163 research outputs found
Regulation of murine B cell development and function
No full textVertebrates have developed an effective and protective, yet complex and dynamic immune system to defend themselves against the many threats from microorganisms. In this thesis some of the questions regarding the regulation of murine B cell development and function have been addressed.
In the first part it was investigated whether the quality of the pairing interaction between the µH chain and the surrogate light (SL) chain of a given pre-B cell receptor may determine the size of the clonal expansion at the large pre-B II cell stage in the bone marrow (BM). VHDHJH-rearrangements derived from pre-B cells having proliferated to different extent in vitro in the absence of any added cytokines were isolated and analysed for their sequences and pairing capacities. To this end, the present study shows a tendency of a better pairing capacity for VHDHJH-rearrangements derived from cells, which have undergone more cell divisions.
In the second part the effect of TSLP in lymphocyte development was investigated. It could be shown that increased TSLP availability through transgene expression fully restored lymphopoiesis in IL-7 deficient mice. It rescued B-cell development, increased the thymic cellularity, and rescued the thymic architecture in IL-7-/- mice. Adult wt bone marrow cells differentiated normally into B and T lineages and restored peripheral compartments when adoptively transferred into lethally irradiated IL-7-/- TSLP Tg recipients. Moreover, it could be shown that B cells generated in IL-7-/- TSLP Tg mice originated from adult precursors as these B cells contained N nucleotide additions in their IgH junctions.
In the third part the expression of BAFF-R on murine BM B cells was investigated with the use of novel monoclonal anti-mBAFF-R antibodies. We found that expression of BAFF-R is first detectable by flow cytometry (FACS) on a fraction of CD19+ CD93+ IgM+ CD23- BM B cells. This BAFF-R+ BM B cell population showed higher levels of surface IgM expression and decreased recombination-activating gene 2 (RAG-2) transcripts than BAFF-R- immature B cells. When cultured in vitro, BAFF-R+ immature B cells did not undergo further B cell receptor rearrangement, while BAFF-R- immature B cells did. However, when cultured in the presence of an anti-kappa light chain antibody, BAFF-R+ immature B cells could be induced to undergo receptor editing and this correlated with the upregulation of RAG-2 and the downregulation of both surface IgM and BAFF-R expression. Addition of BAFF did not inhibit this induced receptor editing. We concluded, that expression of BAFF-R can be used as a marker to identify immature B cells, which under normal conditions no longer undergo BCR editing, but can still be induced to do so by BCR engagement.
In the fourth part a putative new B cell population was phenotypically and functionally characterized. These cells, in this study termed ‘newB’ cells, were defined by the expression of CD19+ CD93- CD21-/low CD23-/low CD5-. The collected data so far supports the idea that newB cells could represent an additional intermediate cell population in the transition of immature to mature B cells. Alternatively, newB cells could represent cells, which for as yet, unknown reasons get selected out from the pool of mature, reactive B cells later, because they do not fulfill criteria to remain and therefore are rendered anergic and regain a more immature phenotype.
In the last part the differential response of splenic mature B cells to the mitogen lipopolysaccharide (LPS) was investigated. It was known that frequencies of LPS-reactive B cells in C57BL/6 mice is higher than in BALB/c mice. In this study, it could be shown that actually the FOB cells of C57BL/6 respond stronger to LPS in vitro than FOB cells of the BALB/c strain. In addition, MZB cells of both mouse strains showed a stronger response to LPS than the FOB cells. However, in contrast to the observation in FOB cells, MZB cells of both tested strains responded equally strong. A genetic approach did not lead to the identification of a responsible locus for the observed differential response in FOB cells, but indicated that most probably multiple genes control the response in a differential fashion in FOB cells in the tested mouse strains. Furthermore, the results suggest that the stronger response of FOB cells from C57BL/6 mice either involves components within the MyD88-dependent pathway or components, which can modulate the MyD88-dependent signaling pathway. In addition and in contrast to another study, we could show that the observed differential response is not determined by the MHC class II haplotype in these strains
Analysis of Rad3 and Chk1 protein kinases defines different checkpoint responses.
