NLRP3 promotes inflammation-induced skin cancer but is dispensable for asbestos-induced mesothelioma.

Asbestos exposure can result in serious and frequently lethal diseases, including malignant mesothelioma. The host sensor for asbestos-induced inflammation is the NLRP3 inflammasome and it is widely assumed that this complex is essential for asbestos-induced cancers. Here, we report that acute interleukin-1β production and recruitment of immune cells into peritoneal cavity were significantly decreased in the NLRP3-deficient mice after the administration of asbestos. However, NLRP3-deficient mice displayed a similar incidence of malignant mesothelioma and survival times as wild-type mice. Thus, early inflammatory reactions triggered by asbestos are NLRP3-dependent, but NLRP3 is not critical in the chronic development of asbestos-induced mesothelioma. Notably, in a two-stage carcinogenesis-induced papilloma model, NLRP3-deficient mice showed a resistance phenotype in two different strain backgrounds, suggesting a tumour-promoting role of NLRP3 in certain chemically-induced cancer types.

Role of defensins and cathelicidin LL37 in auto-immune and auto-inflammatory diseases.

Defensins and cathelicidins are anti-microbial peptides (AMPs) that act as natural antibiotics and are part of the innate immune defence in many species. We consider human defensins and LL37, the only human member of the cathelicidin family. In particular, we refer to the human alpha-defensins called human neutrophil peptides (HNP1 through 4), which are produced by neutrophils, HD5 and HD6, mainly expressed in Paneth cells of intestine, the human beta-defensins HBD1, HBD2 and HBD3, synthesized by epithelial cells and LL37, which is located in granulocytes, but is also produced by epithelial cells of the skin, lungs, and gut. In the last years, the study of AMPs activity and regulation has allowed to understand the important role of these peptides not only in the innate defence mechanisms against bacteria, viruses, fungi, but also in the regulation of immune cell activation and migration. Complementary studies have disclosed a role for AMPs in modulating many physiological processes that involve non-immune cells, such as activation of wound healing, angiogenesis, cartilage remodeling. Due to the pleiotropic tasks of these peptides, many of them are now being discovered to contribute to immune pathology of chronic diseases that affect skin, gut, joints; this is supported by many examples of immune-mediated pathologies in which their expression is disregulated. In this article we review the current literature that suggests a role for human defensins and LL37 in pathogenic mechanisms of several chronic diseases that are considered of auto-immune or auto-inflammatory origin.

Comparison of quantiferon-TB gold with tuberculin skin test for detecting latent tuberculosis infection prior to liver transplantation.

Screening for latent tuberculosis infection (LTBI) is recommended prior to organ transplantation. The Quantiferon-TB Gold assay (QFT-G) may be more accurate than the tuberculin skin test (TST) in the detection of LTBI. We prospectively compared the results of QFT-G to TST in patients with chronic liver disease awaiting transplantation. Patients were screened for LTBI with both the QFT-G test and a TST. Concordance between test results and predictors of a discordant result were determined. Of the 153 evaluable patients, 37 (24.2%) had a positive TST and 34 (22.2%) had a positive QFT-G. Overall agreement between tests was 85.1% (kappa= 0.60, p < 0.0001). Discordant test results were seen in 12 TST positive/QFT-G negative patients and in 9 TST negative/QFT-G positive patients. Prior BCG vaccination was not associated with discordant test results. Twelve patients (7.8%), all with a negative TST, had an indeterminate result of the QFT-G and this was more likely in patients with a low lymphocyte count (p = 0.01) and a high MELD score (p = 0.001). In patients awaiting liver transplantation, both the TST and QFT-G were comparable for the diagnosis of LTBI with reasonable concordance between tests. Indeterminate QFT-G result was more likely in those with more advanced liver disease.

Allergy to betalactam antibiotics in children: a prospective follow-up study in retreated children after negative responses in skin and challenge tests.

