Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines

BACKGROUND The inability of cancer cells to present antigen on the cell surface via MHC class I molecules is one of the mechanisms by which tumor cells evade anti-tumor immunity. Alterations of Jak-STAT components of interferon (IFN)-mediated signaling can contribute to the mechanism of cell resistance to IFN, leading to lack of MHC class I inducibility. Hence, the identification of IFN-gamma-resistant tumors may have prognostic and/or therapeutic relevance. In the present study, we investigated a mechanism of MHC class I inducibility in response to IFN-gamma treatment in human melanoma cell lines. METHODS Basal and IFN-induced expression of HLA class I antigens was analyzed by means of indirect immunofluorescence flow cytometry, Western Blot, RT-PCR, and quantitative real-time RT-PCR (TaqMan(R) Gene Expression Assays). In demethylation studies cells were cultured with 5-aza-2'-deoxycytidine. Electrophoretic Mobility Shift Assay (EMSA) was used to assay whether IRF-1 promoter binding activity is induced in IFN-gamma-treated cells. RESULTS Altered IFN-gamma mediated HLA-class I induction was observed in two melanoma cells lines (ESTDAB-004 and ESTDAB-159) out of 57 studied, while treatment of these two cell lines with IFN-alpha led to normal induction of HLA class I antigen expression. Examination of STAT-1 in ESTDAB-004 after IFN-gamma treatment demonstrated that the STAT-1 protein was expressed but not phosphorylated. Interestingly, IFN-alpha treatment induced normal STAT-1 phosphorylation and HLA class I expression. In contrast, the absence of response to IFN-gamma in ESTDAB-159 was found to be associated with alterations in downstream components of the IFN-gamma signaling pathway. CONCLUSION We observed two distinct mechanisms of loss of IFN-gamma inducibility of HLA class I antigens in two melanoma cell lines. Our findings suggest that loss of HLA class I induction in ESTDAB-004 cells results from a defect in the earliest steps of the IFN-gamma signaling pathway due to absence of STAT-1 tyrosine-phosphorylation, while absence of IFN-gamma-mediated HLA class I expression in ESTDAB-159 cells is due to epigenetic blocking of IFN-regulatory factor 1 (IRF-1) transactivation.

Skin hyperemia induced by local heating : why is it blunted on repeat stimulations ?

Background: In human skin, local heating produces local vasodilatation, a response termed thermal hyperemia. Thermal hyperemia is largely mediated by nitric oxide (NO). It is blunted on repeat stimulations applied to the same skin spot, a phenomenon termed desensitization. As this phenomenon could reflect a desensitization in the vasodilator effects of NO, we investigated whether a prior exposure to exogenous NO would result in an attenuated vasodilatory response to a subsequent thermal challenge. Methods: Thirteen healthy young men were studied. Skin blood flow (SkBF) was mesured on forearm skin with laser Doppler imaging. Exposure to exogenous NO was carried out by iontophoresis of sodium nitroprusside (SNP), a donor of NO. A local thermal stimulus (temperature step from 34 to 41°C maintained for 30 minutes) was applied with temperature-controlled chambers. We tested the influence of a previous transient exposure to exogenous NO on : 1) thermal hyperemia and 2) the response to a second identical exposure to exogeneous NO. Results: Thermal hyperemia (plateau SkBF at 30 minutes minus SkBF at 34°C) obtained on a site preexposed to exogenous NO two hours before was lower than obtained on a site preexposed to iontophoretic current only (mean±SD 395±139 perfusion units [PU] vs 540±79 PU ; p<0.01). When repeated on the same skin site two hours after the first one, exposure to exogenous NO led to a blunted vasodilatory response (298±121 PU vs 394±92 PU), although this difference was not statistically significant (p≈0.09). Conclusion: In forearm human skin, prior exposure to exogenous NO partially inhibits thermal hyperemia. These data support that desensitization of thermal hyperemia depends on a downregulation of the NO-cGMP pathway, possibly downstream from the endogenous production of NO.

