Apoptosis and p53 expression in rat adjuvant arthritis

INTRODUCTION: RA is a chronic inflammatory disorder that is characterized by inflammation and proliferation of synovial tissue. The amount of DNA fragmentation is significantly increased in rheumatoid synovium. Only low numbers of apoptotic cells are present in rheumatoid synovial tissue, however. The proportion of cells with DNA strand breaks is so great that this disparity suggests impaired apoptosis. Therefore, the development of novel therapeutic strategies that are aimed at inducing apoptosis in rheumatoid synovial tissue is an attractive goal. Although animal models for arthritis only approximate RA, they provide a useful test system for the evaluation of apoptosis-inducing therapies. AA in rats is among the most commonly used animal models for RA. For the interpretation of such studies, it is essential to characterize the extent to which apoptosis occurs during the natural course of the disease. Therefore, we evaluated the number of apoptotic cells and the expression of p53 in various phases of AA. MATERIALS AND METHODS: In order to generate the AA rat model, Lewis rats were immunized with Mycobacterium tuberculosis in mineral oil on day 0. Paw swelling usually started around day 10. For the temporal analysis rats were sacrificed on days 0, 5 (prearthritis), 11 (onset of arthritis), 17 (accelerating arthritis), or 23 (chronic arthritis). For the detection of apoptotic cells, the hind paws were harvested on days 0(n=6),5 (n=6), 11 (n=6), 17 (n=6), or 23 (n=4). The right ankle joints were fixed in formalin, decalcified in ethylenediaminetetra-acetic acid, embedded in paraffin, and sectioned. The TUNEL method was applied. The percentage of TUNEL-positive cells of the total inflammatory cell infiltrate was noted. For Western blot analysis, hind paws were harvested on days 0 (n=2), 5 (n=3), 11 (n=4), 17 (n=4), or 23 (n=4). In addition, hind paws of normal rats (n=2) were studied. The right ankle joints were snap frozen and pulverized. Synovial tissue was also obtained by arthroscopy of three patients with longstanding (>5 years) RA. After protein extraction in lysis buffer, equal amounts of protein samples from lysates were pooled and examined by Western bolt analysis using anti-p53 monoclonal antibody D07, which recognizes wild-type and mutant p53 from rodents and humans. For immunohistochemical analysis, six rats were sacrificed on day 23 after immunization and synovial tissue of the right ankle joints was snap frozen and evaluated by immunohistochemistry using anti-p53-pan. The sections were evaluated semi-quantitatively using a 0-4 scale. The kruskal-Wallis test for several group means was used to compare the percentage of TUNEL-positive cells at different time points. RESULTS: The percentages of TUNEL-positive cells were strongly dependent on the stage of the disease. Very few TUNEL-positive cells were detected in normal rats or in the early phases of AA; the number of TUNEL-positive cells was 1% or less of the total cell infiltrate, including neutrophils, from days 0-17 (Table 1). On day 23, however, the percentage of TUNEL-positive cells was significantly increased [15.8±5.1% (mean ± standard error of the mean); P=0.01]. TUNEL-positive cells were observed in the intimal lining layer and synovial sublining of the invasive front, as well as in the articular cartilage (Fig. 1). Subsequently, we examined expression of the tumor suppressor gene p53, because this is a key regulator of apoptosis. Expression of p53 in pooled rat AA joint extracts gradually increased from day 0 (6 arbitrary units) to day 23 (173 arbitrary units), which was markedly higher than p53 levels in RA synovium (32 arbitrary units; Table 1). Overexpression of p53 protein on day 23 was confirmed by immunohistochemistry in a separate experiment in six rats with AA. Overexpression of p53 was observed in the intimal lining layer and synovial sublining in all rats on day 23. In all cases a semiquantitative score of 4 was assigned, indicating that 51% or more of the cells were positive, whereas control sections were negative. DISCUSSION: The results presented here reveal that the number of TUNEL-positive cells remained very low until chronic arthritis developed. This indicates that, although there was sufficient DNA damage to cause an increment in p53 expression in the early phases, DNA strand breaks that can be detected by TUNEL assays only occurred in chronic AA. The observation that TUNEL-positive cells were nearly absent in early AA clearly indicates that only very few cells were undergoing programmed cell death. This is an important observation, which makes it possible to study the effects of apoptosis-inducing therapies in situ in early and accelerating AA. An effective therapy would obviously increase the number of TUNEL-positive cells. There is already some overexpression of p53 in the preclinical phase and during the onset of the arthritis, with an additional increment in p53 expression during accelerating and chronic arthritis. Presumably, this is wild-type p53, because the disease duration is likely too short to allow for the development of p53 mutations. Transcription of p53 is probably increased in response to the toxic environment of the inflamed joint. The increased expression of p53 in the joints of rats with chronic AA was even greater than that observed in synovial tissue of RA patients with long-standing disease. Overexpression of p53 and increased numbers of apoptotic cells did not occur simultaneously in this model; rather p53 overexpression preceded increased apoptosis. Activation of p53 leads to induction of cell growth arrest, allowing time for DNA repair. It appears that DNA damage is only extensive enough to induce apoptosis in the latter stages of AA. Factors other than p53 may also play an important role in the actual induction of apoptosis Taken together, significant apoptosis only occurs late in AA and it follows marked p53 overexpression, making it a useful model for testing proapoptotic therapies. AA is not the best model for p53 gene therapy, however, because dramatic p53 overexpression occurs in the latter stages of the disease

