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The causal association between persistent human papillomavirus (HPV) infection and cervical cancer has been established, but the mechanisms that favor HPV persistence in cervical cells are still unknown. The diminished capability of the immune system to control and resolve HPV infection is one of several hypotheses. The tolerogenic protein HLA-G has shown aberrant expression in a variety of cancers, which has been suggested as a mechanism for tumor escape from immunosurveillance. In the present study we evaluate the role of epigenetic modification (promoter de-methylation) of the HLA-G gene on susceptibility to HPV infection and development of high-grade cervical lesions.
A case–control study was carried out in Curitiba, Brazil, between February and June 2010. A total of 789 women aged 15–47 years were recruited: 510 controls with normal cervical cytology, and 279 cases with histologically confirmed cervical intraepithelial neoplasia grade 2 (CIN2, N = 150) or grade 3 (CIN3, N = 129). All women were administered a questionnaire by interview, which collected information on demographic and lifestyle factors, and a cervical sample was collected. HPV DNA detection was performed by GP5+/GP6+ primer-mediated PCR. HPV-positive samples were genotyped by multiplex PCR. A pilot analysis of HLA-G promoter methylation was carried out in a subset of the study population (96 cases and 76 controls) by pyrosequencing. HLA-G methylation and HPV infection status of cases and controls were compared, and confounding factors were computed by t Student and non-parametric Wilcoxon tests. Comparison of HLA-G methylation between cases and controls was assessed by the Bonferroni correction. The association of HLA-G methylation with CIN2/3 was evaluated by logistic regression.
HPV prevalence was 19.6% in controls and 94.3% in CIN2/3 cases. HPV16, 31, 33, 35 and 18 were the most prevalent types. Methylation analysis of seven CpGs in the HLA-G promoter did not reveal any spontaneous de-methylation events in CIN2/3 cases (mean proportion of methylation: 75.8%) with respect to controls (mean 73.7%; odds ratio 1.01, 95% confidence interval 0.96, 1.07).
This study did not support the hypothesis that spontaneous de-methylation events in the HLA-G promoter play a primary role in promoting escape from immunosurveillance in the development of precancerous cervical lesions.
Keywords:HPV; Cervical cancer; HLA-G; Methylation
Cervical cancer is the third most common cancer and the fourth cause of cancer mortality in women worldwide [1]. Screening programs in industrialized countries have drastically reduced the incidence and mortality of cervical cancer, and research in the last decades has greatly improved our capability to detect the etiologic viral component of the disease, human papillomavirus (HPV), and contributed to a decrease in rates of progression to cervical cancer.
Nevertheless, cervical cancer remains a public health issue. Several unresolved aspects of the natural history of the disease also remain. Among these is the mechanism behind the development of cervical cancer. Indeed, only a small proportion of women infected with HPV ever develop cervical cancer; in most women the infection regresses spontaneously. While the causal association between persistence of HPV infection and risk of developing cervical lesions is recognized [2], the events that promote or prevent this persistence in cervical cells has not yet been identified. Several hypotheses exist, one of which is the capability of the immune system to control and resolve HPV infection. One recently considered hypothesis focuses on human leucocyte antigen-G (HLA-G), a tolerogenic protein involved in the control of immune response, due to its reported aberrant expression in a wide variety of cancer cells [3-6]. HLA-G is a non-classical gene of the major hystocompatibility complex that codes for a protein involved in immunosuppressive mechanisms. HLA-G inhibits cell-mediated immunity through interaction with receptors expressed on lymphoid, myeloid and natural killer cells [7,8]. It plays a primary role in immune tolerance, and has been widely described in fetal-maternal tolerance [9]. The HLA-G protein is physiologically present in fetal [10] and immature (thymus [11]) cells, and in a small number of adult tissues [5,11-13], but it is not commonly expressed in mature normal cells. HLA-G re-expression has been suggested as a mechanism of viral [14] and tumor [3-6] escape from immunosurveillance. Recent evidence [15] showed that HLA-G expression increased with grade of precancerous cervical lesions, with the highest expression found in cervical cancer. Since promoter methylation has been described as one of the crucial mechanisms that regulate gene expression [16], occurrence of spontaneous de-methylation events in the HLA-G promoter was postulated to explain the re-expression of this protein in adult cells. De-methylation events have been reported in the HLA-G promoter of ovarian tumor cells compared to normal ovarian epithelial cells [17].
The present study aimed to investigate the association of HPV infection and development of cervical intraepithelial neoplasia grades 2 (CIN2) and 3 (CIN3) with HLA-G promoter methylation in a Brazilian population. Association with the characteristics of the study population was also evaluated.
Materials and Methods
Study population and sample collection
A case–control study was set up in the framework of the collaboration among the Unit of Cancer Epidemiology in Turin, Italy; the Laboratory of Immunogenetics and Hystocompatibility (LIGH) in Curitiba, Brazil; the Department of Gynecology and Obstetrics, at the Federal University of Paraná, Infectious Diseases in Gynecology and Obstetrics Sector; and the Department of Cervical Pathology, Hospital Erasto Gaertner, in Curitiba, Brazil. The study was approved by the Ethical Committee for Clinical Research of the Hospital Erasto Gaertner (protocol CEP: 81520–060, P.P No 1943). All participating women were informed about the study purpose and signed an informed consent form.
Women were recruited in Curitiba, where the prevalence of HPV infection and cervical lesions has been reported to be higher than in Turin [18-20]. Under the supervision of the LIGH, women aged 15 to 47 years were recruited: local gynecologists working at three reference centers for cervical cancer screening collaborated to enroll women. Some women were also recruited through awareness campaigns for adhesion to cervical screening. Women over 47 years of age were not included to avoid atrophy or dysplasia associated with menopause, although the suggested impact of menopausal hormonal status on cervical dysplasia due to a weakened immune response, specifically in HPV-positive menopausal women, has not been properly documented.
A total of 789 women were recruited: 510 had normal cervical cytology and were classified as controls; 279 had histologically confirmed CIN2 (N = 150) or CIN3 (N = 129) after loop electrosurgical excision procedure or cold knife conization, and were classified as CIN2/3 cases. A cervical sample was collected from all study women. Cervical cell samples were collected from controls at collection for cytology using the cytobrush provided in the collection kit (Digene sample collection kit, Qiagen, Hilden, Germany), which was then placed into a tube containing sample transport medium (STM, Qiagen). A cervical sample was analogously collected in STM from CIN2/3 cases at loop electrosurgical excision procedure or cold-knife conization.
Study women were administered a questionnaire by interview, which collected information on demographic, sexual and lifestyle factors, including age, education level, ethnic group, age at first sexual intercourse, lifetime number of sexual partners, number of full-term pregnancies, smoking status and number of cigarettes smoked per day. Study women were classified into four ethnic groups based on their replies in the questionnaire: Euro-Descendent, Afro-Descendent, Brazilian Mixed and Asian. There is a general consensus as to the definition of a person of Brazilian Mixed ethnicity in the Genetic Department of the Federal University of Paranà in Curitiba, and following that consensus, all study women with a multiracial origin, i.e., a miscegenation of Euro-Descendent (mostly), Afro-Descendent, Amerindian and East Asian, were classified as Brazilian Mixed [21]. Women were assigned to the corresponding ethnic group following interview at recruitment.
DNA extraction
Genomic DNA was extracted from cervical cell samples using the commercial purification system QIAmp DNA Mini Kit (Qiagen) according to the manufacturer's instructions. The final elution in 100 μl of the provided elution buffer was repeated twice to increase the DNA yield. The DNA concentration was evaluated by a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). DNA adequacy was checked by amplification of a β-globin housekeeping gene sequence of 268bp, as previously described [22]. After gel electrophoresis onto a 2% agarose gel stained with ethidium bromide, amplicons were visualized by ultraviolet trans-illumination. The amplicon of the β-globin gene fragment was detected in all the study samples. Purified DNA was stored at −80°C.
HPV detection
HPV detection was performed in Turin, Italy, on the genomic DNA extracted from cervical samples by consensus primer GP5+/6 + −mediated PCR [23], which allows to detect a broad variety of HPV types. PCR reaction was performed in a total volume of 25 μl containing buffer (KCl) 50 mM, Tris-HC1 10 mM pH 8.3, dNTP 200 μM, MgCl2 3.5 mM, Taq polymerase 1U, GP5+/6+ 50 pmol and DNA 5 μl. The following amplification profile was used: 94°C for 9 min, followed by 40 cycles of denaturation at 94°C for 20s, annealing at 38°C for 30s, extension at 71°C for 80 s. A final extension of 4 min at 71°C was performed.
HPV genotyping
HPV-positive samples were genotyped by multiplex PCR, in order to detect the seven oncogenic HPV types that are prevalent, and more associated with cervical cancer in Brazil [24]: HPV16, 18, 31, 33, 35, 45 and 52. Multiplex PCR was performed as previously described [25], with the exception of HPV16, for which a different primer set was used [26]. Briefly, the PCR mix was carried out in a final volume of 25 μl containing buffer (KCl) 1X, MgCl2 2 mM, dNTPs 200 μM, 0.4 μM both primers, Taq polymerase 2.5U, and DNA 3 μl. The amplification profile was as follows: 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 30s, annealing at 56°C for 30 s, extension at 72°C for 45 s. A final extension at 72°C for 4 min was performed.
The set of primers used for the multiplex PCR were as follows: HPV16 sense 5′AAGGGCGTAACCGAAATCGGT3′, antisense 5′CATATACCTCACGTCGCAG3′; HPV18 sense 5′CACTTCACTGCAAGACATAGA3′, antisense 5′GTTGTGAAATCGTCGTTTTTCA3′;
HPV31 sense 5′GAAATTGCATGAACTAAGCTCG3′,antisense 5′ACATATACCTTTGTTT-GTCAA3′; HPV33 sense 5′ACTATACACAACATTGAACTA3′, antisense 5′GTTTTTACACG-TCACAGTGCA3′; HPV35 sense 5′CAACGAGGTAGAAAGC-ATC3′, antisense 5′CCGA-CCTG TCCACCGTCCACCG3′; HPV45 sense 5GATGGAAAAGTGCATTACAGG3, antisense 5′ACCTCTGTGCGTTCCAATGT3′;HPV52 sense 5′TAAGGCTG CAGTGTGTGCAG3′, antisense 5′CTAAT AGTTATTTCACTTAATGGT3′.
Samples positive at HPV detection, but negative at PCR genotyping were re-tested by reverse-line blot hybridization using the Digene HPV Genotyping RH (Qiagen) commercial kit, according to the manufacturer's protocol. The kit employed biotynilated primers (GP5+/GP6+) and the assay targets 18 HPV types [27], including those classified by the International Agency for Research on Cancer (IARC) as carcinogenic (Group 1 carcinogen, HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59), probably carcinogenic (Group 2A carcinogen, HPV68), and as possibly carcinogenic to humans (Group 2B carcinogen, HPV26, 53, 66, 73, 82) [28]. PCR biotinylated products (10 μl) were denatured and hybridized with type-specific oligonucleotide probes immobilized as parallel lines on nitrocellulose membrane strips. The hybrids were detected with alkaline phosphatase–streptavidin conjugate and substrate (5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium), resulting in a purple precipitate at positive probe lines. After drying, the strips were analyzed by visually comparing them with the interpretation grid supplied in the kit; the presence of a clearly visible line was considered a positive reaction.
A biotinylated poly (dT) control for conjugate reaction was applied to each strip to ensure the validity of the test and proper alignment of the strips on the interpretation sheet.
HLA-G methylation – pilot analysis
The analysis of the HLA-G methylation was performed as a pilot analysis on the first set of samples shipped to Italy from Brazil (N = 172, 76 controls and 96 CIN2/3 cases). The analysis was performed through bisulfite modification and pyrosequencing.
Bisulfite modification
