Aptamer selection against cell extracts containing the zoonotic obligate intracellular bacterium, Anaplasma phagocytophilum.

A. phagocytophilum is a zoonotic and tick-borne bacterium, threatening human and animal health. Many questions persist concerning the variability of strains and the mechanisms governing the interactions with its different hosts. These gaps can be explained by the difficulty to cultivate and study A. phagocytophilum because of its strict intracellular location and the lack of specific tools, in particular monoclonal antibodies, currently unavailable. The objective of our study was to develop DNA aptamers against A. phagocytophilum, or molecules expressed during the infection, as new study and/or capture tools. Selecting aptamers was a major challenge due to the strict intracellular location of the bacterium. To meet this challenge, we set up a customized selection protocol against an enriched suspension of A. phagocytophilum NY18 strain, cultivated in HL-60 cells. The implementation of SELEX allowed the selection of three aptamers, characterized by a high affinity for HL-60 cells infected with A. phagocytophilum NY18 strain. Interestingly, the targets of these three aptamers are most likely proteins expressed at different times of infection. The selected aptamers could contribute to increase our understanding of the interactions between A. phagocytophilum and its hosts, as well as permit the development of new diagnostic, therapeutic or drug delivery appliances.

Optimized Lambda Exonuclease Digestion or Purification Using Streptavidin-Coated Beads: Which One Is Best for Successful DNA Aptamer Selection?

The high failure rate of the in vitro aptamer selection process by SELEX (Systematic Evolution of Ligands by EXponential enrichment) limits the production of these innovative oligonucleotides and, consequently, limits their potential applications. The generation of single-stranded DNA (ssDNA) is a critical step of SELEX, directly affecting the enrichment and the selection of potential binding sequences. The main goal of this study was to confirm the best method for generating ssDNA by comparing the purification of ssDNA, using streptavidin-coated beads, and lambda exonuclease digestion, and by improving ssDNA recovery through protocol improvements. In addition, three techniques for quantifying the ssDNA generated (Qubit vs. NanodropTM vs. gel quantification) were compared, and these demonstrated the accuracy of the gel-based quantification method. Lambda exonuclease digestion was found to be more efficient for ssDNA recovery than purification using streptavidin-coated beads, both quantitatively and qualitatively. In conclusion, this work provides a detailed and rigorous protocol for generating ssDNA, improving the chances of a successful aptamer selection process.

Enlisting the Ixodes scapularis Embryonic ISE6 Cell Line to Investigate the Neuronal Basis of Tick-Pathogen Interactions.

Neuropeptides are small signaling molecules expressed in the tick central nervous system, i.e., the synganglion. The neuronal-like Ixodes scapularis embryonic cell line, ISE6, is an effective tool frequently used for examining tick-pathogen interactions. We detected 37 neuropeptide transcripts in the I. scapularis ISE6 cell line using in silico methods, and six of these neuropeptide genes were used for experimental validation. Among these six neuropeptide genes, the tachykinin-related peptide (TRP) of ISE6 cells varied in transcript expression depending on the infection strain of the tick-borne pathogen, Anaplasma phagocytophilum. The immunocytochemistry of TRP revealed cytoplasmic expression in a prominent ISE6 cell subpopulation. The presence of TRP was also confirmed in A. phagocytophilum-infected ISE6 cells. The in situ hybridization and immunohistochemistry of TRP of I. scapularis synganglion revealed expression in distinct neuronal cells. In addition, TRP immunoreaction was detected in axons exiting the synganglion via peripheral nerves as well as in hemal nerve-associated lateral segmental organs. The characterization of a complete Ixodes neuropeptidome in ISE6 cells may serve as an effective in vitro tool to study how tick-borne pathogens interact with synganglion components that are vital to tick physiology. Therefore, our current study is a potential stepping stone for in vivo experiments to further examine the neuronal basis of tick-pathogen interactions.

Bovine Organospecific Microvascular Endothelial Cell Lines as New and Relevant In Vitro Models to Study Viral Infections.