Association study of genetic variants of pro-inflammatory chemokine and cytokine genes in systemic lupus erythematosus

BACKGROUND. Several lines of evidence suggest that chemokines and cytokines play an important role in the inflammatory development and progression of systemic lupus erythematosus. The aim of this study was to evaluate the relevance of functional genetic variations of RANTES, IL-8, IL-1alpha, and MCP-1 for systemic lupus erythematosus. METHODS. The study was conducted on 500 SLE patients and 481 ethnically matched healthy controls. Genotyping of polymorphisms in the RANTES, IL-8, IL-1alpha, and MCP-1 genes were performed using a real-time polymerase chain reaction (PCR) system with pre-developed TaqMan allelic discrimination assay. RESULTS. No significant differences between SLE patients and healthy controls were observed when comparing genotype, allele or haplotype frequencies of the RANTES, IL-8, IL-1alpha, and MCP-1 polymorphisms. In addition, no evidence for association with clinical sub-features of SLE was found. CONCLUSION. These results suggest that the tested functional variation of RANTES, IL-8, IL-1alpha, and MCP-1 genes do not confer a relevant role in the susceptibility or severity of SLE in the Spanish population.

Mutation analysis of genes that control the G1/S cell cycle in melanoma: TP53, CDKN1A, CDKN2A, and CDKN2B.

BACKGROUND The role of genes involved in the control of progression from the G1 to the S phase of the cell cycle in melanoma tumors in not fully known. The aim of our study was to analyse mutations in TP53, CDKN1A, CDKN2A, and CDKN2B genes in melanoma tumors and melanoma cell lines METHODS We analysed 39 primary and metastatic melanomas and 9 melanoma cell lines by single-stranded conformational polymorphism (SSCP). RESULTS The single-stranded technique showed heterozygous defects in the TP53 gene in 8 of 39 (20.5%) melanoma tumors: three new single point mutations in intronic sequences (introns 1 and 2) and exon 10, and three new single nucleotide polymorphisms located in introns 1 and 2 (C to T transition at position 11701 in intron 1; C insertion at position 11818 in intron 2; and C insertion at position 11875 in intron 2). One melanoma tumor exhibited two heterozygous alterations in the CDKN2A exon 1 one of which was novel (stop codon, and missense mutation). No defects were found in the remaining genes. CONCLUSION These results suggest that these genes are involved in melanoma tumorigenesis, although they may be not the major targets. Other suppressor genes that may be informative of the mechanism of tumorigenesis in skin melanomas should be studied.

Transmission blocking vaccines to control insect-borne diseases: a review

Insect-borne diseases are responsible for severe mortality and morbidity worldwide. As control of insect vector populations relies primarily on the use of insecticides, the emergence of insecticide resistance as well to unintended consequences of insecticide use pose significant challenges to their continued application. Novel approaches to reduce pathogen transmission by disease vectors are been attempted, including transmission-blocking vaccines (TBVs) thought to be a feasible strategy to reduce pathogen burden in endemic areas. TBVs aim at preventing the transmission of pathogens from infected to uninfected vertebrate host by targeting molecule(s) expressed on the surface of pathogens during their developmental phase within the insect vector or by targeting molecules expressed by the vectors. For pathogen-based molecules, the majority of the TBV candidates selected as well as most of the data available regarding the effectiveness of this approach come from studies using malaria parasites. However, TBV candidates also have been identified from midgut tissues of mosquitoes and sand flies. In spite of the successes achieved in the potential application of TBVs against insect-borne diseases, many significant barriers remain. In this review, many of the TBV strategies against insect-borne pathogens and their respective ramification with regards to the immune response of the vertebrate host are discussed.

The return of increased blood pressure after discontinuation of antihypertensive treatment is associated with an impaired post-ischemic skin blood flow response.

