Targeted inhibition of the serotonin 5HT 2A receptor improves coronary patency in an in vivo model of recurrent thrombosis

BACKGROUND: Release of serotonin and activation of serotonin 5HT2A receptors on platelet surfaces is a potent augmentative stimulus for platelet aggregation. However, earlier-generation serotonin receptor antagonists were not successfully exploited as antiplatelet agents, possibly owing to their lack of specificity for the 5HT2A receptor subtype. OBJECTIVE: To assess whether targeted inhibition of the serotonin 5HT2A receptor attenuates recurrent thrombosis and improves coronary patency in an in vivo canine model mimicking unstable angina. METHODS: In protocol 1, anesthetized dogs were pretreated with a novel, selective inverse agonist of the 5HT2A receptor (APD791) or saline. Recurrent coronary thrombosis was then initiated by coronary artery injury+stenosis, and coronary patency was monitored for 3 h. Protocol 2 was similar, except that: (i) treatment with APD791 or saline was begun 1 h after the onset of recurrent thrombosis; (ii) template bleeding time was measured; and (iii) blood samples were obtained for in vitro flow cytometric assessment of platelet responsiveness to serotonin. RESULTS: APD791 attenuated recurrent thrombosis, irrespective of the time of treatment: in both protocols, flow-time area (index of coronary patency; normalized to baseline coronary flow) averaged 58-59% (P<0.01) following administration of APD791 vs. 21-28% in saline controls. Moreover, the in vivo antithrombotic effect of APD791 was not accompanied by increased bleeding, but was associated with significant and selective inhibition of serotonin-mediated platelet activation. CONCLUSION: 5HT2A receptor inhibition with APD791, even when initiated after the onset of recurrent thrombosis, improves coronary patency in the in vivo canine model

Mechanism of IL-12 mediated alterations in tumour blood vessel morphology: analysis using whole-tissue mounts

Angiogenesis is a multistep process that is limited and carefully regulated in normal adult tissue, but in tumours this regulation is disrupted and the process remains ‘switched on’ (Hanahan and Folkman, 1996). Ample experimental data support the fact that tumour growth requires access to blood vessels and subsequent expansion of host vessels to provide nutrients for the growing tumour mass (Folkman, 1995a). Furthermore, many studies in a variety of tumour types have reported a correlation between the extent of tumour vasculature and poor prognosis or increased metastases (Weidner et al, 1991; Folkman, 1995b; Weidner and Folkman, 1996). Thus, accurate assessment of the vasculature of tumours could provide valuable information regarding treatment outcomes and the likelihood of metastatic spread to other sites. Angiogenesis can be regulated by a variety of factors. Several cytokines produced by immune cells also have been shown to affect the process of angiogenesis. One of the most noteworthy is interleukin (IL)-12, which is produced by antigen presenting cells (APC), such as macrophages and dendritic cells (DC) in response to bacterial stimuli or other inflammatory cytokines. Thus, IL-12 plays an important role in both the innate and adaptive immune responses (Trinchieri, 1998). Owing to its central role in stimulating immunity, it has been examined for possible therapeutic effects in the treatment of tumours. In addition to its effects on the immune system, IL-12 has also been shown to inhibit angiogenesis (Voest et al, 1995; Sgadari et al, 1996). Despite studies in both experimental models and in patients (reviewed in Trinchieri and Scott, 1999), and clear demonstrations of therapeutic efficacy, relatively little is known about how it alters vessel formation within tumours. In part, this is due to the difficulty in assessing the three-dimensional structure of vessels and other cellular components within the tumour. Assessment of tumour vessels is generally based on immunohistochemistry of tumour sections. Although use of this technique has led to a great deal of important information, these procedures are extremely time consuming and provide only a limited two-dimensional view of the vessels. This makes it very difficult to visualise the structure of the microvasculature and identify differences among different tumour types or changes following treatment regimens. To more easily and accurately visualise vessels within tumours, we developed a whole-tissue mount technique that provides a three-dimensional view of the tumour vasculature relative to other components of the tumour tissue. This technique was first validated by studying vessels from transgenic mice that express green fluorescent protein (GFP) (Wu et al, 2000), and then used to investigate the mechanism by which IL-12 influences the vessel architecture within B16 tumours

