Innate Sex Bias of Staphylococcus aureus Skin Infection Is Driven by α-Hemolysin.

Numerous studies have reported sex bias in infectious diseases, with bias direction dependent on pathogen and site of infection. Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs), yet sex bias in susceptibility to S. aureus SSTI has not been described. A search of electronic health records revealed an odds ratio of 2.4 for S. aureus SSTI in males versus females. To investigate the physiological basis of this bias, we compared outcomes between male and female mice in a model of S. aureus dermonecrosis. Consistent with the epidemiological data, female mice were better protected against SSTI, with reduced dermonecrosis followed later by increased bacterial clearance. Protection in females was disrupted by ovariectomy and restored by short-term estrogen administration. Importantly, this sex bias was mediated by a sex-specific response to the S. aureus-secreted virulence factor α-hemolysin (Hla). Infection with wild-type S. aureus suppressed inflammatory cytokine production in the skin of female, but not male, mice when compared with infection with an isogenic hla deletion mutant. This differential response was conserved following injection with Hla alone, demonstrating a direct response to Hla independent of bacterial burden. Additionally, neutrophils, essential for clearing S. aureus, demonstrated sex-specific S. aureus bactericidal capacity ex vivo. This work suggests that sex-specific skin innate responsiveness to Hla and neutrophil bactericidal capacity play important roles in limiting S. aureus SSTI in females. Understanding the molecular mechanisms controlling this sex bias may reveal novel targets to promote host innate defense against S. aureus skin infection.

Analysis of DNA polymerase ν function in meiotic recombination, immunoglobulin class-switching, and DNA damage tolerance.

DNA polymerase ν (pol ν), encoded by the POLN gene, is an A-family DNA polymerase in vertebrates and some other animal lineages. Here we report an in-depth analysis of pol ν-defective mice and human cells. POLN is very weakly expressed in most tissues, with the highest relative expression in testis. We constructed multiple mouse models for Poln disruption and detected no anatomic abnormalities, alterations in lifespan, or changed causes of mortality. Mice with inactive Poln are fertile and have normal testis morphology. However, pol ν-disrupted mice have a modestly reduced crossover frequency at a meiotic recombination hot spot harboring insertion/deletion polymorphisms. These polymorphisms are suggested to generate a looped-out primer and a hairpin structure during recombination, substrates on which pol ν can operate. Pol ν-defective mice had no alteration in DNA end-joining during immunoglobulin class-switching, in contrast to animals defective in the related DNA polymerase θ (pol θ). We examined the response to DNA crosslinking agents, as purified pol ν has some ability to bypass major groove peptide adducts and residues of DNA crosslink repair. Inactivation of Poln in mouse embryonic fibroblasts did not alter cellular sensitivity to mitomycin C, cisplatin, or aldehydes. Depletion of POLN from human cells with shRNA or siRNA did not change cellular sensitivity to mitomycin C or alter the frequency of mitomycin C-induced radial chromosomes. Our results suggest a function of pol ν in meiotic homologous recombination in processing specific substrates. The restricted and more recent evolutionary appearance of pol ν (in comparison to pol θ) supports such a specialized role.

The R-Enantiomer of Ketorolac Delays Mammary Tumor Development in Mouse Mammary Tumor Virus-Polyoma Middle T Antigen (MMTV-PyMT) Mice.

Epidemiologic studies report improved breast cancer survival in women who receive ketorolac (Toradol) for postoperative pain relief compared with other analgesic agents. Ketorolac is a racemic drug. The S-enantiomer inhibits cyclooxygenases; R-ketorolac is a selective inhibitor of the small GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42), which are signaling molecules up-regulated during breast cancer progression and metastasis. The goal of this study was to determine whether R-ketorolac altered breast cancer development in the mouse mammary tumor virus-polyoma middle T-antigen model. Mice were administered ketorolac orally at 1 mg/kg twice daily to approximate the typical human dose. Mammary glands were analyzed for tumor number and immunohistochemical markers of proliferation and differentiation. R-ketorolac treatment significantly reduced mammary epithelial proliferation, based on Ki67 staining, and suppressed tumor development. Proliferative mammary epithelium from R-ketorolac-treated mice displayed greater differentiation, based on significantly higher total E-cadherin and decreased keratin 5 staining than epithelium of placebo-treated mice. No differences were detected in estrogen receptor, progesterone receptor, ß-catenin, or vimentin expression between placebo and R-ketorolac treatment groups. These findings indicate that R-ketorolac treatment slows tumor progression in an aggressive model of breast cancer. R-ketorolac may thus represent a novel therapeutic approach for breast cancer prevention or treatment based on its pharmacologic activity as a Rac1 and Cdc42 inhibitor.