OBJECTIVE: To assess the post-ischemic skin blood flow response after withdrawal of antihypertensive therapy in hypertensive patients with normal blood pressure during treatment. DESIGN AND METHODS: Twenty hypertensive patients (group A) with a normal clinic blood pressure (<140/ 90 mmHg) receiving antihypertensive treatment (any monotherapy; one pill per day for at least 6 months) had their treatment discontinued. Before medication withdrawal and 2, 4, 12 and 24 weeks thereafter, the following measurements were made: clinic blood pressure, home blood pressure (three times per week, morning and evening) and skin blood flow response to a 5 min forearm arterial occlusion (using laser Doppler flowmetry). The patients were asked to perform an ambulatory blood pressure recording at any time if home blood pressure was > or =160/95 mmHg on two consecutive days, and treatment was initiated again, after determination of the skin hyperemic response, if daytime ambulatory blood pressure was > or =140/90 mmHg. The same studies were performed in 20 additional hypertensive individuals in whom antihypertensive treatment was not withdrawn (group B). The allocation of patients to groups A and B was random. RESULTS: The data fom 18 patients in group A who adhered strictly to the procedure were available for analysis. Seven of them had to start treatment again within the first 4 weeks of follow-up; four additional patients started treatment again during the next 8 weeks (group A1). The seven other patients remained untreated (group A2). The skin hyperemic response decreased significantly in patients in group A1 and returned to baseline values at the end of the study, when there were again receiving antihypertensive treatment. In patients in group A2 a significant attenuation of the hyperemic response was also observed. This impaired response was present even at the end of the 6 month follow-up, at which time the patients were still untreated but exhibited a significantly greater blood pressure than before drug discontinuation. The hyperemic response of patients who did not stop treatment (group B) did not change during the course of the study. CONCLUSIONS: Our findings show a decrease in the postischemic skin blood flow response after withdrawal of antihypertensive treatment in hypertensive patients. This impaired response may be due to the development of endothelial dysfunction, vascular remodeling, or both, and might contribute to the return of blood pressure to hypertensive values after withdrawal of antihypertensive therapy.

Kératose précancéreuse palpébrale [Eyelid precancerous keratosis].

A 25-year-old-male patient had one keratinized tumor on the inferior right eyelid, first observed 5 months before. Its size had been increasing for the last three weeks. The visual function was normal. Clinical examination disclosed a papillomatous lesion on the cutaneous middle part of the eyelid. A surgical excision was performed under local anesthesia. The histopathological study found a papillomatous epidermal proliferation with both structural and cytological atypias, concluding with a precancerous keratosis of the eyelid.