Modeling flow cytometry data for cancer vaccine immune monitoring

Flow cytometry (FCM) is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. In all these applications, the challenge is to detect extremely rare cell subsets while avoiding spurious positive events. To achieve this objective, it helps to be able to analyze FCM data using multiple markers simultaneously, since the additional information provided often helps to minimize the number of false positive and false negative events, hence increasing both sensitivity and specificity. However, with manual gating, at most two markers can be examined in a single dot plot, and a sequential strategy is often used. As the sequential strategy discards events that fall outside preceding gates at each stage, the effectiveness of the strategy is difficult to evaluate without laborious and painstaking back-gating. Model-based analysis is a promising computational technique that works using information from all marker dimensions simultaneously, and offers an alternative approach to flow analysis that can usefully complement manual gating in the design of optimal gating strategies. Results from model-based analysis will be illustrated with examples from FCM assays commonly used in cancer immunotherapy laboratories

Acidic Extracellular pH Promotes Activation of Integrin αvβ3

Acidic extracellular pH is characteristic of the cell microenvironment in several important physiological and pathological contexts. Although it is well established that acidic extracellular pH can have profound effects on processes such as cell adhesion and migration, the underlying molecular mechanisms are largely unknown. Integrin receptors physically connect cells to the extracellular matrix, and are thus likely to modulate cell responses to extracellular conditions. Here, we examine the role of acidic extracellular pH in regulating activation of integrin [alpha]v[beta]3. Through computational molecular dynamics simulations, we find that acidic extracellular pH promotes opening of the [alpha]v[beta]3 headpiece, indicating that acidic pH can thereby facilitate integrin activation. This prediction is consistent with our flow cytometry and atomic force microscope-mediated force spectroscopy assays of integrin [alpha]v[beta]3 on live cells, which both demonstrate that acidic pH promotes activation at the intact cell surface. Finally, quantification of cell morphology and migration measurements shows that acidic extracellular pH affects cell behavior in a manner that is consistent with increased integrin activation. Taken together, these computational and experimental results suggest a new and complementary mechanism of integrin activation regulation, with associated implications for cell adhesion and migration in regions of altered pH that are relevant to wound healing and cancer.National Institute of Biomedical Imaging and Bioengineering (U.S.) (Award Number T32EB006348)Massachusetts Institute of Technology (Collamore-Rogers Fellowship)National Institutes of Health (U.S.) (NIH Cell Migration Consortium Grant U54-GM069668)National Science Foundation (U.S.) (CAREER Award)Singapore-MIT Alliance for Research and Technology (BioSystem and Micromechanics (BioSyM) Interdisciplinary Research Group

Static platelet adhesion, flow cytometry and serum TXB2 levels for monitoring platelet inhibiting treatment with ASA and clopidogrel in coronary artery disease: a randomised cross-over study

<p>Abstract</p> <p>Background</p> <p>Despite the use of anti-platelet agents such as acetylsalicylic acid (ASA) and clopidogrel in coronary heart disease, some patients continue to suffer from atherothrombosis. This has stimulated development of platelet function assays to monitor treatment effects. However, it is still not recommended to change treatment based on results from platelet function assays. This study aimed to evaluate the capacity of a static platelet adhesion assay to detect platelet inhibiting effects of ASA and clopidogrel. The adhesion assay measures several aspects of platelet adhesion simultaneously, which increases the probability of finding conditions sensitive for anti-platelet treatment.</p> <p>Methods</p> <p>With a randomised cross-over design we evaluated the anti-platelet effects of ASA combined with clopidogrel as well as monotherapy with either drug alone in 29 patients with a recent acute coronary syndrome. Also, 29 matched healthy controls were included to evaluate intra-individual variability over time. Platelet function was measured by flow cytometry, serum thromboxane B₂(TXB₂)-levels and by static platelet adhesion to different protein surfaces. The results were subjected to Principal Component Analysis followed by ANOVA, t-tests and linear regression analysis.</p> <p>Results</p> <p>The majority of platelet adhesion measures were reproducible in controls over time denoting that the assay can monitor platelet activity. Adenosine 5'-diphosphate (ADP)-induced platelet adhesion decreased significantly upon treatment with clopidogrel compared to ASA. Flow cytometric measurements showed the same pattern (r²= 0.49). In opposite, TXB₂-levels decreased with ASA compared to clopidogrel. Serum TXB₂and ADP-induced platelet activation could both be regarded as direct measures of the pharmacodynamic effects of ASA and clopidogrel respectively. Indirect pharmacodynamic measures such as adhesion to albumin induced by various soluble activators as well as SFLLRN-induced activation measured by flow cytometry were lower for clopidogrel compared to ASA. Furthermore, adhesion to collagen was lower for ASA and clopidogrel combined compared with either drug alone.</p> <p>Conclusion</p> <p>The indirect pharmacodynamic measures of the effects of ASA and clopidogrel might be used together with ADP-induced activation and serum TXB₂for evaluation of anti-platelet treatment. This should be further evaluated in future clinical studies where screening opportunities with the adhesion assay will be optimised towards increased sensitivity to anti-platelet treatment.</p

