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1.  Creation of an Open-Access, Mutation-Defined Fibroblast Resource for Neurological Disease Research 
PLoS ONE  2012;7(8):e43099.
Our understanding of the molecular mechanisms of many neurological disorders has been greatly enhanced by the discovery of mutations in genes linked to familial forms of these diseases. These have facilitated the generation of cell and animal models that can be used to understand the underlying molecular pathology. Recently, there has been a surge of interest in the use of patient-derived cells, due to the development of induced pluripotent stem cells and their subsequent differentiation into neurons and glia. Access to patient cell lines carrying the relevant mutations is a limiting factor for many centres wishing to pursue this research. We have therefore generated an open-access collection of fibroblast lines from patients carrying mutations linked to neurological disease. These cell lines have been deposited in the National Institute for Neurological Disorders and Stroke (NINDS) Repository at the Coriell Institute for Medical Research and can be requested by any research group for use in in vitro disease modelling. There are currently 71 mutation-defined cell lines available for request from a wide range of neurological disorders and this collection will be continually expanded. This represents a significant resource that will advance the use of patient cells as disease models by the scientific community.
PMCID: PMC3428297  PMID: 22952635
2.  Whole-exome sequencing of DNA from peripheral blood mononuclear cells (PBMC) and EBV-transformed lymphocytes from the same donor 
BMC Genomics  2011;12:464.
The creation of lymphoblastoid cell lines (LCLs) through Epstein-Barr virus (EBV) transformation of B-lymphocytes can result in a valuable biomaterial for cell biology research and a renewable source of DNA. While LCLs have been used extensively in cellular and genetic studies, the process of cell transformation and expansion during culturing may introduce genomic changes that may impact their use and the interpretation of subsequent genetic findings.
We performed whole exome sequencing on a tetrad family using DNA derived from peripheral blood mononuclear cells (PBMCs) and LCLs from each individual. We generated over 4.7 GB of mappable sequence to a 125X read coverage per sample. An average of 19,354 genetic variants were identified. Comparison of the two DNA sources from each individual showed an average concordance rate of 95.69%. By lowering the variant calling parameters, the concordance rate between the paired samples increased to 99.82%. Sanger sequencing of a subset of the remaining discordant variants did confirm the presence of de novo mutations arising in LCLs.
By varying software stringency parameters, we identified 99% concordance between DNA sequences derived from the two different sources from the same donors. These results suggest that LCLs are an appropriate representation of the genetic material of the donor and suggest that EBV transformation can result in low-level generation of de novo mutations. Therefore, use of PBMC or early passage EBV-transformed cells is recommended. These findings have broad-reaching implications, as there are thousands of LCLs in public biorepositories and individual laboratories.
PMCID: PMC3203102  PMID: 21943378
3.  Small Intestinal Cannabinoid Receptor Changes Following a Single Colonic Insult with Oil of Mustard in Mice 
Cannabinoids are known to be clinically beneficial for control of appetite disorders and nausea/vomiting, with emerging data that they can impact other GI disorders, such as inflammation. Post-inflammatory irritable bowel syndrome (PI-IBS) is a condition of perturbed intestinal function that occurs subsequent to earlier periods of intestinal inflammation. Cannabinoid 1 receptor (CB1R) and CB2R alterations in GI inflammation have been demonstrated in both animal models and clinically, but their continuing role in the post-inflammatory period has only been implicated to date. Therefore, to provide direct evidence for CBR involvement in altered GI functions in the absence of overt inflammation, we used a model of enhanced upper GI transit that persists for up to 4 weeks after a single insult by intracolonic 0.5% oil of mustard (OM) in mice. In mice administered OM, CB1R immunostaining in the myenteric plexus was reduced at day 7, when colonic inflammation is subsiding, and then increased at 28 days, compared to tissue from age-matched vehicle-treated mice. In the lamina propria CB2R immunostaining density was also increased at day 28. In mice tested 28 day after OM, either a CB1R-selective agonist, ACEA (1 and 3 mg/kg, s.c.) or a CB2R-selective agonist, JWH-133 (3 and 10 mg/kg, s.c.) reduced the enhanced small intestinal transit in a dose-related manner. Doses of ACEA and JWH-133 (1 mg/kg), alone or combined, reduced small intestinal transit of OM-treated mice to a greater extent than control mice. Thus, in this post-colonic inflammation model, both CBR subtypes are up-regulated and there is increased efficacy of both CB1R and CB2R agonists. We conclude that CBR remodeling occurs not only during GI inflammation but continues during the recovery phase. Thus, either CB1R- or CB2-selective agonists could be efficacious for modulating GI motility in individuals experiencing diarrhea-predominant PI-IBS.
