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Bacterial mastitis is caused by invasion of the udder, bacterial multiplication and induction of
inflammatory responses in the bovine mammary gland. Disease severity and the cause of disease are
influenced by environmental factors, the cow’s immune response as well as bacterial traits. Escherichia coli (E. coli) is one of the main causes of acute bovine mastitis, but although pathogenic E. coli strains can be classified into different pathotypes, E. coli causing mastitis cannot unambiguously be distinguished from commensal E. coli nor has a common set of virulence factors
been described for mastitis isolates. This project focussed on the characterization of virulence-
associated traits of E. coli mastitis isolates in comprehensive analyses under conditions either
mimicking initial pathogenesis or conditions that E. coli mastitis isolates should encounter while entering the udder. Virulence-associated traits as well as fitness traits of selected bovine mastitis or faecal E. coli strains were identified and analyzed in comparative phenotypic assays. Raw milk whey was introduced to
test bacterial fitness in native mammary secretion known to confer antimicrobial effects.
Accordingly, E. coli isolates from bovine faeces represented a heterogeneous group of which some
isolates showed reduced ability to survive in milk whey whereas others phenotypically resembled
mastitis isolates that represented a homogeneous group in that they showed similar survival and
growth characteristics in milk whey. In contrast, mastitis isolates did not exhibit such a uniform phenotype when challenged with iron shortage, lactose as sole carbon source and lingual
antimicrobial peptide (LAP) as a main defensin of milk. Reduced bacterial fitness could be related to LAP suggesting that bacterial adaptation to an intramammary lifestyle requires resistance to host
defensins present in mammary secretions, at least LAP.
E. coli strain 1303 and ECC-1470 lack particular virulence genes associated to mastitis isolates. To find out whether differences in gene expression may contribute to the ability of E. coli variants to cause mastitis, the transcriptome of E. coli model mastitis isolates 1303 and ECC-1470 were analyzed to
identify candidate genes involved in bacterium-host interaction, fitness or even pathogenicity during bovine mastitis.
DNA microarray analysis was employed to assess the transcriptional response of E. coli 1303 and
ECC-1470 upon cocultivation with MAC-T immortalized bovine mammary gland epithelial cells to
identify candidate genes involved in bacterium-host interaction. Additionally, the cell adhesion and invasion ability of E. coli strain 1303 and ECC-1470 was investigated. The transcriptonal response to the presence of host cells rather suggested competition for nutrients and oxygen between E. coli and MAC-T cells than marked signs of adhesion and invasion. Accordingly, mostly fitness traits that may also contribute to efficient colonization of the E. coli primary habitat, the gut, have been utilized by the mastitis isolates under these conditions. In this study, RNA-Seq was employed to assess the bacterial transcriptional response to milk whey.
According to our transcriptome data, the lack of positively deregulated and also of true virulence-associated determinants in both of the mastitis isolates indicated that E. coli might have adapted by other means to the udder (or at least mammary secretion) as an inflammatory site. We identified traits that promote bacterial growth and survival in milk whey. The ability to utilize citrate promotes fitness and survival of E. coli that are thriving in mammary secretions. According to our results, lactoferrin has only weak impact on E. coli in mammary secretions. At the same time bacterial determinants involved in iron assimilation were negatively regulated, suggesting that, at least during the first hours, iron assimilation is not a challenge to E. coli colonizing the mammary gland. It has been hypothesized that cellular iron stores cause temporary independency to extracellular accessible iron. According to our transcriptome data, this hypothesis was supported and places iron uptake
systems beyond the speculative importance that has been suggested before, at least during early
phases of infection. It has also been shown that the ability to resist extracytoplasmic stress, by oxidative conditions as well as host defensins, is of substantial importance for bacterial survival in mammary secretions.
In summary, the presented thesis addresses important aspects of host-pathogen interaction and
bacterial conversion to hostile conditions during colonization of the mastitis inflammatory site, the mammary gland.