Oral Tolerance to Environmental Mycobacteria Interferes with Intradermal, but Not Pulmonary, Immunization against Tuberculosis.

Bacille Calmette-Guérin (BCG) is currently the only approved vaccine against tuberculosis (TB) and is administered in over 150 countries worldwide. Despite its widespread use, the vaccine has a variable protective efficacy of 0-80%, with the lowest efficacy rates in tropical regions where TB is most prevalent. This variability is partially due to ubiquitous environmental mycobacteria (EM) found in soil and water sources, with high EM prevalence coinciding with areas of poor vaccine efficacy. In an effort to elucidate the mechanisms underlying EM interference with BCG vaccine efficacy, we exposed mice chronically to Mycobacterium avium (M. avium), a specific EM, by two different routes, the oral and intradermal route, to mimic human exposure. After intradermal BCG immunization in mice exposed to oral M. avium, we saw a significant decrease in the pro-inflammatory cytokine IFN-γ, and an increase in T regulatory cells and the immunosuppressive cytokine IL-10 compared to naïve BCG-vaccinated animals. To circumvent the immunosuppressive effect of oral M. avium exposure, we vaccinated mice by the pulmonary route with BCG. Inhaled BCG immunization rescued IFN-γ levels and increased CD4 and CD8 T cell recruitment into airways in M. avium-presensitized mice. In contrast, intradermal BCG vaccination was ineffective at T cell recruitment into the airway. Pulmonary BCG vaccination proved protective against Mtb infection regardless of previous oral M. avium exposure, compared to intradermal BCG immunization. In conclusion, our data indicate that vaccination against TB by the pulmonary route increases BCG vaccine efficacy by avoiding the immunosuppressive interference generated by chronic oral exposure to EM. This has implications in TB-burdened countries where drug resistance is on the rise and health care options are limited due to economic considerations. A successful vaccine against TB is necessary in these areas as it is both effective and economical.

Dual role for mammalian DNA polymerase ζ in maintaining genome stability and proliferative responses.

DNA polymerase ζ (polζ) is critical for bypass of DNA damage and the associated mutagenesis, but also has unique functions in mammals. It is required for embryonic development and for viability of hematopoietic cells, but, paradoxically, skin epithelia appear to survive polζ deletion. We wished to determine whether polζ functions in a tissue-specific manner and how polζ status influences skin tumorigenesis. Mice were produced in which Rev3L (the catalytic subunit of polζ) was deleted in tissues expressing keratin 5. Efficient epidermal deletion of Rev3L was tolerated but led to skin and hair abnormalities, accompanied by evidence of DNA breaks. Unchallenged mice developed tumors in keratin 5-expressing tissues with age, consistent with the chromosomal instability accompanying a polζ defect. Unexpectedly, mice with the Rev3L deletion were much more sensitive to UVB radiation than mice defective in other DNA repair genes. Following irradiation, polζ-defective mice failed to mount skin-regenerative responses and responded to stress by mobilizing melanocytes to the epidermis. However, they did not develop skin tumors after chronic UVB irradiation. To determine the proliferative potential of polζ-deficient skin epithelia, keratinocytes were isolated and examined. These keratinocytes harbored chromosomal gaps and breaks and exhibited a striking proliferation defect. These results can be unified by a model in which slowly dividing cells accumulate replication-associated DNA breaks but otherwise survive Rev3L deletion, but functional polζ is essential for responses requiring rapid proliferation, both in cell culture and in vivo. The results reveal a biological role for mammalian polζ in tolerating DNA damage and enabling proliferative responses in vivo.

Modeling gene-environment interactions in oral cavity and esophageal cancers demonstrates a role for the p53 R72P polymorphism in modulating susceptibility.