Functional similarities between pigeon \u27milk\u27 and mammalian milk : induction of immune gene expression and modification of the microbiota

Pigeon &lsquo;milk&rsquo; and mammalian milk have functional similarities in terms of nutritional benefit and delivery of immunoglobulins to the young. Mammalian milk has been clearly shown to aid in the development of the immune system and microbiota of the young, but similar effects have not yet been attributed to pigeon &lsquo;milk&rsquo;. Therefore, using a chicken model, we investigated the effect of pigeon &lsquo;milk&rsquo; on immune gene expression in the Gut Associated Lymphoid Tissue (GALT) and on the composition of the caecal microbiota. Chickens fed pigeon &lsquo;milk&rsquo; had a faster rate of growth and a better feed conversion ratio than control chickens. There was significantly enhanced expression of immune-related gene pathways and interferon-stimulated genes in the GALT of pigeon &lsquo;milk&rsquo;-fed chickens. These pathways include the innate immune response, regulation of cytokine production and regulation of B cell activation and proliferation. The caecal microbiota of pigeon &lsquo;milk&rsquo;-fed chickens was significantly more diverse than control chickens, and appears to be affected by prebiotics in pigeon &lsquo;milk&rsquo;, as well as being directly seeded by bacteria present in pigeon &lsquo;milk&rsquo;. Our results demonstrate that pigeon &lsquo;milk&rsquo; has further modes of action which make it functionally similar to mammalian milk. We hypothesise that pigeon &lsquo;lactation&rsquo; and mammalian lactation evolved independently but resulted in similarly functional products

The role of platelets in the recruitment of leukocytes during vascular disease

Besides their role in the formation of thrombus during haemostasis, it is becoming clear that platelets contribute to a number of other processes within the vasculature. Indeed, the integrated function of the thrombotic and inflammatory systems, which results in platelet-mediated recruitment of leukocytes, is now considered to be of great importance in the propagation, progression and pathogenesis of atherosclerotic disease of the arteries. There are three scenarios by which platelets can interact with leukocytes: (1) during haemostasis, when platelets adhere to and are activated on sub-endothelial matrix proteins exposed by vascular damage and then recruit leukocytes to a growing thrombus. (2) Platelets adhere to and are activated on stimulated endothelial cells and then bridge blood borne leukocytes to the vessel wall and. (3) Adhesion between platelets and leukocytes occurs in the blood leading to formation of heterotypic aggregates prior to contact with endothelial cells. In the following review we will not discuss leukocyte recruitment during haemostasis, as this represents a physiological response to tissue trauma that can progress, at least in its early stages, in the absence of inflammation. Rather we will deal with scenarios 2 and 3, as these pathways of platelet–leukocyte interactions are important during inflammation and in chronic inflammatory diseases such as atherosclerosis. Indeed, these interactions mean that leukocytes possess means of adhesion to the vessel wall under conditions that may not normally be permissive of leukocyte–endothelial cell adhesion, meaning that the disease process may be able to bypass the regulatory pathways which would ordinarily moderate the inflammatory response