No full textEukaryotic cells respond to DNA damage and S phase replication blocks by arresting cell-cycle progression through the DNA structure checkpoint pathways. In Schizosaccharomyces pombe, the Chk1 kinase is essential for mitotic arrest and is phosphorylated after DNA damage. During S phase, the Cds1 kinase is activated in response to DNA damage and DNA replication blocks. The response of both Chk1 and Cds1 requires the six 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1). We demonstrate that DNA damage-dependent phosphorylation of Chk1 is also cell-cycle specific, occurring primarily in late S phase and G2, but not during M/G1 or early S phase. We have also isolated and characterized a temperature-sensitive allele of rad3. Rad3 functions differently depending on which checkpoint pathway is activated. Following DNA damage, rad3 is required to initiate but not maintain the Chk1 response. When DNA replication is inhibited, rad3 is required for both initiation and maintenance of the Cds1 response. We have identified a strong genetic interaction between rad3 and cds1, and biochemical evidence shows a physical interaction is possible between Rad3 and Cds1, and between Rad3 and Chk1 in vitro. Together, our results highlight the cell-cycle specificity of the DNA structure-dependent checkpoint response and identify distinct roles for Rad3 in the different checkpoint responses
Characterization of a novel human SMC heterodimer homologous to the Schizosaccharomyces pombe Rad18/Spr18 complex
No full textThe structural maintenance of chromosomes (SMC) protein encoded by the fission yeast rad18 gene is involved in several DNA repair processes and has an essential function in DNA replication and mitotic control. It has a heterodimeric partner SMC protein, Spr18, with which it forms the core of a multiprotein complex. We have now isolated the human orthologues of rad18 andspr18 and designated them hSMC6 andhSMC5. Both proteins are about 1100 amino acids in length and are 27–28% identical to their fission yeast orthologues, with much greater identity within their N- and C-terminal globular domains. The hSMC6 and hSMC5 proteins interact to form a tight complex analogous to the yeast Rad18/Spr18 heterodimer. In proliferating human cells the proteins are bound to both chromatin and the nucleoskeleton. In addition, we have detected a phosphorylated form of hSMC6 that localizes to interchromatin granule clusters. Both the total level of hSMC6 and its phosphorylated form remain constant through the cell cycle. Both hSMC5 and hSMC6 proteins are expressed at extremely high levels in the testis and associate with the sex chromosomes in the late stages of meiotic prophase, suggesting a possible role for these proteins in meiosis
First results of the Auger Radio Infill SKALA Extension (ARISE)
No full textRadio detection of extensive air showers has become a powerful technique for studying high-energy cosmic rays. To further enhance these measurements, the Pierre Auger Observatory in Argentina, one of the world`s largest cosmic-ray experiments, has been upgraded with radio antenna stations. This upgrade aims to improve the precision of air-shower energy measurements in the energy range of several tens of PeV and above.
Within this framework, a new experiment, ARISE (``Auger Radio Infill SKALA Extension\u27\u27), has been deployed at the Pierre Auger Observatory. ARISE consists of six stations, each comprising three SKALA antennas installed around a surface detector in the denser infill region.