A large number of epidemiological studies have linked a common single-nucleotide polymorphism (SNP) in the human p53 gene to risk for developing a variety of cancers. This SNP encodes either an arginine or proline at position 72 (R72P) of the p53 protein, which can alter the apoptotic activity of p53 via transcriptional and non-transcriptional mechanisms. This SNP has also been reported to modulate the development of human papilloma virus (HPV)-driven cancers through differential targeting of the p53 variant proteins by the E6 viral oncoprotein. Mouse models for the p53 R72P polymorphism have recently been developed but a role for this SNP in modifying cancer risk in response to viral and chemical carcinogens has yet to be established experimentally. Here, we demonstrate that the p53 R72P polymorphism modulates the hyperprolferative, apoptotic and inflammatory phenotypes caused by expression of the HPV16 E6 and E7 oncoproteins. Moreover, the R72P SNP also modifies the carcinogenic response to the chemical carcinogen 4NQO, in the presence and absence of the HPV16 transgene. Our findings confirm several human epidemiological studies associating the codon 72 proline variant with increased risk for certain cancers but also suggest that there are tissue-specific differences in how the R72P polymorphism influences the response to environmental carcinogens.

Preventative topical diclofenac treatment differentially decreases tumor burden in male and female Skh-1 mice in a model of UVB-induced cutaneous squamous cell carcinoma.

Ultraviolet B (UVB) light is the major environmental carcinogen contributing to non-melanoma skin cancer (NMSC) development. There are over 3.5 million NMSC diagnoses in two million patients annually, with men having a 3-fold greater incidence of squamous cell carcinoma (SCC) compared with women. Chronic inflammation has been linked to tumorigenesis, with a key role for the cyclooxygenase-2 (COX-2) enzyme. Diclofenac, a COX-2 inhibitor and non-steroidal anti-inflammatory drug, currently is prescribed to patients as a short-term therapeutic agent to induce SCC precursor lesion regression. However, its efficacy as a preventative agent in patients without evidence of precursor lesions but with significant UVB-induced cutaneous damage has not been explored. We previously demonstrated in a murine model of UVB-induced skin carcinogenesis that when exposed to equivalent UVB doses, male mice had lower levels of inflammation but developed increased tumor multiplicity, burden and grade compared with female mice. Because of the discrepancy in the degree of inflammation between male and female skin, we sought to determine if topical treatment of previously damaged skin with an anti-inflammatory COX-2 inhibitor would decrease tumor burden and if it would be equally effective in the sexes. Our results demonstrated that despite observed sex differences in the inflammatory response, prolonged topical diclofenac treatment of chronically UVB-damaged skin effectively reduced tumor multiplicity in both sexes. Unexpectedly, tumor burden was significantly decreased only in male mice. Our data suggest a new therapeutic use for currently available topical diclofenac as a preventative intervention for patients predisposed to cutaneous SCC development before lesions appear.

Slug expression during melanoma progression.

Slug (Snai2), a member of the Snail family of zinc finger transcription factors, plays a role in the epithelial-to-mesenchymal transformation (EMT) that occurs during melanocyte emigration from the neural crest. A role for Slug in the EMT-like loss of cell adhesion and increased cell motility exhibited during melanoma progression has also been proposed. Our immunohistochemical studies of melanoma arrays, however, revealed that Slug expression was actually higher in nevi than in primary or metastatic melanomas. Moreover, Slug expression in melanomas was not associated with decreased expression of E-cadherin, the canonical Slug target in EMT. Comparisons of endogenous Slug and E-cadherin expression in cultured normal human melanocytes and melanoma cell lines supported our immunohistochemical findings. Expression of exogenous Slug in melanocytes and melanoma cells in vitro, however, suppressed E-cadherin expression, enhanced N-cadherin expression, and stimulated cell migration and invasion. Interestingly, both in tumors and cultured cell lines, there was a clear relationship between expression of Slug and MITF, a transcription factor known to regulate Slug expression during development. Taken together, our findings suggest that Slug expression during melanomagenesis is highest early in the process and that persistent Slug expression is not required for melanoma progression. The precise role of Slug in melanomagenesis remains to be elucidated and may be related to its interactions with other drivers of EMT, such as Snail.

