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

Small- bowel mucosal changes and antibody responses after low- and moderate-dose gluten challenge in celiac disease

<p>Abstract</p> <p>Background</p> <p>Due to the restrictive nature of a gluten-free diet, celiac patients are looking for alternative therapies. While drug-development programs include gluten challenges, knowledge regarding the duration of gluten challenge and gluten dosage is insufficient.</p> <p>We challenged adult celiac patients with gluten with a view to assessing the amount needed to cause some small-bowel mucosal deterioration.</p> <p>Methods</p> <p>Twenty-five celiac disease adults were challenged with low (1-3 g) or moderate (3-5g) doses of gluten daily for 12 weeks. Symptoms, small-bowel morphology, densities of CD3+ intraepithelial lymphocytes (IELs) and celiac serology were determined.</p> <p>Results</p> <p>Both moderate and low amounts of gluten induced small-bowel morphological damage in 67% of celiac patients. Moderate gluten doses also triggered mucosal inflammation and more gastrointestinal symptoms leading to premature withdrawals in seven cases. In 22% of those who developed significant small- intestinal damage, symptoms remained absent. Celiac antibodies seroconverted in 43% of the patients.</p> <p>Conclusions</p> <p>Low amounts of gluten can also cause significant mucosal deterioration in the majority of the patients. As there are always some celiac disease patients who will not respond within these conditions, sample sizes must be sufficiently large to attain to statistical power in analysis.</p

Cigarette smoke induces β2-integrin-dependent neutrophil migration across human endothelium

<p>Abstract</p> <p>Background</p> <p>Cigarette smoking induces peripheral inflammatory responses in all smokers and is the major risk factor for neutrophilic lung disease such as chronic obstructive pulmonary disease. The aim of this study was to investigate the effect of cigarette smoke on neutrophil migration and on β₂-integrin activation and function in neutrophilic transmigration through endothelium.</p> <p>Methods and results</p> <p>Utilizing freshly isolated human PMNs, the effect of cigarette smoke on migration and β₂-integrin activation and function in neutrophilic transmigration was studied. In this report, we demonstrated that cigarette smoke extract (CSE) dose dependently induced migration of neutrophils <it>in vitro</it>. Moreover, CSE promoted neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that Mac-1 (CD11b/CD18) is responsible for the cigarette smoke-induced firm adhesion of neutrophils to fibrinogen. Furthermore, neutrophils transmigrated through endothelium by cigarette smoke due to the activation of β₂-integrins, since pre-incubation of neutrophils with functional blocking antibodies against CD11b and CD18 attenuated this transmigration.</p> <p>Conclusion</p> <p>This is the first study to describe that cigarette smoke extract induces a direct migratory effect on neutrophils and that CSE is an activator of β₂-integrins on the cell surface. Blocking this activation of β₂-integrins might be an important target in cigarette smoke induced neutrophilic diseases.</p

Whole-chromosome hitchhiking driven by a male-killing endosymbiont.

Neo-sex chromosomes are found in many taxa, but the forces driving their emergence and spread are poorly understood. The female-specific neo-W chromosome of the African monarch (or queen) butterfly Danaus chrysippus presents an intriguing case study because it is restricted to a single 'contact zone' population, involves a putative colour patterning supergene, and co-occurs with infection by the male-killing endosymbiont Spiroplasma. We investigated the origin and evolution of this system using whole genome sequencing. We first identify the 'BC supergene', a broad region of suppressed recombination across nearly half a chromosome, which links two colour patterning loci. Association analysis suggests that the genes yellow and arrow in this region control the forewing colour pattern differences between D. chrysippus subspecies. We then show that the same chromosome has recently formed a neo-W that has spread through the contact zone within approximately 2,200 years. We also assembled the genome of the male-killing Spiroplasma, and find that it shows perfect genealogical congruence with the neo-W, suggesting that the neo-W has hitchhiked to high frequency as the male-killer has spread through the population. The complete absence of female crossing-over in the Lepidoptera causes whole-chromosome hitchhiking of a single neo-W haplotype, carrying a single allele of the BC supergene and dragging multiple non-synonymous mutations to high frequency. This has created a population of infected females that all carry the same recessive colour patterning allele, making the phenotypes of each successive generation highly dependent on uninfected male immigrants. Our findings show how hitchhiking can occur between the physically unlinked genomes of host and endosymbiont, with dramatic consequences

Habitat properties are key drivers of Borrelia burgdorferi (s.l.) prevalence in Ixodes ricinus populations of deciduous forest fragments