Microvascular endothelial cells constitute potential targets for exogenous microorganisms, in particular for vector-borne pathogens. Their phenotypic and functional variations according to the organs they are coming from provide an explanation of the organ selectivity expressed in vivo by pathogens. In order to make available relevant tools for in vitro studies of infection mechanisms, our aim was to immortalize bovine organospecific endothelial cells but also to assess their permissivity to viral infection. Using transfection with SV40 large T antigen, six bovine microvascular endothelial cell lines from various organs and one macrovascular cell line from an umbilical cord were established. They display their own panel of endothelial progenitor/mature markers, as assessed by flow cytometry and RT-qPCR, as well as the typical angiogenesis capacity. Using both Bluetongue and foot-and-mouth disease viruses, we demonstrate that some cell lines are preferentially infected. In addition, they can be transfected and are able to express viral proteins such as BTV8-NS3. Such microvascular endothelial cell lines bring innovative tools for in vitro studies of infection by viruses or bacteria, allowing for the study of host-pathogen interaction mechanisms with the actual in vivo target cells. They are also suitable for applications linked to microvascularization, such as anti-angiogenic and anti-tumor research, growing fields in veterinary medicine.

Experimental Ixodes ricinus-Sheep Cycle of Anaplasma phagocytophilum NV2Os Propagated in Tick Cell Cultures.

The causative agent of tick-borne fever and human granulocytic anaplasmosis, Anaplasma phagocytophilum, is transmitted by Ixodes ricinus, and is currently considered an emerging disease throughout Europe. In this study, we established a model of A. phagocytophilum sheep infection and I. ricinus transmission using the European Norway variant 2 ovine strain (NV2Os) propagated in both IDE8 and ISE6 tick cells. Two sheep were inoculated with IDE8 tick cells infected with NV2Os. Both sheep developed A. phagocytophilum infection as determined by qPCR and PCR, the presence of fever 4 days post inoculation (dpi), the observation of morulae in granulocytes at 6 dpi, and the detection of A. phagocytophilum antibodies at 14 dpi. A. phagocytophilum was detected by PCR in skin, lung, small intestine, liver, spleen, uterus, bone marrow, and mesenteric lymph node from necropsies performed at 14 and 15 dpi. One sheep was infested during the acute phase of infection with I. ricinus nymphs from a pathogen-free colony. After molting, A. phagocytophilum transstadial transmission in ticks was validated with qPCR positive bacterial detection in 80% of salivary glands and 90% of midguts from female adults. Infected sheep blood collected at 14 dpi was demonstrated to be able to infect ISE6 tick cells, thus enabling the infection of two additional naive sheep, which then went on to develop similar clinical signs to the sheep infected previously. One of the sheep remained persistently infected until 115 dpi when it was euthanized, and transmitted bacteria to 70 and 2.7% of nymphs engorged as larvae during the acute and persistent infection stages, respectively. We then demonstrated that these infected nymphs were able to transmit the bacteria to one of two other naive infested sheep. As expected, when I. ricinus females were engorged during the acute phase of infection, no A. phagocytophilum transovarial transmission was detected. The development of this new experimental model will facilitate future research on this tick-borne bacterium of increasing importance, and enable the evaluation of any new tick/transmission control strategies.

Co-circulation of different A. phagocytophilum variants within cattle herds and possible reservoir role for cattle.

BACKGROUND: Anaplasma phagocytophilum is a zoonotic tick-borne intracellular alpha-proteobacterium causing tick-borne fever, which leads to significant economic losses in domestic ruminants in Europe. Its epidemiological cycles are complex and reservoir host species of bovine strains have not yet been identified. Given that little genetic information is available on strains circulating within a defined bovine environment, our objective was to assess the genetic diversity of A. phagocytophilum obtained from the same farms over time. METHODS: Blood samplings were performed several times in two European herds. In the French herd, 169 EDTA-blood samples were obtained from 115 cows (32 were sampled two to four times). In the German herd, 20 cows were sampled six times (120 EDTA-blood samples). The presence of A. phagocytophilum DNA was assessed using a qPCR targeting msp2. The positive DNA samples underwent MLST at nine genetic markers (typA, ctrA, msp4, pleD, recG, polA, groEL, gyrA, and ankA). For each locus, sequences were aligned with available bacterial sequences derived from cattle, horse, dog, and roe deer hosts, and concatenated neighbor joining trees were constructed using three to six loci. RESULTS: Around 20% (57/289) of samples were positive. Forty positive samples from 23 French and six German cows (11 of them being positive at two time points) were sequenced. Six loci (typA, ctrA, msp4, pleD, recG, and polA) allowed to build concatenated phylogenetic trees, which led to two distinct groups of bovine variants in the French herd (hereafter called A and B), whereas only group A was detected in the German herd. In 42% of French samples, double chromatogram peaks were encountered in up to four loci. Eleven cows were found infected three weeks to 17 months after first sampling and harboured a new variant belonging to one or the other group. CONCLUSIONS: Our results demonstrate the occurrence of two major bovine strain groups and the simultaneous infection of single cows by more than one A. phagocytophilum strain. This challenges the role of cattle as reservoirs for A. phagocytophilum. This role may be facilitated via long-term bacterial persistence in individual cows and active circulation at the herd scale.

