Is there an ideal way to initiate antiplatelet therapy with aspirin? A crossover study on healthy volunteers evaluating different dosing schemes with whole blood aggregometry

<p>Abstract</p> <p>Background</p> <p>Guidelines recommend an early initiation of aspirin treatment in patients with acute cerebral ischemia. Comparative studies on the best starting dose for initiating aspirin therapy to achieve a rapid antiplatelet effect do not exist. This study evaluated the platelet inhibitory effect in healthy volunteers by using three different aspirin loading doses to gain a model for initiating antiplatelet treatment in acute strokes patients.</p> <p>Methods</p> <p>Using whole blood aggregometry, this study with a prospective, uncontrolled, open, crossover design examined 12 healthy volunteers treated with three different aspirin loading doses: intravenous 500 mg aspirin, oral 500 mg aspirin, and a course of 200 mg aspirin on two subsequent days followed by a five-day course of 100 mg aspirin. Aspirin low response was defined as change of impedance exceeding 0 Ω after stimulation with arachidonic acid.</p> <p>Results</p> <p>Sufficient antiplatelet effectiveness was gained within 30 seconds when intravenous 500 mg aspirin was used. The mean time until antiplatelet effect was 74 minutes for 500 mg aspirin taken orally and 662 minutes (11.2 hours) for the dose scheme with 200 mg aspirin with a high inter- and intraindividual variability in those two regimes. Platelet aggregation returned to the baseline range during the wash-out phase within 4 days.</p> <p>Conclusion</p> <p>Our study reveals that the antiplatelet effect differs significantly between the three different aspirin starting dosages with a high inter- and intraindividual variability of antiplatelet response in our healthy volunteers. To ensure an early platelet inhibitory effect in acute stroke patients, it could be advantageous to initiate the therapy with an intravenous loading dose of 500 mg aspirin. However, clinical outcome studies must still define the best way to initiate antiplatelet treatment with aspirin.</p

Apoptosis Inducing Effect of Plumbagin on Colonic Cancer Cells Depends on Expression of COX-2

Plumbagin, a quinonoid found in the plants of the Plumbaginaceae, possesses medicinal properties. In this study we investigated the anti-proliferative and apoptotic activity of plumbagin by using two human colonic cancer cell lines, HT29 and HCT15. IC50 of Plumbagin for HCT15 and HT29 cells (22.5 µM and 62.5 µM, respectively) were significantly different. To study the response of cancer cells during treatment strategies, cells were treated with two different concentrations, 15 µM, 30 µM for HCT15 and 50 µM, 75 µM for HT29 cells. Though activation of NFκB, Caspases-3, elevated levels of TNF-α, cytosolic Cytochrome C were seen in both HCT15 cells HT29 treated with plumbagin, aberrant apoptosis with decreased level of pEGFR, pAkt, pGsk-3β, PCNA and Cyclin D1was observed only in 15 µM and 30 µM plumbagin treated HCT15 and 75 µM plumbagin treated HT29 cells. This suggests that plumbagin induces apoptosis in both HCT15 cells and HT29 treated, whereas, proliferation was inhibited only in 15 µM and 30 µM plumbagin treated HCT15 and 75 µM plumbagin treated HT29 cells, but not in 50 µM plumbagin treated HT29 cells. Expression of COX-2 was decreased in 75 µM plumbagin treated HT29 cells when compared to 50 µM plumbagin treated HT29 cells, whereas HCT15 cells lack COX. Hence the observed resistance to induction of apoptosis in 50 µM plumbagin treated HT29 cells are attributed to the expression of COX-2. In conclusion, plumbagin induces apoptosis in colonic cancer cells through TNF-α mediated pathway depending on expression of COX-2 expression

An organelle-specific protein landscape identifies novel diseases and molecular mechanisms

Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine

In Silico Prediction and Analysis of Caenorhabditis EF-hand Containing Proteins

Calcium (Ca+2) is a ubiquitous messenger in eukaryotes including Caenorhabditis. Ca+2-mediated signalling processes are usually carried out through well characterized proteins like calmodulin (CaM) and other Ca+2 binding proteins (CaBP). These proteins interact with different targets and activate it by bringing conformational changes. Majority of the EF-hand proteins in Caenorhabditis contain Ca+2 binding motifs. Here, we have performed homology modelling of CaM-like proteins using the crystal structure of Drosophila melanogaster CaM as a template. Molecular docking was applied to explore the binding mechanism of CaM-like proteins and IQ1 motif which is a ∼25 residues and conform to the consensus sequence (I, L, V)QXXXRXXXX(R,K) to serve as a binding site for different EF hand proteins. We made an attempt to identify all the EF-hand (a helix-loop-helix structure characterized by a 12 residues loop sequence involved in metal coordination) containing proteins and their Ca+2 binding affinity in Caenorhabditis by analysing the complete genome sequence. Docking studies revealed that F165, F169, L29, E33, F44, L57, M61, M96, M97, M108, G65, V115, F93, N104, E144 of CaM-like protein is involved in the interaction with IQ1 motif. A maximum of 170 EF-hand proteins and 39 non-EF-hand proteins with Ca+2/metal binding motif were identified. Diverse proteins including enzyme, transcription, translation and large number of unknown proteins have one or more putative EF-hands. Phylogenetic analysis revealed seven major classes/groups that contain some families of proteins. Various domains that we identified in the EF-hand proteins (uncharacterized) would help in elucidating their functions. It is the first report of its kind where calcium binding loop sequences of EF-hand proteins were analyzed to decipher their calcium affinities. Variation in Ca+2-binding affinity of EF-hand CaBP could be further used to study the behaviour of these proteins. Our analyses postulated that Ca+2 is likely to be key player in Caenorhabditis cell signalling