Genomic SNP array as a gold standard for prenatal diagnosis of foetal ultrasound abnormalities

Background: We have investigated whether replacing conventional karyotyping by SNP array analysis in cases of foetal ultrasound abnormalities would increase the diagnostic yield and speed of prenatal diagnosis in clinical practice. Findings/results. From May 2009 till June 2011 we performed HumanCytoSNP-12 array (HCS) (http://www.Illumina.com) analysis in 207 cases of foetal structural abnormalities. HCS allows detecting unbalanced genomic abnormalities with a resolution of about 150/200 kb. All cases were selected by a clinical geneticist after excluding the most common aneuploidies by RAD (rapid aneuploidy detection). Pre-test genetic counselling was offered in all cases. In 24/207 (11,6%) foetuses a clinically relevant genetic abnormality was detected. Only 8/24 abnormalities would have been detected if only routine karyotyping was performed. Submicroscopic abnormalities were found in 16/207 (7,7%) cases. The array results were achieved within 1-2 weeks after amniocentesis. Conclusions: Prenatal SNP array testing is faster than karyotyping and allows detecting much smaller aberrations (∼0.15 Mb) in addition to the microscopic unbalanced chromosome abnormalities detectable with karyotyping (∼ > 5 Mb). Since karyotyping would have missed 66% (16/24) of genomic abnormalities in our cohort, we propose to perform genomic high resolution array testing assisted by pre-test counselling as a primary prenatal diagnostic test in cases of foetal ultrasound abnormalities

Corruption in the Middle East and the Limits of Conventional Approaches

Die Unzufriedenheit mit der verbreiteten Korruption war 2011/2012 eine wesentliche Ursache für die arabischen Unruhen und weitere Aufstände weltweit. Der Fall Jordanien zeigt allerdings, dass konventionelle Ansätze zur Bekämpfung von Korruption nicht ausreichen. Eine angemessene Strategie gegen Korruption muss diese als ein Problem der Verteilungsgerechtigkeit und nicht des Strafrechts verstehen. Wie in allen anderen arabischen Staaten ist die Unzufriedenheit in der Bevölkerung über die offensichtliche Korruption auch in Jordanien beträchtlich. Allerdings wird im Allgemeinen nicht über Fälle von Bestechung und Erpressung geklagt, die weniger häufig vorkommen, sondern über lokale Praktiken politischer Patronage und Begünstigung, die unter dem Begriff "Wasta" zusammengefasst werden. "Wasta" wurde bislang als Form der Korruption und strafrechtliches Problem angesehen, weshalb Versuche zur Eindämmung überwiegend ineffizient blieben: "Wasta"-Praktiken werden in der Regel nicht mit Rechtsverstößen verbunden, sondern bewegen sich innerhalb formal legaler Verfahren. Konventionelle Ansätze zur Bekämpfung von Korruption, die sich an rechtsstaatlichen Grundsätzen und Transparenz orientieren, sind deshalb nicht zielführend. Demokratisierung allein ist ebenfalls ungeeignet, das Problem „Wasta” zu lösen. In der parlamentarischen Praxis macht "Wasta" den Großteil der Aktivitäten aller Parlamentsmitglieder aus. Diese werden deshalb als persönliche Dienstleister für ihre Wahlbezirke und nicht als Mitglieder einer gesetzgebenden Körperschaft wahrgenommen. Gleichzeitig hält die Bevölkerung das Parlament für eine zutiefst korrupte Institution. "Wasta" wird problematisch, wenn diese Praxis zu einem ungleichen Zugang der Bürger zu öffentlichen Ressourcen führt. Statt sich nur auf politische und administrative Reformen zu konzentrieren, muss der Fokus der Bekämpfung auf den (Wieder-)Aufbau wohlfahrtsstaatlicher Strukturen gelegt werden, zu denen alle Bürger gleichermaßen Zugang haben

Identification of regulatory variants associated with genetic susceptibility to meningococcal disease.