Aurora B is a mitotic kinase that is essential for cell division. Because it is mutated or overexpressed in a range of cancer types, it has been suggested as a novel therapeutic target. Currently chemical inhibitors against Aurora B are in various phases of clinical trials for treatment of solid tumors and leukemia. Information regarding the molecular requirements for the reported phenotypes of Aurora B inhibition such as cell cycle arrest, activation of the tumor suppressor p53 and its target p21 are not well understood.
In this study, I investigated the requirements for p21 induction after Aurora B inhibition. I found that p38 is phosphorylated and activated when Aurora B is inhibited. Experiments with chemical inhibitors against p38 indicate that p38 is required for p21 induction and cell cycle arrest in response to Aurora B inhibition. p53 induction after impairment of Aurora B function and the recruitment of p53 to its binding site in the p21 gene promoter occur independently of p38 signaling. Instead, I found that p38 is required for the enrichment of the elongating RNA Polymerase II in the coding region of the p21 gene. Furthermore, p38 is required for formation of the full-length p21 mRNA transcript. These data indicate that p38 promotes the transcriptional elongation of p21 gene in response to Aurora B inhibition. In further experiments I could show that the p21 causes cell cycle arrest due to a decrease in E2F-dependent transcription by promoting the dephosphorylation of the retinoblastoma protein.
Using synchronized cells I could show that the induction of p21 in response to Aurora B inhibition requires transition through an aberrant mitosis and does not occur in cells that are arrested in interphase. Interestingly, p38, p53 and p21 are already induced by partial inhibition of Aurora B, which results in aneuploidy but not in cytokinesis failure and in tetraploidy. This supports the notion that activation of p38-p53-p21 signaling correlates with aneuploidy but not with tetraploidy or binucleation. Partial inhibition of Aurora B also leads to increased generation of reactive oxygen species (ROS), which are required for the activation of p38, p21 and cell cycle arrest. Based on these observations I propose the following model: Inhibition of Aurora B leads to chromosome missegregation resulting in aneuploidy. This results in increased generation of ROS (reactive oxygen species) possibly through proteotoxic stress caused by an imbalance of protein synthesis in aneuploid cells. ROS triggers the activation of p38, which then stimulates the transcriptional elongation of p21 resulting in cell cycle arrest.
Aneuploidy, proteotoxic stress and oxidative stress are hallmarks of cancer cells. Based on my results reported in this study, I suggest that the combination of Aurora B inhibitors with drugs that specifically target aneuploid cells might be a novel strategy for cancer therapy, as this is a lethal combination for proliferation of cancer cells.
MicroRNAs are endogenous ≈22 nt long non coding RNA molecules that modulate gene expression
at the post transcriptional level by targeting mRNAs for cleavage or translational repression.
MicroRNA-mRNA interaction involves a contiguous and perfect pairing within complementary
sites usually in the 3’ UTR of the target mRNA. Heart failure is associated with myocyte
hypertrophy and death, due to compensatory pathological remodeling and minimal functional repair
along with microRNA deregulation.
In this study, we identified candidate microRNAs based on their expression strength in
cardiomyocytes and by their ability to regulate hypertrophy. Expression profiling from early and
late stages of heart failure showed several deregulated microRNAs. Of these microRNAs, miR-378
emerged as a potentially interesting microRNA that was highly expressed in the mouse heart and
downregulated in the failing heart. Antihypertrophic activity of miR-378 was first observed by
screening a synthetic miR library for morphologic effects on cardiomyocytes, and validated in vitro proving the tight control of hypertrophy by this miR. We combined bioinformatic target prediction analysis and microarray analysis to identify the targets of miR-378. These analyses suggested that factors of the MAP kinase pathway were enriched among miR-378 targets, namely MAPK1 itself (also termed ERK2), the insulin-like growth factor receptor 1 (IGF1R), growth factor receptor bound protein 2 (GRB2) and kinase suppressor of ras 1 (KSR1). Regulation of these targets by miR-378 was then confirmed by mRNA and protein expression analysis. The use of luciferase reporter constructs with natural or mutated miR-378 binding sites further validated these four proteins as direct targets of miR-378. RNA interference with MAPK1 and the other three targets prevented the prohypertrophic effect of antimiR-378, suggesting that they functionally relate to miR-378. In vivo restoration of disease induced loss of miR-378 in a pressure overload mouse model of hypertrophy using adeno associated virus resulted in partial attenuation cardiac hypertrophy and significant improvement in cardiac function along with reduced expression of the four targets in heart.