BACKGROUND: Up to 10% of the patients in whom suspected betalactam hypersensitivity (HS) has been excluded by skin and challenge tests report suspected allergic reactions during subsequent treatments with the same or very similar betalactams. It has been suggested that the reactions may result from a resensitization induced by the challenge performed at the time of the allergological work-up. However, most patients did not undergo a second allergological work-up, to determine if the reactions resulted from betalactam HS or not. OBJECTIVES: We aimed to determine if children diagnosed nonallergic to betalactams have tolerated subsequent treatments with the initially suspected and/or other betalactams, and, in case of a reaction, if the reaction resulted from betalactam HS. Methods: We sent a questionnaire concerning the clinical history of their children to the parents of 256 children previously diagnosed nonallergic to betalactams. A second allergological work-up was performed in the children reporting suspected allergic reactions during subsequent treatments with the same and/or other betalactams. Skin tests were performed with the soluble form of the suspected (or very similar) betalactams and other betalactams from the same and other classes. Skin test responses were assessed at 15-20 min (immediate), 6-8 h (semi-late) and 48-72 h (late). Oral challenge (OC) was performed in children with negative skin tests, either at the hospital (immediate and accelerated reactions), or at home (delayed reactions). RESULTS: A response was obtained from 141 children (55.3%). Forty-eight (34%) of those children had not been treated with the betalactams for whom a diagnosis of allergy had been ruled out previously. Seven (7.5%) of the 93 children who had been treated again reported suspected allergic reactions. Skin tests and OC were performed in six of those children, and gave negative results in five children. In one child previously diagnosed nonallergic to amoxicillin associated with clavulanic acid, we diagnosed a delayed HS to clavulanic acid and a serum sickness-like disease to cefaclor. Thus, the frequency of reactions resulting from betalactam HS in children with negative skin and challenge tests is very low, and does not exceed 2.1% (2/93) if we consider that the child which refused a second allergological work-up is really allergic to betalactams. CONCLUSION: Our results in a very large number of children show that reactions presumed to result from betalactam HS are rare in children in whom the diagnosis of betalactam allergy has been ruled out previously. Moreover, they suggest that, as shown for the initial reactions, most of the reactions during subsequent treatments are rather a consequence of the infectious diseases for whom betalactams have been prescribed than a result of betalactam HS. Finally, they suggest that the risk of resensitization by OC is very low, and do not support the notion that skin testing should be repeated in children diagnosed nonallergic to betalactams.

Skin fibroblast model to study an impaired glutathione synthesis: consequences of a genetic polymorphism on the proteome.

An impaired glutathione (GSH) synthesis was observed in several multifactorial diseases, including schizophrenia and myocardial infarction. Genetic studies revealed an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL). Disease-associated genotypes of this polymorphism correlated with a decrease in GCLC protein expression, GCL activity and GSH content. To clarify consequences of a decreased GCL activity at the proteome level, three schizophrenia patients and three controls have been selected based on the GCLC GAG TNR polymorphism. Fibroblast cultures were obtained by skin biopsy and were challenged with tert-butylhydroquinone (t-BHQ), a substance known to induce oxidative stress. Proteome changes were analyzed by two dimensional gel electrophoresis (2-DE) and results revealed 10 spots that were upregulated in patients following t-BHQ treatment, but not in controls. Nine corresponding proteins could be identified by MALDI mass spectrometry and these proteins are involved in various cellular functions, including energy metabolism, oxidative stress response, and cytoskeletal reorganization. In conclusion, skin fibroblasts of subjects with an impaired GSH synthesis showed an altered proteome reaction in response to oxidative stress. Furthermore, the study corroborates the use of fibroblasts as an additional mean to study vulnerability factors of psychiatric diseases.

Whole-cell bioprocessing of human fetal cells for tissue engineering of skin.