This presentation will present first air-shower measurements from ARISE recorded in coincidence with the Auger surface detector array
European eel (Anguilla anguilla): prediction of spawner escapement from continental population parameters
No full textThis paper describes the assessment of silver European eel (Anguilla anguilla) escapement based on a “sedentary” population fraction analysis in a 60-km2 watershed of northern Brittany (France). Downstream migration fluxes were monitored using eel traps and related to environmental factors. Intensive electrofishing and fyke-net fishing were conducted to assess eel biomass, densities, and population structure. A total of 564 eels, including 81 silver eels, were PIT tagged. In 1996, 616 eels were caught in the catchment including 68 silver eels (11%). During the following downstream migration period, 12 of the PIT-tagged silver eels, among a total of 678, were recaptured in the downstream traps. Seven were recaptured in the catchment in 1997. It was shown that (i) only about 20% of the silver eels present in the catchment emigrated during the following year, (ii) 12% stayed in the catchment including two (3.4%) that recovered yellow eel characteristics, and (iii) the other eels either died or settled in the catchment but were not recaptured. It was also estimated that 2% (650 eels) of the population (34 000 eels) among 3000 silver eels considered “emigration candidates” emigrated each year
The yellow European eel (Anguilla anguilla L.) may adopt a sedentary lifestyle in inland freshwaters
No full textWe analysed the movements of the growing yellow phase using a long-term mark–recapture programme on European eels in a small catchment (the Frémur, France). The results showed that of the yellow eels (>200 mm) recaptured, more than 90% were recaptured at the original marking site over a long period before the silvering metamorphosis and downstream migration. We conclude that yellow European eels >200 mm may adopt a sedentary lifestyle in freshwater area, especially in small catchment
Towards a standardized characterization of the potentially migrating silver European eel (Anguilla anguilla L.)
No full textWe defined a standardized method for discriminating candidate silver eels
that may undergo catadromous migration in the following season from the sedentary
fraction of a population. A combination of two qualitative criteria (state of differentiation
of the lateral line and colour contrast) and one quantitative criterion (Ocular Index
OI) was used to determine the development toward silvering. In the non-migratory
phase, we found a gradient of the three criteria between yellow (0 criterion), presilver
(1 to 2 criteria) and silver (3 criteria) eels. In the migrant phase, silver eels had ended
their metamorphosis process and were characterized at the same time by the presence
of the 3 silvering criteria. A mark-recapture survey using PIT-tags provided evidence
that only identified silver eels (3 silvering criteria present) in the catchment actually
emigrated the following season. Moreover, the use of a single criterion of silvering
among the three generated large variation in the estimated proportion of candidates for
emigration which varied between –22% and +63 %. Such a result confirmed that a
multicriteria approach is needed to characterize in a standard way the potentially migrating
silver eel
A study of the modulation of Toll-like receptor signalling in macrophages by Annexin-1
No full textPhDAnnexin-A1 (AnxA1) is an endogenous anti-inflammatory protein that has been shown to exert a protective role against the lethal effects induced by the Toll-like receptor (TLR) 4 agonist lipopolysaccharide (LPS). The aim of this PhD studentship was to expand these observations and investigate the possible cross-talk between AnxA1 and other TLR signalling pathways in macrophages. To this aim, we compared the response in vitro of AnxA1-/- to AnxA1+/+ bone marrow-derived macrophages (BMDMs) after stimulation with TLR2, 3, 5, 7 and 9 ligands. AnxA1-/- BMDMs exhibited higher expression of the activation markers MHC II and co-stimulatory molecules CD40, CD80, CD86 at the basal level, but a similar upregulation after stimulation with different TLR agonists. Stimulation of AnxA1-/- BMDMs with MyD88-dependent TLR agonists caused an increased production of TNF- and IL-6 compared to AnxA1+/+. Conversely, stimulation with the TRIF-dependent ligand poly (I:C) caused a decreased production of IL-6, but not TNF-, by these cells. Interestingly, comparison of MyD88 and TRIF-dependent downstream signalling pathways in AnxA1+/+ and AnxA1-/- BMDMs showed different kinetics of NF-B DNA-binding activity, IB- degradation and ERK1/2 phosphorylation. Consistent with this, measurement of MyD88-dependent or TRIF-regulated genes in AnxA1+/+ and AnxA1-/- BMDMs