Non-coding genetic variants play an important role in driving susceptibility to complex diseases but their characterization remains challenging. Here, we employed a novel approach to interrogate the genetic risk of such polymorphisms in a more systematic way by targeting specific regulatory regions relevant for the phenotype studied. We applied this method to meningococcal disease susceptibility, using the DNA binding pattern of RELA - a NF-kB subunit, master regulator of the response to infection - under bacterial stimuli in nasopharyngeal epithelial cells. We designed a custom panel to cover these RELA binding sites and used it for targeted sequencing in cases and controls. Variant calling and association analysis were performed followed by validation of candidate polymorphisms by genotyping in three independent cohorts. We identified two new polymorphisms, rs4823231 and rs11913168, showing signs of association with meningococcal disease susceptibility. In addition, using our genomic data as well as publicly available resources, we found evidences for these SNPs to have potential regulatory effects on ATXN10 and LIF genes respectively. The variants and related candidate genes are relevant for infectious diseases and may have important contribution for meningococcal disease pathology. Finally, we described a novel genetic association approach that could be applied to other phenotypes

Validity of the Patient Experiences and Satisfaction with Medications (PESaM) Questionnaire

Background: This study assessed the validity and reliability of the generic module of the recently developed Patient Experiences and Satisfaction with Medications (PESaM) questionnaire in a sample of patients in the Netherlands. Methods: The generic module of the PESaM questionnaire consists of 18 items related to the domains effectiveness, side effects and ease of use of medications. It assesses patients’ experiences regarding the impact of the medication on daily life, health and satisfaction. In 2017, the PESaM questionnaire was sent out to idiopathic pulmonary fibrosis patients using pirfenidone or nintedanib, atypical haemolytic uraemic syndrome patients receiving eculizumab and patients using tacrolimus after kidney transplantation. Mean scores for each domain were calculated applying a scoring algorithm. Construct validity and reliability were assessed using recommended methods. Results: 188 participants completed the generic module, of whom 48% used pirfenidone, 36% nintedanib, 11% tacrolimus and 5% eculizumab. The generic module has good structural properties. Internal consistency values of the domains were satisfactory (i.e. Cronbach’s coefficient alpha above 0.7). Confirmatory factor analysis provided further evidence for construct validity, with good convergent and discriminant validity. The PESaM questionnaire also showed different scores for patients using different medications, in line with expectations, and was therefore able to differentiate between patient groups. Test–retest reliability of the items and domains were rated as moderate to fair (i.e. intraclass coefficients ranged between 0.18 and 0.76). Conclusions: The PESaM questionnaire is a unique patient-reported outcome measure evaluating patient experiences and satisfaction with medications. It has been developed in conjunction with patients, ensuring coverage of domains and issues relevant from the patient’s perspective. This study has shown promising validity of the generic module of the PESaM questionnaire. Further research is recommended to assess reliability in greater detail as well as the responsiveness of the measure. Trial registration: The study

Plasma lipid profiles discriminate bacterial from viral infection in febrile children

Fever is the most common reason that children present to Emergency Departments. Clinical signs and symptoms suggestive of bacterial infection are often non-specific, and there is no definitive test for the accurate diagnosis of infection. The 'omics' approaches to identifying biomarkers from the host-response to bacterial infection are promising. In this study, lipidomic analysis was carried out with plasma samples obtained from febrile children with confirmed bacterial infection (n = 20) and confirmed viral infection (n = 20). We show for the first time that bacterial and viral infection produces distinct profile in the host lipidome. Some species of glycerophosphoinositol, sphingomyelin, lysophosphatidylcholine and cholesterol sulfate were higher in the confirmed virus infected group, while some species of fatty acids, glycerophosphocholine, glycerophosphoserine, lactosylceramide and bilirubin were lower in the confirmed virus infected group when compared with confirmed bacterial infected group. A combination of three lipids achieved an area under the receiver operating characteristic (ROC) curve of 0.911 (95% CI 0.81 to 0.98). This pilot study demonstrates the potential of metabolic biomarkers to assist clinicians in distinguishing bacterial from viral infection in febrile children, to facilitate effective clinical management and to the limit inappropriate use of antibiotics

Plasma lipid profiles discriminate bacterial from viral infection in febrile children