The antiviral effects of a MEK1/2 inhibitor promote tumor regression in a preclinical model of human papillomavirus infection-induced tumorigenesis.

Human papillomaviruses (HPVs) are a significant public health concern due to their widespread transmission, morbidity, and oncogenic potential. Despite efficacious vaccines, millions of unvaccinated individuals and those with existing infections will develop HPV-related diseases for the next two decades and beyond. The continuing burden of HPV-related diseases is exacerbated by the lack of effective therapies or cures for infections, highlighting the need to identify and develop antivirals. The experimental murine papillomavirus type 1 (MmuPV1) model provides opportunities to study papillomavirus pathogenesis in cutaneous epithelium, the oral cavity, and the anogenital tract. However, to date the MmuPV1 infection model has not been used to demonstrate the effectiveness of potential antivirals. We previously reported that inhibitors of cellular MEK/ERK signaling suppress oncogenic HPV early gene expression in three-dimensional tissue cultures. Herein, we adapted the MmuPV1 infection model to determine whether MEK inhibitors have anti-papillomavirus properties in vivo. We demonstrate that oral delivery of a MEK1/2 inhibitor promotes papilloma regression in immunodeficient mice that otherwise would have developed persistent infections. Quantitative histological analyses reveal that inhibition of MEK/ERK signaling reduces E6/E7 mRNA, MmuPV1 DNA, and L1 protein expression within MmuPV1-induced lesions. These data suggest that MEK1/2 signaling is essential for both early and late MmuPV1 replication events supporting our previous findings with oncogenic HPVs. We also provide evidence that MEK inhibitors protect mice from developing secondary tumors. Thus, our data suggest that MEK inhibitors have potent antiviral and anti-tumor properties in a preclinical mouse model and merit further investigation as papillomavirus antiviral therapies.

Inhibition of Cellular MEK/ERK Signaling Suppresses Murine Papillomavirus Type 1 Replicative Activities and Promotes Tumor Regression.

Human papillomaviruses (HPVs) are a significant public health concern due to their widespread transmission, morbidity, and oncogenic potential. Despite efficacious vaccines, millions of unvaccinated individuals and those with existing infections will develop HPV-related diseases for the next two decades. The continuing burden of HPV-related diseases is exacerbated by the lack of effective therapies or cures for most infections, highlighting the need to identify and develop antivirals. The experimental murine papillomavirus type 1 (MmuPV1) model provides opportunities to study papillomavirus pathogenesis in cutaneous epithelium, the oral cavity, and the anogenital tract. However, to date the MmuPV1 infection model has not been used to demonstrate the effectiveness of potential antivirals. We previously reported that inhibitors of cellular MEK/ERK signaling suppress oncogenic HPV early gene expression in vitro . Herein, we adapted the MmuPV1 infection model to determine whether MEK inhibitors have anti-papillomavirus properties in vivo . We demonstrate that oral delivery of a MEK1/2 inhibitor promotes papilloma regression in immunodeficient mice that otherwise would have developed persistent infections. Quantitative histological analyses revealed that inhibition of MEK/ERK signaling reduces E6/E7 mRNAs, MmuPV1 DNA, and L1 protein expression within MmuPV1-induced lesions. These data suggest that MEK1/2 signaling is essential for both early and late MmuPV1 replication events supporting our previous findings with oncogenic HPVs. We also provide evidence that MEK inhibitors protect mice from developing secondary tumors. Thus, our data suggest that MEK inhibitors have potent anti-viral and anti-tumor properties in a preclinical mouse model and merit further investigation as papillomavirus antiviral therapies. Significance Statement: Persistent human papillomavirus (HPV) infections cause significant morbidity and oncogenic HPV infections can progress to anogenital and oropharyngeal cancers. Despite the availability of effective prophylactic HPV vaccines, millions of unvaccinated individuals, and those currently infected will develop HPV-related diseases over the next two decades and beyond. Thus, it remains critical to identify effective antivirals against papillomaviruses. Using a mouse papillomavirus model of HPV infection, this study reveals that cellular MEK1/2 signaling supports viral tumorigenesis. The MEK1/2 inhibitor, trametinib, demonstrates potent antiviral activities and promotes tumor regression. This work provides insight into the conserved regulation of papillomavirus gene expression by MEK1/2 signaling and reveals this cellular pathway as a promising therapeutic target for the treatment of papillomavirus diseases.