indicated a different time course of expression following stimulation with TLR3 (TRIF-dependent), TLR9 (MyD88-dependent) and TLR4 (TRIF and MyD88-dependent) ligands. Finally, AnxA1-/- mice showed an increased survival after challenge with poly (I:C), in contrast to increased lethality after injection of LPS, compared to AnxA1+/+ mice. These results suggest that endogenous AnxA1 influences mainly the MyD88-dependent pathway and to a lesser extent the TRIF-dependent pathway both in vitro and in vivo. In addition, this study provides future venues for the investigation of molecular mechanisms by which endogenous AnxA1 preferentially interferes with specific TLR signalling pathways.Saint Bartholomew's and the royal London Charitable Foundatio
Impact of the magnetic horizon on the interpretation of the Pierre Auger Observatory spectrum and composition data
No full textThe flux of ultra-high energy cosmic rays reaching Earth above the ankle energy (5 EeV) can be described as a mixture of nuclei injected by extragalactic sources with very hard spectra and a low rigidity cutoff. Extragalactic magnetic fields existing between the Earth and the closest sources can affect the observed CR spectrum by reducing the flux of low-rigidity particles reaching Earth. We perform a combined fit of the spectrum and distributions of depth of shower maximum measured with the Pierre Auger Observatory including the effect of this magnetic horizon in the propagation of UHECRs in the intergalactic space. We find that, within a specific range of the various experimental and phenomenological systematics, the magnetic horizon effect can be relevant for turbulent magnetic field strengths in the local neighbourhood in which the closest sources lie of order B-rms similar or equal to (50-100) nG(20Mpc/ds)(100 kpc/L-coh)(1/2), with d(s) the typical intersource separation and L-coh the magnetic field coherence length. When this is the case, the inferred slope of the source spectrum becomes softer and can be closer to the expectations of diffusive shock acceleration, i.e., proportional to E-2. An additional cosmic-ray population with higher source density and softer spectra, presumably also extragalactic and dominating the cosmic-ray flux at EeV energies, is also required to reproduce the overall spectrum and composition results for all energies down to 0.6 EeV
Characterisation of the anti-inflammatory and anti-proliferative effects of natriuretic peptides in rodents.
No full textNatriuretic peptides are a family of vasoactive hormones that play important roles in cardiovascular homeostasis. These peptides exert biological effects primarily via activation of guanylate cyclase (GC)-coupled natriuretic peptide receptors (NPR) that generate the intracellular messenger cyclic guanosine 3',5'-monophosphate (cGMP). Activation of the cytosolic GC by NO is well-established to mediate cGMP-dependent, anti-atherogenic effects however, an analogous cytoprotective role for natriuretic peptides has yet to be fully elucidated. Since many cardiovascular disorders (e.g. atherosclerosis, septic shock) are now accepted as inflammation-based diseases, identification of potential roles for natriuretic peptides in regulating vascular inflammation might assist in the prevention and treatment of cardiovascular pathology. The studies described in this thesis investigated the hypothesis that natriuretic peptides (i.e. atrial natriuretic peptide ANP , C-type natriuretic peptide CNP ) affect pro-inflammatory protein expression (i.e. inducible NO synthase, iNOS) and cell proliferation via activation of GC-linked NPR. Herein, it is demonstrated that in NPR-A knockout mice, iNOS expression and NO production in response to intravenous administration of bacterial lipopolysaccharide is significantly reduced compared to wild-type controls this difference is mirrored in the ex vivo functional reactivity of vessels from such animals. However, neither ANP nor CNP were able to alter iNOS expression or NO production in vitro in RAW264.7 murine macrophages or primary rat aortic smooth muscle cells. CNP, but not ANP, transiently enhanced phosphorylation of extracellular signal-regulated kinase (ERK)1/2. CNP-induced ERK 1/2 phosphorylation was blocked by the selective ERK 1/2 inhibitor PD98059, the Gj-protein inhibitor Pertussis toxin, and the selective NPR-C antagonist M372049. Accordingly, CNP inhibited vascular smooth muscle proliferation in a PD98059- and M372049-reversible manner. These observations suggest that part of the anti-atherogenic profile of CNP is mediated via NPR-C, Gi-dependent ERK 1/2 phosphorylation and inhibition of vascular smooth muscle proliferation. Moreover, my findings identify a potential pro-inflammatory role for ANP/NPR-A-dependent signalling in vivo
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