Sodium bisulfite modification converts unmethylated cytosine into uracyl, but leaves the methylated cytosines unchanged. Genomic DNA (1μg) from cervical samples was converted using the EpiTect Bisulfite commercial kit (Qiagen) according to the manufacturer's instructions.
Pyrosequencing was used to evaluate HLA-G methylation status and to quantify the methylation of each individual CpG investigated. It was performed on a Pyromark Q24 using PyroMark Gold Q24 (Qiagen) reagents. The assay allowed the quantification of methylation of the seven CpGs we sited in the HLA-G promoter, using primers designed with the Pyromark Assay Design software (Qiagen) according to the HLA-G reference sequence GenBank J03027.1. Preliminary PCR was performed, targeting a 441 bp sequence of the HLA-G promoter, using primers with the following sequences: sense 5′GGGAGGTAGGGAGTTTAGTT-TA3′, antisense Biotin-5′CCATAACCACCATCCTTAAC3′. The primer antisense is biotinylated to allow binding to sepharose beads during the subsequent pyrosequencing process. To improve efficiency, three different pairs of sequencing primers that targeted 2, 3 and 2 CpGs, respectively, were employed: Sequencing primer 1 (2 CpGs, positions 350 and 428) 5′GGAGTTTAGTTTAGGGATAG3′, Sequencing primer 2 (3 CpGs, positions 494, 512 and 523) 5′ATTTAGGGAGATATTGAGA3′, Sequencing primer 3 (2 CpGs, positions 573 and 598) 5′GGGTTTTAGGTTTTATAGG3′.
The preliminary PCR reaction was performed in a total volume of 35 μl containing buffer (KCl) 1X, MgCl2 2 mM, dNTPs 200 μM, 0.5 μM each primer (antisense biotinylated), Taq polymerase 1.75U and 6 μl converted DNA with the following cycling profile: 95°C for 1 min followed by 45 denaturation cycles at 54°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 1 min, final extension at 72°C for 10 min. Amplicons were analyzed by gel electrophoresis on a 2% agarose gel stained with ethidium bromide and visualized by ultraviolet trans-illumination. The residual PCR product (28 μl) was added to 12 μl of dH2O and incubated under shaking with 37 μl of binding buffer pH 7.6 (10 mM Tris–HCl; 2 M NaCl; 1 mM EDTA; 0.1% Tween 20) and 3 ml sepharose beads covered with streptavidin. PCR products were washed with ethanol 70%, denatured with NaOH 0.2 M and re-washed with Tris-Acetate 10 mM pH 7.6. Pyrosequencing reaction was performed in 45 μl of annealing buffer [44.82 μl of 20 mM Tris-Acetate + 5 mM MgAc2 and 0.18 μl of sequencing primer (0.3 μM)].
Quantitative methylation results were expressed as the mean of the methylation percentage of all seven CpGs investigated.
The individual methylation percentage for two CpGs, one located in a binding site for the transcription factor specificity protein 1 (Sp1), and the other located in the enhancer region, were also evaluated for their association with HPV infection and CIN2/3.
Statistical analyses
Analyses investigating the association between HLA-G methylation, HPV status, and demographic and lifestyle factors were restricted to controls, due to the fact that almost all CIN2/3 cases were HPV-positive. For HLA-G methylation analyses with sufficiently high frequency, the t Student test was used, or alternatively the non-parametric Wilcoxon test. To evaluate whether there was a difference in the percentage of methylation between CIN2/3 cases and controls, and to further compare subgroups of the study population (i.e., controls vs. CIN2 cases, controls vs. CIN3 cases, CIN2 cases vs. CIN3 cases), the Bonferroni correction for multiple comparisons was used. Logistic regression models, adjusted for age, education level, ethnic group and smoking status, were fitted to evaluate the effect of HLA-G methylation on the development of cervical cancer [crude and adjusted odds ratios (ORs) are reported].
Characteristics of the study population
During the study period 789 women were recruited, including 510 controls and 279 CIN2/3 cases (150 CIN2, 129 CIN3). CIN2/3 cases and controls had a comparable mean age (32 years for both), education level, age at first sexual intercourse (16 years for cases, 17 years for controls) and number of full-term pregnancies (Table 1). The ethnic group Brazilian Mixed showed the highest prevalence of HPV infection in both CIN2/3 cases and controls. Slight differences emerged in the distribution of CIN2/3 cases and controls by ethnic group, sexual factors, and smoking status. The most represented ethnic group in CIN2/3 cases was Brazilian Mixed (58.1%), while in controls it was Euro-Descendent (46.9%). Among CIN2/3 cases, 74.6% reported a total number of partners between 2 and 10, while the corresponding percentage among controls was 53.9%. Current smokers were more common among CIN2/3 cases (34.4%) than controls (15.9%) (Table 1).
Table 1. Characteristics of the study population
HPV detection and type distribution
We found a HPV prevalence of 19.6% in controls and 94.3% in CIN2/3 cases (Table 2). HPV-positive CIN2/3 cases and controls showed a higher frequency of single HPV infections (cases 66.2%, controls 65%) than multiple infections (cases 32.7%, controls 29%). A proportion of samples could not be typed (cases 1.14%, controls 6%) by our methods. Among the most frequent genotypes (HPV16, 18, 31, 33, 35, 45 and 52, IARC Group 1 carcinogen [28]) a higher prevalence was observed for HPV16, 31 and 33 for both CIN2/3 cases and controls, followed by HPV35, 52, 18 and 45 in CIN2/3 cases, and HPV18, 35, 52 and 45 in controls (Table 3). A cumulative frequency of 9.3% and 4.9% was detected in CIN2/34 cases and controls respectively, for HPV types with a lower prevalence: HPV39, 51, 56, 58 and 59, IARC Group 1 carcinogen; HPV68, IARC Group 2A carcinogen; and HPV26, 53, 66, 73 and 82, IARC Group 2B carcinogen [28].
Table 2. Frequency of human papillomavirus (HPV) infection in the study population
Table 3. Distribution of selected human papillomavirus (HPV) types in the study population
Frequency of HPV infection by selected characteristics in controls
Table 4 reports the frequency of HPV infection by selected characteristics among controls. HPV infection was inversely associated with age (p < 0.001), and was positively associated with lifetime number of sexual partners (p = 0.001), smoking status (p = 0.09) and Brazilian Mixed ethnic group (p = 0.09). There was no evidence of association with education level (p = 0.31).
Table 4. Frequency of HPV infection by characteristics of controls
HLA-G methylation status – pilot analysis
We calculated the mean percentage of methylation of the seven evaluated CpGs of the HLA-G promoter, among the 76 controls and 96 CIN2/3 cases included in the pilot analysis. We did not find lower methylation levels among CIN2/3 cases as expected, instead they were slightly higher (Table 5). Demographic and lifestyle factors, specifically smoking status and ethnicity, were not associated with mean HLA-G methylation (data not shown). Moreover, no decrease was found in overall methylation when HPV-positive and HPV-negative controls were compared (data not shown).
Table 5. Overall mean methylation percentage in the study population
The CpGs located in regulatory sites of the HLA-G promoter, i.e., the binding site for the transcription factor Sp1 (sequence position 573), and the enhancer region (sequence position 598), were also evaluated individually with the aim of highlighting any relevant decrease in methylation that could directly affect gene transcription. The results, stratified for HPV positivity in controls, and for CIN2 and CIN3 in cases, are shown in Table 6. There was no evidence of any differences in the mean methylation percentage between CIN2/3 cases and controls for the CpG located in the binding site for transcription factor Sp1 (crude OR = 1.01, 95% confidence interval [CI]: 0.97,1.06; adjusted OR = 1.01, 95% CI: 0.96,1.07), while the mean methylation percentage for the CpG located in the enhancer region was slightly higher in CIN2/3 cases than in controls (crude OR = 1.04, 95% CI: 1.00,1.08; adjusted OR = 1.03, 95% CI: 0.99,1.08). These results were confirmed when the analysis was restricted to HPV-positive controls for both individually analyzed CpGs (binding site for transcription factor Sp1: crude OR = 1.01, 95% CI: 0.96,1.07; adjusted OR = 0.99, 95% CI: 0.93,1.07; enhancer region: crude OR = 1.03, 95% CI: 0.98,1.08; adjusted OR = 1.01, 95% CI: 0.95,1.06).
Table 6. Mean methylation percentage in specific CpG sites
When a threshold of the median percentage of methylation (33%) was applied for the CpG located in the enhancer region (Table 7), we obtained a higher proportion of CIN2/3 cases (61.5%) than controls (47.4%) with methylation levels over the threshold (crude OR = 1.77, 95% CI: 0.96,3.26; adjusted OR = 1.40, 95% CI: 0.71,2.76).
Table 7. Distribution of the study population according to methylation level of the CpG located in the enhancer
A case–control study was set up in a Brazilian female population to investigate the relationships between HPV infection, prevalence of HPV types, methylation status in the gene promoter of the tolerogenic HLA-G protein and high-grade cervical lesions.
We explored the occurrence of spontaneous de-methylation in the HLA-G promoter as a surrogate of re-expression of the HLA-G protein in HPV-infected cells, as the HLA-G protein is a recognized inducer of a tolerogenic effect and tumor escape from immunosurveillance. By exploring this in a case–control study, our goal was to try and highlight any association of decreased methylation with the carcinogenic process. Indeed, according to the hypothesis of the association with, and role of de-methylation of the HLA-G protein on oncogenic progression, our controls were expected to show high HLA-G methylation, and our CIN2/3 cases were expected to show low HLA-G methylation. We did not include CIN1 in our study, as it is less informative given its high rate of spontaneous regression [29-33]. Similarly, a recent publication exploring the association between HLA-G expression and cervical cancer progression also focused on high-grade lesions only [34].
We did not consider invasive cervical cancer in the present study, since the occurrence of HLA-G expression in cervical cancer cells is still controversial in the scientific literature. Some authors reported variable HLA-G expression in cervical cancer cells [15,35,36], others very low or no expression [37,38], suggesting that if methylation status plays a role in promoting carcinogenesis, it probably acts in the early phases, rather than in the advanced phases of the process. For these reasons we focused our investigation on comparing normal cervical cells with high-grade cervical lesions, in which the carcinogenic process, if it has started, is more frequently active.
The HLA-G gene is silenced under physiological conditions independently from proliferative or differentiative status of normal cells [10,11,39,40]. Therefore the collection of cervical cells by cytobrush should not have biased the results of our methylation analyses even if many dead epithelial cells were present. As expected, we did find high HLA-G methylation levels in normal cervical cells, which in this context can be considered appropriate controls.
The percentage of HPV-positive CIN2/3 cases was very high, as expected. The low proportion of HPV-negative samples among CIN2/3 cases is consistent with previous reports of HPV DNA-negative CIN2 and CIN3, even though an incorrect histological diagnosis was suspected [41-43]. Among controls, HPV positivity was about 20%, which is in agreement with the mean prevalence described in Brazilian populations (10.4%-24.5%) [44]. Frequency analysis of population characteristics and HPV infection were conducted in controls only, as almost all CIN2/3 cases were HPV-positive. This analysis confirmed the risk factors for HPV infection already described in the literature, including young age, low education level, smoking and a higher lifetime number of sexual partners. Brazilian Mixed, was the ethnic group that showed the highest prevalence of HPV infection, both in CIN2/3 cases and controls.
The analyses of HPV type distribution in our study population showed a slight increase in the prevalence of some types compared to the distribution that has been previously described in Brazil [24], but were in agreement with the reported prevalence for South America in a worldwide analysis of HPV type distribution [45].
To our knowledge, this is the first study on HLA-G methylation and its association with high-grade cervical lesions. We found a high mean percentage of methylation in both CIN2/3 cases and controls, without substantial differences. This is not in line with data reported for some other cancer types. In ovarian cancer, malignant cells were reported to show higher levels of methylation than normal control cells in some CpG sites, even though expression of the protein did not properly correlate with the methylation status [17]. In renal cancer cells, HLA-G expression via partial de-methylation of its promoter was counted among the strategies used by malignant cells to escape immune response [46]. Indeed HLA-G expression is widely documented in renal cancer cells, while no expression has been reported in normal renal cells [47-49]. Although HLA-G expression has been documented in several other cancer sites, i.e. cervical cancer [15,34-37], melanoma [49-51], breast [49,52-54], colorectal [55-57], gastric [57-60], esophageal [57,61,62], lung [57,63,64], and other cancers [49], the implications of HLA-G methylation on the expression of the protein have not been described.