Background: The tick Ixodes ricinus has considerable impact on the health of humans and other terrestrial animals because it transmits several tick-borne pathogens (TBPs) such as B. burgdorferi (sensu lato), which causes Lyme borreliosis (LB). Small forest patches of agricultural landscapes provide many ecosystem services and also the disservice of LB risk. Biotic interactions and environmental filtering shape tick host communities distinctively between specific regions of Europe, which makes evaluating the dilution effect hypothesis and its influence across various scales challenging. Latitude, macroclimate, landscape and habitat properties drive both hosts and ticks and are comparable metrics across Europe. Therefore, we instead assess these environmental drivers as indicators and determine their respective roles for the prevalence of B. burgdorferi in I. ricinus. Methods: We sampled I. ricinus and measured environmental properties of macroclimate, landscape and habitat quality of forest patches in agricultural landscapes along a European macroclimatic gradient. We used linear mixed models to determine significant drivers and their relative importance for nymphal and adult B. burgdorferi prevalence. We suggest a new prevalence index, which is pool-size independent. Results: During summer months, our prevalence index varied between 0 and 0.4 per forest patch, indicating a low to moderate disservice. Habitat properties exerted a fourfold larger influence on B. burgdorferi prevalence than macroclimate and landscape properties combined. Increasingly available ecotone habitat of focal forest patches diluted and edge density at landscape scale amplified B. burgdorferi prevalence. Indicators of habitat attractiveness for tick hosts (food resources and shelter) were the most important predictors within habitat patches. More diverse and abundant macro- and microhabitat had a diluting effect, as it presumably diversifies the niches for tick-hosts and decreases the probability of contact between ticks and their hosts and hence the transmission likelihood.[br/] Conclusions: Diluting effects of more diverse habitat patches would pose another reason to maintain or restore high biodiversity in forest patches of rural landscapes. We suggest classifying habitat patches by their regulating services as dilution and amplification habitat, which predominantly either decrease or increase B. burgdorferi prevalence at local and landscape scale and hence LB risk. Particular emphasis on promoting LB-diluting properties should be put on the management of those habitats that are frequently used by humans. In the light of these findings, climate change may be of little concern for LB risk at local scales, but this should be evaluated further

Conscious perception of errors and its relation to the anterior insula

To detect erroneous action outcomes is necessary for flexible adjustments and therefore a prerequisite of adaptive, goal-directed behavior. While performance monitoring has been studied intensively over two decades and a vast amount of knowledge on its functional neuroanatomy has been gathered, much less is known about conscious error perception, often referred to as error awareness. Here, we review and discuss the conditions under which error awareness occurs, its neural correlates and underlying functional neuroanatomy. We focus specifically on the anterior insula, which has been shown to be (a) reliably activated during performance monitoring and (b) modulated by error awareness. Anterior insular activity appears to be closely related to autonomic responses associated with consciously perceived errors, although the causality and directions of these relationships still needs to be unraveled. We discuss the role of the anterior insula in generating versus perceiving autonomic responses and as a key player in balancing effortful task-related and resting-state activity. We suggest that errors elicit reactions highly reminiscent of an orienting response and may thus induce the autonomic arousal needed to recruit the required mental and physical resources. We discuss the role of norepinephrine activity in eliciting sufficiently strong central and autonomic nervous responses enabling the necessary adaptation as well as conscious error perception

Risico’s van Vibrio-besmetting in zwemwater, schelpdierproductiewater en schelpdieren

Vibrio-bacteriën komen van nature in zout en brak oppervlaktewater voor. Er zijn veel Vibrio-soorten, die verschillende gezondheidsklachten kunnen veroorzaken. Mensen kunnen infecties met Vibrio oplopen door te zwemmen in besmet oppervlaktewater. Ze krijgen dan vooral oor- en wondinfecties door Vibrio alginolyticus. Mensen kunnen ook infecties oplopen door schelpdieren te eten, zoals oesters. Als daar Vibrio parahaemolyticus in zitten, krijgen ze vooral maagdarmklachten. Uit eerder onderzoek van het RIVM in 2009–2012 bleek dat er verschillende Vibrio-soorten zaten in Nederlands zwemwater en in Nederland gekweekte schelpdieren. Bekend is dat het aantal Vibrio-bacteriën in water toeneemt bij hogere watertemperaturen (18 tot 25 graden Celsius). Hierdoor komen er naar verwachting in de toekomst door klimaatverandering meer Vibrio-bacteriën in het oppervlaktewater. Om te onderzoeken of dat gaat gebeuren, heeft het RIVM het eerdere onderzoek in 2019–2021 herhaald. Daaruit bleek dat het aantal Vibrio-bacteriën nu nog niet is gestegen. De watertemperatuur verschilde tussen 2009 en 2021 wat per jaar, maar werd per saldo niet steeds hoger. Op alle zwemlocaties zijn dezelfde Vibrio-soorten gevonden als in het eerdere onderzoek. Dat was ook zo op de meeste schelpdierkweeklocaties en in de meeste opgeviste schelpdieren. Net als in het vorige onderzoek kwam Vibrio alginolyticus het meest voor, gevolgd door Vibrio parahaemolyticus. De aantallen bacteriën waren ook ongeveer hetzelfde. Voor de toekomst (2030 en 2050) zijn de aantallen Vibrio-bacteriën berekend voor verschillende klimaatscenario’s. Als het water warmer wordt, zullen de aantallen in zwemwater en schelpdieren toenemen. Omdat de bacteriën van nature in zout en brak water zitten, is deze stijging niet te voorkomen. Het is daarom belangrijk dat zwemwaterbeheerders mensen zo nodig informeren over de risico’s bij zwemmen in zeewater. Ook moeten mensen zich bewust zijn van de risico’s die ze lopen bij het eten van rauwe oesters. Nu en in de toekomst. In de twee onderzoeken zijn zwemlocaties in de Noordzee, Waddenzee en Oosterschelde en schelpdierkweeklocaties in de Noordzee, Waddenzee, Oosterschelde en het Veerse Meer onderzocht. Verder zijn schelpdieren (oesters, mosselen) van deze kweeklocaties onderzocht