Molecular analysis of non-melanoma skin cancer

SUMMARY:Cylindroma, trichoepithelioma and spiradenoma are benign tumors of hair follicle. They are caused by mutations and loss of heterozygosity in the CYLD gene. CYLD is a ubiquitously expressed, but the tumors are restricted to skin, suggesting that the tumorigenesis is influenced by skin-specific regulators and probably by mutations in other genes. The objectives of the thesis were to analyze the molecular mechanisms leading to the aforementioned tumors. In the first project, we have identified five new mutations in CYLD gene in tive families affected with different combinations of these skin appendage tumors. F our of these mutations caused the introduction of a premature stop codon in CYLD protein sequence, but one was a missense mutation changing aspartic acid 681 into glycine (D68lG), in patients exhibiting multiple trichoepitheliomas. CYLD is a deubiquitinase which can downregulate NF-κB and INK pathways through the deubiquitination of TRAF2, for example. We showed that the CYLD-D681G mutant was unable to remove polyubiquitin chains from TRAF2. We also proved that CYLD-D68lG could not inhibit TRAP 2- or TNFα- mediated NF-κB or INK activations in 293T cells. These results underlined the importance of the D68l residue for the enzymatic activity of CYLD. TRAP-interacting protein (TRIP), which is a E3-Ubiquitin ligase, is a partner of CYLD. In the second project of the thesis, we studied the function of TRIP in the epidermis. We found that TRIP was a nucleolar protein in cultured human primary keratinocytes (HEK) and HeLa cells, and was detected in the midbody of HeLa cells. Moreover, TRIP expression was shown to be downregulated through a PKC-dependent mechanism before induction of keratinocyte differentiation. We also proved that TRIP was upregulated in basal cell carcinomas. Furthermore, TRIP was found to be important for keratinocyte survival and proliferation through the regulation of the Gl/S transition. Our results suggest that TRIP may be involved in keratinocyte tumorigenesis.RÉSUMÉ :Les cylindromes, trichoépithéliomes et spiradénomes sont des tumeurs bénignes du follicule pileux causées par des mutations et une perte d'hétérozygotie du gène CYLD. CYLD est ubiquitaire mais les tumeurs sont limitées à la peau, suggérant que la tumorigénèse est influencée par des protéines spécifiques de la peau et par des mutations dans d'autres gènes. Les objectifs de la thèse étaient d'2malyser les mécanismes moléculaires aboutissant à la formation de ces tumeurs. Dans le premier projet, cinq nouvelles mutations du gène CYLD ont été identifiées chez cinq familles présentant différentes combinaisons des tumeurs citées ci- dessus. Quatre de ces mutations causaient I' introduction d'un codon stop prématuré dans la séquence protéique, mais une était une mutation «misser1se» changeant l'aspartate 681 en résidu glycine (D68lG) chez des patients présentant des trichoépithéliomes multiples. CYLD est une déubiquitinase qui inhibe les voies de signalisation de NF-κB et JNK, en déubiquitinant notamment TRAF2. Nous avons montré que la protéine mutante CYLD- D68lG ne pouvait pas cliver la chaîne de poly-ubiquitines liée à TRAF2. CYLD-D68lG était aussi incapable d'inhiber l'activation de NF-κB ou de JNK induite par TRAF2 ou TNF-o dans les cellules 293T. Ces résultats ont donc souligné l'impo1tance du résidu D68l pour l'activité de CYLD. «TRAF-interacting protein (TRIP)», qui est une «E3-ubiquitin-ligase», est un partenaire de CYLD. Dans le second proj et de la thèse, nous avons étudié la fonction de TRIP dans l'épidenne. Nous avons montrépque TRIP était nucléolaire dans les cellules HeLa et les kératinocytes primaires humains en culture et était détectée dans le «midbody» des cellules HeLa. Nous avons prouvé que l'ARNm de TRIP était diminué avant l'induction de la différentiation des kératinocytes, par un mécanisme dépendent de la protéine kinase C, tandis qu'il était augmenté dans les carcinomes baso-cellulaires. Nous avons aussi montré que TRIP influençait la prolifération et la survie des kératinocytes en régulant la transition G1/S, Nos résultats suggèrent que TRIP est peut-être impliquée dans la tumorigénèse des kératinocytes. 7

Uptake of macromolecules by cercariae during skin penetration and transformation to schistosomula (Schistosoma mansoni)

Here, we observed the uptake of membrane-impermeant molecules by cercariae as they penetrate the skin and are transformed into schistosomula. We propose that membrane-impermeant molecules, Lucifer Yellow, Propidium iodide and Hoechst 33258 enter the parasite through both thenephridiopore and the surface membrane and then diffuse throughout the body of the parasite. We present a hypothesis that the internal cells of the body of the schistosomulum represent a new host-parasite interface, at which skin-derived growth factors may stimulate receptors on internal membranes during transformation of the cercariae into the schistosomulum.

High Prevalence of Systemic Autoimmune Diseases in Patients with Meniere's Disease

BACKGROUND. Autoimmunity appears to be associated with the pathophysiology of Meniere's disease (MD), an inner ear disorder characterized by episodes of vertigo associated with hearing loss and tinnitus. However, the prevalence of autoimmune diseases (AD) in patients with MD has not been studied in individuals with uni or bilateral sensorineural hearing loss (SNHL). METHODS AND FINDINGS. We estimated the prevalence of AD in 690 outpatients with MD with uni or bilateral SNHL from otoneurology clinics at six tertiary referral hospitals by using clinica criteria and an immune panel (lymphocyte populations, antinuclear antibodies, C3, C4 and proinflammatory cytokines TNFα, INFγ). The observed prevalence of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and ankylosing spondylitis (AS) was higher than expected for the general population (1.39 for RA, 0.87 for SLE and 0.70 for AS, respectively). Systemic AD were more frequently observed in patients with MD and diagnostic criteria for migraine than cases with MD and tension-type headache (p = 0.007). There were clinical differences between patients with uni or bilateral SNHL, but no differences were found in the immune profile. Multiple linear regression showed that changes in lymphocytes subpopulations were associated with hearing loss and persistence of vertigo, suggesting a role for the immune response in MD. CONCLUSIONS. Despite some limitations, MD displays an elevated prevalence of systemic AD such as RA, SLE and AS. This finding, which suggests an autoimmune background in a subset of patients with MD, has important implications for the treatment of MD.