PMCID: PMC3134866  PMID: 21779244
colitis; pathophysiology; motility; receptor up-regulation; endocannabinoids; inflammation; cannabinoid receptors; enteric nervous system
4.  Mandatory role of proteinase-activated receptor 1 in experimental bladder inflammation 
BMC Physiology  2007;7:4.
In general, inflammation plays a role in most bladder pathologies and represents a defense reaction to injury that often times is two edged. In particular, bladder neurogenic inflammation involves the participation of mast cells and sensory nerves. Increased mast cell numbers and tryptase release represent one of the prevalent etiologic theories for interstitial cystitis and other urinary bladder inflammatory conditions. The activity of mast cell-derived tryptase as well as thrombin is significantly increased during inflammation. Those enzymes activate specific G-protein coupled proteinase-activated receptors (PAR)s.
Four PARs have been cloned so far, and not only are all four receptors highly expressed in different cell types of the mouse urinary bladder, but their expression is altered during experimental bladder inflammation. We hypothesize that PARs may link mast cell-derived proteases to bladder inflammation and, therefore, play a fundamental role in the pathogenesis of cystitis.
Here, we demonstrate that in addition to the mouse urinary bladder, all four PA receptors are also expressed in the J82 human urothelial cell line. Intravesical administration of PAR-activating peptides in mice leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS), substance P, and antigen was strongly attenuated by PAR1-, and to a lesser extent, by PAR2-deficiency.
Our results reveal an overriding participation of PAR1 in bladder inflammation, provide a working model for the involvement of downstream signaling, and evoke testable hypotheses regarding the role of PARs in bladder inflammation. It remains to be determined whether or not mechanisms targeting PAR1 gene silencing or PAR1 blockade will ameliorate the clinical manifestations of cystitis.
PMCID: PMC1853108  PMID: 17397548
5.  Regulatory network of inflammation downstream of proteinase-activated receptors 
BMC Physiology  2007;7:3.
Protease-activated receptors (PAR) are present in the urinary bladder, and their expression is altered in response to inflammation. PARs are a unique class of G protein-coupled that carry their own ligands, which remain cryptic until unmasked by proteolytic cleavage. Although the canonical signal transduction pathway downstream of PAR activation and coupling with various G proteins is known and leads to the rapid transcription of genes involved in inflammation, the effect of PAR activation on the downstream transcriptome is unknown.
We have shown that intravesical administration of PAR-activating peptides leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS), substance P (SP), and antigen was strongly attenuated by PAR1- and to a lesser extent by PAR2-deficiency.
Here, cDNA array experiments determined inflammatory genes whose expression is dependent on PAR1 activation. For this purpose, we compared the alteration in gene expression in wild type and PAR1-/- mice induced by classical pro-inflammatory stimuli (LPS, SP, and antigen). 75 transcripts were considered to be dependent on PAR-1 activation and further annotated in silico by Ingenuity Pathways Analysis (IPA) and gene ontology (GO). Selected transcripts were target validated by quantitative PCR (Q-PCR). Among PAR1-dependent transcripts, the following have been implicated in the inflammatory process: b2m, ccl7, cd200, cd63, cdbpd, cfl1, dusp1, fkbp1a, fth1, hspb1, marcksl1, mmp2, myo5a, nfkbia, pax1, plaur, ppia, ptpn1, ptprcap, s100a10, sim2, and tnfaip2. However, a balanced response to signals of injury requires a transient cellular activation of a panel of genes together with inhibitory systems that temper the overwhelming inflammation. In this context, the activation of genes such as dusp1 and nfkbia seems to counter-balance the inflammatory response to PAR activation by limiting prolonged activation of p38 MAPK and increased cytokine production. In contrast, transcripts such as arf6 and dcnt1 that are involved in the mechanism of PAR re-sensitization would tend to perpetuate the inflammatory reaction in response to common pro-inflammatory stimuli.
The combination of cDNA array results and genomic networks reveals an overriding participation of PAR1 in bladder inflammation, provides a working model for the involvement of downstream signaling, and evokes testable hypotheses regarding the transcriptome downstream of PAR1 activation.
It remains to be determined whether or not mechanisms targeting PAR1 gene silencing or PAR1 blockade will ameliorate the clinical manifestation of cystitis.
PMCID: PMC1853107  PMID: 17397547
6.  Proteinase-activated receptor-1 (PAR-1) activation contracts the isolated human renal artery in vitro 
British Journal of Pharmacology  2003;139(1):21-27.