Protective role of cathepsin L in mouse skin carcinogenesis.

Lysosomal cysteine protease cathepsin L (CTSL) is believed to play a role in tumor progression and is considered a marker for clinically invasive tumors. Studies from our laboratory using the classical mouse skin carcinogenesis model, with 7,12-dimethyl-benz[a]anthracene (DMBA) for initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) for promotion, showed that expression of CTSL is increased in papillomas and squamous cell carcinomas (SCC). We also carried out carcinogenesis studies using Ctsl-deficient nackt (nkt) mutant mice on three different inbred backgrounds. Unexpectedly, the multiplicity of papillomas was significantly higher in Ctsl-deficient than in wild-type mice on two unrelated backgrounds. Topical applications of TPA or DMBA alone to the skin of nkt/nkt mice did not induce papillomas, and there was no increase in spontaneous tumors in nkt/nkt mice on any of the three inbred backgrounds. Reduced epidermal cell proliferation in Ctsl-deficient nkt/nkt mice after TPA treatment suggested that they are not more sensitive than wild-type mice to TPA promotion. We also showed that deficiency of CTSL delays terminal differentiation of keratinocytes, and we propose that decreased elimination of initiated cells is at least partially responsible for the increased papilloma formation in the nackt model.

SKHIN/Sprd, a new genetically defined inbred hairless mouse strain for UV-induced skin carcinogenesis studies.

Strains of mice vary in their susceptibility to ultra-violet (UV) radiation-induced skin tumors. Some strains of hairless mice (homozygous for the spontaneous Hr(hr) mutation) are particularly susceptible to these tumors. The skin tumors that develop in hairless mice resemble, both at the morphologic and molecular levels, UV-induced squamous cell carcinomas (SCC) and their precursors in human. The most commonly employed hairless mice belong to the SKH1 stock. However, these mice are outbred and their genetic background is not characterized, which makes them a poor model for genetic studies. We have developed a new inbred strain from outbred SKH1 mice that we named SKHIN/Sprd (now at generation F31). In order to characterize the genetic background of this new strain, we genotyped a cohort of mice at F30 with 92 microsatellites and 140 single nucleotide polymorphisms (SNP) evenly distributed throughout the mouse genome. We also exposed SKHIN/Sprd mice to chronic UV irradiation and showed that they are as susceptible to UV-induced skin carcinogenesis as outbred SKH1 mice. In addition, we proved that, albeit with low efficiency, inbred SKHIN/Sprd mice are suitable for transgenic production by classical pronuclear microinjection. This new inbred strain will be useful for the development of transgenic and congenic strains on a hairless inbred background as well as the establishment of syngeneic tumor cell lines. These new tools can potentially help elucidate a number of features of the cutaneous response to UV irradiation in humans, including the effect of genetic background and modifier genes.

Cancer vaccines from cryogenically silicified tumour cells functionalized with pathogen-associated molecular patterns.

The production of personalized cancer vaccines made from autologous tumour cells could benefit from mechanisms that enhance immunogenicity. Here we show that cancer vaccines can be made via the cryogenic silicification of tumour cells, which preserves tumour antigens within nanoscopic layers of silica, followed by the decoration of the silicified surface with pathogen-associated molecular patterns. These pathogen-mimicking cells activate dendritic cells and enhance the internalization, processing and presentation of tumour antigens to T cells. In syngeneic mice with high-grade ovarian cancer, a cell-line-based silicified cancer vaccine supported the polarization of CD4+ T cells towards the T-helper-1 phenotype in the tumour microenvironment, and induced tumour-antigen-specific T-cell immunity, resulting in complete tumour eradication and in long-term animal survival. In the setting of established disease and a suppressive tumour microenvironment, the vaccine synergized with cisplatin. Silicified and surface-modified cells from tumour samples are amenable to dehydration and room-temperature storage without loss of efficacy and may be conducive to making individualized cancer vaccines across tumour types.