Current restrictions for human cell-based therapies have been related to technological limitations with regards to cellular proliferation capacity (simple culture conditions), maintenance of differentiated phenotype for primary human cell culture and transmission of communicable diseases. Cultured primary fetal cells from one organ donation could possibly meet the exigent and stringent technical aspects for development of therapeutic products. Master and working cell banks from one fetal organ donation (skin) can be developed in short periods of time and safety tests can be performed at all stages of cell banking. For therapeutic use, fetal cells can be used up to two thirds of their life-span in an out-scaling process and consistency for several biological properties includes protein concentration, gene expression and biological activity. As it is the intention that banked primary fetal cells can profit from the prospected treatment of hundreds of thousands of patients with only one organ donation, it is imperative to show consistency, tracability and safety of the process including donor tissue selection, cell banking, cell testing and growth of cells in out-scaling for the preparation of whole-cell tissue-engineering products.

Identification of the first nonsense CDSN mutation with expression of a truncated protein causing peeling skin syndrome type B.

BACKGROUND: Peeling skin disease (PSD), a generalized inflammatory form of peeling skin syndrome, is caused by autosomal recessive nonsense mutations in the corneodesmosin gene (CDSN). OBJECTIVES: To investigate a novel mutation in CDSN. METHODS: A 50-year-old white woman showed widespread peeling with erythema and elevated serum IgE. DNA sequencing, immunohistochemistry, Western blot and real-time polymerase chain reaction analyses of skin biopsies were performed in order to study the genetics and to characterize the molecular profile of the disease. RESULTS: Histology showed hyperkeratosis and acanthosis of the epidermis, and inflammatory infiltrates in the dermis. DNA sequencing revealed a homozygous mutation leading to a premature termination codon in CDSN: p.Gly142*. Protein analyses showed reduced expression of a 16-kDa corneodesmosin mutant in the upper epidermal layers, whereas the full-length protein was absent. CONCLUSIONS: These results are interesting regarding the genotype-phenotype correlations in diseases caused by CDSN mutations. The PSD-causing CDSN mutations identified heretofore result in total corneodesmosin loss, suggesting that PSD is due to full corneodesmosin deficiency. Here, we show for the first time that a mutant corneodesmosin can be stably expressed in some patients with PSD, and that this truncated protein is very probably nonfunctional.

Mandatory infectious diseases consultation for MRSA bacteremia is associated with reduced mortality.

OBJECTIVES: Although infectious disease (ID) consultation has been associated with lower mortality in Staphylococcus aureus bloodstream infections, it is still not mandatory in many centers. This study aimed at assessing the impact of ID consultation on diagnostic and therapeutic management of methicillin-resistant S. aureus (MRSA) bacteremia. METHODS: Retrospective cohort study of all patients with MRSA bacteremia from 2001 to 2010. ID consultations were obtained on request between 2001 and 2006 and became mandatory since 2007. RESULTS: 156 episodes of MRSA bacteremia were included, mostly from central venous catheter (32%) and skin and soft tissue (19%) infections. ID consultation coverage was 58% between 2001 and 2006 and 91% between 2007 and 2010. ID consultation was associated with more echocardiography (59% vs. 26%, p < 0.01), vancomycin trough level measurements (99% vs. 77%, p < 0.01), follow-up blood cultures (71% vs. 50%, p = 0.05), deep-seated infections (43% vs. 16%, p < 0.01), more frequent infection source control (83% vs. 57%, p = 0.03), a longer duration of MRSA-active therapy (median and IQR: 17 days, 13-30, vs. 12, 3-14, p < 0.01) and a 20% reduction in 7-day, 30-day and in-hospital mortality. CONCLUSIONS: ID consultation was associated with a better management of patients with MRSA bacteremia and a reduced mortality.

Peroxisome proliferator activated receptor BETA/DELTA as a central player in the skin response to ultra-violets radiation