It has been suggested that even a single CpG dinucleotide could represent a regulatory sequence highly predictive of the explored outcome [65]. Thus we also restricted our association analyses to two specific CpGs that might play a regulatory role, one located in the binding site of transcription factor Sp1, and one in the enhancer region. However, no significant differences were found in methylation between CIN2/3 cases and controls. Evidence of de-methylation events in CIN2/3 cases with respect to controls, which could suggest a re-expression of the HLA-G protein in the cells of cervical high-grade lesions, was not observed. If anything, we found a slight increase in the overall methylation percentage in CIN2/3 cases. This increase was more evident when the analysis was restricted to the CpG located in the enhancer region, specifically when a threshold was set for the methylation level. Although it seems paradoxical, this could be explained by the concomitant global DNA methylation induced by persistent HPV infection [66]. Recent studies have suggested that HPV can modulate DNA methylation patterns in order to control cell proliferation. The oncogenic HPV E7 protein can bind DNA methyltransferases, stimulating their activity [67]. Indeed, many genes have been shown to be hypermethylated in neoplastic cervical lesions [68]. We cannot exclude that the overlap of these hypermethylation events could overshadow low levels of de-methylation in the HLA-G promoter that may be present, or that may occur earlier in the carcinogenesis process. However the impact of low levels of de-methylation is unlikely to be functionally relevant.
Our findings of low HLA-G hypermethylation in CIN2/3 cases also suggested that alterations in methylation can be detected in the cervical samples of subjects with disease despite contamination of the sample by normal cells. This suggests that the results we obtained in our pilot analysis on HLA-G methylation are sufficiently suggestive of the absence of detectable de-methylation events in the HLA-G promoter, without requiring an extension of the analyses to the entire study population. Realistically, as has been previously reported, other mechanisms like histone modifications [69], polymorphisms [70] or miRNA [71] may modify HLA-G expression.
We compared population characteristics by HPV status and HLA-G promoter methylation, as some characteristics, including ethnicity, have been reported to affect either one or both of them [72]. If we had found an association, it would have been appropriate to evaluate our results in relation to the demographic and lifestyle characteristics of both cases and controls. However, while we could confirm known associations of some characteristics with HPV infection, we did not find any association with HLA-G methylation; we did not observe significant differences between CIN2/3 cases and controls, nor between HPV-positive and HPV-negative control women.
This study did not support the hypothesis that spontaneous de-methylation events in the HLA-G promoter play a primary role in the development of precancerous cervical lesions through HLA-G re-expression and consequential promotion of viral and tumor escape from immunosurveillance. Nor was any association found between HLA-G methylation and various demographic and lifestyle factors.
CI: Confidence interval; CIN: Cervical intraepithelial neoplasia; HLA-G: Human leucocyte antigen-G; HPV: Human papillomavirus; LIGH: Laboratory of Immunogenetics and Hystocompatibility; OR: Odds ratio.
Competing interests
The authors declare no competing interets.
Authors' contributions
AGT, MGB, VF, LDM, HML, LR and FM participated in the design of the study. MBSX and RS extracted cervical DNA samples. AGT and VT set up the study database. NSC, CAM enrolled study women and provided cytological and histological diagnoses. VT, CG, MT, VF performed molecular analyses. AGT, VF, VT, LDM interpreted the results. LR, DZ performed statistical analyses. AGT drafted the manuscript. All authors read and approved the final manuscript.
We thank all those in the multidisciplinary group in Curitiba who collaborated in the recruitment of the study population. The study was partially supported by the Alliance FUNPAR-LIGH, the Compagnia di San Paolo/Firms and the Piedmont Region.
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Yoshinouchi M, Hongo A, Nakamura K, Kodama J, Itoh S, Sakai H, Kudo T: Analysis by multiplex PCR of the physical status of human papillomavirus type 16 DNA in cervical cancers.
Geraets DT, Heideman DA, de Koning MN, Snijders PJ, Meijer CJ, van Doorn LJ, Quint WG: High genotyping concordance between the Digene HPV Genotyping RH Test and the Reverse Line Blot genotyping assay on GP5+/6 + −PCR products.
Bouvard V, Baan R, Straif K, El Ghissassi F, Benbrahim-Tallaa L, Guha N, Freeman C, Galichet L, Gogliano V, on behalf of the WHO IARC in Cancer Monograph working group: A review of human carcinogens –Part B: biological agents.
Bansal N, Wright JD, Cohen CJ, Herzog TJ: Natural history of established low-grade cervical intraepithelial (CIN 1) lesions.
Sastre-Garau X, Cartier I, Jourdan-Da Silva N, De Crémoux P, Lepage V, Charron D: Regression of low-grade cervical intraepithelial neoplasia in patients with HLA-DRB1*13 genotype.
Cox JT, Schiffman M, Solomon D: Prospective follow-up suggests similar risk of subsequent cervical intraepithelial neoplasia grade 2 or 3 among women with cervical intraepithelial neoplasia grade 1 or negative colposcopy and directed biopsy.
Melnikow J, Nuovo J, Willan AR, Chan BK, Howell LP: Natural history of cervical squamous intraepithelial lesions: a meta-analysis.
Ostor AG: Natural history of cervical intraepithelial neoplasia: a critical review.
Li XJ, Zhang X, Lin A, Ruan YY, Yan WH: Human leukocyte antigen-G (HLA-G) expression in cervical cancer lesions is associated with disease progression.
Rodríguez JA, Galeano L, Palacios DM, Serrano ML, Bravo MM, Combita AL: Altered HLA Class I and HLA-G Expression Is Associated with IL-10 Expression in Patients with Cervical Cancer.
Zheng N, Wang CX, Zhang X, Du LT, Zhang J, Kan SF, Zhu CB, Dong ZG, Wang LL, Wang S, Li W: Up-regulation of HLA-G expression in cervical premalignant and malignant lesions.
Zhou JH, Ye F, Chen HZ, Zhou CY, Lu WG, Xie X: Altered expression of cellular membrane molecules of HLA-DR, HLA-G and CD99 in cervical intraepithelial neoplasias and invasive squamous cell carcinoma.
Poláková K, Russ G: Expression of the nonclassical HLA-G anti- gen in tumor cell lines is extremely restricted.
Amiot L, Ferrone S, Grosse-Wilde H, Seliger B: Biology of HLA-G in cancer: a candidate molecule for therapeutic intervention?
Carosella ED, Favier B, Rouas-Freiss N, Moreau P, LeMaoult J: Beyond the increasing complexity of the immunomodulatory HLA-G molecule.
González-Bosquet E, Esteva C, Muñoz-Almagro C, Ferrer P, Pérez M, Lailla JM: Identification of vaccine human papillomavirus genotypes in squamous intraepithelial lesions (CIN2-3).
Castle PE, Cox JT, Jeronimo J, Solomon D, Wheeler CM, Gravitt PE, Schiffman M: An analysis of high-risk human papillomavirus DNA-negative cervical precancers in the ASCUS-LSIL Triage Study (ALTS).
Sigurdsson K, Taddeo FJ, Benediktsdottir KR, Olafsdottir K, Sigvaldason H, Oddsson K, Rafnar T: HPV genotypes in CIN 2–3 lesions and cervical cancer: a population-based study.
Roteli-Martins CM, de Carvalho NS, Naud P, Teixeira J, Borba P, Derchain S, Tyring S, Gall S, Diaz A, Blatter M, Shier RM, Romanowski B, Quint WG, Issam J, Galindo C, Schuind A, Dubin G: Prevalence of human papillomavirus infection and associated risk factors in young women in Brazil, Canada, and the United States: a multicenter cross-sectional study.
Clifford GM, Gallus S, Herrero R, Muñoz N, Snijders PJ, Vaccarella S, Anh PT, Ferreccio C, Hieu NT, Matos E, Molano M, Rajkumar R, Ronco G, de Sanjosé S, Shin HR, Sukvirach S, Thomas JO, Tunsakul S, Meijer CJ, Franceschi S, IARC HPV Prevalence Surveys Study Group: Worldwide distribution of human papillomavirus types in cytologically normal women in the International Agency for Research on Cancer HPV prevalence surveys: a pooled analysis.
Seliger B: Immune escape mechanisms of renal cell carcinoma.
Seliger B, Schlaf G: Structure, expression and function of HLA-G in renal cell carcinoma.
Bukur J, Malenica B, Huber C, Seliger B: Altered expression of nonclassical HLA class 1B antigens in hu- man renal cell carcinoma and its association with impaired immune response.
Rouas-Freiss N, Moreau P, Menier C, LeMaoult J, Carosella ED: Expression of tolerogenic HLA-G molecules in cancer prevents antitumor responses.
Rebmann V, Wagner S, Grosse-Wilde H: HLA-G expression in malignant melanoma.
Paul P, Rouas-Freiss N, Khalil-Daher I, Moreau P, Riteau B, Le Gal FA, Avril MF, Dausset J, Guillet JG, Carosella ED: HLA-G expression in melanoma: a way for tumor cells to escape from immunosurveillance.
Dong DD, Yie SM, Li K, Li F, Xu Y, Xu G, Song L, Yang H: Importance of HLA-G expression and tumor infiltrating lymphocytes in molecular subtypes of breast cancer.
Provatopoulou X, Kalogera E, Sagkriotis A, Zagouri F, Nonni A, Zografos GC, Gounaris A: Soluble human leukocyte antigen-G expression in patients with ductal and lobular breast malignancy.
de Kruijf EM, Sajet A, van Nes JG, Natanov R, Putter H, Smit VT, Liefers GJ, van den Elsen PJ, van de Velde CJ, Kuppen PJ: HLA-E and HLA-G expression in classical HLA class I-negative tumors is of prognostic value for clinical outcome of early breast cancer patients.
Ye SR, Yang H, Li K, Dong DD, Lin XM, Yie SM: Human leukocyte antigen G expression: as a significant prognostic indicator for patients with colorectal cancer.
Zhu CB, Wang CX, Zhang X, Zhang J, Li W: Serum sHLA-G levels: a useful indicator in distinguishing colorectal cancer from benign colorectal diseases.
Cao M, Yie SM, Liu J, Ye SR, Xia D, Gao E: Plasma soluble HLA-G is a potential biomarker for diagnosis of colorectal, gastric, esophageal and lung cancer.
Yie SM, Yang H, Ye SR, Li K, Dong DD, Lin XM: Expression of human leukocyte antigen G (HLA-G) correlates with poor prognosis in gastric carcinoma.
Du L, Xiao X, Wang C, Zhang X, Zheng N, Wang L, Zhang X, Li W, Wang S, Dong Z: Human leukocyte antigen-G is closely associated with tumor immune escape in gastric cancer by increasing local regulatory T cells.
Ishigami S, Natsugoe S, Miyazono F, Nakajo A, Tokuda K, Matsumoto M, Okumura H, Douchi T, Hokita S, Aikou T: HLA-G expression in gastric cancer.
Lin A, Zhang X, Zhou WJ, Ruan YY, Xu DP, Wang Q, Yan WH: Human leukocyte antigen-G expression is associated with a poor prognosis in patients with esophageal squamous cell carcinoma.
Yie SM, Yang H, Ye SE, Li K, Dong DD, Lin XM: Expression of HLA-G is associated with prognosis in esophageal squamous cell carcinoma.
Schütt P, Schütt B, Switala M, Bauer S, Stamatis G, Opalka B, Eberhardt W, Schuler M, Horn PA, Rebmann V: Prognostic relevance of soluble human leukocyte antigen-G and total human leukocyte antigen class I molecules in lung cancer patients.
Yie SM, Yang H, Ye SR, Li K, Dong DD, Lin XM: Expression of human leucocyte antigen G (HLA-G) is associated with prognosis in non-small cell lung cancer.
Claus R, Lucas DM, Stilgenbauer S, Ruppert AS, Yu L, Zucknick M, Mertens D, Bühler A, Oakes CC, Larson RA, Kay NE, Jelinek DF, Kipps TJ, Rassenti LZ, Gribben JG, Döhner H, Heerema NA, Marcucci G, Plass C, Byrd JC: Quantitative DNA methylation analysis identifies a single CpG dinucleotide important for ZAP-70 expression and predictive of prognosis in chronic lymphocytic leukemia.
Szalmás A, Kónya J: Epigenetic alterations in cervical carcinogenesis.
Burgers WA, Blanchon L, Pradhan S, de Launoit Y, Kouzarides T, Fuks F: Viral oncoproteins target the DNA methyltransferases.
Wentzensen N, Sherman ME, Schiffman M, Wang SS: Utility of methylation markers in cervical cancer early detection: appraisal of the state-of-the-science.
Polakova K, Bandzuchova E, Tirpakova J, Kuba D, Russ G: Modulation of HLA-G expression.
Castelli EC, Mendes-Junior CT, Veiga-Castelli LC, Roger M, Moreau P, Donadi EA: A comprehensive study of polymorphic sites along the HLA-G gene: implication for gene regulation and evolution.
Veit TD, Chies JA: Tolerance versus immune response – microRNAs as important elements in the regulation of the HLA-G gene expression.
Wiley KL, Treadwell E, Manigaba K, Word B, Lyn-Cook BD: Ethnic Differences in DNA Methyltransferases Expression in Patients with Systemic Lupus Erythematosus.
J Clin Immunol 2012, : .
Epub October 9
Pre-publication history
The pre-publication history for this paper can be accessed here:


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Can we just cuddle instead :D ?
Gf and I are trying this tomorrow. Should be interesting!
I wanna fuck, now
Too much frosting and by itself would make me sick, then I wouldn't want to fuck anyone 😓
funfetti XD...I prefer fudge icing...makes the tongue work harder
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calorie compensation. clever.
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As soon as he heard that a massive tornado ripped apart his hometown, Brent Beshore was ready to hop into his car that Sunday night and drive to Joplin.
Beshore's father, a business executive and fifth-generation Joplin resident, persuaded him not to make the trip into the disaster area so soon.
Feeling desperate for information about family and friends and figuring that many others were in the same predicament, the entrepreneur and marketing whiz kid created a Facebook page and called it, "Joplin, MO Tornado Recovery."
Late that night and into the early morning hours, Beshore watched the messages flow through the social network and the "likes" accumulate at an exponential pace — from dozens to hundreds to thousands to 40,000 after one day and more than 100,000 after a few days.
Donations to aid organizations kept pace and rose to more than $1 million in four days.
"In the past I've questioned the value of social media," the 28-year-old CEO of AdVentures wrote on his blog at innovateandcurate.com. "Like any form of communication, it can be misused and abused. It can be a distraction. It can give one's life the illusion of value. After this week, I will never challenge the power of social media."
Most significantly, Beshore began seeing Joplin residents using the page as a community bulletin board — a headquarters of sorts — for posting updates and relaying valuable information faster than news media and support services on the ground.
"I watched the Facebook page as one person posted asking for someone to check in on her elderly grandfather," Beshore wrote. "Someone responded, checked on him and reported back. He was OK. I couldn't believe what I was watching as this scenario was replayed hundreds of times. People helping people."
After setting up the Facebook page, Beshore emailed Tim Rich, a friend and the executive director Heart of Missouri United Way, where Beshore is also a board member. He sent the following message: "You should consider opening up a special fund for the Joplin, Mo., tornado disaster."
Rich responded that night, pledged his support and agreed to set up a special fund into which 100 percent of the proceeds would go to Joplin's tornado relief with zero overhead. That greased the wheels for what would become the first international relief fund for Joplin tornado victims.
By Monday morning, Rich had the text message campaign and the donation PayPal page already up and running.
The rest of the week was a blur for Rich and Beshore, who learned that at least four of his friends had died and many more lost their homes. The duo and Marty Siddall, general manager of KOMU TV 8, organized a primetime telethon that aired that Thursday night.
National news media outlets picked up the story of the Facebook page and the ensuing fundraising campaign. Oprah Winfrey told her Twitter followers about the campaign, and the Oakridge Boys promoted it. Other musical groups, some with national reach, pledged to dedicate songs, do benefit concerts and spread the word via social media.
Donations came in from around the world: Saudi Arabia, Sweden and South Africa, to name a few. The Sam's Club Foundation pitched in $200,000 targeting long-term small-business recovery efforts.
Rich also credited KOMU's quick response.
"The stunning thing was that KOMU pulled together that telethon in two days," he said. "I've worked with them in the past, and they're a great crew, but normally it takes us three or four days to put together a telethon."
Rich said Matt Garrett, KOMU's director of audience development, told him that in his 20 years of doing telethons, this was the first time he'd heard "a phone bank ring steady from beginning to the end." The calls were still coming in when the telethon was scheduled to end, so they extended the show by an hour.
Combined with online and text pledges, the campaign raised, as of last Tuesday, $1.34 million. The "Joplin, MO Tornado Recovery" Facebook page had 172,234 "likes."
Rich estimated that mid-Missouri residents contributed at least $100,000 to the campaign.
Lessons learned
"No. 1, we were successful because we were up within six hours of the disaster, and it was being cited by all the news outlets as the place to go," Rich said. "The other thing that really made this a go for us, and put us in the lead, was we said out of the gate — and I made the executive decision — that we were going to take 100 percent of all proceeds and direct that to Joplin. We weren't going to charge any overhead or 'admin' fees."
Typically the United Way charges 12 to 13 percent in overhead and administration fees.
"Most of the donations came from online," Rich said. "This fundraiser was a phenomenon of online social networking and social media,"
In previous fundraising campaigns, the United Way has struggled to incorporate social media.
"We've been on Facebook for at least two years, and we've been trying to do Text to Give for the last year, but it hasn't been very successful," he said. "I think it was partly because we didn't have staff that were properly trained to engage it. There's been an ongoing conversation in the nonprofit world, especially among fundraisers, that social media is hyped as the way, and yet nobody's figured out how to raise dollars with it."
After witnessing the success of social media's role in the Joplin fundraiser, Rich said he has a better grasp on what needs to happen.
"Our United Way system has been based pretty much on corporate employee campaigns and payroll deduction," he said. "We have watched, across the country, those sort of campaigns slide down because the employee base is shrinking. You don't have large employers with a captive audience and a chance to speak with them.
"This suggests to me — and this was the theme this year from the national conference of the United Way — that we have to get beyond just engaging the companies," he said. "We've got to be engaging people on an individual level, and that's what social media does. It gives everybody a chance to do something and feel a part of something bigger than themselves."
Emily Eldridge, senior vice president of Pure (a media and marketing firm founded by Beshore), said social media directly buoyed the enormous response.
"You gather intel from social media; you get connected with people," she said. "It's been invaluable for us in this effort, especially because our Facebook page grew from six fans to 40,000 people overnight. So you had 40,000 people talking about, 'where are my family members, how can I get involved.' You know what your call to actions are."
Social media — part of mix
But Eldridge said social media, in itself, is not enough.
"If you look at a lot of failed relief efforts via social media, it's because they were only social-media-based," she said. "So with this effort, we tried to get the right media mix in place, use every outlet available and just get organized."
The windfall of donations bolstered by the social media campaign still pleasantly baffles Rich.
"I think the thing that was striking for me is that our annual campaign in the mid-Missouri community is about $3.5 million for ongoing programs and services in the community — health and human services," he said. "We've raised more than $1.3 million now. To raise that in five to seven days is unbelievable."
Social media also helped snag the $200,000 donation from the Sam's Club Foundation to fund a targeted grant.
"They heard about us through social networking and online marketing," Rich said. "We got a call from them on Tuesday requesting a conference call. They were looking for a way to make a contribution that would help small businesses recover long-term."
So Rich spent two days down in Joplin and worked with its United Way, city manager and Chamber of Commerce along with organizers of rebuildjoplin.org. The website bills itself as "a collaborative local effort of community, education, government and business organizations to support the long-term recovery of Joplin."
"We basically handed the torch off the them and said, 'We want an investment review team of people who will make sure these dollars go where they're most needed,'" Rich said. "We're passing that money through, and we want it to get to work as soon as possible."
The view from Joplin
Helping run rebuildjoplin.org is Mark Kinsley, a 29-year-old advertising executive in Joplin and lifelong resident.
Although Kinsley lives on the north side of town, which was unaffected by the tornado, he said the devastation spared no one.
"I've not met a single person that doesn't know someone who was affected, completely lost their home or lost their life," Kinsley said. "I lost a guy I went to high school with. He didn't make it through the storm. And another nice young man who spent some time here at the advertising agency, his father's business was next door. He had just graduated high school. He didn't make it."
Rebuildjoplin.org aggregated information from radio stations and other media sources along with social networks.
"Now it's just a process for people to try and figure out what's the next step," he said. "That's what we're helping people with, connecting them with the agencies that can help with their FEMA paperwork or help them get their birth certificate back. These people's identities were wiped away."
Kinsley added: "We're looking at the long term. When the cameras disappear and the news dies down, how do we get the resources for some of these unforeseen needs? And I think it's going to be a process of raising money."
Emily Eldridge of Pure said accountability is fundamental for a successful relief campaign.
"It's very easy to give someone $10,000 and say, 'Rebuild your business with this,' and never check in with them again; the United Way here is all about responsible giving," she said.
Rich toured Joplin with Beshore's relatives and described the landscape as "reminiscent of the aftermath of Hiroshima." The disaster, he added, "has really galvanized the community. That was clear to me."
Now, "Joplin, MO Tornado Recovery" — the Facebook page that started it all — is joining forces and pooling resources with rebuildjoplin.org, which Rich expects to stay active for at least two years.
"Six months from now, that's when the really important stuff will happen," Rich said. "To put this in perspective, Joplin's city manager said that on May 25 they started clearing debris. To get it all moved out, they will have to move one semi truck, every three minutes, around the clock, until the end of August. And that's before anyone can do any rebuilding."
Here are some of the relief efforts of local businesses and organizations for victims of the Joplin tornado:
- Crockett Engineering helped organize a group of local builders who donated, at the request of a Joplin organization, a semi-truck load of plywood.
- Central Bank, Jefferson Bank, Hawthorn Bank and Providence Bank teamed with the local chapter of the American Red Cross to match all individual donations up to a combined total of more than $13,500.
- The Columbia Board of Realtors collected at least 4,000 pairs of shoes for Joplin residents.
- Telephone and Internet service provider Socket Telecom provided free access to the Internet to help Joplin tornado victims connect to family members. The company's technical service also was made available to everyone using the free connection. Socket reports that as of June 3, more than 165 hours of free Internet access have been used. Socket also gathered more than $1,200 in cash donations as well as clothes and supplies.
- Campus Bar and Grill and its employees donated $1,200 to the Heart of Missouri United Way.
- The Callaway Bank is accepting donations of its members' reward points to help the bank's relief effort. Donations are given to either the Heart of Missouri United Way or the American Red Cross.
- Viewfinder Graphix of Rocheport will repair or restore for free photos damaged in either the Joplin or Sedalia tornadoes. Visit viewfindergraphix.com for more information.
- Geosyntec Consultants donated $10,000 to the American Red Cross.
- The District held a benefit show at The Blue Note with proceeds going to the Red Cross Greater Ozarks Regional Chapter.
- The University of Missouri partnered with Heart of Missouri United Way to sell tornado relief T-shirts. More than 15,000 T-shirts have been sold, totaling more than $165,000.
- In addition to personal contributions from Poppy employees, the store also placed a collection jar on its sales counter to raise funds from customers for the Heart of Missouri United Way.
- The employees of ABC Laboratories conducted a companywide fundraiser and donated $2,425 to the Heart of Missouri United Way.
- A group of 11 members of Blue Ridge Christian Church took two vans and two horse trailers full of donated supplies to Joplin, then stayed for an additional eight hours and volunteered at Joplin Family Worship Center as well as at the Red Cross Shelter at Missouri Southern State University.
- The Food Bank for Central and Northeast Missouri is sending weekly truckloads of donations collected in mid-Missouri.
- Harpo's Bar held its Joplin Relief Fundraiser with a happy hour and live bank. Donations were accepted at the door, bartenders donated their tips and 20 percent of the evenings sales were donated to the American Red Cross.
- Fletcher Honda is serving as a drop-off point for donations and has collected more than $500 and more than $3,500 worth of supplies.
- Check Please Loans collected more than 500 pounds of food and baby and pet items.
- University Subaru teamed with Chase Daniel, a New Orleans Saints and former MU quarterback, to raise money for the tornado relief efforts.


