Memory B Cell Activation Induced by Pertussis Booster Vaccination in Four Age Groups of Three Countries

Background: Immunogenicity of acellular pertussis (aP) vaccines is conventionally assessed by measuring antibody responses but antibody concentrations wane quickly after vaccination. Memory B cells, however, are critical in sustaining long-term protection and therefore may be an important factor when assessing pertussis immunity after vaccination. Aim: We studied pertussis specific memory B cell (re)activation induced by an aP booster vaccination in four different age groups within three countries. Materials and methods: From a phase IV longitudinal interventional study, 268 participants across Finland, the Netherlands and the United Kingdom were included and received a 3-component pertussis booster vaccine: children (7-10y, n=53), adolescents (11-15y, n=66), young adults (20-34y, n=74), and older adults (60-70y, n=75). Memory B cells at baseline, day 28, and 1 year post-vaccination were measured by a pertussis toxin (Ptx), filamentous haemagglutinin (FHA), and pertactin (Prn) specific ELISpot assay. Antibody results measured previously were available for comparison. Furthermore, study participants were distributed into groups based on their baseline memory B cell frequencies, vaccine responses were monitored between these groups. Results: Geometric mean (GM) memory B cell frequencies for pertussis antigens at baseline were low. At 28 days post-vaccination, these frequencies increased within each age group and were still elevated one year post-booster compared to baseline. Highest frequencies at day 28 were found within adolescents (GM: 5, 21, and 13, for Ptx, FHA and Prn, respectively) and lowest within older adults (GM: 2, 9, and 3, respectively). Moderate to strong correlations between memory B cell frequencies at day 28 and antibody concentrations at day 28 and 1 year were observed for Prn. Memory B cell frequencies > 1 per 100,000 PBMCs at baseline were associated with significantly higher memory responses after 28 days and 1 year. Conclusions: An aP booster vaccine (re)activated memory B cells in all age groups. Still elevated memory B cell frequencies after one year indicates enhanced immunological memory. However, antigen specific memory B cell activation seems weaker in older adults, which might reflect immunosenescence. Furthermore, the presence of circulating memory B cells at baseline positively affects memory B cell responses. This study was registered at www.clinicaltrialsregister.eu: No. 2016-003678-42.</p

Loss of DNMT1o Disrupts Imprinted X Chromosome Inactivation and Accentuates Placental Defects in Females

The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1omat-/- mouse embryos born to Dnmt1Δ1o/Δ1o female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1Δ1o/Δ1o mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation. © 2013 McGraw et al

The role of the gluconeogenic enzyme PCK2 in endothelial cells

Blood vessels supply tissues with vital nutrients and oxygen and they take part in controling systemic pH and temperature homeostasis. As such they are crucial for maintaining tissue homeostasis and normal health. Endothelial cells (ECs) form the inner lining of blood vessels, however, they are more than inert lining material of the vasculature. Instead, they are active players in the formation of new blood vessels from pre-existing ones (a process called vessel sprouting or angiogenesis) both in health and (life-threatening) diseases. Remarkably, aberrant EC behavior, either by dysfunctionality or by excessive vessel sprouting, contributes to the etiology of more diseases than any other tissue in the body. ECs display a remarkable behavioral plasticity; while quiescent for years, ECs can switch almost instantaneously to an activated, highly proliferative, and migratory state during vessel sprouting in response to growth factor stimuli, primarily through vascular endothelial growth factor (VEGF) signaling. Emerging evidence, mainly from the host lab, reveals that EC metabolism drives vessel sprouting (angiogenesis) in parallel to well-established growth factor-based signaling. Moreover, proof-of-principle studies have shown that targeting EC metabolism can inhibit pathological angiogenesis and can be exploited as an alternative for growth factor-based therapies, with a potentially advantageous reduction in resistance and escape mechanisms (as they occur for example in tumor vasculature upon anti-VEGF treatment). This highlights the therapeutic potential of targeting EC metabolism. Angiogenesis is an energy- and biomass-demanding process. It has been reported by the host lab that ECs are highly glycolytic and metabolize glucose for the synthesis of glycolytic intermediates, precursors of energy and biomass production and redox homeostasis. In mature vessels, quiescent ECs are exposed to ample glucose in the plasma (5.5 mM). However, angiogenic ECs constantly face a fluctuating nutrient supply and sprout into avascular areas, such as in tumor tissue in which glucose concentrations can be as low as 0.12 mM. Whether and how ECs synthesize glycolytic intermediates in such glucose-starved conditions, remains a fundamental question in vascular biology. For this doctoral research thesis, I explored whether ECs in glucose-deprived conditions switch to de novo synthesis of glycolytic intermediates, required for normal EC function, via gluconeogenesis (GNG). The gluconeogenic pathway has been considered to be for the most part the reversal of the glycolytic pathway. Here, I report that glucose-deprived ECs upregulate the expression of the key gluconeogenic enzyme PCK2 and rely on a PCK2-dependent abbreviated GNG pathway, whereby lactate and glutamine are used for the synthesis of lower glycolytic intermediates that enter the serine and glycerophospholipid biosynthesis pathways for respectively redox homeostasis and phospholipid synthesis. I show that endothelial PCK2 is essential for vessel sprouting and barrier integrity; and that increased oxidative stress and impaired glycerol-phospholipid synthesis in glucose-deprived PCK2KD ECs may be the underlying cause for their impaired angiogenic behavior and vascular barrier integrity. Unexpectedly, however, PCK2KD impaired the angiogenic behavior of ECs even in normal glucose conditions. Mechanistically, irrespective of extracellular glucose concentrations, PCK2KD ECs have an impaired unfolded protein response, leading to accumulation of misfolded proteins, which due to defective proteasomes and impaired autophagy, results in the accumulation of toxic protein aggregates in lysosomes and cellular demise. Overall, these results provide novel insights regarding the metabolic profile of ECs, as they show that glucose-deprived ECs have an active yet abbreviated GNG pathway under the control of PCK2 and that endothelial PCK2 is essential for vessel sprouting. Moreover, this study identifies an additional and previously unrecognized role of PCK2 in cellular proteostasis, beyond its traditional metabolic role in GNG.status: publishe