We conclude from these findings that miR-378 is an antihypertrophic microRNA in cardiomyocytes, and the main mechanism underlying this effect is the suppression of the MAP kinase-signaling pathway on four distinct levels. Restoration of disease-associated loss of miR-378 through cardiomyocyte-targeted AAV-miR-378 may prove as an effective therapeutic strategy in myocardial disease.
Theories of attention deficit hyperactivity disorder (ADHD) aetiology have placed a focus on impaired behavioural inhibition presumably leading to executive function (EF) deficits. Neuroimaging studies report neurophysiological findings consistent with these hypothesised impairments, and investigations of functional brain activation from a network perspective report hypoactivation in the frontoparietal network as well as hyperactivation in the dorsal attention network. Studies investigating the acute effects of stimulant medication on EF show an improvement on behavioural EF measures including working memory. In addition, methylphenidate (MPH) was shown to up-regulate the task-positive/ frontoparietal network in children and adolescents with ADHD. So far, there are only few studies investigating the impact of ADHD on behavioural and neurophysiological EF measures as well as the effect of several weeks of stimulant medication in adult patients.
The importance of the catechol-O-methyltransferase (COMT) enzyme for subcortical and cortical dopaminergic and noradrenergic functioning furthermore led to studies investigating a potential interactive impact of COMT genotype and ADHD on neuropsychological functioning, with a particular focus on working memory. The results of these studies were very heterogeneous. In addition, as none of the studies compared the results of ADHD patients to those of a healthy control group, possible differential effects of COMT in patients and healthy controls could not be examined.
The aim of this dissertation was to investigate selective attention properties of the central executive component during a working memory task and to transfer this task to fMRI. A third study then aimed to investigate the effects of adult ADHD (aADHD), MPH, and COMT genotype on working memory with a particular focus on activation of the task-positive network during the analysis of the fMRI data.
The first study (EEG) could replicate and extend the results from previous research. This study could furthermore connect the overall activation in frontal areas to suppression efficiency in posterior visual areas as well as establish the impact of hyperactive/ impulsive ADHD symptoms on task performance. The second study (fMRI) allowed the successful transfer of the paradigm to fMRI, and the further replication and extension of previous findings. In addition, this study showed the sensitivity of the task to the effects of the COMT genotype. The third study (fMRI) was one of the first studies that exploratorily investigated the effects COMT in a sample of aADHD patients and a comparable healthy control group. This study showed an interactive effect of these two factors on neuropsychological measures as well as on fMRI activation during a classic n-back working memory task. In addition, this task led to more activation in the task-positive network of the aADHD group compared to a healthy control group in the absence of performance differences, pointing towards compensatory activation in the aADHD group. Furthermore, activation in the frontal cortex was increased in patients taking MPH compared to a placebo. The fMRI data from the selective attention task moreover showed decreased activation in the right DLPFC of the patient group, which was associated with reduced suppression efficiency across all participants. The clinical effect of MPH in the third study was visible but did not reach significance, which is probably attributable to a lack of experimental power.
The studies in this dissertation could successfully replicate and extend previous findings. A goal for future studies should be the further investigation of the interactive effects of COMT genotype and aADHD on neuropsychological test results and fMRI activation, but also on medication response and adverse effects. In this context, the adaptation of a network perspective during the analysis of fMRI data seems to be the best way to detect existing between-group differences.
Attention-deficit/hyperactivity disorder (ADHD) is a highly prevalent childhood-onset neurodevelopmental disorder that involves a substantial risk of persisting into adolescence and adulthood. A number of genome-wide screening studies in ADHD have been conducted in recent years, giving rise to the discovery of several variants at distinct chromosomal loci, thus emphasising the genetically complex and polygenic nature of this disorder. Accordingly, promising novel candidate genes have emerged, such as the gene encoding the glucose transporter isoform 3 (SLC2A3) and the gene encoding the latrophilin isoform 3 (LPHN3).