text:
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text:
Starbucks Offering Free Coffee and Tea on Earth Day
Categories: This Caffeinated Orange
Select Starbucks locations will be pouring your choice of hot coffee or tea that day just for bringing in your own travel mug. The website does not list specific hours, nor if a purchase is required, so the best thing to do is call your local caffeine fix and ask. Chances are it will be a win-win for everyone: you, Starbucks and the environment.
Nothing more to say--go get it!
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Village Pointe Wine
Nikkie Boulay will be broadcasting LIVE from noon-2pm, Saturday, June 11th, 2011 at the Village Pointe Wine Festival located on 168th & Dodge. Come out and stop by the Star 104.5 table for your chance to win prizes! Also, from 1pm-9pm don't miss out on your chance to sample more than 100 wine samples & food offerings from Firebirds Wood Fired Grill, Kona Grill and Brix restaurants. Tickets are available at Brix. For more information please visit villagepointeshopping.com.
Occurs once: Jun 11, 2011 (12:00 PM to 2:00 PM)


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What you'd do?
Assuming managerial responsibility for a team of professionals to deliver quality audit and other assurance services, you have to ensure client satisfaction through high quality service deliverables.
You will be responsible for participation and management of audit engagements, audit of financial statements, preparation / review of consolidated financial statements, performing planning and control procedures, reviewing clients' accounting system and internal control system, preparation of audit reports, managing, appraising and training of audit teams.You will gain exposure to working on a wide variety of clients & potentially allow you the opportunity to gain exposure to special work e.g. stock exchange transactions, due diligence & acquisition work.