The Estrada Real project and endemic diseases: the case of schistosomiasis, geoprocessing and tourism

Geographical Information System (GIS) is a tool that has recently been applied to better understand spatial disease distributions. Using meteorological, social, sanitation, mollusc distribution data and remote sensing variables, this study aimed to further develop the GIS technology by creating a model for the spatial distribution of schistosomiasis and to apply this model to an area with rural tourism in the Brazilian state of Minas Gerais (MG). The Estrada Real, covering about 1,400 km, is the largest and most important Brazilian tourism project, involving 163 cities in MG with different schistosomiasis prevalence rates. The model with three variables showed a R² = 0.34, with a standard deviation of risk estimated adequate for public health needs. The main variables selected for modelling were summer vegetation, summer minimal temperature and winter minimal temperature. The results confirmed the importance of Remote Sensing data and the valuable contribution of GIS in identifying priority areas for intervention in tourism regions which are endemic to schistosomiasis.

Role of Notch signaling in the skin and cornea

Résumé : La voie de signalisation Notch est essentielle pour la différentiation de l'épiderme lors du développement embryonnaire de la peau. Il a été démontré que l'inactivation de Notch1 dans la peau de souris conduit à une hyperplasie de l'épiderme ainsi qu'à la formation subséquente de carcinomes basocellulaires ainsi que de plaques cornéennes. L'inactivation de Notch1 dans la cornée combinée à des lésions mécaniques démontre que les cellules progénitrices de la cornée se différentient en un épithélium hyperplasique et kératinisé comme la peau. Ce changement de destinée cellulaire conduit à une cécité cornéenne et implique des processus non-autonomes aux cellules épithéliales, caractérisés par la sécrétion de FGF-2 par l'épithélium Notch1-/- suivi d'une vascularisation et d'un remaniement du stroma sous-jacent. La déficience en vitamine A est connu comme cause de lésions cornéennes humaines (xérophtalmie sévère). En accord, nous avons trouvé que la signalisation Notch1 était liée au métabolisme de la vitamine A par la régulation de l'expression de CRBP1, nécessaire pour générer un pool de rétinol intracellulaire. La perte de Notch1 dans l'épiderme, l'autre récepteur de la famille présent dans la peau marine, ne conduit pas à un phénotype manifeste. Cependant, l'inactivation dans l'épiderme de Notch1 et Notch2 ensemble, ou de RBP-J, induit une dermatite atopique (DA) sévère chez les souris. De même, les patients souffrants de DA mais pas ceux souffrant de psoriasis ou de lichen plan, ont une réduction marquée de l'expression des récepteurs Notch dans la peau. La perte de Notch dans les keratinocytes conduit à une activation de la voie NF-κB, ce qui ensuite induit la production de TSLP, une cytokine profondément impliquée dans la pathogenèse de la DA. Nous démontrons génétiquement que TSLP est responsable de la DA ainsi que du développent d'un syndrome myéloprolifératif non-autonome aux cellules induit par le G-CSF. Cependant, ces souris avec une inactivation dans l'épiderme de Notch1 et Notch2 et aussi incapables de répondre au TSLP développent des tumeurs invasive sévères caractérisées par une haute activité de signalisation ß-catenin. TSLPR est identifié comme un potentiel suppresseur de tumeur non-autonome aux cellules tumorales; la transplantation de cellules hématopoïétiques TSLPR-/- dans des souris déficientes pour Notch est suffisant pour causer des tumeurs. Summary : The Notch pathway is essential for proper epidermal differentiation during embryonic skin development. It has previously been demonstrated that Notch1 inactivation in marine skin results in epidermal hyperplasia and subsequent formation of basal cell carcinoma-like (BCC-like) tumors as well as corneal plaques. Inducible ablation of Notch1 in the cornea combined with mechanical wounding show that Notch1 deficient corneal progenitor cells differentiate into a hyperplasic, keratinized, skin-like epithelium. This cell fate switch leads to corneal blindness and involves cell non-autonomous processes, characterized by secretion of FGF-2 through Notch1-/- epithelium followed by vascularisation and remodelling of the underlying stroma. Vitamin A deficiency is known to induce a similar corneal defect in humans (severe xerophthalmia). Accordingly, we found that Notch1 signaling is linked to vitamin A metabolism by regulating the expression of CRBP1, required to generate a pool of intracellular retinol. Epidermal loss of Notch2, the other Notch receptor present in marine skin, doesn't lead to any overt phenotypes. However, postnatal epidermis-specific inactivation of both Notch1 and Notch2, or of RBP-J, induces the development of a severe form of atopic dermatitis (AD) in mice. Likewise, patients suffering from AD, but not psoriasis or lichen planas, have a marked reduction of Notch receptor expression in the skin. Loss of Notch in keratinocytes leads to an activation of NF-κB signaling which in turn induces the production of Thymic stromal lymphopoietin (TSLP), a cytokine deeply implicated in the pathogenesis of AD. We genetically demonstrate that TSLP is responsible for AD as well as the development of a cell non-autonomous G-CSF induced myeloproliferative disorder (MPD) in mice. However, these mice with conditional epidermal inactivation of Notch1 and Notch2 as well as incapable to respond to TSLP develop severe invasive tumors characterized by high ß-catenin signaling activity. TSLPR is identified as a potential cell non-autonomous tumor suppressor; transplantation of TSLPR-/- hematopoietic cells into epidermal Notch deficient mice is sufficient to cause tumors.