Endothelial cell metabolism in normal and diseased vasculature

Higher organisms rely on a closed cardiovascular circulatory system with blood vessels supplying vital nutrients and oxygen to distant tissues. Not surprisingly, vascular pathologies rank among the most life-threatening diseases. At the crux of most of these vascular pathologies are (dysfunctional) endothelial cells (ECs), the cells lining the blood vessel lumen. ECs display the remarkable capability to switch rapidly from a quiescent state to a highly migratory and proliferative state during vessel sprouting. This angiogenic switch has long been considered to be dictated by angiogenic growth factors (eg, vascular endothelial growth factor) and other signals (eg, Notch) alone, but recent findings show that it is also driven by a metabolic switch in ECs. Furthermore, these changes in metabolism may even override signals inducing vessel sprouting. Here, we review how EC metabolism differs between the normal and dysfunctional/diseased vasculature and how it relates to or affects the metabolism of other cell types contributing to the pathology. We focus on the biology of ECs in tumor blood vessel and diabetic ECs in atherosclerosis as examples of the role of endothelial metabolism in key pathological processes. Finally, current as well as unexplored EC metabolism-centric therapeutic avenues are discussed.status: publishe

DNA methylation and allelic expression analyses of <i>Dnmt1o^mat−/−</i> female placentae.

<p>(A) Sex-specific restriction landmark genomic scanning (RLGS) analysis of single-copy gene DNA methylation in <i>Dnmt1o^mat−/−</i> 9.5dpc placentae. Bars indicate the number of spots showing hypo- and hypermethylation changes in each <i>Dnmt1o^mat−/−</i> placenta (P). (B) Evidence of relaxation of paternal imprinted XCI in female <i>Dnmt1o^mat−/−</i>offspring. Allele specific expression assay (RT-PCR) on X chromosome linked genes. The RNA used was extracted from the visceral endoderm layer of yolk sacs from 9.5dpc <i>Dnmt1o^mat−/−</i> and wild-type control (<i>Dnmt1o^mat+/+</i>) extraembryonic tissues. Maternal (129(M)) and paternal (Cast(P)) control fragments were derived from 129/sv and <i>Mus musculus castaneus</i> embryo RNA respectively. The 129× Cast control fragments were generated from F1 hybrid embryo RNA (129/sv × <i>Mus musculus castaneus</i>). Schematic on the right shows approximate gene positions (Mb) on the X chromosome.</p

Map of selected elements within or surrounding the X-inactivation center region.

<p>The <i>Xite</i> (yellow) regulatory element is a positive enhancer of <i>Tsix</i> (green) expression, which is an antisense transcript that represses <i>Xist</i> (red). Numbers below arrows indicate regions amplified for DNA methylation studies. 1- <i>Xist</i>; 2-<i>Tsix</i>-CTCFc; 3- <i>Tsix</i> CGI, (major promoter); 4- <i>Tsix</i> (upstream major promoter); 5- <i>Xite</i>-DHS2; 6- <i>Xite-</i>DHS4, 7- <i>Xite</i>-DHS6 (<i>Tsix</i> minor promoter) and 8- <i>Chic1</i> promoter.</p

Model of the dynamic regulation of methylation maintenance by DNMT1o and establishment of imprinted XCI during preimplantation development.