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Gem had a vet appointment at 9am today. There was heat in both front feet and a lot of sensitivity in his left hoof. He wouldn't pick up either of his front feet. Last time the vet was out, it was only his right front. So the vet thinks that he has some form of laminitis :( We're going to be taking radiographs on Monday when the farrier comes out so we can have a game plan on how to trim his feet. The vet also said that his coffin bone could either be rotating or sinking
So until then, he's on stall rest with no grain and only hay. We are going to hose his two hooves for about 20 minutes each to try and alleviate the heat and hopefully the pain too.
I brought up barefoot trimming yesterday to Tom and he wasn't really all for it, but after doing some research, he says that's the way to go (Yay!) So we're looking into getting a barefoot trimmer for Gem and Vega, but mostly Gem for right now.
I don't know if i'm more upset that he may have laminitis and may founder, or the fact that before we bought Gem, Spring Valley did absolutely nothing for him!
If anyone has had a horse that had laminitis and/or founder, can you please share you experience? Thanks so much


text: Firefighters in Kingston, Ontario battled a major building fire this afternoon that may have been caused by a propane explosion. Worst of all, it left a man trapped on a crane at the site of the blaze — and the play-by-play of his dramatic rescue was captured on Twitter. »


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- Filed Under
April is knocking at the door, and avid anglers, birders and hunters can't wait to let her in.
One of the coldest, snowiest months of March in memory has sparked an epidemic of spring fever, and sunshine Friday and highs in the 40s over the weekend are a welcome change from the ice and snow overload.
A gradual meltdown of the snow is perfect for river steelheaders, bay ice anglers and those with an interest in the soil moisture in fields and forests. ...


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We created Pandora to put the Music Genome Project directly in your hands
It's a new kind of radio –
stations that play only music you like
Zero Boys
The EP got some very minor attention, given that there were only 500 copies pressed, but forces both external and internal to the Zero Boys were to lay the groundwork for their follow-up, which was to be the acclaimed mark they had wished to leave. Internally, the band had a roster change in the form of bassist David "Tufty" Clough replacing John Mitchell. Clough was a faster, more frenetic player, taking the Zero Boys' sound to blazing new places. Externally, by the turn of the decade the West Coast hardcore scene had begun to fully develop, influencing new scenes in Minneapolis, Detroit, and -- more specifically -- Indianapolis. The speed and polemics of bands such as Black Flag and the Circle Jerks were backed up by an indie label and word of mouth network with zines like Flipside and Maximum Rock and Roll that would allow bands like the Zero Boys to play places as far as Los Angeles, New York, and Vancouver, British Columbia.
The Zero Boys began rehearsing in the garage for recording sessions that were to begin in summer of 1981. Two days of recording and 16 songs later, the band's landmark full-length, Vicious Circle, was completed and released on minor-league label Nimrod Records in 1982. Even though the record was to garner critical acclaim and turn the band into one of the legends of early-'80s hardcore, the band was to fold a year later, citing being "landlocked" in Indianapolis in comparison to the larger national scene. Bassist Clough was to join up with Toxic Reasons, and Mahern formed two short-lived bands, Dandelion Abortion and Datura Seeds, before focusing his time as a producer and engineer. The band found new life after the positive reception the reissue of Vicious Circle (with bonus tracks) received upon its release in 1988 on the Toxic Shock label. Three-quarters of the band got back together (with guitarist Hollywood replaced by Vess Ruthenberg), and toured and released two further albums, 1991's Make It Stop and 1993's The Heimlich Maneuver (released on Bitzcore and Skyclad, respectively). The band would fade out, and by the turn of the 21st century had re-formed to tour in support of the release for History Of. ~ Chris True


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Easy hairstyling tip: quick curls with no fuss
Want controlled, fun curls with a minimum of hassle? It's super easy with this quick hair styling trick. All you need is some styling product and about 30 minutes to let hair dry.
After hair is washed, divide into four sections, one on each side of the face, and one behind each of those. Coat hand lightly with a touch of molding or styling wax (Aveda Light Elements Defining Whip is great for this), grab one complete section of hair and twirl in the direction you want the curl to go. When hair is twisted all the way to the end, let it rest and move on to the next section and repeat.
So it looks like you have four big dreadlocks on your head, and that's okay. When hair has dried, bend at the waist and shake hair out of the twirls loosely. Curls will be controlled with no frizz, and heat-damage free.
You can find Aveda Light Elements Defining Whip at Aveda.com.


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Such a real and substantial interest in specific property as will sustain a contract to indemnify the person interested against its loss. Mutual F. Ins. Co. v. Wagner (Pa.) 7 Atl. 104; Insurance Co. v. Brooks, 131 Ala. 014, 30 South. 870; Berry v. Insurance Co., 132 N. Y. 49, 30 N. E. 254, 28 Am. St. Rep. 548; Strong v. Insurance Co., 10 Pick. (Mass.) 43, 20 Am. Dec. 507; Insurance Co. v. Winsmore, 124 Pa. 61, 16 Atl. 510. If the assured had no BL.LAw DICT.(2D ED.)-41 real interest, the contract would be a mere wager policy. Every interest in property, or any relation thereto, or liability in respect thereof, of such a nature that a contemplated peril might directly damnify the insured, is an in- surable interest. Civil Code, Cal.
What is INSURABLE INTEREST?
Edited and fact checked by James: Google +


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Where do I register to run?
Click here
OR the day before race at Mission 500 table top outside main entrance of Expo hall
What is the cost involved?
A $30 registration fee
What does the entry fee include?
T-Shirt (for the first 750 participants)
Goody Bag (for the first 750 participants)
Post walk/run snacks and beverages
Does all of it go to charity?
Yes, net proceeds collected from race registration go to sponsor needy children
Where is the starting location?
What is the starting time?
7:30 am sharp
What is the course - is there a map?
The course is along the beautiful Sunset Park Trail running path, less than 5 miles from the Sands Convention Center.
Click here to view a PDF of the course map.
Where do we finish - is finishing line different to starting line?
Will there be shuttle buses taking us from hotels to the starting location?
YES, buses pick up at the Taxi Ramp at the Sands Convention Center - 201 Sands Ave
Will there be food after the race?
Will there be awards after the race?
Yes, there will be an awards reception that afternoon, April 16, at 5:00 pm at the "media-stage" in the concourse (close to Expo hall entrance).
Are there award categories?
Yes, 20-30, 31-40, 41-50, 50+ age categories, and one each for first place male and female runners
Where do I get my race bib and package?
On site before the race
Will there be a bag storage service on site?
No, important to come only with your running gear and race bags will be given out after the race
Can I register on site?
Yes, the day before the show at Mission 500 table top which will be in the Show Registration area.
When does online registration close?
The day before the event at 5:00 pm
Who can I contact with any other questions?
Tim Purpura at
© 2015 United Publications, Inc. | security5k.com