Previous studies in the lab of Dr. Liliane Michalik, have shown thai the nuclear hormone receptor Peroxisome Proliferator Activated Receptor beta/delta (PPARß/ö) is an important regulator of skin homeostasis, being involved in the regulation of keratinocyte differentiation, inflammation, apoptosis, arid mouse skin wound healing. Studies of PPARß/ö knock out mice have suggested a possible role for this receptor in cancer. However, contradictory observations of the role for PPARß/ö on tumor growth have been published, depending on cellular contexts and biological models. Given the controversial role of PPARß/ö in skin carcinoma development, the main aim of this PhD work has been to further explore the implication of PPARß/ö in skin response to UV and skin tumor growth. This PhD dissertation is divided in four chapters. The first chapter describes the core part of the project, where I explored the changes in miRNA expression in the skin upon chronic UV irradiation of PPARß/ö wild type and knock-out mice. This analysis shed light on a miRNA- PPARß/ö signature and also predicted thai miR-21-3p (previously named miR-21*) is a key regulator of the PPARß/ö-dependent UV response in the pre-lesiona! skin. Using mice acutely UV-irradiated, ! further demonstrated that miR-21-3p is indirectly regulated by PPARß/ö through activation of Transforming Growth Factor (TGFß)-1 under UV exposure. I also show that miR-21-3p is deregulated in human cutaneous squamous celi carcinoma. In cultured keratinocytes, application of a miR-21 -3p mimic oligonucleotide sequence leads to the regulation of lipid metabolism-related pathway. In the second chapter, I demonstrate that the usage of an mRNA/miRNA combined bioinformatics analysis leads to the discovery of important pathways involved in the PPARß/ö-miRNA response of the skin to chronic UV irradiation, indeed, I validated angiogenesis and lipid metabolism as important functions regulated by PPARß/ö in this context. In the third chapter, we demonstrate that PPARß/5 knockout mice have decreased cutaneous squamous cell carcinomas incidence compared to wild type mice and that PPARß/5 directly activates the cSrc kinase gene. In the last chapter, we review novel insights into PPAR functions in keratinocytes and liver, with emphasis on PPARß/ö but also on PPARa. In summary, this PhD study shows that i) PPARß/5 is able to regulate biological function through regulation of miRNAs, and specifically through miR-21-3p, the passenger miRNA of the oncomiR miR-21, and that ii) the PPARß/5-dependent skin response to UV involves the regulation of angiogenesis and lipid metabolism. Furthermore, the bioinformatics study highlights the relevance of performing integrated mRNA and miRNA genome-wide studies in order to better screen mRNAs and/or miRNAs of interest in the biological context of diseases. - Des études préalables dans le laboratoire du Dr. Liliane Michalik ont démontré que le récepteur nucléaire PPARß/5 est un régulateur important de l'homéostasie de la peau, étant impliqué dans la régulation de la différenciation des keratinocytes, dans l'inflammation, dans l'apoptose et dans la cicatrisation de la peau chez !a souris. L'étude de souris knock-out pour le gène PPARß/5, ont suggérées un rôle possible de ce récepteur dans le cancer. Cependant, des observations opposées ont été publiées suggérant un rôle pro- ou anti- cancer selon le tissue impliqué et le type- cellulaire. En considérant cette controverse autour du rôle de PPARß/5 dans le développement des cancers de la peau, le but principal de mon projet de recherche aura été d'approfondir l'exploration du rôle de PPARß/5 dans la réponse de la peau aux UVs et dans le développement du cancer. Cette dissertation de thèse est divisée en quatre parties. Une première partie, représentant le coeur de mon travail de recherche, décrit la découverte de l'implication des microRNAs (rniRNAs) dans la réponse aux UVs de PPARß/ö et plus spécifiquement l'implication du miRNA miR- 21 -3p (précédemment nommé miR-21*). En étudiant un modèle de souris irradiées de manière aigüe aux UVs, nous montrons que ia régulation de miR-21-3p est PPARß/ö-däpenaante et que cette régulation à lieu par l'intermédiaire du facteur de transcription TGFß-1. Dans des cultures de keratinocytes Humains, la transfecticn d'une séquence oligonucléotidique similaire à celle de miR-21-3p (mimic), montre l'implication de rniR-21-3p dans des fonctions importantes pour le développement des cancers telles que le métabolisme des lipides. Dans un second chapitre, nous montrons que l'usage d'une méthode bioinformatique combinant l'expression des ARN messagers et des miRNAs permet de mettre en évidence des fonctions biologiques importantes lors de ia réponse de PPARß/ö à l'irradiation chronique. L'angiogenèse, le stress oxydatif et le métabolisme des lipides font partie de ces fonctions régulées par PPARß/5 dans la peau irradiée aux UVs. Nous mettons également en évidence la régulation du gène LpcatS par PPARß/5 dans la peau irradiée aux UV ainsi que dans des keratinocytes humains suggérant un rôle pour PPARß/5 dans le remodelage des lipides membranaires. Dans une troisième partie, nous établissons un lien entre la régulation de l'oncogène Src et l'activation de PPARß/5 dans les carcinomes spinocellulaires de la peau. Finalement dans un quatrième chapitre, nous faisons une revue des dernières recherches portées sur le rôle de PPARß/5 et de PPARa dans le foie et ia peau. En résumé ce projet de thèse représente un avancement pour la recherche sur rimplication de PPARß/5 dans la réponse aux UVs de la peau. Pour la première fois, un lien est établi entre ce facteur de transcription et la régulation de microRNAs dans le cadre du carcinome spinocellulare. Jusqu'alors resté dans l'ombre de rniR-21-5p, miR-21-3p est en fait fortement augmenté à la fois dans un modèle de souris d'irradiation aux UVs ainsi que dans ie carcinome spinocellulare chez i'humain. De nouvelles fonctions biologiques pour PPARß/5 ont été également mises en évidence dans ce travail, comme la régulation de l'angiogenèse ou du métabolisme des lipides dans Sa peau. De plus cette dissertation valorise l'intérêt d'une association entre le travail de laboratoire et celui de la bioinformatique.

