically significantly less apoptosis in p53 −/− mice than in p53+/+ mice at 72 hours after PUVA. In addition, immunohistochemical analysis revealed more Fas and FasL expression in p53+/+ mice than in p53−/− mice, suggesting that p53 is required to transcriptionally activate Fas, which in turn causes the cells to undergo apoptosis. Studies with gld mice confirmed a role for Fas/FasL interactions in PUVA-induced apoptosis. There was statistically significantly less apoptosis in gld mice compared with wild-type mice 24, 48, and 72 hours after PUVA. These results demonstrate that PUVA-induced apoptosis in mouse epidermal cells requires p53 and Fas/FasL interactions. These findings may be important for designing effective treatments for diseases such as psoriasis without increasing the patient's risk for skin cancer. ^

Chemopreventive function of retinoid X receptors in human squamous cell carcinoma of the skin

Retinoid therapy has been successful for the treatment of skin squamous cell carcinoma (SCC). A suppression of the predominant retinoid X receptor expressed in skin, retinoid X receptor α (RXRα), has been reported in skin SCC. These observations have led to the hypothesis that retinoid receptor loss contributes to the tumorigenic phenotype of epithelial cancers. To test this hypothesis, the RXRα gene was mapped in order to generate a targeting construct. Additionally the transcriptional regulation of the human RXRα a gene in keratinocytes was characterized after identifying the transcription initiation sites, the promoter, and enhancer regions of this gene. The structure is highly conserved between human and mouse. A nontumorigenic human skin-derived cell line called near diploid immortalized keratinocytes (NIKS) has the advantage of growing as organotypic raft cultures, under physiological conditions closely resembling in-vivo squamous stratification. We have exploited the raft culture technique to develop an in-vitro model for skin SCC progression that includes the NIKS cells, HaCaT cells, a premalignant cell line, and SRB 12-p9 cells, a tumorigenic SCC skin cell line. The differentiation, proliferation and nuclear receptor ligand response characteristics of this system were studied and significant and novel results were obtained. RXRs are obligate heterodimerization partners with many of the nuclear hormone receptors, including retinoic acid receptors (RARs), vitamin D3 receptors (VDR), thyroid hormone receptors (T3 R) and peroxisome proliferator activate receptors (PPARs), which are all known to be active in skin. Treatment of the three cell lines in raft culture with the RXR specific ligand BMS649, BMS961 (RARγ-specific), vitamin D3 (VDR ligand), thryoid hormone (T3R ligand) and clofibrate (PPARa ligand), and the combination of BMS649 with each of the 4 receptor partner ligands, resulted in distinct effects on differentiation, proliferation and apoptosis. The effects of activation of RXRs in each of the four-receptor pathways; in the context of skin SCC progression, with an emphasis on the VDR/RXR pathway, are discussed. These studies will lead to a better understanding of RXRα action in human skin and will help determine its role in SCC tumorigenesis, as well as its potential as a target for the prevention, treatment, and control of skin cancer. ^

ROLE OF STAT3 IN KERATINOCYTE STEM CELLS DURING SKIN TUMORIGENESIS

STATs play crucial roles in a wide variety of biological functions, including development, proliferation, differentiation, migration and in cancer development. In the present study, we examined the impact of Stat3 deletion or activation on behavior of keratinocytes, including keratinocyte stem cells (KSCs). Deletion of Stat3 specifically in the bulge region of the hair follicle using K15.CrePR1 X Stat3fl/fl mice led to decreased tumor development by altering survival of bulge region KSCs. To further understand the role of KSCs in skin tumorigenesis, K5.Stat3C transgenic (Tg) mice which express a constitutively active/dimerized form of Stat3 called Stat3C via the bovine keratin 5 (K5) promoter were studied. The number of CD34 and α6 integrin positive cells was significantly reduced in Tg mice as compared to non-transgenic (NTg) littermates. There was a concomitant increase in the progenitor populations (Lgr-6, Lrig-1 and Sca-1) in the Tg mice vs. the stem cell population (CD34 and Keratin15). To investigate the mechanism underlying the increase in the progenitor population at the expense of bulge region KSCs we examined if Stat3C expression was involved in inducing migration of the bulge region KSCs. There was altered β-catenin and α6-integrin expression in the hair follicles of Tg mice, which may have contributed to reduced adhesive interactions between the epithelial cells and the basement membrane facilitating migration out of the niche. To further study the effect of Stat3 on differentiation of keratinocytes we analyzed the epidermal keratinocytes in K5.Cre X Stat3fl/fl mice. There was an increase in the expression of epidermal differentiation markers in the Stat3 knockout mice. These data suggest that deletion of Stat3 in the epidermis and hair follicle induced differentiation in these cells. Preliminary studies done with the BK5.Stat3C mouse model suggests that multiple hair follicle stem/progenitor populations may be involved in skin tumor development and progression in this model of skin tumorigenesis. Overall, these data suggest that Stat3 plays an important role in differentiation as well as migration of keratinocytes and that these effects may play a role during epithelial carcinogenesis.

A Novel Mechanism of Skin Tumor Promotion Involving Interferon-gamma (IFNγ)/Signal Transducer and Activator of Transcription-1 (Stat1) Signaling in Epidermis

The JAK-STAT pathway is a major signaling pathway involved in many biological processes including proliferation, apoptosis, and differentiation. Aberrant expression of STATs has been reported in multiple human cancers and murine mouse models of tumorigenesis. Previous studies from our lab and others have established a critical role for Stat3 in epithelial tumorigenesis, but the role of Stat1 is largely unknown. The current study was designed to explore the role of Stat1 during multistage skin carcinogenesis. Topical treatment with both TPA and the anthrone derivative chrysarobin (CHRY) led to rapid phosphorylation of Stat1 on both tyrosine (Tyr701) and serine (Ser727) residues in epidermis. CHRY treatment also led to upregulation of unphosphorylated Stat1 (uStat1) at later time points. In addition, CHRY treatment also led to upregulation of IRF-1 mRNA and protein which was dependent on Stat1. Further analyses demonstrated that topical treatment with CHRY but not TPA upregulated interferon-gamma (IFNg) mRNA in the epidermis and that the induction of both IRF-1 and uStat1 was dependent on IFNg signaling. Stat1 deficient (Stat1-/-) mice were highly resistant to skin tumor promotion by CHRY. In contrast, the tumor response (in terms of both papillomas and squamous cell carcinomas) was similar in Stat1-/- mice and wild-type littermates with TPA as the promoter. Histological evaluation of the proliferative response confirmed the data obtained from the tumor study for both TPA and CHRY. In addition, maximal induction of both cyclooxygenase-2 and inducible nitric oxide synthase in epidermis following treatment with CHRY was also dependent on the presence of functional Stat1. Following CHRY treatment, Stat1-/- mice exhibited reduced macrophage infiltration and reduced production of many immune cell derived chemokines/cytokines. These studies define a novel mechanism associated with skin tumor promotion by the anthrone class of tumor promoters involving upregulation of IFNg signaling in the epidermis and downstream signaling through activated (phosphorylated) Stat1 and subsequent upregulation of IRF-1 and uStat1.