Risks of Vibrio infection in bathing water, shellfish production water and shellfish

Vibrio-bacteriën komen van nature in zout en brak oppervlaktewater voor. Er zijn veel Vibrio-soorten, die verschillende gezondheidsklachten kunnen veroorzaken. Mensen kunnen infecties met Vibrio oplopen door te zwemmen in besmet oppervlaktewater. Ze krijgen dan vooral oor- en wondinfecties door Vibrio alginolyticus. Mensen kunnen ook infecties oplopen door schelpdieren te eten, zoals oesters. Als daar Vibrio parahaemolyticus in zitten, krijgen ze vooral maagdarmklachten. Uit eerder onderzoek van het RIVM in 2009–2012 bleek dat er verschillende Vibrio-soorten zaten in Nederlands zwemwater en in Nederland gekweekte schelpdieren. Bekend is dat het aantal Vibrio-bacteriën in water toeneemt bij hogere watertemperaturen (18 tot 25 graden Celsius). Hierdoor komen er naar verwachting in de toekomst door klimaatverandering meer Vibrio-bacteriën in het oppervlaktewater. Om te onderzoeken of dat gaat gebeuren, heeft het RIVM het eerdere onderzoek in 2019–2021 herhaald. Daaruit bleek dat het aantal Vibrio-bacteriën nu nog niet is gestegen. De watertemperatuur verschilde tussen 2009 en 2021 wat per jaar, maar werd per saldo niet steeds hoger. Op alle zwemlocaties zijn dezelfde Vibrio-soorten gevonden als in het eerdere onderzoek. Dat was ook zo op de meeste schelpdierkweeklocaties en in de meeste opgeviste schelpdieren. Net als in het vorige onderzoek kwam Vibrio alginolyticus het meest voor, gevolgd door Vibrio parahaemolyticus. De aantallen bacteriën waren ook ongeveer hetzelfde. Voor de toekomst (2030 en 2050) zijn de aantallen Vibrio-bacteriën berekend voor verschillende klimaatscenario’s. Als het water warmer wordt, zullen de aantallen in zwemwater en schelpdieren toenemen. Omdat de bacteriën van nature in zout en brak water zitten, is deze stijging niet te voorkomen. Het is daarom belangrijk dat zwemwaterbeheerders mensen zo nodig informeren over de risico’s bij zwemmen in zeewater. Ook moeten mensen zich bewust zijn van de risico’s die ze lopen bij het eten van rauwe oesters. Nu en in de toekomst. In de twee onderzoeken zijn zwemlocaties in de Noordzee, Waddenzee en Oosterschelde en schelpdierkweeklocaties in de Noordzee, Waddenzee, Oosterschelde en het Veerse Meer onderzocht. Verder zijn schelpdieren (oesters, mosselen) van deze kweeklocaties onderzocht.Vibrio bacteria naturally occur in salt and brackish surface water. There are many Vibrio species, all of which can cause various health problems. People can contract Vibrio infections by swimming in contaminated surface water. In this case, they mainly contract ear and wound infections due to Vibrio alginolyticus. People can also become infected by eating shellfish, such as oysters. Consumption of shellfish that contain Vibrio parahaemolyticus typically leads to gastrointestinal symptoms. Previous research by RIVM in 2009–2012 showed that various Vibrio species were present in Dutch bathing water and in shellfish produced in the Netherlands. It is known that the number of Vibrio bacteria in water increases at higher water temperatures (18–25 °C). That is why climate change is expected to result in more Vibrio bacteria in surface water in the future. To investigate whether this is likely to happen, RIVM repeated the previous study in 2019–2021. The findings showed that the amount of Vibrio bacteria had not yet increased. Water temperatures varied somewhat over the years between 2009 and 2021, but did not increase overall. The same Vibrio species were found at all bathing sites as in the previous study. This was also the case at most shellfish production sites and in most shellfish harvested. As in the previous study, Vibrio alginolyticus was the most common strain, followed by Vibrio parahaemolyticus. The bacteria counts were about the same as well. RIVM calculated the number of Vibrio bacteria for different climate scenarios in the future (2030 and 2050). If the water gets warmer, the bacteria counts in bathing water and shellfish will increase. Because the bacteria are naturally present in salt and brackish water, this increase cannot be prevented. That is why it is important for managers of bathing water sites to inform people about the risks of swimming in the sea, when necessary. People should also be aware of the risks of eating raw oysters. Now and in the future. The two studies examined bathing sites in the North Sea, Wadden Sea and Eastern Scheldt, and shellfish production sites in the North Sea, Wadden Sea, Eastern Scheldt and Lake Veere. Shellfish (including oysters and mussels) from these farming sites were examined as well