Comparison of interferon-gamma release assay versus tuberculin skin test for tuberculosis screening in inflammatory bowel disease.

OBJECTIVES: Reactivation of latent tuberculosis (TB) in inflammatory bowel disease (IBD) patients treated with antitumor necrosis factor-alpha medication is a serious problem. Currently, TB screening includes chest x-rays and a tuberculin skin test (TST). The interferon-gamma release assay (IGRA) QuantiFERON-TB Gold In-Tube (QFT-G-IT) shows better specificity for diagnosing TB than the skin test. This study evaluates the two test methods among IBD patients. METHODS: Both TST and IGRA were performed on 212 subjects (114 Crohn's disease, 44 ulcerative colitis, 10 indeterminate colitis, 44 controls). RESULTS: Eighty-one percent of IBD patients were under immunosuppressive therapy; 71% of all subjects were vaccinated with Bacille Calmette Guérin; 18% of IBD patients and 43% of controls tested positive with the skin test (P < 0.0001). Vaccinated controls tested positive more often with the skin test (52%) than did vaccinated IBD patients (23%) (P = 0.011). Significantly fewer immunosuppressed patients tested positive with the skin test than did patients not receiving therapy (P = 0.007); 8% of patients tested positive with the QFT-G-IT test (14/168) compared to 9% (4/44) of controls. Test agreement was significantly higher in the controls (P = 0.044) compared to the IBD group. CONCLUSIONS: Agreement between the two test methods is poor in IBD patients. In contrast to the QFT-G-IT test, the TST is negatively influenced by immunosuppressive medication and vaccination status, and should thus be replaced by the IGRA for TB screening in immunosuppressed patients having IBD.

Clinicopathological features of eyelid skin tumors. A retrospective study of 5504 cases and review of literature.

Eyelid tumors are the most common neoplasm in daily ophthalmology practice and encompass a wide variety of benign and malignant tumors. In this retrospective study, we report the clinical and histological features of 5504 eyelid skin tumors diagnosed at the Laboratory of Ophthalmopathology of the Hôpital Ophtalmique Jules Gonin, Lausanne, Switzerland, between January 1989 and December 2007. Benign tumors largely predominated over malignant ones, representing 84% of cases in this series, and the 5 most frequent subtypes were squamous cell papilloma (26%), seborrheic keratosis (21%), melanocytic nevus (20%), hidrocystoma (8%), and xanthoma/xanthelasma (6%). Basal cell carcinoma was the most frequent malignant tumor (86%), followed by squamous cell carcinoma (7%) and sebaceous carcinoma (3%). For several tumor subtypes, there was a poor correlation between clinical and histological diagnosis, stressing the numerous pitfalls in the diagnosis of eyelid tumors. We further discuss our results with reference to previously published series.