In this thesis, both genes were investigated in form of two separated projects. The first focused on SLC2A3 polymorphisms associated with ADHD and their potential physiological impact. For this purpose, gene expression analyses in peripheral cell models were performed as well as functional EEG measurements in humans. The second project concerned the murine gene Lphn3 including the goal of developing a mouse line containing a genetically modified Lphn3 with conditional knockout potential. In this respect, a specific DNA vector was applied to target the Lphn3 gene locus in murine embryonic stem (ES) cells as a prerequisite for the generation of appropriate chimeric mice.
The results of the first project showed that SLC2A3 duplication carriers displayed increased SLC2A3 mRNA expression in peripheral blood cells and significantly altered event-related potentials (ERPs) during tests of cognitive response control and working memory, possibly involving changes in prefrontal brain activity and memory processing. Interestingly, ADHD patients with the rs12842 T-allele, located within and tagging the SLC2A3 gene, also exhibited remarkable effects during these EEG measurements. However, such effects reflected a reversed pattern to the aforementioned SLC2A3 duplication carriers with ADHD, thus indicative of an opposed molecular mechanism. Besides, it emerged that the impact of the aforementioned SLC2A3 variants on different EEG parameters was generally much more pronounced in the group of ADHD patients than the healthy control group, implying a considerable interaction effect. Concerning the second project, preliminary results were gathered including the successful targeting of Lphn3 in murine ES cells as well as the production of highly chimeric, phenotypically unremarkable and
mostly fertile mouse chimeras. While germline transmission of the modified Lphn3 allele has not yet occurred, there are still several newborn chimeric mice that will be tested in the near future.
In conclusion, the findings suggest that SLC2A3 variants associated with ADHD are accompanied by transcriptional and functional changes in humans. Future research will help to elucidate the molecular network and neurobiological basis involved in these effects and apparently contributing to the complex clinical picture of ADHD. Moreover, given the increasing number of publications concerning latrophilins in recent years and the multitude of research opportunities provided by a conditional knockout of Lphn3 in mice, the establishment of a respective mouse line, which currently is in progress, constitutes a promising approach for the investigation of this gene and its role in ADHD.
TRAIL is a member of TNF superfamily and mediates apoptosis by binding to two DRs, TRAILR1 and TRAILR2. Despite the fact that there are other TRAILRs, TRAILR1 and TRAILR2 receive the major research interest due to their ability to trigger apoptosis and their possible use as targets in tumor therapy. Due to the potential advantages of TRAILR1- or TRAILR2-specific targeting, we investigated recently published TRAIL DR-specific mutants, one conferring specificity for TRAILR1 (TRAILmutR1) and one for TRAILR2 (TRAILmutR2). It was well proved in this work that TRAILmutR1 shows specific binding to TRAILR1 and no specific binding to TRAILR2. TRAILmutR2 vice versa shows specific binding to TRAILR2 and no significant binding to TRAILR1. Moreover, these mutants were able to induce caspase activation and cell death in a TRAILR1/2-specific manner. Moreover, the enhancement of TRAILR2-induced apoptosis by secondary oligomerization of soluble wild-type TRAIL was confirmed for the TRAILR2-specifc TRAIL mutant and similar findings were made with the TRAILR1-specific TRAIL mutant.