Two hypomorphic alleles of mouse Ass1 as a new animal model of citrullinemia type I and other hyperammonemic syndromes.

Citrullinemia type I (CTLN1, OMIM# 215700) is an inherited urea cycle disorder that is caused by an argininosuccinate synthetase (ASS) enzyme deficiency. In this report, we describe two spontaneous hypomorphic alleles of the mouse Ass1 gene that serve as an animal model of CTLN1. These two independent mouse mutant alleles, also described in patients affected with CTLN1, interact to produce a range of phenotypes. While some mutant mice died within the first week after birth, others survived but showed severe retardation during postnatal development as well as alopecia, lethargy, and ataxia. Notable pathological findings were similar to findings in human CTLN1 patients and included citrullinemia and hyperammonemia along with delayed cerebellar development, epidermal hyperkeratosis, and follicular dystrophy. Standard treatments for CTLN1 were effective in rescuing the phenotype of these mutant mice. Based on our studies, we propose that defective cerebellar granule cell migration secondary to disorganization of Bergmann glial cell fibers cause cerebellar developmental delay in the hyperammonemic and citrullinemic brain, pointing to a possible role for nitric oxide in these processes. These mouse mutations constitute a suitable model for both mechanistic and preclinical studies of CTLN1 and other hyperammonemic encephalopathies and, at the same time, underscore the importance of complementing knockout mutations with hypomorphic mutations for the generation of animal models of human genetic diseases.

The skinny on Slug.

The zinc finger transcription factor Slug (Snai2) serves a wide variety of functions in the epidermis, with roles in skin development, hair growth, wound healing, skin cancer, and sunburn. Slug is expressed in basal keratinocytes and hair follicles where it is important in maintaining epidermal homeostasis. Slug also helps coordinate the skin response to exogenous stimuli. Slug is rapidly induced by a variety of growth factors and injurious agents and Slug controls, directly or indirectly, a variety of keratinocyte responses, including changes in differentiation, adhesion, motility, and production of inflammatory mediators. Slug thus modulates the interactions of the keratinocyte with its environment and with surrounding cells. The function of Slug in the epidermis appears to be distinct from that of the closely related Snail transcription factor.

Microarray analysis of cutaneous squamous cell carcinomas reveals enhanced expression of epidermal differentiation complex genes.

Gene expression profiles were determined for 12 cutaneous squamous cell carcinomas (SCC) removed from sun-exposed sites on nonimmunosuppressed patients. Gene expression in each SCC was compared to that in sun-exposed skin from the same patient using the Affymetrix HGU133 2.0 PlusGeneChip. We identified 440 genes with increased expression in SCC and 738 with decreased expression; overall we identified a large number of small changes in gene expression rather than a few marked changes that distinguished SCC from sun-exposed skin. Analyzing this robust data set according to biofunctional pathways using DAVID, transcriptional control elements using oPOSSUM, and chromosomal location using GSEA suggested genetic and epigenetic mechanisms of gene expression regulation in SCC. Some altered patterns of gene expression in SCC were consistent with regulation of spatially separated genes by a number of developmentally important transcription factors (forkhead, HMG, and homeo factors) that negatively regulated gene expression and to a few factors that positively regulated expression (Creb-1, NFkappaB, RelA, and Sp-1). We also found that coordinately enhanced expression of epidermal differentiation complex genes on chromosome 1q21 was a hallmark of SCC. A novel finding in our study was enhanced expression of keratin 13 in SCC, a result validated by immunohistochemical staining of an SCC tumor tissue array.

Macrophage migration inhibitory factor (MIF) plays a critical role in pathogenesis of ultraviolet-B (UVB) -induced nonmelanoma skin cancer (NMSC).