The stellate vascular smooth muscle cell phenotype is induced by IL-1ß via the secretion of PGE2 and subsequent cAMP-dependent protein kinase A activation.

Atherosclerosis development is associated with morphological changes to intimal cells, leading to a stellate cell phenotype. In this study, we aimed to determine whether and how key pro-atherogenic cytokines present in atherosclerotic plaques (IL-1ß, TNFα and IFNγ) could induce this phenotype, as these molecules are known to trigger the transdifferentiation of vascular smooth muscle cells (VSMCs). We found that, IL-1ß was the only major inflammatory mediator tested capable of inducing a stellate morphology in VSMCs. This finding was confirmed by staining for F-actin and vinculin at focal adhesions, as these two markers were disrupted only by IL-1ß. We then investigated the possible association of this IL-1ß-dependent change in morphology with an increase in intracellular cAMP concentration ([cAMP]), using the FRET-based biosensor for cAMP (T)Epac(VV). Experiments in the presence of IL-1ß or medium conditioned by IL-1ß-treated VSMCs and pharmacological tools demonstrated that the long-term increase in intracellular cAMP concentration was induced by the secretion of an autocrine/paracrine mediator, prostaglandin E2(PGE2), acting through the EP4 receptor. Finally, by knocking down the expression of the regulatory subunit PKAR1α, thereby reproducing the effects of IL-1ß and PGE2 on VSMCs, we demonstrated the contribution of PKA activity to the observed behavior of VSMCs.

Expression of sarco (endo) plasmic reticulum calcium ATPase (SERCA) system in normal mouse cardiovascular tissues, heart failure and atherosclerosis.

UNLABELLED: The sarco(endo)plasmic reticulum Ca(2+)ATPases (SERCA) system, a key regulator of calcium cycling and signaling, is composed of several isoforms. We aimed to characterize the expression of SERCA isoforms in mouse cardiovascular tissues and their modulation in cardiovascular pathologies (heart failure and/or atherosclerosis). Five isoforms (SERCA2a, 2b, 3a, 3b and 3c) were detected in the mouse heart and thoracic aorta. Absolute mRNA quantification revealed SERCA2a as the dominant isoform in the heart (~99%). Both SERCA2 isoforms co-localized in cardiomyocytes (CM) longitudinal sarcoplasmic reticulum (SR), SERCA3b was located at the junctional SR. In the aorta, SERCA2a accounted for ~91% of total SERCA and SERCA2b for ~5%. Among SERCA3, SERCA3b was the most expressed (~3.3%), mainly found in vascular smooth muscle cells (VSMC), along with SERCA2a and 2b. In failing CM, SERCA2a was down-regulated by 2-fold and re-localized from longitudinal to junctional SR. A strong down-regulation of SERCA2a was also observed in atherosclerotic vessels containing mainly synthetic VSMCs. The proportion of both SERCA2b and SERCA3b increased to 9.5% and 8.3%, respectively. IN CONCLUSION: 1) SERCA2a is the major isoform in both cardiac and vascular myocytes; 2) the expression of SERCA2a mRNA is ~30 fold higher in the heart compared to vascular tissues; and 3) nearly half the amount of SERCA2a mRNA is measured in both failing cardiomyocytes and synthetic VSMCs compared to healthy tissues, with a relocation of SERCA2a in failing cardiomyocytes. Thus, SERCA2a is the principal regulator of excitation-contraction coupling in both CMs and contractile VSMCs.

ß-Amyloid context intensifies vascular smooth muscle cells induced inflammatory response and de-differentiation.

Several studies have shown that the accumulation of ß-amyloid peptides in the brain parenchyma or vessel wall generates an inflammatory environment. Some even suggest that there is a cause-and-effect relationship between inflammation and the development of Alzheimer's disease and/or cerebral amyloid angiopathy (CAA). Here, we studied the ability of wild-type Aß1-40 -peptide (the main amyloid peptide that accumulates in the vessel wall in sporadic forms of CAA) to modulate the phenotypic transition of vascular smooth muscle cells (VSMCs) toward an inflammatory/de-differentiated state. We found that Aß1-40 -peptide alone neither induces an inflammatory response, nor decreases the expression of contractile markers; however, the inflammatory response of VSMCs exposed to Aß1-40 -peptide prior to the addition of the pro-inflammatory cytokine IL-1ß is greatly intensified compared with IL-1ß-treated VSMCs previously un-exposed to Aß1-40 -peptide. Similar conclusions could be drawn when tracking the decline of contractile markers. Furthermore, we found that the mechanism of this potentiation highly depends on an Aß1-40 preactivation of the PI3 Kinase and possibly NFκB pathway; indeed, blocking the activation of these pathways during Aß1-40 -peptide treatment completely suppressed the observed potentiation. Finally, strengthening the possible in vivo relevance of our findings, we evidenced that endothelial cells exposed to Aß1-40 -peptide generate an inflammatory context and have similar effects than the ones described with IL-1ß. These results reinforce the idea that intraparietal amyloid deposits triggering adhesion molecules in endothelial cells, contribute to the transition of VSMCs to an inflammatory/de-differentiated phenotype. Therefore, we suggest that acute inflammatory episodes may increase vascular alterations and contribute to the ontogenesis of CAA.