The soluble form of TRAIL exhibits weak apoptotic activity as compared to transmembrane TRAIL. Therefore, there is the challenge in clinical research to improve the activity of soluble TRAIL. A second strategy besides the above mentioned oligomerization to improve soluble TRAIL activity is anchoring of the molecule to the cell surface, e.g. through the genetic fusion with a scFv domain recognizing a cell surface antigen. In this work, we generated fusion proteins of TRAIL, TRAILmutR1 and TRAILmutR2 with a scFv recognizing CD40 (scFv:G28). Initially, we analyzed the functionality of both the TRAIL domain and the scFv:G28 domain of the corresponding fusion proteins. TRAIL functionality was well proved through its ability to induce cell death in TRAIL sensitive cells such as Jurkat cells, provided that scFv:G28-TRAIL fusion proteins were oligomerized by anti-Flag mAb M2. Concerning the scFv:G28 domain, the fusion proteins showed enhanced binding affinity to cell lines expressing CD40 as compared to their parental CD40-negative cells. Consistent with previous studies investigating TRAIL fusion proteins with other cell surface antigen-targeting scFvs, the scFv:G28 fusion proteins with TRAIL, TRAILmutR1 and TRAILmutR2 showed enhanced induction of cell death in a CD40-dependent manner. Moreover, our results revealed that these fusion proteins have a significant paracrine apoptotic effect on CD40-negative bystander cells upon anchoring to CD40-positive cells which are TRAIL resistant. Thus, the current work provides for the first time scFv fusion proteins of TRAIL and TRAILR1- and TRAILR2-specific TRAIL mutants with CD40-restricted activity. These fusion proteins provide the advantage of attenuating the off-target effects and the potential side effects of per se highly active TRAIL variants on one hand due to the CD40-binding dependent enhancement of activity and on the other hand due to the differential use of TRAILR1 and TRAILR2.
CD40 represents a tumor associated marker which is expressed on many tumor cells but also on immune cells. Therefore, the last part of this work focused on the analysis of the ability of scFv:G28-TRAIL fusion proteins to induce CD40 signaling both in tumor cells and also in immune cells. It turned out that the scFv:G28-TRAIL fusion proteins are able to induce CD40 signaling in CD40-positive tumor cells but especially also in immune cells such as iDCs leading to their maturation and further activation of immune responses.
Taken together, this work provides novel bifunctional scFv-TRAIL fusion proteins which combine the induction of apoptosis via TRAIL DR with stimulation of CD40 signaling which possibly enhances antitumor immunity.
Evolution of Vγ9Vδ2 T-cells
(2014)
Human Vγ9Vδ2 T cells are the major subset of blood γδ T cells and account for 1-5% of blood T cells. Pyrophosphorylated metabolites of isoprenoid biosynthesis are recognized by human Vγ9Vδ2 T cells and are called as phosphoantigens (PAg). Isopentenyl pyrophosphate (IPP) and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) are among the few well studied PAg. IPP is found in all organisms while HMBPP is a precursor of IPP found only in eubacteria, plants and apicomplexaen parasite. Interestingly, the PAg reactive Vγ9Vδ2 T cells are so far identified only in human and higher primates but not in rodents. Hence, Vγ9Vδ2 T cells are believed to be restricted to primates. With regard to PAg recognition, a Vγ9JP recombined TCRγ chain and certain CDR3 motifs of the TCR chain are mandatory. The BTN3A1 molecule is essential for a response to PAg. BTN3 is a trans-membrane protein belonging to butyrophilin family of proteins. Though BTN3A1 was found to be essential for PAg presentation, the exact molecular basis of PAg presentation still remains unclear.
This thesis presents new data on the evolution of Vγ9Vδ2 TCR and its ligands (BTN3) as well as the genetic basis of PAg presentation to Vγ9Vδ2 TCR.
The comprehensive analysis of genomic database sequences at NCBI and other public domain databases revealed for the first time that Vγ9, Vδ2 and BTN3 genes emerged and co-evolved along with the placental mammals. Vγ9, Vδ2 and BTN3 genes are scattered across mammalian species and not restricted to primates. But interestingly, all three genes are highly conserved between phylogenetically distinct species. Moreover, the distribution pattern of Vγ9, Vδ2 TCR genes and BTN3 genes suggests a functional association between these genes representing the TCR - ligand relationship. Alpaca (Vicugna pacos), a member of the camelid family, is one among the 6 candidate non-primate species which were found to possess functional Vγ9, Vδ2 and BTN3 genes.