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The holidays just got a little merrier for children in Wayne County, thanks to the Red Cross and its supporters.
The holidays just got a little merrier for children in Wayne County, thanks to the Red Cross and its supporters.
Throughout the past few weeks, the Wayne County Chapter of the American Red Cross encouraged businesses and organizations to donate new, unopened toys and games which parents could then "shop for" at the Red Cross Chapter in Newark. The toys were free of charge to qualifying program participants.
"We were able to assist many families with gifts this year," said James Love, Executive Director of the American Red Cross of Wayne County. "I am extremely proud of the efforts that our Wayne County community partners put forth in providing a happy holiday season for so many children."
On Dec. 10 and 11, 152 families shopped for gifts for their children. More than 400 children will receive gifts through this program.
The toys were distributed by Red Cross staff and volunteers, as well as volunteers from Newark Rotary Club, Lyons National Bank, and Walgreens.
This program has been a Wayne County Red Cross staple for more than 20 years. This year's toy drive yielded more than 1,100 items for parents to choose from, with any leftover items going to the St. Mark's Church Baby Closet in Newark, as well as to organizations that provide assistance to needy families located in Palmyra, Sodus, and Newark.
The Red Cross wishes to express its gratitude to the 22 Wayne County businesses, organizations, and social service groups who collected items in support of the program. These included: Word of God Ministries, Woodlane Community Church, Lyons National Bank in Newark, Maco Bag, IEC Electronics, Dr. Pepper Snapple Group, Blossom View Nursing Home, Williamson Library, Newark Public Library, Wayne County Bar Associates, Redeemer Lutheran Church, St. Marks Church Episcopal Church, Emmanuel United Church, Rebecca/Ruth Circle of the First United Methodist Church, Monroe Title and Abstract, the Doebler family, Newark Wegmans, Newark Wal-Mart, the Herbertus & Helmi Behrla Fund, Walgreens in Newark, Newark Rotary Club, United Way of Wayne County, the Wayne County Red Cross Board of Directors, and Red Cross volunteers.
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Location, ST | website.com


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FILE - In this May 6, 2013 file photo, TV personality Katie Couric, left, and John Molner attend The Metropolitan Museum of Art's Costume Institute benefit celebrating 'PUNK: Chaos to Couture' in New York. Couric is engaged to her financier boyfriend John Molner. Couric?s spokesman Matthew Hiltzik confirmed the engagement Tuesday morning following a report by People magazine. Molner gave 56-year-old Couric, the former host of ?Today,? a diamond ring over the weekend in East Hampton. (Photo by Charles Sykes/Invision/AP, File)
- Filed Under
Katie Couric celebrated her Labor Day weekend by saying yes to the dress.
The 56-year-old TV host accepted a proposal from her boyfriend of nearly two years, banker John Molner, People magazine reports. He reportedly popped the question with a diamond ring at sunset on the beach in East Hampton, N.Y.
The host of "Katie," which returns for a second season on Sept. 9, has been a widow since her husband, Jay Monahan, died of colon cancer in 1998.
Couric and Molner, 50, who is head of mergers and acquisitions at Brown Brothers Harriman, first went public with their romance in April 2012.


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ok so i have jumped on the slimming world wagon, again. after losing nearly four stone last year i have put nearly all of it back on after having my gorgeous baby boy on new years day, and i have a bridesmaid dress to wear in july that doesnt currently fit. so i will be using this diary to keep track and as a motivational tool for myself so will probably not interest any one else in the slightest but if anyone does read this and has any advice for me i would be very grateful!
So for breakfast today i have had
total 0% greek yoghurt with frozen berries and acacia honey 1 syn