Eligibility for and outcome of treatment of latent tuberculosis infection in a cohort of HIV-infected people in Spain

BACKGROUND Previous studies have demonstrated the efficacy of treatment for latent tuberculosis infection (TLTBI) in persons infected with the human immunodeficiency virus, but few studies have investigated the operational aspects of implementing TLTBI in the co-infected population.The study objectives were to describe eligibility for TLTBI as well as treatment prescription, initiation and completion in an HIV-infected Spanish cohort and to investigate factors associated with treatment completion. METHODS Subjects were prospectively identified between 2000 and 2003 at ten HIV hospital-based clinics in Spain. Data were obtained from clinical records. Associations were measured using the odds ratio (OR) and its 95% confidence interval (95% CI). RESULTS A total of 1242 subjects were recruited and 846 (68.1%) were evaluated for TLTBI. Of these, 181 (21.4%) were eligible for TLTBI either because they were tuberculin skin test (TST) positive (121) or because their TST was negative/unknown but they were known contacts of a TB case or had impaired immunity (60). Of the patients eligible for TLTBI, 122 (67.4%) initiated TLTBI: 99 (81.1%) were treated with isoniazid for 6, 9 or 12 months; and 23 (18.9%) with short-course regimens including rifampin plus isoniazid and/or pyrazinamide. In total, 70 patients (57.4%) completed treatment, 39 (32.0%) defaulted, 7 (5.7%) interrupted treatment due to adverse effects, 2 developed TB, 2 died, and 2 moved away. Treatment completion was associated with having acquired HIV infection through heterosexual sex as compared to intravenous drug use (OR:4.6; 95% CI:1.4-14.7) and with having taken rifampin and pyrazinamide for 2 months as compared to isoniazid for 9 months (OR:8.3; 95% CI:2.7-24.9). CONCLUSIONS A minority of HIV-infected patients eligible for TLTBI actually starts and completes a course of treatment. Obstacles to successful implementation of this intervention need to be addressed.