From peripheral lymphocytes of alpaca, Vγ9 chain transcripts with a characteristic JP rearrangement and transcripts of Vδ2 chains with a CDR3 typical for PAg-reactive TCR were identified. The transduction of αβ TCR negative mouse thymoma BW cells with alpaca Vγ9 and Vδ2 TCR chains resulted in surface expression of the TCR complex as it was deduced from detection of cell surface expression of mouse CD3. Cross-linking of alpaca Vγ9Vδ2 TCR transductants with anti-CD3ε led to IL-2 production which confirmed that alpaca Vγ9 and Vδ2 TCR chains pair to form a functional TCR. Besides the conservation of human like Vγ9 and Vδ2 TCR chains, alpaca has conserved an orthologue for human BTN33A1 as well. Interestingly, the predicted PAg binding sites of human BTN3A1 was 100% conserved in deduced amino acid sequence of alpaca BTN3A1. All together alpaca is a promising candidate for further studies as it might have preserved Vγ9Vδ2 T cells to function in surveillance of stress and infections.
This thesis also provides the sequence of Vγ9Vδ2 TCR of African green monkey (Chlorocebus aethiops), which was previously unknown. Moreover, our data indicates the lack of any species specific barrier which could hinder the PAg presentation by African monkey derived COS cells to human Vγ9Vδ2 TCR and vice versa of human cells to African green monkey Vγ9Vδ2 TCR which was in contradiction to previously reported findings.
Apart from the above, the thesis also presents new data on the genetic basis of PAg presentation to Vγ9Vδ2 T cells, which revealed that human chromosome 6 is sufficient for the presentation of exogenous and endogenous PAg. By employing human/mouse somatic hybrids, we identified the role of human chromosome 6 in PAg presentation and in addition, we observed the lack of capacity of human chromosome 6 positive hybrids to activate Vγ9Vδ2 TCR transductants in the presence of the alkylamine sec-butylamine (SBA). Investigation of Chinese hamster ovary (CHO) cells containing the human chromosome 6 also yielded similar results. This suggests that aminobisphosphonates (zoledronate) and alkylamines employ different mechanisms for activation of Vγ9Vδ2 T cells although both have been described to act by inhibition of farnesyl pyrophosphate synthase activity which is known to increase intracellular levels of the IPP.
In conclusion, this thesis suggests that Vγ9, Vδ2 and BTN3 genes controlling Vγ9Vδ2 TCR- ligand relationship emerged and co-evolved along with placental mammals; and also identified candidate non-primate species which could possess Vγ9Vδ2 T cells. Furthermore, it suggests alpaca as a promising non-primate species to investigate the physiological function of Vγ9Vδ2 T cells. With respect to PAg antigen presentation it was shown that chromosome 6 is essential and sufficient for exogenous and endogenous PAg presentation. Moreover, the alkylamine SBA and aminobisphosphonate zoledronate may engage different cellular mechanism to exert inhibition over IPP consumption. The thesis raises interesting questions which need to be addressed in future: 1) What are the environmental and evolutionary factors involved in preservation of Vγ9Vδ2 T cells only by few species? 2) What could be the functional nature and antigen recognition properties of such a conserved T cell subset? 3) What is the genetic and molecular basis of the differential capacity of human chromosome 6 bearing rodent-human hybridoma cells in activating Vγ9Vδ2 T cells in presence of SBA and aminobisphosphonates?
Platelets are important players in haemostasis and their activation is essential to limit post-traumatic blood loss upon vessel injury. On the other hand, pathological platelet activation may lead to thrombosis resulting in myocardial infarction and stroke. Platelet activation and subsequent thrombus formation are, therefore, tightly regulated and require a well-defined interplay of platelet surface receptors, intracellular signalling molecules, cytoskeletal rearrangements and the activation of the coagulation cascade.