Mounting evidence suggests that macrophage migration inhibitory factor (MIF) may serve as an important link between chronic inflammation and cancer development. The proinflammatory and proangiogenic activities of MIF position it as a potentially important player in the development and progression of nonmelanoma skin cancer (NMSC). To assess the role of MIF in the development and progression of NMSC, we exposed MIF(-/-) BALB/c mice to acute and chronic ultraviolet B (UVB) irradiation. Our studies demonstrate that MIF(-/-) BALB/c mice have a significantly diminished acute inflammatory response to UVB exposure compared to wild-type mice, as measured by myeloperoxidase activity, dermal neutrophil infiltration, and edematous response. Relative to wild-type mice, MIF(-/-) mice also show significantly lower vascular endothelial growth factor (VEGF) concentrations in whole skin and significantly lower 8-oxo-dG adduct concentrations in epidermal DNA following UVB exposure. Furthermore, MIF(-/-) mice showed significant increases in p53 activity, epidermal thickness, and epidermal cell proliferation following acute UVB insult. In response to chronic UVB exposure, MIF(-/-) mice showed a 45% reduction in tumor incidence, significantly less angiogenesis, and delayed tumor progression when compared to their wild-type counterparts. These data indicate that MIF plays an important role in UVB-induced NMSC development and progression.

Pyrazole-Based Lactate Dehydrogenase Inhibitors with Optimized Cell Activity and Pharmacokinetic Properties.

Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate, with concomitant oxidation of reduced nicotinamide adenine dinucleotide as the final step in the glycolytic pathway. Glycolysis plays an important role in the metabolic plasticity of cancer cells and has long been recognized as a potential therapeutic target. Thus, potent, selective inhibitors of LDH represent an attractive therapeutic approach. However, to date, pharmacological agents have failed to achieve significant target engagement in vivo, possibly because the protein is present in cells at very high concentrations. We report herein a lead optimization campaign focused on a pyrazole-based series of compounds, using structure-based design concepts, coupled with optimization of cellular potency, in vitro drug-target residence times, and in vivo PK properties, to identify first-in-class inhibitors that demonstrate LDH inhibition in vivo. The lead compounds, named NCATS-SM1440 (43) and NCATS-SM1441 (52), possess desirable attributes for further studying the effect of in vivo LDH inhibition.

The hairless mouse in skin research.

The hairless (Hr) gene encodes a transcriptional co-repressor highly expressed in the mammalian skin. In the mouse, several null and hypomorphic Hr alleles have been identified resulting in hairlessness in homozygous animals, characterized by alopecia developing after a single cycle of relatively normal hair growth. Mutations in the human ortholog have also been associated with congenital alopecia. Although a variety of hairless strains have been developed, outbred SKH1 mice are the most widely used in dermatologic research. These unpigmented and immunocompetent mice allow for ready manipulation of the skin, application of topical agents, and exposure to UVR, as well as easy visualization of the cutaneous response. Wound healing, acute photobiologic responses, and skin carcinogenesis have been extensively studied in SKH1 mice and are well characterized. In addition, tumors induced in these mice resemble, both at the morphologic and molecular levels, UVR-induced skin malignancies in man. Two limitations of the SKH1 mouse in dermatologic research are the relatively uncharacterized genetic background and its outbred status, which precludes inter-individual transplantation studies.

Immunohistochemical analysis of ocular hemangiomas and hemangiosarcomas in dogs.

PURPOSE: To determine if molecular markers typically associated with ultraviolet exposure could be detected in canine ocular hemangiomas (HA) and hemangiosarcomas (HSA). METHODS: Paraffin-embedded samples of canine ocular HA (n = 6) and HSA (n = 6) were examined for the presence of p53, p21, p16, cyclin D, PCNA, pAkt, telomerase, and estrogen receptor (ER)-alpha using immunohistochemistry. RESULTS: p53 and cyclin D protein were not detected in any of the canine HA or HSA samples. The majority of the HA and HSA were negative for both p21 and telomerase. pAkt immunoreactivity was absent in one HA, one HSA, but was present in five HA and five HSA. All of the HA or HSA samples were strongly positive for p16 and PCNA. ERalpha was expressed in all of the samples examined; there was more intense staining in the HSA samples compared to the HA samples. CONCLUSIONS: Results from this study describe the protein expression, via immunohistochemistry, that might be altered in UV exposure in HA and HAS formation. p53 may not play an important role in tumor development; rather, in the tumors examined, expression of cell cycle regulators independent of the p53 pathway appear central in HA and HSA formation and progression. In addition, this study finds that ERalpha may be involved in promoting the invasive behavior associated with HSA.