<p>Wild-type XX: Maternally produced DNMT1o translocates into the nucleus of 8-cell embryos. Nuclear DNMT1o produces a ‘boost’ to maintain methylation marks on DMDs, and sequences on the X chromosome, repeats and other specific sequences. In the blastocyst, a reprogramming and <i>de novo</i> methylation phase takes place in the inner cell mass (ICM) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003873#pgen.1003873-Sado4" target="_blank">[65]</a> and high levels of global genome methylation are observed in this cell lineage. Imprinted XCI is maintained on the Xp in the extraembryonic derivatives, while in the ICM the reprogramming activity reactivates the Xp and random XCI causes inactivation of either the Xm or Xp. <i>Dnmt1o^mat−/−</i> XY and <i>Dnmt1o^mat−/−</i> XX: Lack of DNMT1o at the 8-cell stage prevents the methylation ‘boost’ and causes a failure in the maintenance of methylation marks on DMDs and other sequences including repeat sequences and X-linked genes. This failure of maintenance methylation at the 8-cell stage results in expression from both Xp and Xm in the extraembryonic lineage (relaxation of imprinted XCI). Activation of both X chromosomes in extraembryonic tissues is associated with methylation loss at repeat elements as well as other sequences across the genome. In contrast, the reprogramming event in the ICM restores proper epigenetic patterns and normal random XCI is established in XX embryos. Following the reprogramming and <i>de novo</i> methylation phase, the global DNA methylation levels in the XY and XX cells derived from ICM are similar to wild-type.</p

Early <i>Dnmt1o^mat−/−</i> blastocysts exhibit abnormal methylation of <i>Xite</i> but normal <i>Xist</i> expression.

<p>(A) Perturbed imprinted XCI model following <i>Dnmt1o</i>-deficiency. Lack of DNMT1o activity initiates hypomethylation events on <i>Xite</i> sequences, which activates and sustains the <i>Tsix</i> expression on the paternal X chromosome. This chain of events leads to repression of <i>Xist</i> expression on the paternal X chromosome. (B) Bisulfite cloning and sequencing results in sexed control and <i>Dnmt1o^mat−/−</i> blastocysts for two regions that exhibited hypomethylation in female <i>Dnmt1o^mat−/−</i> extraembryonic tissues (9.5dpc). Each line represents one sequenced allele, and the number at the left indicates the number of clones sequenced for that allele. Filled circles refer to methylated CpG dinucleotides. Percentages of methylated CpGs are shown. (C) Graphs represent the means of methylation percentages obtained for single sexed blastocysts. Mean ± SEM. **p<0.001. (D) Combined confocal microscopy images from RNA-DNA FISH experiments. Localization of <i>Xist</i> RNA (green) marks the inactive X chromosome. Specific X chromosome staining with Dxwas70 (red). Nuclei were counterstained with DAPI (blue).</p

DNA methylation of X-CGI and <i>Xic</i> loci in <i>Dnmt1o^mat−/−</i> female placentae.

<p>(A) Analysis of X-CGI and <i>Xic</i> loci in <i>Dnmt1o^mat−/−</i> female placentae using MassARRAY. The positions of 17 CpG islands and 7 regions of the <i>Xic</i> that were analysed are depicted on the left of the figure. Each tick mark equals 10 Mb. Methylation values are displayed relative to the average of the control samples for each gene (blue, hypomethylation; yellow, hypermethylation). Samples are clustered according to their methylation values revealing two distinct groups (green, control; pink, <i>Dnmt1o^mat−/−</i> normal morphology; red, <i>Dnmt1o^mat−/−</i> abnormal morphology). (*p<0.05 control versus <i>Dnmt1o^mat−/−</i>; ^†p<0.05 control versus <i>Dnmt1o^mat−/−</i> abnormal morphology). Loci marked with “<b>#</b>” were identified through RLGS experiments. (B) Histograms displaying the absolute methylation values for <i>Xite</i>-DHS6, <i>Tsix</i>-CTCFc and <i>Xist</i> genes separated into placenta morphology groups (*p<0.05 control versus normal or abnormal <i>Dnmt1o^mat−/−</i>).</p

Identified loci that display altered DNA methylation in <i>Dnmt1o^mat−/−</i> embryos and placentae.

a<p>Number of profiles with methylation change for specific loci (total placenta samples : 4XX + 4XY).</p>b<p>Methylation changed in all XX profiles and unchanged in all XY profiles.</p>c<p>Loci methylation changed in all profiles.</p