In vivo thrombosis and haemostasis models mimic thrombus formation at sites of vascular lesions and are frequently used to assess thrombotic and haemostatic functions of platelets. In this dissertation, different in vivo models were used in mice to address the question at what level a reduced platelet count (PC) compromises stable thrombus formation. To study this, mice were rendered thrombocytopenic by low-dose anti-GPIbα antibody treatment and subjected to a tail bleeding time assay as well as to four different in vivo thrombosis models. Haemostasis and occlusive thrombus formation in small vessels were only mildly affected even at severe reductions of the PC. In contrast, occlusive thrombus formation in larger arteries required higher PCs demonstrating that considerable differences in the sensitivity for PC reductions exist between these models.
In a second part of this study, mice were rendered thrombocytopenic by injection of high-dose anti-GPIbα antibody which led to the complete loss of all platelets from the circulation for several days. During recovery from thrombocytopenia, the newly generated platelet population was characterised and revealed a defect in immunoreceptor tyrosine-based activation motif (ITAM)-signalling. This defect translated into impaired arterial thrombus formation.
To further investigate ITAM-signalling in vivo, genetically modified mice were analysed which display a positive or negative regulation of platelet ITAM-signalling in vitro. Whereas mice lacking the adapter Grb2 in platelets showed a delayed thrombus formation in vivo after acetylsalicylic acid treatment, Clp36ΔLIM bone marrow chimeric mice and SLAP/SLAP2-deficient mice displayed pro-thrombotic properties in vivo. Finally, mice lacking the adapter protein EFhd2 were analysed in vitro and in vivo. However, EFhd2-deficient platelets showed only a minor increase in the procoagulant activity compared to control.
Function and regulation of phospholipase D in blood platelets: in vitro and in vivo studies in mice
(2014)
Summary
Platelet activation and aggregation are crucial for primary hemostasis but can also result in occlusive thrombus formation. Agonist induced platelet activation involves different signaling pathways leading to the activation of phospholipases (PL) which produce second messengers. While the role of PLCs in platelet activation is well established, less is known about the relevance of PLDs. In the current study, the function and regulation of PLD in platelets was investigated using genetic and pharmacological approaches.
In the first part of this thesis, adhesion, activation and aggregation of platelets from mice lacking PLD2 or both PLD1 and PLD2 were analyzed in vitro and in vivo. While the absence of PLD2 resulted in slightly reduced PLD activity in platelets, it had no detectable effect on the platelet function in vitro and in vivo. However, the combined deficiency of both PLD isoforms resulted in defective alpha-granule release and protection in a model of ferric chloride induced arteriolar thrombosis, effects that were not observed in mice lacking only one PLD isoform. These results revealed, for the first time, redundant roles of PLD1 and PLD2 in platelet alpha-granule secretion and indicate that this may be relevant for pathological thrombus formation. Thus, PLD might represent a promising target for antithrombotic therapy.
Thus, this hypothesis was tested more directly in the second part of this thesis. The effects of pharmacological inhibition of PLD activity on hemostasis, thrombosis and thrombo-inflammatory brain infarction in mice were assessed. Treatment of platelets with the reversible, small molecule PLD inhibitor 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI) led to a specific blockade of PLD activity that was associated with reduced -granule release and integrin activation. Mice that received FIPI at a dose of 3 mg/kg displayed reduced occlusive thrombus formation upon chemical injury of carotid arteries or mesenterial arterioles. Similarly, FIPI-treated mice had smaller infarct sizes and significantly better motor and neurological function 24 hours after transient middle cerebral artery occlusion. This protective effect was not associated with major intracerebral hemorrhage or prolonged tail bleeding times. Thus, pharmacological PLD inhibition might represent a safe therapeutic strategy to prevent arterial thrombosis or ischemic stroke.
After revealing a central role for PLD in thrombo-inflammation, the regulation of PLD activity in platelets was analyzed in the last part of the thesis. Up to date, most studies made use of inhibitors potentially exerting off-target effects and consequently PLD regulation is discussed controversially. Therefore, PLD activity in mice genetically lacking potential modulators of PLD activity was determined to address these controversies. These studies revealed that PLD is tightly regulated during initial platelet activation. While integrin outside-in signaling and Gi signaling was dispensable for PLD activation, it was found that PLC dependent pathways were relevant for the regulation of PLD enzyme activity.