Profiling CpG island field methylation in both morphologically normal and neoplastic human colonic mucosa

Aberrant CpG island (CGI) methylation occurs early in colorectal neoplasia. Quantitative methylation-specific PCR profiling applied to biopsies was used to quantify low levels of CGI methylation of 18 genes in the morphologically normal colonic mucosa of neoplasia-free subjects, adenomatous polyp patients, cancer patients and their tumours. Multivariate statistical analyses distinguished tumour from mucosa with a sensitivity of 78.9% and a specificity of 100% (P=3 × 10−7). In morphologically normal mucosa, age-dependent CGI methylation was observed for APC, AXIN2, DKK1, HPP1, N33, p16, SFRP1, SFRP2 and SFRP4 genes, and significant differences in CGI methylation levels were detected between groups. Multinomial logistic regression models based on the CGI methylation profiles from normal mucosa correctly identified 78.9% of cancer patients and 87.9% of non-cancer (neoplasia-free+polyp) patients (P=4.93 × 10−7) using APC, HPP1, p16, SFRP4, WIF1 and ESR1 methylation as the most informative variables. Similarly, CGI methylation of SFRP4, SFRP5 and WIF1 correctly identified 61.5% of polyp patients and 78.9% of neoplasia-free subjects (P=0.0167). The apparently normal mucosal field of patients presenting with neoplasia has evidently undergone significant epigenetic modification. Methylation of the genes selected by the models may play a role in the earliest stages of the development of colorectal neoplasia

Colorectal Hyperplasia and Dysplasia Due to Human Carcinoembryonic Antigen (CEA) Family Member Expression in Transgenic Mice

CEA and CEACAM6 are immunoglobulin family intercellular adhesion molecules that are up-regulated without structural mutations in approximately 70% of human cancers. Results in in vitro systems showing tumorigenic effects for these molecules suggest that this correlation could indicate an instrumental role in tumorigenesis. To test whether this applies in vivo, transgenic mice harboring 187 kb of the human genome containing four CEA family member genes including the CEA and CEACAM6 genes were created and their copy numbers increased by mating until colonocyte expression levels reached levels seen in human colorectal carcinomas. The colonocyte surface level of integrin α5 and the activation of AKT increased progressively with the expression levels of CEA/CEACAM6. Colonic crypts showed a progressive increase in colonocyte proliferation, an increase in crypt fission, and a strong inhibition of both differentiation and anoikis/apoptosis. All transgenic mice showed massively enlarged colons comprising a continuous mosaic of severe hyperplasia, dysplasia and serrated adenomatous morphology. These results suggest that up-regulated non-mutated adhesion molecules could have a significant instrumental role in human cancer

Activation Status of Wnt/ß-Catenin Signaling in Normal and Neoplastic Breast Tissues: Relationship to HER2/neu Expression in Human and Mouse

Wnt/ß-catenin signaling is strongly implicated in neoplasia, but the role of this pathway in human breast cancer has been controversial. Here, we examined Wnt/ß-catenin pathway activation as a function of breast cancer progression, and tested for a relationship with HER2/neu expression, using a human tissue microarray comprising benign breast tissues, ductal carcinoma in situ (DCIS), and invasive carcinomas. Cores were scored for membranous ß-catenin, a key functional component of adherens junctions, and for nucleocytoplasmic ß-catenin, a hallmark of Wnt/ß-catenin pathway activation. Only 82% of benign samples exhibited membrane-associated ß-catenin, indicating a finite frequency of false-negative staining. The frequency of membrane positivity was similar in DCIS samples, but was significantly reduced in carcinomas (45%, P<0.001), consistent with loss of adherens junctions during acquisition of invasiveness. Negative membrane status in cancers correlated with higher grade (P = 0.04) and estrogen receptor-negative status (P = 0.03), both indices of poor prognosis. Unexpectedly, a substantial frequency of nucleocytoplasmic ß-catenin was observed in benign breast tissues (36%), similar to that in carcinomas (35%). Positive-staining basal nuclei observed in benign breast may identify putative stem cells. An increased frequency of nucleocytoplasmic ß-catenin was observed in DCIS tumors (56%), suggesting that pathway activation may be an early event in human breast neoplasia. A correlation was observed between HER2/neu expression and nucleocytoplasmic ß-catenin in node-positive carcinomas (P = 0.02). Furthermore, cytoplasmic ß-catenin was detected in HER2/neu-induced mouse mammary tumors. The Axin2NLSlacZ mouse strain, a previously validated reporter of mammary Wnt/ß-catenin signaling, was utilized to define in vivo transcriptional consequences of HER2/neu-induced ß-catenin accumulation. Discrete hyperplastic foci observed in mammary glands from bigenic MMTV/neu, Axin2NLSlacZ mice, highlighted by robust ß-catenin/TCF signaling, likely represent the earliest stage of mammary intraepithelial neoplasia in MMTV/neu mice. Our study thus provides provocative evidence for Wnt/ß-catenin signaling as an early, HER2/neu-inducible event in breast neoplasia