The Notch pathway attenuates interleukin 1ß (IL1ß)-mediated induction of adenylyl cyclase 8 (AC8) expression during vascular smooth muscle cell (VSMC) trans-differentiation.

Vascular smooth muscle cell (VSMC) trans-differentiation, or their switch from a contractile/quiescent to a secretory/inflammatory/migratory state, is known to play an important role in pathological vascular remodeling including atherosclerosis and postangioplasty restenosis. Several reports have established the Notch pathway as tightly regulating VSMC response to various stress factors through growth, migration, apoptosis, and de-differentiation. More recently, we showed that alterations of the Notch pathway also govern VSMC acquisition of the inflammatory state, one of the major events accelerating atherosclerosis. We also evidenced that the inflammatory context of atherosclerosis triggers a de novo expression of adenylyl cyclase isoform 8 (AC8), associated with the properties developed by trans-differentiated VSMCs. As an initial approach to understanding the regulation of AC8 expression, we examined the role of the Notch pathway. Here we show that inhibiting the Notch pathway enhances the effect of IL1ß on AC8 expression, amplifies its deleterious effects on the VSMC trans-differentiated phenotype, and decreases Notch target genes Hrt1 and Hrt3. Conversely, Notch activation resulted in blocking AC8 expression and up-regulated Hrt1 and Hrt3 expression. Furthermore, overexpressing Hrt1 and Hrt3 significantly decreased IL1ß-induced AC8 expression. In agreement with these in vitro findings, the in vivo rat carotid balloon-injury model of restenosis evidenced that AC8 de novo expression coincided with down-regulation of the Notch3 pathway. These results, demonstrating that the Notch pathway attenuates IL1ß-mediated AC8 up-regulation in trans-differentiated VSMCs, suggest that AC8 expression, besides being induced by the proinflammatory cytokine IL1ß, is also dependent on down-regulation of the Notch pathway occurring in an inflammatory context.

Wild-type amyloid beta 1-40 peptide induces vascular smooth muscle cell death independently from matrix metalloprotease activity.

Cerebral amyloid angiopathy (CAA) is an important cause of intracerebral hemorrhages in the elderly, characterized by amyloid-ß (Aß) peptide accumulating in central nervous system blood vessels. Within the vessel walls, Aß-peptide deposits [composed mainly of wild-type (WT) Aß(1-40) peptide in sporadic forms] induce impaired adhesion of vascular smooth muscle cells (VSMCs) to the extracellular matrix (ECM) associated with their degeneration. This process often results in a loss of blood vessel wall integrity and ultimately translates into cerebral ischemia and microhemorrhages, both clinical features of CAA. In this study, we decipher the molecular mechanism of matrix metalloprotease (MMP)-2 activation in WT-Aß(1-40) -treated VSMC and provide evidence that MMP activity, although playing a critical role in cell detachment disrupting ECM components, is not involved in the WT-Aß(1-40) -induced degeneration of VSMCs. Indeed, whereas this peptide clearly induced VSMC apoptosis, neither preventing MMP-2 activity nor hampering the expression of membrane type1-MMP, or preventing tissue inhibitors of MMPs-2 (TIMP-2) recruitment (two proteins evidenced here as involved in MMP-2 activation), reduced the number of dead cells. Even the use of broad-range MMP inhibitors (GM6001 and Batimastat) did not affect WT-Aß(1-40) -induced cell apoptosis. Our results, in contrast to those obtained using the Aß(1-40) Dutch variant suggesting a link between MMP-2 activity, VSMC mortality and degradation of specific matrix components, indicate that the ontogenesis of the Dutch familial and sporadic forms of CAAs is different. ECM degradation and VSMC degeneration would be tightly connected in the Dutch familial form while being two independent processes in sporadic forms of CAA.