The in vitro motor function of protease-activated recepter-1 (PAR-1), PAR-2 and PAR-4 and the presence by immunohistochemistry of PAR-1 in the human renal artery have been investigated.Thrombin and the human PAR-1 (SFLLRN-NH2) activating peptide, but not the PAR-1 reverse peptide (NRLLFS-NH2), contracted both endothelial-intact and endothelial-denuded human renal artery strips, whereas no relaxation was observed either in strips non-precontracted or precontracted with phenylephrine. Maximum contraction by thrombin or SFLLRN-NH2 was about 60% of phenylephrine. However, thrombin was approximately 1000-fold more potent than SFLLRN-NH2.PAR-1 desensitisation, using repeated applications of SFLLRN-NH2, almost completely blocked the response to thrombin. The contractile effect produced by thrombin and SFLLRN-NH2 was not affected by nitric oxide synthase inhibition, but was significantly reduced by cyclooxygenase blockade.Trypsin, the PAR-2 (SLIGKV-NH2 and SLIGRL-NH2) and PAR-4 (GYPGQV-NH2 and AYPGKF-NH2) activating peptides did not produce any significant contraction or relaxation.In agreement with the motor function data immunohistochemistry showed specific staining patterns for PAR-1 in the human renal artery.Combined, these studies would suggest a possible role for PAR-1 in renal vascular homeostasis.
PMCID: PMC1573818  PMID: 12746219
Protease-activated receptor-1 (PAR-1); thrombin; human renal artery
7.  Agonists of proteinase-activated receptor 1 induce plasma extravasation by a neurogenic mechanism 
British Journal of Pharmacology  2001;133(7):975-987.
Thrombin, generated in the circulation during injury, cleaves proteinase-activated receptor 1 (PAR1) to stimulate plasma extravasation and granulocyte infiltration. However, the mechanism of thrombin-induced inflammation in intact tissues is unknown. We hypothesized that thrombin cleaves PAR1 on sensory nerves to release substance P (SP), which interacts with the neurokinin 1 receptor (NK1R) on endothelial cells to cause plasma extravasation.PAR1 was detected in small diameter neurons known to contain SP in rat dorsal root ganglia by immunohistochemistry and in situ hybridization.Thrombin and the PAR1 agonist TFLLR-NH2 (TF-NH2) increased [Ca2+]i >50% of cultured neurons (EC50s 24 mu ml−1 and 1.9 μM, respectively), assessed using Fura-2 AM. The PAR1 agonist completely desensitized responses to thrombin, indicating that thrombin stimulates neurons through PAR1.Injection of TF-NH2 into the rat paw stimulated a marked and sustained oedema. An NK1R antagonist and ablation of sensory nerves with capsaicin inhibited oedema by 44% at 1 h and completely by 5 h.In wild-type but not PAR1−/− mice, TF-NH2 stimulated Evans blue extravasation in the bladder, oesophagus, stomach, intestine and pancreas by 2–8 fold. Extravasation in the bladder, oesophagus and stomach was abolished by an NK1R antagonist.Thus, thrombin cleaves PAR1 on primary spinal afferent neurons to release SP, which activates the NK1R on endothelial cells to stimulate gap formation, extravasation of plasma proteins, and oedema. In intact tissues, neurogenic mechanisms are predominantly responsible for PAR1-induced oedema.
PMCID: PMC1572861  PMID: 11487506
Thrombin; proteinase-activated receptor 1; neurogenic inflammation; sensory nerves; substance P; neurokinin receptors; oedema
8.  Arginine-Specific Protease from Porphyromonas gingivalis Activates Protease-Activated Receptors on Human Oral Epithelial Cells and Induces Interleukin-6 Secretion 
Infection and Immunity  2001;69(8):5121-5130.
Periodontitis is a chronic inflammatory disease affecting oral tissues. Oral epithelial cells represent the primary barrier against bacteria causing the disease. We examined the responses of such cells to an arginine-specific cysteine proteinase (RgpB) produced by a causative agent of periodontal disease, Porphyromonas gingivalis. This protease caused an intracellular calcium transient in an oral epithelial cell line (KB), which was dependent on its enzymatic activity. Since protease-activated receptors (PARs) might mediate such signaling, reverse transcription-PCR was used to characterize the range of these receptors expressed in the KB cells. The cells were found to express PAR-1, PAR-2, and PAR-3, but not PAR-4. In immunohistochemical studies, human gingival epithelial cells were found to express PAR-1, PAR-2, and PAR-3 on their surface, but not PAR-4, indicating that the cell line was an effective model for the in vivo situation. PAR-1 and PAR-2 expression was confirmed in intracellular calcium mobilization assays by treatment of the cells with the relevant receptor agonist peptides. Desensitization experiments strongly indicated that signaling of the effects of RgpB was occurring through PAR-1 and PAR-2. Studies with cells individually transfected with each of these two receptors confirmed that they were both activated by RgpB. Finally, it was shown that, in the oral epithelial cell line, PAR activation by the bacterial protease-stimulated secretion of interleukin-6. This induction of a powerful proinflammatory cytokine suggests a mechanism whereby cysteine proteases from P. gingivalis might mediate inflammatory events associated with periodontal disease on first contact with a primary barrier of cells.
PMCID: PMC98608  PMID: 11447194

Results 1-8 (8)