Fever is the most common reason that children present to Emergency Departments. Clinical signs and symptoms suggestive of bacterial infection ar

The importance of relationships with parents and best friends for adolescents' romantic relationship quality: Differences between indigenous and ethnic Dutch adolescents

Contains fulltext : 90600.pdf (publisher's version ) (Closed access)This study examined how the quality of relationships with parents and friends were related to intimacy, commitment, and passion in adolescents' romantic relationships for indigenous Dutch and ethnic Dutch adolescents. Self-report survey data were used from 444 (88.9%) indigenous Dutch and 55 (11.1%) ethnic Dutch adolescents between 12 and 18 years of age (M = 15.34, SD = 1.14), all of whom were involved in a romantic relationship. About 61.6% (n = 307) were girls. For both indigenous and ethnic Dutch youths, the quality of the parent-adolescent relationship was positively associated with romantic relationship intimacy, whereas the quality of one's best friendship was related to higher commitment to one's romantic partner. Significant interactions in hierarchical regression analyses revealed that only ethnic Dutch adolescents who experienced low-quality parent-adolescent relationships showed more commitment to their romantic relationships. Additionally, ethnic Dutch adolescents who experienced low-quality best friendships showed more passion in their romantic relationships as compared to indigenous Dutch adolescents. These findings are indicative of ethnic differences in the roles that parents and friends play in the romantic lives of Dutch adolescents.7 p

Isolated elevated serum transaminases leading to the diagnosis of asymptomatic Pompe disease

An asymptomatic boy, aged 1.5 years, was referred with presumed liver disease because of persistently increased transaminase. Ultimately Pompe disease was confirmed, without specific abnormalities in muscle biopsy. This case demonstrates that increased transaminases do not always suggest liver disease. It is hard to determine prognosis and to decide whether enzyme replacement therapy should be started in asymptomatic patients with Pompe disease

The Impact of the COVID-19 Pandemic in The Netherlands on Primary Healthcare Use and Clinical Outcomes in Persons with Type 2 Diabetes

The COVID-19 pandemic has had a significant impact on healthcare systems worldwide, including the postponing or canceling of appointments and procedures for type 2 diabetes (T2D) care by general practitioners (GPs) in the Netherlands. The aim of this study was to investigate the impact of the COVID-19 pandemic on primary healthcare use and clinical measurements for people with T2D. Additionally, we aimed to determine if changes were observed among specific risk groups: (1) persons 70 years or older, or below 70 years, (2) patients who were meeting their HbA1c targets and those who were not, and (3) patients with high-risk and non-high-risk T2D. This retrospective cohort study among persons with T2D was conducted using data from the DIAbetes MANagement and Treatment (DIAMANT) data infrastructure, deriving data from electronic medical records of Dutch GPs. The study assessed GP visit counts, and counts and values of clinical measurements, including hemoglobin A1c (HbA1c), body mass index (BMI), low-density lipoprotein (LDL) cholesterol, and systolic blood pressure (SBP). Adjusted negative binomial (NB) regression and generalized estimating equations (GEE) models were used to estimate GP visit counts and population averages of clinical measurements, respectively, comparing 2019 (pre-pandemic) with 2020 (during the pandemic). Changes in specific groups were examined by stratifying outcomes for the aforementioned subgroups. The cohort consisted of 182,048 patients with T2D (47% female, mean age 69 ± 13 years) on 1 March 2019, of which 168,097 persons (92%) still contributed follow-up data in 2020. We observed an increase in total GP visits in 2020, with an adjusted rate ratio (RR) of 1.09 (95% CI 1.08–1.09). The frequency increased for office visits (RR 1.06; 1.06–1.07) and phone calls (RR 1.33; 1.31–1.35) but remained stable for home visits (RR 1.02; 0.99–1.04). On both population and individual levels, HbA1c values increased in 2020 by 1.65 (1.59–1.70) mmol/mol compared to 2019. Observed changes in 2020 for BMI, LDL, and SBP values were also statistically significant but small. Subgroup stratifications showed higher scores of all clinical measurements in younger persons (<70 years), those who met their HbA1c target, and non-high-risk T2D patients than their respective high-risk subgroups. During the first year of the COVID-19 pandemic in the Netherlands, changes in primary healthcare use were observed among persons with T2D, with an increase in GP office visits and phone calls and a decreased number of clinical measurements and GP home visits. HbA1c levels increased among patients with T2D in 2020. Further research is necessary to determine the impact of the COVID-19 pandemic on long-term clinical outcomes and (long-term) T2D complications