Selective inhibition of JNK with a peptide inhibitor attenuates pain hypersensitivity and tumor growth in a mouse skin cancer pain model.

Cancer pain significantly affects the quality of cancer patients, and current treatments for this pain are limited. C-Jun N-terminal kinase (JNK) has been implicated in tumor growth and neuropathic pain sensitization. We investigated the role of JNK in cancer pain and tumor growth in a skin cancer pain model. Injection of luciferase-transfected B16-Fluc melanoma cells into a hindpaw of mouse induced robust tumor growth, as indicated by increase in paw volume and fluorescence intensity. Pain hypersensitivity in this model developed rapidly (<5 days) and reached a peak in 2 weeks, and was characterized by mechanical allodynia and heat hyperalgesia. Tumor growth was associated with JNK activation in tumor mass, dorsal root ganglion (DRG), and spinal cord and a peripheral neuropathy, such as loss of nerve fibers in the hindpaw skin and induction of ATF-3 expression in DRG neurons. Repeated systemic injections of D-JNKI-1 (6 mg/kg, i.p.), a selective and cell-permeable peptide inhibitor of JNK, produced an accumulative inhibition of mechanical allodynia and heat hyperalgesia. A bolus spinal injection of D-JNKI-1 also inhibited mechanical allodynia. Further, JNK inhibition suppressed tumor growth in vivo and melanoma cell proliferation in vitro. In contrast, repeated injections of morphine (5 mg/kg), a commonly used analgesic for terminal cancer, produced analgesic tolerance after 1 day and did not inhibit tumor growth. Our data reveal a marked peripheral neuropathy in this skin cancer model and important roles of the JNK pathway in cancer pain development and tumor growth. JNK inhibitors such as D-JNKI-1 may be used to treat cancer pain.

Mutations in NDUFB11, Encoding a Complex I Component of the Mitochondrial Respiratory Chain, Cause Microphthalmia with Linear Skin Defects Syndrome.

Microphthalmia with linear skin defects (MLS) syndrome is an X-linked male-lethal disorder also known as MIDAS (microphthalmia, dermal aplasia, and sclerocornea). Additional clinical features include neurological and cardiac abnormalities. MLS syndrome is genetically heterogeneous given that heterozygous mutations in HCCS or COX7B have been identified in MLS-affected females. Both genes encode proteins involved in the structure and function of complexes III and IV, which form the terminal segment of the mitochondrial respiratory chain (MRC). However, not all individuals with MLS syndrome carry a mutation in either HCCS or COX7B. The majority of MLS-affected females have severe skewing of X chromosome inactivation, suggesting that mutations in HCCS, COX7B, and other as-yet-unidentified X-linked gene(s) cause selective loss of cells in which the mutated X chromosome is active. By applying whole-exome sequencing and filtering for X-chromosomal variants, we identified a de novo nonsense mutation in NDUFB11 (Xp11.23) in one female individual and a heterozygous 1-bp deletion in a second individual, her asymptomatic mother, and an affected aborted fetus of the subject's mother. NDUFB11 encodes one of 30 poorly characterized supernumerary subunits of NADH:ubiquinone oxidoreductase, known as complex I (cI), the first and largest enzyme of the MRC. By shRNA-mediated NDUFB11 knockdown in HeLa cells, we demonstrate that NDUFB11 is essential for cI assembly and activity as well as cell growth and survival. These results demonstrate that X-linked genetic defects leading to the complete inactivation of complex I, III, or IV underlie MLS syndrome. Our data reveal an unexpected role of cI dysfunction in a developmental phenotype, further underscoring the existence of a group of mitochondrial diseases associated with neurocutaneous manifestations.