A precious treasure in traditional Chinese medicine (TCM), acupuncture played a vital and irreplaceable role in contributing to people’s health in the thousands of years of Chinese history, and in 2010 was officially added to the “Representative List of the Intangible Cultural Heritage of Humanity” by the United Nations. Because of the side-effects of long-term drug therapy for pain, and the risks of dependency, acupuncture has been widely accepted as one of the most important alternative choice therapies for treating varieties of acute and chronic pain-related disorders. The clinical application and scientific mechanism research of acupuncture have therefore increased intensively in the last few decades. Besides hand acupuncture, other treatment approaches e.g. electroacupuncture (EA) have been widely accepted and applied as an important acupuncture-related technique for acupuncture analgesia (AA) research. The involvement of opioid peptides and receptors in acute AA has been shown via pre-EA application of opioid receptor/peptide antagonists. However, existing publications still cannot illuminate the answer to the following question: how does sustained antinociception happen by EA treatment? The hypothesis of opioid peptide-mediated tonic AA might be able to answer the question.
In the first part of this thesis, the institution of a reproducible acupuncture treatment model as well as the endogenous opioid-related mechanisms was demonstrated. An anatomically-based three-dimensional (3D) rat model was established to exhibit a digital true-to-life organism, accurate acupoint position and EA treatment protocol on bilateral acupoint GB-30 Huantiao. The optimal EA treatment protocol (100 Hz, 2-3 mA, 0.1 ms, 20 min) at 0 and 24 h after induction of inflammatory pain by complete Freund’s adjuvant (CFA) on conscious free-moving rats was then established. EA elicited significant sustained mechanical and thermal antinociception up to 144 h. Post-EA application of opioid receptors (mu opioid receptor, MOR; delta opioid receptor, DOR) antagonists naloxone (NLX) and naltrindole (NTI), or opioid peptide antibodies anti-beta-endorphin (anti-END), met-enkephalin (anti-ENK) or -dynorphin A (anti-DYN) could also block this effect at a late phase (96 h) of CFA post-EA, which suggested opioid-dependent tonic analgesia was produced by EA. Meanwhile, EA also reduced paw temperature and volume at 72-144 h post CFA indicating anti-inflammatory effects. Nociceptive thresholds were assessed by paw pressure threshold (Randall-Sellito) or paw withdrawal latency (Hargreaves) and an anti-inflammatory effect was evaluated by measurement of plantar temperature and volume of inflamed paw.
The second part of the thesis further suggests the correlation between the chemokine CXCL10 (= interferon-gamma inducible protein 10, IP-10) and opioid peptides in EA-induced antinociception. Based on a comprehensive Cytokine Array of 29 cytokines, targeted cytokines interleukin (IL)-1alpha, interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, interleukin (IL)-13, interferon (IFN)-gamma as well as CXCL10 were selected and quantified by enzyme-linked immunosorbent assay (ELISA), and real time reverse transcription-polymerase chain reaction (RT-PCR) quantification confirmed upregulation of CXCL10 mRNA at both 72 and 96 h. The following hyperalgesic assessment suggested the antinociceptive effect of CXCL10. The double immunostaining localizing opioid peptides with macrophages expressed the evident upregulation of CXCR3-receptor of CXCL10 in EA treated samples as well as the significant upregulation or downregulation of opioid peptides by repeated treatment of CXCL10 or antibody of CXCL10 via behavioral tests and immune staining. Subsequent immunoblotting measurements showed non-alteration of opioid receptor level by EA, indicating that the opioid receptors did not apparently contribute to AA in the present studies. In vitro, CXCL10 did not directly trigger opioid peptide END release from freshly isolated rat macrophages. This might implicate an indirect property of CXCL10 in vitro stimulating the opioid peptide-containing macrophages by requiring additional mediators in inflammatory tissue.
In summary, this project intended to explore the peripheral opioid-dependent analgesic mechanisms of acupuncture with a novel 3D treatment rat model and put forward new information to support the pivot role of chemokine CXCL10 in mediating EA-induced tonic antinociception via peripheral opioid peptides.