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Catholic schools week celebrates pride, tradition of service
February 9, 2010
By Chantelle Lusebrink
Throngs of students filled the doorway to St. Joseph School's cafeteria.
It's the lunch rush and the third-grade girls manning the table hollered out the names of delectable treats for purchase like the best of auctioneers.
"What do you want?" third-grader Celeste Veitch shouted over a sea of younger students. "We have cookies, brownies, cupcakes. Let me know when you're ready."
For 50 cents each, the endless buffet beckoned to nearly every student, preschool to third grade. Unfortunately for the students, there was a three-item limit.
The school hosted the bake sale at both the Issaquah and Snoqualmie campuses, as part of its celebration of Catholic Schools Week, meant to celebrate Catholic schools and faculty.
But more than a sweet treat for students, the bake sale was a fundraiser for Haitian children.
"We're doing it for Haiti because of the earthquake," said third-grader Rachael Goodwin, "to help the children of Haiti, because they don't have as much as we do."
They need "food, water and medical supplies," Celeste added. "We want them to be able to buy strong stuff and live in places that don't fall down."
Between the two campuses, the students raised more than $1,000 for one of their favorite organizations, Friends of the Orphans, which operates in Haiti and several other South American countries, said Vice Principal Jackie Olund.
The organization provides housing, food and education for orphans and children whose parents can't afford to support them. However, the organization doesn't promote overseas adoption, Olund said.
"Their goal is to educate and make them strong citizens for their country," she said. "We've been making donations every year, for about the last seven years."
The school's campuses collect loose change at lunches during Lent, Feb. 17 – March 28, to donate to Friends of the Orphans. Organizers from the nonprofit organization have come to speak to students at St. Joseph's in prior years, to help them understand their mission and why it's important to help others, Olund said.
"When we called them to see what we could do, they said they really needed money," she said. "We thought Catholic Schools Week would be a great opportunity for the kids to do their service, because it is a big part of our mission."
The organization's volunteer dormitory collapsed during the 7.0-magnitude earthquake; its epicenter was about 12 miles from the country's capital Port-au-Prince Jan. 12. In that collapse, Molly Hightower, a Port Orchard woman and a volunteer, died.
The school made the bake sale a special event of Student Appreciation Day during the weeklong celebration.
The week started in 1974, as an annual national celebration of the important role that Catholic elementary and secondary schools play in providing a values-added education, according to a press release from the National Catholic Educational Association.
At both campuses, students participated in a variety of activities, including an all-school Mass and assembly, and student, faculty and parent appreciation days.
Students in each grade have a variety of community service and learning projects they do throughout the year. For instance, one grade holds a pet food drive and pet blessing in the spirit of St. Francis, the patron saint of animals, while another makes sandwiches for Lazarus House in Seattle, which provides sandwiches to people without lunches. Older grades in Snoqualmie have coordinated efforts to donate food and clothing to Issaquah Community Services in Issaquah and for Encompass in Snoqualmie.
The week also serves to market Catholic schools and education to families, so the schools were open for a variety of open houses.
"Catholic schools are all about outreach and how to get ourselves involved in our community," said Ann Shikany, a spokeswoman for the schools. "This is a wonderful opportunity to get involved with that at a time we're celebrating nationally."
On the Web
Friends of the Orphans
Chantelle Lusebrink: 392-6434, ext. 241, or email@example.com. Comment at www.issaquahpress.com.
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Hi everyone,
I'm expecting my Model S (85) in Feb/March, and I was hoping to make my first long distance (relatively speaking) trip from San Francisco Bay area down to Disneyland for my 2.5 year old sometime after I get my car.
I've done some research on chargepoint and recargo, and haven't found too many places to charge around that area. Hilton and Conventions center were only 2 places and they charg $16/$24 for parking--charging free. The hotel I plan to stay (Candy cane Inn) doesn't have any way to charge my car overnight.
Has anyone been to Disneyland with Model S yet? and if so, any suggestions on charging? I don't need much...just enough to get to the supercharging station once we're done.
Also, is there a supercharging station on the way to San Diego?
I don't think there is a Tesla supercharging station on way to San Diego. According to the plugshare app, there are 110 plugs at the Disneyland Mickey & Friends parking lot. One of the reviewers recommended to go to the Mitsubishi HQ in nearby Cybress. Then again you could just use the Hawthorne supercharger.
Better yet, stop by my house in Laguna Niguel and plug into my NEMA 14-50. We'll compare cars and share stories. Beer is on me.
Nice! This is why Tesla owners still look for others on the road, we are like family.
I can't provide any evidence of it, but I seem to remember seeing J1772 chargers at the main parking structure in Disneyland with special parking spaces and everything. They're on the ground floor.
When we were there last Fall, I recall thinking that it would have been really nice to be driving my Model S since I would have had a much better parking space than the terrible one I ended up with on the top floor as far as possible from the escalators.
Does anyone know if the Disneyland Grand Californian has any EV parking spots with charging? I was hoping to take the kids from the SF bay area to Disneyland via the superchargers and then hopefully have a place to charge while parked at the hotel. If anyone has done this, would love to hear about it also.
I have been told that there are several in the "Down Town Disney" parking lot. Not sure what their configuration is for plugging in. Thinking of going to the park tonight. If we make It, I will research it and let you know what I find.
Try the blink network, too. Can't wait until we have superchargers in the pacific northwest so I can drive to Disneyland!
@michael...thanks for the offer....I may take you up on that. hehe.
thanks for all the info everyone.
when I make it down there, I'll post as much info as I can.
If you Google "rv parks near disneyland", you'll see there are several. Might be worth a call to them to see if they have a NEMA 14-50 you could charge from with the UMC, and shuttle service to Disneyland.
@steven...thanks for the link. missed that on the website earlier
Hi everyone! This is our first post on here! Please keep us posted about your experience as we plan a trip to Disney as well.
We are the new (and proud!) owners of the Model S (85) Signature Performance. We are planning a similar trip, except we are leaving from BC, Canada, going all the way to Disneyland. So far, our challenges are with the Portland-Sacramento corridor. We are planning where to stop/sleep to maximize our time during the charging. Any experience or feedback welcome.
Louis and Marie, BC.
@ L & M: There are many choices, one of which is RV parks most of which have 14-50 plugins. Some also have rental cabins for the night. Here is a good web site for finding them.
Have a great trip and enjoy the new S.
Now here's a thread that has me interested. I'm planning a Disneyland visit in February -- my first trip of any real length.
While it's less than ideal, the Anaheim City Hall charger looks doable, about two miles from the park, and should be reasonably accessible via public transit (to get back to the house of the mouse).
It'll be my day-before-the-trip-home charging station if nothing better turns up before February.
I've had troubles with public charger cables being too short to work with the Model S unless you back in, and this site mentions in particular that there are big fine$ if you back in. Catch-22. Can anybody headed that way take a look?
Dave LV, where did you see the fine for parking tail in?
@DTsea: See the Check-Ins log for the Anaheim City Hall Recargo page for Feb. 28, 2012.
SoCal lot operators seem to really boost their income with backing-in parking space fines. Take a look at the logs for LAX, they're all over the place!
Sent an e-mail to the Disney folks - here is the reply
Thank you for your e-mail to the DISNEYLAND® Resort.
Unfortunately, we do not offer a charging station for electric vehicles.
We apologize for any disappointment this may cause you and your family.
Again, thank you for taking the time to write. We hope you will have the
opportunity to visit the DISNEYLAND® Resort soon and trust your visit
will be pleasant in all regards.
Linda Trump
DISNEYLAND® Resort
Guest Experience Services
Please note all information is subject to change without notice and
should be confirmed just prior to your visit.
Received date: 1/11/13
The owner of the Castle Inn does not yet have chargers, but he is very interested in Tesla. He asked a lot of questions about ours, but I have not had a chance to show him yet. I suspect that if several people ask about charging he will put them in. The Castle Inn is directly across the street from Disneyland on Harbor Blvd. They are planning on room renovations over the next couple of months. I will send him an email when I have a chance.
Louis and Marie,
I'm a bit southeast of seattle. You can use the 14-50 and be our guests. Email is above.
We are planning an August trip to Disneyland, staying at the Grand Californian. We normally go for at least 4 days (mostly staying within the park)so being able to charge for the trip home to the Bay Area is a concern.
As much as Disney touts itself as being a green company, and considering they are forward thinkers, I have to believe they will be installing chargers in their garages. Maybe they just need some encouragement.
This thread is a good indicator of how many Model S owners are already planning a Disneyland trip. If we have to go off-site to charge our car, we will most likely eat a meal outside of the Disney property. Maybe we will see another place of interest and want to go outside the park again. Keeping people on site is a good business decision.
I suggest Tesla owners start a campaign for getting either a Tesla Supercharger or at least 50 amp chargers in the Disney garages. I plan to make contact with Guest Services in the next few days. Maybe we can make the Tesla grin a common feature at the Happiest Place on Earth!
I informed Disneyland that we will be choosing an outside hotel due to charging availability.
I'm driving to Disneyland this weekend.
I just posted a new thread asking if there is real-time information on whether the Superchargers along the route are actually "up and working". I'm a bit concerned that a supercharger that I'm relying on my be "down" - then I'd be stuck there until it's fixed.
@jchangyy, I've stayed at the Candy Cane Inn. It's a great place for a reasonable price and includes the continental breakfast and is walking distance to the park entrance (but has a good shuttle that runs every 30 minutes).
If you've been there already, you probably know already that the parking spaces are a bit tight. With the width of the Model S, that would be my concern - that's why I'm not staying there this time.
I'm staying at a Double Tree a bit south and hoping that with business travelers mostly, they would be a bit more considerate in not banging their door into my new baby.
Regarding charging....
I'm have the route planned out and will charge at the Hawthorne supercharger. It's only about 30 miles from Disneyland. I don't plan on charging overnight at Disneyland, but do plan to use/lose about 15 miles of range per day/night when I'm there. I also plan to go to Irvine on that same charge and expect to have about 55 miles of range remaining when I get back to the Hawthorne supercharger.
My assumption is that I will consume power at 1.4x the EPA rated power. So, I'm expecting no more than 170 miles of range on a standard charge during my trip. This will allow me to drive reasonably fast on I-5 and also not poke along in So-Cal.
I hope to keep good notes of my experience over this weekend and post to this thread when I get back.
Shopping centers in SoCal seem to be on the ball even if Disney isn't, or at least so my minimal web research suggests.
The Block at Orange, an outlet mall with shops, restaurants and movies, is only about 3½ miles from Disneyland. Looks excellent. Just do dinner there the evening before departure, and the 107 miles to the Barstow Supercharger (allow ~20 miles for the awful climb up Cajon Pass, plus some "safety" miles plus a possible detour or stop en route) and the trip home to Las Vegas should be plain sailing.
Backup shopping center locations a few miles further from D'land include South Coast Plaza in Costa Mesa and Newport Fashion Island in Newport Beach.
I think I'm set for 2013! But I'll still hope (and maybe agitate a little) for charging locations at Disneyland proper and/or at the nearby hotels in future.
If I remember to do it, I'll post how this works out for me. I'm planning on the Washington's Birthday holiday weekend next month.
@dahtye...I'm very much looking forward to your report on your experience. We did stay at Candy cane Inn and we enjoyed it very much, especially having the option to come back to the inn at 1pm for a nice nap before heading back out.
we're planning to go sometime in the summer.
Becareful driving through the grapevine...it's been very cold recently.
My daughter called the Grand Californian today to inquire about chargers in their parking lot. She was told they have one (don't know the amps)and it gets very little use. It is 1st come 1st serve, so you can't count on it. I predict they will need more very soon.
The person she talked to seemed interested in Teslas, but didn't know much about them. We need to make Disney aware of these vehicles of the future!
How about converting the Autopia ride to electric vehicles? Tesla could sponsor the ride & get great publicity! That area of the park would smell a lot better too.
Sounds like a fine plan! Let us know how it 'comes together'.
--Mr. T. & friends
I sent an e-mail to Guest Services letting them know that there are many new Tesla owners in California and several are requesting information regarding chargers in Disney garages. I will report back witht their response.
They really should add charging stations on the first floor and close to the tram.
I just posted my trip summary on this forum.
I asked the parking attendant in the Mickey and Friends parking structure about the location of AC outlets. He didn't know of any. The reason I asked is one of the phone apps for chargers showed that there are outlets on the north end of the parking structure. But it doesn't mention which level. I quickly checked as I was driving on the level that I was on (named Donald) and didn't see any.
The guy was somewhat oblivious to our plight.
See my post of Jan 16 - answer from Disneyland
I actually am very familiar with this issue as a Disneyland enthusiast and a 3 year Tesla owner.
Nora-te: The Grand California and Disneyland Hotel valets will plug you into a 110 outlet. They have a few of these outlets, but they dont publicize this option. I have done this many times (more than I care to admit, but lets just say plenty enough to be confident of what I am saying) and the only question was if the valet wanted me to go with them to make sure it was done right. Not once has there been any question of whether I could actually plug in. This is what they were referring to when your daughter called. Of course if this gets well known, they may get regularly tied up (so I'm going against short term self interest here, but you are fellow Tesla owners.)
I'm pretty confident that there is currently no working J1772 or other 220+ v charger or outlet anywhere at Disneyland except the employee only structure. I have pushed on this issue and have heard they are working on a plan.
I have charged at the Anaheim Civic lot. This option works fine, but it is a $10 cab ride to the park. If you park during the day, don't bother calling for a cab. Its faster to just walk out to Anaheim Blvd and wave one down.
As of a few months ago, the Hilton had a working Avcon charger (same as J1772 but with a different connector) for guests, but you need an adapter for this. I have an Avcon to NEMA 14-50 adapter which I used there successfully. You may find one on eBay but they aren't cheap.
If you are coming from the north, and using a 110 outlet long enough at your hotel is not an option, the Hawthorne supercharger is a great solution. I would recommend this if you are in a hurry. If you're lucky, you'll strike up a conversation with SpaceX employees and get a factory tour. The mall solution is also fine if you have more time.
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December 1, 2015
GELSENKIRCHEN, Germany (AP) - German soccer team Schalke will unveil a memorial plaque in front of its stadium to honor nine Jewish club members and supporters who perished in the Holocaust.
The Bundesliga club says the plaque will be unveiled Jan. 30. Players scheduled to attend include captain Benedikt Hoewedes, Christoph Metzelder and Julian Draxler.
Copyright 2015 Associated Press. All rights reserved. This material may not be published, broadcast, rewritten, or redistributed.
Click Here to access the online Public Inspection File
Viewers with disabilities can get assistance accessing this station's FCC Public Inspection File by contacting the station with the information listed below. Questions or concerns relating to the accessibility of the FCC's online public file system should be directed to the FCC at 888-225-5322, 888-835-5322 (TTY), or firstname.lastname@example.com.


text:
The Muscular Dystrophy Association (MDA) is the nonprofit health agency dedicated to finding treatments and cures for muscular dystrophy, ALS and related diseases by funding worldwide research. The Association also provides comprehensive health care and support services, advocacy and education.
MDA was created in 1950 by a group of adults with muscular dystrophy, parents of children with muscular dystrophy and a physician-scientist studying the disorder.
MDA combats neuromuscular diseases through programs of worldwide research, comprehensive medical and community services, and far-reaching professional and public health education. With national headquarters in Tucson, Ariz., MDA has 120 local offices across the country, sponsors 200 medical clinics and supports some 300 research projects around the world. The Association's programs are funded almost entirely by the voluntary contributions of concerned individuals and cooperating organizations.
Some of MDA's special events and volunteer opportunities include: